U.S. patent application number 13/792414 was filed with the patent office on 2013-11-21 for method for detecting human papillomavirus mrna.
This patent application is currently assigned to Norchip A/S. The applicant listed for this patent is Norchip A/S. Invention is credited to Frank Karlsen.
Application Number | 20130309658 13/792414 |
Document ID | / |
Family ID | 26246916 |
Filed Date | 2013-11-21 |
United States Patent
Application |
20130309658 |
Kind Code |
A1 |
Karlsen; Frank |
November 21, 2013 |
METHOD FOR DETECTING HUMAN PAPILLOMAVIRUS mRNA
Abstract
An in vitro method is provided for screening human female
subjects to assess their risk of developing cervical carcinoma
which comprises screening the subject for expression of mRNA
transcripts from the E6 and optionally the L1 gene of human
papillomavirus, wherein subjects positive for expression of L1
and/or E6 mRNA are scored as being at risk of developing cervical
carcinoma. Kits for carrying out such methods are also
provided.
Inventors: |
Karlsen; Frank;
(Klokkarstua, NO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Norchip A/S |
Klokkarstua |
|
NO |
|
|
Assignee: |
Norchip A/S
Klokkarstua
NO
|
Family ID: |
26246916 |
Appl. No.: |
13/792414 |
Filed: |
March 11, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12483860 |
Jun 12, 2009 |
8420314 |
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13792414 |
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10500832 |
Jan 6, 2005 |
7553623 |
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PCT/GB2003/000034 |
Jan 7, 2003 |
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12483860 |
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Current U.S.
Class: |
435/5 |
Current CPC
Class: |
C12Q 1/708 20130101;
Y10S 435/975 20130101; C12Q 1/6886 20130101; C12Q 2600/16
20130101 |
Class at
Publication: |
435/5 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 7, 2002 |
GB |
0200239.2 |
Jun 19, 2002 |
GB |
0214124.0 |
Claims
1. An in vitro method of screening human subjects to assess their
risk of developing cervical carcinoma, which method comprises
screening the subject for expression of mRNA transcripts of the E6
gene of HPV and sorting the subject into one of two categories of
risk for development of cervical carcinoma based on expression of
E6 mRNA, wherein individuals positive for expression of E6 mRNA are
scored as carrying integrated HPV or a modified episomal HPV genome
and are therefore classified as high risk for development of
cervical carcinoma, whereas individuals negative for expression of
E6 mRNA are scored as not carrying integrated HPV or a modified
episomal HPV genome and are therefore classified as no detectable
risk for development of cervical carcinoma.
2. An in vitro method of identifying human subjects having abnormal
cell changes in the cervix, which method comprises screening the
subject for expression of mRNA transcripts of the E6 gene of HPV,
wherein individuals positive for expression of E6 mRNA are
identified as having abnormal cell changes in the cervix.
3. A method according to claim 1 wherein the human subjects are
subjects previously identified as infected with human
papillomavirus DNA in cells of the cervix.
4. A method according to claim 1 wherein the human subjects are
subjects having a previous diagnosis ASCUS, CIN 1 lesions or
condyloma.
5. A method according to claim 1 wherein individuals positive for
expression of E6 mRNA from at least one of HPV types 16, 18, 31, 33
or 45 are scored as carrying integrated HPV.
6. A method according to claim 1 which comprises screening for E6
mRNA expression using a technique which is able to detect E6 mRNA
from at least one cancer-associated HPV type,
7. A method according to claim 1 which comprises screening for E6
mRNA expression using a technique which is able to detect E6 mRNA
from HPV types 16, 18, 31, 33, and 45.
8. A method according to claim 1 wherein screening for E6 mRNA
expression is carried using an amplification reaction to amplify of
a region of the mRNA, together with real-time detection of the
products of the amplification reaction.
9. A method according to claim 1 wherein screening for E6 mRNA
expression is carried using real-time NASBA.
10. A kit for use in the detection of mRNA transcripts of the E6
genes of HPV, the kit comprising primer-pairs which enable
amplification of a region of E6 transcripts from HPV types 16, 18,
31, 33, and 45.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/483,860, filed Jun. 12, 2009, and now pending, which is a
continuation of U.S. application Ser. No. 10/500,832, filed Jan. 6,
2005, and now U.S. Pat. No. 7,553,623, which is a national stage
filing under 35 U.S.C. .sctn.371 of PCT International application
PCT/GB2003/000034, filed Jan. 7, 2003, which was published under
PCT Article 21(2) in English.
FIELD OF THE INVENTION
[0002] The present invention relates to in vitro methods of
screening human subjects in order to assess their risk of
developing cervical carcinoma.
BACKGROUND TO THE INVENTION
[0003] Cervical carcinoma is one of the most common malignant
diseases world-wide and is one of the leading causes of morbidity
and mortality among women (Parkin D M, Pisani P, Ferlay J (1993)
Int J Cancer 54: 594-606; Pisani P, Parkin D M, Ferlay J (1993) Int
J Cancer 55: 891-903). 15,700 new cases of invasive cervical cancer
were predicted in the United States in 1996, and the annual
world-wide incidence is estimated to be 450,000 by the World Health
Organization (1990). The annual incidence rate differs in different
parts of the world, ranging from 7.6 per 100,000 in western Asia to
46.8 per 100,000 in southern Africa (Parkin et al., 1993 ibid).
[0004] The current conception of cervical carcinoma is that it is a
multistage disease, often developing over a period of 10-25 years.
Invasive squamous-cell carcinoma of the cervix is represented by
penetration through the basal lamina and invading the stroma or
epithelial lamina propria. The clinical course of cervical
carcinoma shows considerable variation. Prognosis has been related
to clinical stage, lymph node involvement, primary tumour mass,
histology type, depth of invasion and lymphatic permeation (Delgado
G, et al., (1990) Gynecol Oncol 38: 352-357). Some patients with
less favourable tumour characteristics have a relatively good
outcome, while others suffer a fatal outcome of an initially
limited disease. This shows a clear need for additional markers to
further characterise newly diagnosed cervical carcinomas, in order
to administer risk-adapted therapy (Ikenberg H, et al., Int. J.
Cancer 59:322-6. 1994).
[0005] The epidemiology of cervical cancer has shown strong
association with religious, marital and sexual patterns. Almost 100
case-control studies have examined the relationship between HPV and
cervical neoplasia and almost all have found positive associations
(IARC monographs, 1995). The association is strong, consistent and
specific to a limited number of viral types (Munoz N, Bosch F X
(1992) HPV and cervical neoplasia: review of case-control and
cohort studies. IARC Sci Publ 251-261). Among the most informative
studies, strong associations with HPV 16 DNA have been observed
with remarkable consistency for invasive cancer and high-grade CIN
lesions, ruling out the possibility that this association can be
explained by chance, bias or confounding (IARC monographs, 1995).
Indirect evidence suggested that HPV DNA detected in cancer cells
is a good marker for the role of HPV infection earlier in the
carcinogenesis. Dose-response relationship has been reported
between increasing viral load and risk of cervical carcinoma (Munoz
and Bosch, 1992 ibid). In some larger series up to 100% of the
tumours were positive for HPV but the existence of virus-negative
cervical carcinomas is still debatable (Meijer C J, et al., (1992)
Detection of human papillomavirus in cervical scrapes by the
polymerase chain reaction in relation to cytology: possible
implications for cervical cancer screening. IARC Sci Publ 271-281;
Das B C, et al., (1993) Cancer 72: 147-153).
[0006] The most frequent HPV types found in squamous-cell cervical
carcinomas are HPV 16 (41%-86%) and 18 (2%-22%). In addition HPV
31, 33, 35, 39, 45, 51, 52, 54, 56, 58, 59, 61, 66 and 68 are also
found (IARC, monographs, 1995). In the HPV2000 International
conference in Barcelona HPV 16, 18, 31 and 45 were defined as high
risk, while HPV 33, 35, 39, 51, 52, 56, 58, 59, 68 were defined as
intermediate risk (Keerti V. Shah. P71). The 13 high risk plus
intermediate risk HPVs are together often referred to as
cancer-associated HPV types.
[0007] A number of studies have explored the potential role of HPV
testing in cervical screening (see Cuzick et al. A systematic
review of the role of human papillomavirus testing withing a
cervical screening programme. Health Technol Assess 3:14.
1999).
[0008] Reid et al., (Reid R, et al., (1991) Am J Obstet Gynecol
164: 1461-1469) where the first to demonstrate a role for HPV
testing in a screening context. This study was carried out on
high-risk women from sexually transmitted disease clinics and
specialist gynaecologists, and used a sensitive (low stringency)
Southern blot hybridisation for HPV detection. A total of 1012
women were enrolled, and cervicography was also considered as a
possible adjunct to cytology. Twenty-three CIN II/III lesions were
found altogether, but only 12 were detected by cytology
(sensitivity 52%, specificity 92%). HPV testing found 16 high-grade
lesions.
[0009] Bauer et al. (Bauer H M, et al., (1991) JAMA 265: 472-477)
report an early PCR-based study using MY09/11 primers (Manos M, et
al., (1990) Lancet 335: 734) in young women attending for routine
smears (college students). They found a positive rate of 46% in 467
women, which was much higher than for dot blot assay (11%).
[0010] In a study using PCR with GP5/6 primers (Van Den Brule A J,
et al., (1990) J Clin Microbiol 28: 2739-2743) van der Brule et al.
(Van Den Brule A J, et al., (1991) Int J Cancer 48: 404-408) showed
a very strong correlation of HPV positivity with cervical neoplasia
as assessed by cytology. In older women (aged 35-55 years) with
negative cytology the HPV positivity rate was only 3.5%, and this
was reduced to 1.5% if only types 16, 18, 31 and 33 were
considered, while women with histological carcinoma in situ were
all HPV-positive, and 90% had one of the four above types. Women
with less severe cytological abnormalities had lower HPV positivity
rates in a graded way, showing a clear trend.
[0011] Roda Housman et al. (Roda Housman A M, et al., (1994) Int J
Cancer 56: 802-806) expanded these observations by looking at a
further 1373 women with abnormal smears. This study also confirmed
increasing positivity rate with increasing severity of smear
results. They also noted that the level of HPV heterogeneity
decreased from 22 types for low-grade smears to ten "high-risk"
types for high grade smears. This paper did not include any
cytologically negative women, nor was cytological disease confirmed
histologically.
[0012] Cuzick et al. (Cuzick J, et al., (1992) Lancet 340: 112-113;
Cuzick J, et al., (1994) Br J Cancer 69: 167-171) were the first to
report that HPV testing provided useful information for the triage
of cytological abnormalities detected during random screening. In a
study of 133 women, referral for coloposcopy they found a positive
predictive value of 42%, which was similar to that for moderate
dyskaryosis. The results were most striking for HPV 16, where 39 of
42 HPV 16 positive women were found to have high-grade CIN on
biopsy. This study pointed out the importance of assessing viral
load and only considered high levels of high-risk types as
positive.
[0013] Cox et al. (Cox J T, et al., (1995) Am J Obstet Gynecol 172:
946-954) demonstrated a role for HPV testing using the Hybrid
Capture.TM. system (DIGENE Corporation, Gaithersburg, Md., USA) for
triaging women with borderline smears. This test was performed on
217 such women from a college referral service, and a sensitivity
of 93% was found for CINII/III compared with 73% for repeat
cytology. High viral load was found to further improve performance
by reducing false positives. When 5 RLU was taken as a cut-off, a
PPV of approximately 24% was found with no loss of sensitivity.
[0014] Cuzick et al. (Cuzick J, et al., (1995) Lancet 345:
1533-1536) evaluated HPV testing in a primary screening context in
1985 women attending for routine screening at a family planning
clinic. Sensitivity using type-specific PCR for the four common HPV
types (75%) exceeded that of cytology (46%), and the PPV for a
positive HPV test (42%) was similar to that for moderate
dyskaryosis (43%).
[0015] WO 91/08312 describes methods for determining the prognosis
of individuals infected with HPV which comprise measuring the level
of HPV activity by detecting transcripts of all or a portion of the
E6 and/or E7 HPV genes in a sample and comparing the measurements
of HPV activity with a previously established relationship between
activity and risk of progression to serious cervical dysplasia or
carcinoma.
[0016] WO 99/29890 describes methods for the assessment of HPV
infection based on the measurement and analysis of gene expression
levels. In particular, WO 99/29890 describes methods which are
based on measuring the levels of expression of two or more HPV
genes (e.g. HPV E6, E7, L1 and E2) and then comparing the ratio of
expression of combinations of these genes to provide an indication
of the stage of HPV-based disease in a patient.
[0017] The present inventors have determined that it is possible to
make a clinically useful assessment of HPV-associated disease based
only on a simple positive/negative determination of expression of
HPV L1 and E6 mRNA transcripts, with no requirement for accurate
quantitative measurements of expression levels or for determination
of differences in the levels of expression of the two transcripts.
This method is technically simple and, in a preferred embodiment,
is amenable to automation in a mid-to-high throughput format.
Furthermore, on the basis of results obtained using the method of
the invention the inventors have defined a novel scheme for
classification of patients on the basis of risk of developing
cervical carcinoma which is related to disease-relevant molecular
changes in the pattern of HPV gene expression and is independent of
CIN classification.
[0018] Therefore, in a first aspect the invention provides an in
vitro method of screening human subjects to assess their risk of
developing cervical carcinoma which comprises screening for
expression of mRNA transcripts from the L1 gene and the E6 gene of
human papillomavirus, wherein subjects positive for expression of
L1 and/or full length E6 mRNA are scored as being at risk of
developing cervical carcinoma.
[0019] A positive screening result in the method of the invention
is indicated by positive expression of L1 mRNA and/or E6 mRNA in
cells of the cervix. Positive expression of either one of these
mRNAs or both mRNAs is taken as an indication that the subject is
"at risk" for development of cervical carcinoma. Women who express
E6 mRNA are at high risk of developing cell changes because
oncogenic E6 and E7 bind to cell cycle regulatory proteins and act
as a switch for cell proliferation. Clear expression of E6 mRNA
provides a direct indication of cell changes in the cervix.
Expression of L1 mRNA, with or without expression of E6 mRNA is
also indicative of the presence of an active HPV.
[0020] In the wider context of cervical screening, women identified
as positive for L1 and/or E6 mRNA expression may be selected for
further investigation, for example using cytology. Thus, at one
level the method of the invention may provide a technical simple
means of pre-screening a population of women in order to identify
HPV-positive subjects who may be selected for further
investigation.
[0021] In a specific embodiment, the method of the invention may be
used to classify subjects into four different classes of risk for
developing cervical carcinoma on the basis of positive/negative
scoring of expression of L1 and E6 mRNA.
[0022] Accordingly, in a further aspect the invention provides an
in vitro method of screening human subjects to assess their risk of
developing cervical carcinoma which comprises screening the subject
for expression of mRNA transcripts of the L1 gene of HPV and mRNA
transcripts of the E6 gene of HPV, and sorting the subject into one
of four categories of risk for development of cervical carcinoma
based on expression of L1 and/or E6 mRNA according to the following
classification:
Risk category 1: subjects negative for expression of L1 mRNA but
positive for expression of E6 mRNA from at least one of HPV types
16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 66 or 68. Those
individuals positive for expression of E6 mRNA from at least one of
HPV types 16, 18, 31 or 33 are scored as being at higher risk, for
example in comparison to individuals negative for these types but
positive for expression of E6 mRNA from at least one of HPV types
35, 39, 45, 52, 56, 58, 59, 66 or 68. Risk category 2: subjects
positive for expression of L1 mRNA and positive for expression of
E6 mRNA from at least one of HPV types 16, 18, 31, 33, 35, 39, 45,
52, 56, 58, 59, 66 or 68. Those individuals positive for expression
of E6 mRNA from at least one of HPV types 16, 18, 31 or 33 are
scored as being at higher risk, for example in comparison to
individuals negative for these types but positive for expression of
E6 mRNA from at least one of HPV types 35, 39, 45, 52, 56, 58, 59,
66 or 68. Risk category 3: subjects positive for expression of L1
mRNA but negative for expression of E6 mRNA from the
cancer-associated HPV types, (e.g. negative for expression of E6
mRNA from HPV types 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 66
and 68). Risk category 4: subjects negative for expression of L1
mRNA and negative for expression of E6 mRNA.
[0023] In a preferred embodiment, positive expression is indicated
by the presence of more than 50 copies of the transcript per ml (or
total volume of the sample) and negative expression is indicated by
the presence of less than 1 copy of the transcript per ml (or total
volume of the sample).
[0024] The above classification is based on molecular events which
are relevant to risk of developing cervical carcinoma and is
independent of the CIN status of the subjects. Thus, this method of
classification may provide an alternative to the use of cytology in
the routine screening of women to identify those at potential risk
of developing cervical carcinoma. The method may also be used as an
adjunct to cytology, for example as a confirmatory test to confirm
a risk assessment made on the basis of cytology.
[0025] Women positive for expression of high risk E6 mRNA from one
of HPV types 16, 18, 31 or 33 but negative for expression of L1 are
in the highest level of risk of developing severe cell changes and
cell abnormalities. This is due to the fact that a negative result
for L1 mRNA expression is directly indicative of integrated HPV,
and therefore a higher probability of high and constant expression
of E6 and E7. Integration of a virus in the human genome has also a
direct impact on the stability of the cells. Integration of HPV
also reduces the possibility of regression of cell changes.
[0026] Women positive for expression of E6 mRNA from one of HPV
types 16, 18, 31 or 33 and positive for expression of L1 mRNA have
a "high risk" HPV expression and it is still possible that the HPV
has been integrated. However, the risk of these women is not
classed as high as those who are L1 negative and E6 positive, since
there is a reasonable probability that they do not have integrated
HPV.
[0027] Women negative for expression of E6 mRNA from HPV types 16,
18, 31 or 33 but positive for expression of E6 mRNA from another
HPV type, e.g. 35, 39, 45, 52, 56, 58, 59, 66 and 68, are still
considered "at risk" and may therefore be placed in risk categories
1 or 2 (as defined above) depending on whether they are positive or
negative for expression of L1 mRNA.
[0028] Women positive for L1 mRNA but negative for E6 mRNA are
scored as being at moderate risk. There may be high-risk HPV types
in the sample and L1 expression is indicative of lytic activity.
There may also be integrated HPV types but only with viruses that
are rare. However, detection of lytic activity may show that the
cell may soon develop some changes.
[0029] In the wider context of cervical screening the method of the
invention may be used to classify women according to risk of
developing cervical carcinoma and therefore provide a basis for
decisions concerning treatment and/or further screening. By way of
example: women in risk category 1, particularly those who exhibit
positive expression of E6 mRNA from at least one of HPV types 16,
18, 31 or 33, might be identified as requiring "immediate action",
meaning conisation or colposcopy, including a biopsy and
histology.
[0030] Women in risk category 2, as defined above, might be scored
as requiring immediate attention, meaning colposcopy alone or
colposcopy including a biopsy and histology.
[0031] Women in risk category 3, as defined above, might be scored
as requiring immediate re-test, meaning recall for a further test
for HPV expression immediately or after a relatively short
interval, e.g. six months.
[0032] Women in risk category 4, as defined above, might be
returned to the screening program, to be re-tested for HPV
expression at a later date.
[0033] In a further embodiment the invention provides an in vitro
method of screening human subjects for the presence of integrated
HPV or a modified episomal HPV genome, which method comprises
screening the subject for expression of mRNA transcripts from the
L1 gene and the E6 gene of human papillomavirus, wherein subjects
negative for expression of L1 mRNA but positive for expression of
E6 mRNA are scored as carrying integrated HPV.
[0034] The term "integrated HPV" refers to an HPV genome which is
integrated into the human genome.
[0035] The term "modified episomal HPV genome" is taken to mean an
HPV genome which is retained within a cell of the human subject as
an episome, i.e. not integrated into the human genome, and which
carries a modification as compared to the equivalent wild-type HPV
genome, which modification leads to constitutive or persistent
expression of transcripts of the E6 and/or E7 genes. The
"modification" will typically be a deletion, a multimerisation or
concatermerisation of the episome, a re-arrangement of the episome
etc affecting the regulation of E6/E7 expression.
[0036] As aforesaid, the presence of integrated HPV or a modified
episomal HPV genome is indicated by a negative result for L1 mRNA
expression, together with a positive result for expression of E6
mRNA in cells of the cervix. Therefore, the ability to predict the
presence of integrated HPV or a modified episomal HPV genome in
this assay is critically dependent on the ability to score a
negative result for L1 mRNA expression. This requires a detection
technique which has maximal sensitivity, yet produces minimal
false-negative results. In a preferred embodiment this is achieved
by using a sensitive amplification and real-time detection
technique to screen for the presence or absence of L1 mRNA. The
most preferred technique is real-time NASBA amplification using
molecular beacons probes, as described by Leone et al., Nucleic
Acids Research., 1998, Vol 26, 2150-2155. Due to the sensitivity of
this technique the occurrence of false-negative results is
minimised and a result of "negative L1 expression" can be scored
with greater confidence.
[0037] In a further embodiment, a method of screening human
subjects for the presence of integrated HPV or a modified episomal
HPV genome may be based on screening for expression of E6 mRNA
alone. Thus, the invention relates to an in vitro method of
screening human subjects for the presence of integrated HPV or a
modified episomal HPV genome, which method comprises screening the
subject for expression of mRNA transcripts from the E6 gene of
human papillomavirus, wherein subjects positive for expression of
E6 mRNA are scored as carrying integrated HPV or a modified
episomal HPV genome.
[0038] Moreover, individuals may be sorted into one of two
categories of risk for development of cervical carcinoma based on
an "on/off" determination of expression of E6 mRNA alone.
Therefore, the invention provides an in vitro method of screening
human subjects to assess their risk of developing cervical
carcinoma, which method comprises screening the subject for
expression of mRNA transcripts of the E6 gene of HPV and sorting
the subject into one of two categories of risk for development of
cervical carcinoma based on expression of E6 mRNA, wherein
individuals positive for expression of E6 mRNA are scored as
carrying integrated HPV or a modified episomal HPV genome and are
therefore classified as "high risk" for development of cervical
carcinoma, whereas individuals negative for expression of E6 mRNA
are scored as not carrying integrated HPV or a modified episomal
HPV genome and are therefore classified as "no detectable risk" for
development of cervical carcinoma.
[0039] Subjects are sorted into one of two categories of risk for
development of cervical carcinoma based on an "on/off"
determination of expression of E6 mRNA in cells of the cervix.
Individuals positive for expression of E6 mRNA are scored as
carrying integrated HPV or a modified episomal HPV genome and are
therefore classified "high risk" for development of cervical
carcinoma, whereas individuals negative for expression of E6 mRNA
are scored as not carrying integrated HPV a modified episomal HPV
genome and are therefore classified as "no detectable risk" for
development of cervical carcinoma.
[0040] In the context of cervical screening classification of
subjects into the two groups having "high risk" or "no detectable
risk" for development of cervical carcinoma provides a basis for
decisions concerning treatment and/or further screening. For
example subjects in the high risk category may be scored as
requiring immediate further analysis, e.g. by histological
colposcopy, whilst those in the no detectable risk category may be
referred back to the screening program at three or five year
intervals.
[0041] These methods are particularly useful for assessing risk of
developing carcinoma in subjects known to be infected with HPV,
e.g. those testing positive for HPV DNA, or subjects who have
previously manifested a cervical abnormality via cytology or pap
smear. Subjects placed in the "no detectable risk" category on the
basis of E6 mRNA expression may have HPV DNA present but the
negative result for E6 expression indicates that HPV is unrelated
to oncogene activity at the time of testing.
[0042] The presence of integrated HPV or a modified episomal HPV
genome, as indicated by a positive result for E6 mRNA expression,
is itself indicative that the subject has abnormal cell changes in
the cervix. Therefore, the invention also relates to an in vitro
method of identifying human subjects having abnormal cell changes
in the cervix, which method comprises screening the subject for
expression of mRNA transcripts of the E6 gene of HPV, wherein
individuals positive for expression of E6 mRNA are identified as
having abnormal cell changes in the cervix.
[0043] The term "abnormal cell changes in the cervix" encompasses
cell changes which are characteristic of more severe disease than
low-grade cervical lesions or low squamous intraepithelial lesions,
includes cell changes which are characteristic of disease of equal
or greater severity than high-grade CIN (defined as a neoplastic
expansion of transformed cells), CIN (cervical intraepithelial
neoplasia) III, or high squamous intraepithelial neoplasia (HSIL),
including lesions with multiploid DNA profile and "malignant" CIN
lesions with increased mean DNA-index values, high percentage of
DNA-aneuploidy and 2.5 c Exceeding Rates (Hanselaar et al., 1992,
Anal Cell Pathol., 4:315-324; Rihet et al., 1996, J. Clin Pathol
49:892-896; and McDermott et al., 1997, Br. J. Obstet. Gynaecol.
104:623-625).
[0044] Cervical Intraepithelial Neoplasia (abbreviated "CIN"), also
called Cervical Dysplasia, is a cervical condition caused Human
Papilloma Virus. CIN is classified as I, II or III depending on its
severity. It is considered a pre-cancerous abnormality, but not an
actual cancer. The mildest form, CIN I, usually goes away on its
own, although rarely it can progress to cancer. The more severe
forms, CIN II and CIN III, most often stay the same or get worse
with time. They can become a cancer, but almost never do if treated
adequately.
[0045] HPV has been identified as a causative agent in development
of cellular changes in the cervix, which may lead to the
development of cervical carcinoma. These cellular changes are
associated with constitutive or persistent expression of E6/E7
proteins from the HPV viral genome. Thus, it is possible to
conclude that subjects in which expression of E6 mRNA can be
detected, particularly those subjects who exhibit persistent E6
expression when assessed over a period of time, already manifest
cellular changes in the cervix. These changes may have taken place
in only a very few cells of the cervix, and may not be detectable
by conventional cytology. Nevertheless, with the use of sensitive,
specific and accurate methods for detection of E6 mRNA it is
possible to identify those subjects who already exhibit cellular
changes in the cervix at a much earlier stage than would be
possible using conventional cytological screening. This will allow
earlier intervention with treatments aimed at preventing the
development of cervical carcinoma.
[0046] As a result of HPV integration into the human genome or as a
result of the "modification" in a modified episomal HPV genome,
normal control of the viral E6/E7 oncogene transcription is lost
(Durst et al., 1985, J Gen Virol, 66(Pt 7): 1515-1522; Pater and
Pater, 1985 Virology 145:313-318; Schwarz et al., 1985, Nature 314:
111-114; Park et al., 1997, ibid). In contrast, in premalignant
lesions and HPV-infected normal epithelium papillomaviruses
predominate in "unmodified" episomal forms, hence oncogene (E6/E7)
transcription may be absent or efficiently down-regulated (Johnson
et al., 1990, J Gen Virol, 71(Pt 7): 1473-1479; Falcinelli et al.,
1993, J Med Virol, 40: 261-265). Integration of human
papillomavirus type 16 DNA into the human genome is observed to
lead to a more unstable cell activity/genome, and increased
stability of E6 and E7 mRNAs (Jeon and Lambert, 1995, Proc Natl
Acad Sci USA 92: 1654-1658). Thus HPV integration, typically found
in cervical cancers but only infrequently found in CIN lesions
(Carmody et al., 1996, Mol Cell Probes, 10: 107-116), appears to be
an important event in cervical carcinogenesis.
[0047] The present methods detect E6/E7 viral mRNA expression in
the cervix instead of DNA. E6/E7 viral expression in cervical cells
is a much more accurate assessment of the risk of developing cancer
than simply showing that the HPV virus is present. Furthermore, the
detection of HPV oncogene transcripts may be a more sensitive
indicator of the direct involvement of viral oncogenes in
carcinogenesis (Rose et al., 1994, Gynecol Oncol, 52: 212-217; Rose
et al., 1995, Gynecol Oncol, 56: 239-244). Detection of E6/E7
transcripts by amplification and detection is a useful diagnostic
tool for risk evaluations regarding the development of CIN and its
progression to cervical cancer, especially in high-risk HPV
type-infected patients with ASCUS and CIN I (Sotlar et al., 1998,
Gynecol Oncol, 69: 114-121; Selinka et al., 1998, Lab Invest, 78:
9-18).
[0048] The expression of E6/E7 transcripts of HPV-16/18 is
uniformly correlated with the physical status of HPV DNAs (Park et
al., 1997, Gynecol Oncol, Vol:65(1), 121-9). In most cervical
carcinoma cells the E6 and E7 genes of specific human
papillomaviruses are transcribed from viral sequences integrated
into host cell chromosomes (von Kleben Doeberitz et al., 1991, Proc
Natl Acad Sci USA. Vol:88(4), 1411-5). Viral load and integration
has been evaluated in a large series of CIN lesions (Pietsaro et
al., 2002, J Clin Microbiol, Vol:40(3), 886-91). Only one sample
contained exclusively episomal HPV16 DNA, and this lesion regressed
spontaneously. Seventeen of 37 invasive cervical carcinoma samples
were identified previously as containing the completely integrated
HPV 16 genome by using PCR covering the entire E1/E2 gene, and this
was confirmed by rliPCR in 16 cases. One case, however, showed a
low level of episomal deoxyribonucleic acid in addition to the
predominant integrated form. Of the remaining 20 carcinoma samples
showing episomal forms in the previous analysis, 14 were found to
contain integrated forms using rliPCR, and four contained
multimeric (modified) episomal forms. Thus, in total, 31 of 37 of
the carcinomas (84%) showed integrated HPV16 genome, while absence
of integration could not be detected. (Kalantari et al., 2001,
Diagn Mol Pathol, Vol:10(1), 46-54).
[0049] There have been virtually no observations that cervical
carcinoma cells exist without integrated HPV or modified episomal
HPV DNA (Kalantari et al. 2001; Pietsaro et al., 2002, ibid). It
has further been shown that E6 and E7 may only be transcribed from
integrated or modified episomal HPV DNA (von Kleben Doeberitz et
al., 1991, ibid). Therefore, the inventors surmise that detection
of E6/E7 expression provides a direct indication of integrated HPV
or modified episomal HPV and high oncogene activity, and conclude
that in a clinical context detection of E6 (E6/E7) expression alone
is sufficient to identify subjects at "high risk" of developing
cervical carcinoma. In other words, if E6/E7 mRNA expression can be
detected in a cervical sample, this is directly indicative of
cellular abnormalities in the cervix and there is a very high risk
of development of cervical carcinoma due to persistent HPV oncogene
activity. Therefore, detection of E6/E7 mRNA in a human subject
indicates that the subject has a very high risk of developing
cervical carcinoma and should undergo immediate further screening,
e.g. by colposcopy.
[0050] If HPV E6/E7 mRNA expression is not detected, the subject
may still have an HPV infection. However due to absence of
integration and oncogene activity, it may regress spontaneously (as
observed by Pietsaro et al., 2002, ibid).
[0051] In a clinical context the performance of methods which rely
on screening for expression of E6 mRNA alone is critically
dependent on the ability to score a negative result for E6 mRNA
expression with confidence. This again requires a detection
technique which has maximal sensitivity, yet produces minimal
false-negative results. In a preferred embodiment this is achieved
by using a sensitive amplification and real-time detection
technique to screen for the presence or absence of E6 mRNA. The
most preferred technique is real-time NASBA amplification using
molecular beacons probes, as described by Leone et al., Nucleic
Acids Research., 1998, Vol 26, 2150-2155. Due to the sensitivity of
this technique the occurrence of false-negative results is
minimised and a result of "negative E6 expression" can be scored
with greater confidence. This is extremely important if the assays
are to be used in the context of a clinical screening program.
[0052] In the methods based on detection of E6 mRNA alone it is
preferred to detect at least types HPV 16, 18, 31, 33 and 45, and
in a preferred embodiment the assay may detect only these HPV
types. DNA from HPV types 16, 18, 31 and 33 has been detected in
more than 87% of cervical carcinoma samples (Karlsen et al., 1996,
J Clin Microbiol, 34:2095-2100). Other studies have shown that E6
and E7 are almost invariably retained in cervical cancers, as their
expression is likely to be necessary for conversion to and
maintenance of the malignant state (Choo et al., 1987, J Med Virol
21:101-107; Durst et al., 1995, Cancer Genet Cytogenet, 85:
105-112). In contrast to HPV detection systems which are based on
detection of the undamaged genome or the L1 gene sequence,
detection of HPV mRNA expressed from the E6/E7 area may detect more
than 90% of the patients directly related to a risk of developing
cervical carcinoma.
[0053] In the clinic, methods based on detection of E6 mRNA are
preferred for use in post-screening, i.e. further analysis of
individuals having a previous diagnosis of ASCUS, CIN 1 or
Condyloma. The method may be used to select those with a high risk
of developing cervical carcinoma from amongst the group of
individuals having a previous diagnosis of ASCUS, CIN 1 or
Condyloma. ASCUS, Condyloma and CIN I may be defined as more or
less the same diagnosis due to very low reproducibility between
different cytologists and different cytological departments. Ostor
(Int J. Gyn Path. 12:186-192. 1993) found that only around 1% of
the CIN 1 cases may progress to cervical carcinoma. Thus, there is
a genuine need for an efficient method of identifying the subset of
individuals with ASCUS, Condyloma or CIN I who are at substantial
risk of developing cervical carcinoma. One of HPV types 16, 18, 31
or 33 was detected in 87% of the cervical carcinoma cases study by
Karlsen et al., 1996. By inclusion of HPV 45, nearly 90% of the
cervical carcinoma samples are found to be related to these five
HPV types. Therefore, calculated from the data provided by Ostor
(Int J. Gyn Path. 12:186-192. 1993) more than 99.9% are detected
cases with ASCUS, CIN I or condyloma are missed by our HPV-Proofer
kit.
[0054] In the methods of the invention "positive expression" of an
mRNA is taken to mean expression above background. There is no
absolute requirement for accurate quantitative determination of the
level of mRNA expression or for accurate determination of the
relative levels of expression of L1 and E6 mRNA.
[0055] In certain embodiments, the methods of the invention may
comprise a quantitative determination of levels of mRNA expression.
In a preferred embodiment in order to provide a clear distinction
between "positive expression" and "negative expression" a
determination of "positive expression" may require the presence of
more than 50 copies of the relevant mRNA (per ml of sample or per
total volume of sample), whereas a determination of "negative
expression" may require the presence of less than 1 copy of the
relevant mRNA (per ml of sample or per total volume of sample).
[0056] The methods of the invention will preferably involve
screening for E6 mRNA using a technique which is able to detect
specifically E6 mRNA from cancer-associated HPV types, more
preferably "high risk" cancer-associated HPV types. In the most
preferred embodiment the methods involve screening for E6 mRNA
using a technique which is able to detect E6 mRNA from HPV types
16, 18, 31 and 33, and preferably also 45. Most preferably, the
method will specifically detect expression of E6 mRNA from at least
one of HPV types 16, 18, 31, 33, and preferably also 45, and most
preferably all five types. However, women positive for positive for
expression of E6 from other types than 16, 18, 31, 33 and 45, e.g.
35, 39, 45, 52, 56, 58, 59, 66 and 68 may still be "at risk" of
developing cervical carcinoma. Thus, the method may encompass
screening for expression of E6 mRNA from one or more of these HPV
types, most preferably in addition to screening for E6 mRNA from
HPV types 16, 18, 31, 33 and 45. Certain HPV types exhibit a marked
geographical/population distribution. Therefore, it may be
appropriate to include primers specific for an HPV type known to be
prevalent in the population/geographical area under test, for
example in addition to screening for HPV types 16, 18, 31, 33 and
45.
[0057] For the avoidance of doubt, unless otherwise stated the term
"E6 mRNA" as used herein encompasses all naturally occurring mRNA
transcripts which contain all or part of the E6 open reading frame,
including naturally occurring splice variants, and therefore
includes transcripts which additionally contain all or part of the
E7 open reading frame (and indeed further open reading frames). The
terms "E6/E7 mRNA", "E6/E7 transcripts" etc are used
interchangeably with the terms "E6 mRNA", "E6 transcripts" and also
encompass naturally occurring mRNA transcripts which contain all or
part of the E6 open reading frame, including naturally occurring
splice variants, and transcripts which contain all or part of the
E7 open reading frame. The term "oncogene expression", unless
otherwise stated, also refers to naturally occurring mRNA
transcripts which contain all or part of the E6 open reading frame,
including naturally occurring splice variants, and transcripts
which contain all or part of the E7 open reading frame.
[0058] Four E6/E7 mRNA species have so far been described in cells
infected with HPV 16, namely an unspliced E6 transcript and three
spliced transcripts denoted E6*I, E6*II and E6*III (Smotkin D, et
al., J Virol. 1989 March 63(3):1441-7; Smotkin D, Wettstein F O.
Proc Natl Acad Sci USA. 1986 July 83(13):4680-4; Doorbar J. et al.,
Virology. 1990 September 178(1):254-62; Cornelissen M T, et al. J
Gen Virol. 1990 May 71(Pt 5):1243-6; Johnson M A, et al. J Gen
Virol. 1990 July 71(Pt 7):1473-9; Schneider-Maunoury S, et al. J
Virol. 1987 October 61(10):3295-8; Sherman L, et al. Int J Cancer.
1992 February 50(3):356-64). All four transcripts are transcribed
from a single promoter (p97) located just upstream of the second
ATG of the E6 ORF.
[0059] In one embodiment the methods may comprise screening for E6
transcripts which contain all or part of the E7 open reading frame,
This may be accomplished, for example, using primers or probes
specific for the E7 coding region.
[0060] In a further embodiment, the methods may comprise screening
for the presence of "full length" E6 transcripts. In the case of
HPV 16 the term "full length E6 transcripts" refers to transcripts
which contain all of the region from nucleotide (nt) 97 to nt 880
in the E6 ORF, inclusive of nt 97 and 880. Nucleotide positions are
numbered according to standard HPV nomenclature (see Human
Papillomavirus Compendium OnLine, available via the internet or in
paper form from HV Database, Mail Stop K710, Los Alamos National
Laboratory, Los Alamos, N. Mex. 87545, USA). Specific detection of
full length transcripts may be accomplished, for example, using
primers or probes which are specific for the region which is
present only in full length E6 transcripts, not in splice variants.
Different HPV types exhibit different patterns of E6/E7 mRNA
expression. Transcript maps for various HPV types, including HPV
types 16 and 31, which may be used to assist in the design of
probes or primers for detection of E6/E7 transcripts are publicly
available via the Human Papillomavirus Compendium (as above).
[0061] E6 oligonucleotide primers are described herein which are
suitable for use in amplification of regions of the E6 mRNA from
various HPV types by NASBA or PCR.
[0062] In a preferred embodiment methods which involve screening
for L1 mRNA expression may comprise screening for L1 mRNA
expression using a technique which is able to detect L1 mRNA from
substantially all known HPV types or at least the major
cancer-associated HPV types (e.g. preferably all of HPV types 16,
18, 31 and 33). L1 primers and probes are described herein which
are capable of detecting L1 mRNA from HPV types 6, 11, 16, 18, 31,
33, 35 and 51 in cervical samples.
[0063] Detection of L1 transcripts can be said to detect HPV
"virulence", meaning the presence of HPV lytic activity. Detection
of E6/E7 transcripts can be said to detect HPV "pathogenesis" since
expression of these mRNAs is indicative of molecular events
associated with risk of developing carcinoma.
[0064] In a study of 4589 women it was possible to detect all
except one case of CIN III lesions or cancer using a method based
on screening for expression of E6 and L1 mRNA (see accompanying
Examples).
[0065] In further embodiments, the above-described methods of the
invention may comprise screening for expression of mRNA transcripts
from the human p16.sup.ink4a gene, in addition to screening for
expression of HPV L1 and/or E6 transcripts.
[0066] A positive result for expression of p16.sup.ink4a mRNA is
taken as a further indication of risk of developing cervical
carcinoma.
[0067] P16.sup.ink4a, and the related family members, may function
to regulate the phosphorylation and the growth suppressive activity
of the restinoblastoma gene product (RB). In support of this, it
has been found that there is an inverse relationship between the
expression of p16.sup.ink4a protein and the presence of normal RB
in selected cancer cell lines; p16.sup.ink4a protein is detectable
when RB is mutant, deleted, or inactivated, and it is markedly
reduced or absent in cell lines that contain a normal RB. Kheif et
al. (Kheif S N et al., Proc. Natl. Acad. Sci. USA 93:4350-4354.
1996), found that p16.sup.ink4a protein is expressed in human
cervical carcinoma cells that contain either a mutant RB or a
wild-type RB that is functionally inactivated by E7. They also show
that the inactivation of RB correlates with an upregulation of
p16.sup.ink4a confirming a feedback loop involving p16.sup.ink4a
and RB. Milde-Langosch et al. (Milde-Langosch K, et al., (2001)
Virchows Arch 439: 55-61) found that there were significant
correlations between strong p16 expression and HPV16/18 infection
and between strong p16 expression and HPV 16/18 E6/E7 oncogene
expression. Klaes et al., (Klaes R, et al., (2001) Int J Cancer 92:
276-284) observed a strong over expression of the p16.sup.ink4a
gene product in 150 of 152 high-grade dysplastic cervical lesions
(CIN II to invasive cancer), whereas normal cervical epithelium or
inflammatory or metaplastic lesions were not stained with the
p16.sup.ink4a specific monoclonal antibody E6H4. All CIN I scored
lesions associated with LR-HPV types displayed no or only focal or
sporadic reactivity, whereas all but two CIN I scored lesions
associated with HR-HPV types showed strong and diffuse staining for
p16.sup.ink4a.
[0068] The disclosed screening methods may be carried out on a
preparation of nucleic acid isolated from a clinical sample or
biopsy containing cervical cells taken from the subject under test.
Suitable samples which may be used as a source of nucleic acid
include (but not exclusively) cervical swabs, cervical biopsies,
cervical scrapings, skin biopsies/warts, also paraffin embedded
tissues, and formalin or methanol fixed cells.
[0069] The preparation of nucleic acid to be screened using the
disclosed method must include mRNA, however it need not be a
preparation of purified poly A+ mRNA and preparations of total RNA
or crude preparations of total nucleic acid containing both RNA and
genomic DNA, or even crude cell lysates are also suitable as
starting material for a NASBA reaction. Essentially any technique
known in the art for the isolation of a preparation of nucleic acid
including mRNA may be used to isolate nucleic acid from a test
sample. A preferred technique is the "Boom" isolation method
described in U.S. Pat. No. 5,234,809 and EP-B-0389,063. This
method, which can be used to isolate a nucleic acid preparation
containing both RNA and DNA, is based on the nucleic acid binding
properties of silicon dioxide particles in the presence of the
chaotropic agent guanidine thiocyanate (GuSCN).
[0070] The methods of the invention are based on assessment of
active transcription of the HPV genome in cervical cells. The
methods are not limited with respect to the precise technique used
to detect mRNA expression. Many techniques for detection of
specific mRNA sequences are known in the art and may be used in
accordance with the invention. For example, specific mRNAs may be
detected by hybridisation, amplification or sequencing
techniques.
[0071] It is most preferred to detect mRNA expression by means of
an amplification technique, most preferably an isothermal
amplification such as NASBA, transcription-mediated amplification,
signal-mediated amplification of RNA technology, isothermal
solution phase amplification, etc. All of these methods are well
known in the art More preferably mRNA expression is detected by an
isothermal amplification in combination with real-time detection of
the amplification product. The most preferred combination is
amplification by NASBA, coupled with real-time detection of the
amplification product using molecular beacons technology, as
described by Leone et al., Nucleic Acids Research, 1998, Vol 26,
2150-2155.
[0072] Methods for the detection of HPV in a test sample using the
NASBA technique will generally comprise the following steps:
[0073] (a) assembling a reaction medium comprising suitable
primer-pairs, an RNA directed DNA polymerase, a ribonuclease that
hydrolyses the RNA strand of an RNA-DNA hybrid without hydrolysing
single or double stranded RNA or DNA, an RNA polymerase that
recognises said promoter, and ribonucleoside and
deoxyribonucleoside triphosphates;
[0074] (b) incubating the reaction medium with a preparation of
nucleic acid isolated from a test sample suspected of containing
HPV under reaction conditions which permit a NASBA amplification
reaction; and
[0075] (c) detecting and/or quantitatively measuring any
HPV-specific product of the NASBA amplification reaction.
[0076] Detection of the specific product(s) of the NASBA reaction
(i.e. sense and/or antisense copies of the target RNA) may be
carried out in a number of different ways. In one approach the
NASBA product(s) may be detected with the use of an HPV-specific
hybridisation probe capable of specifically annealing to the NASBA
product. The hybridisation probe may be attached to a revealing
label, for example a fluorescent, luminescent, radioactive or
chemiluminescent compound or an enzyme label or any other type of
label known to those of ordinary skill in the art. The precise
nature of the label is not critical, but it should be capable of
producing a signal detectable by external means, either by itself
or in conjunction with one or more additional substances (e.g. the
substrate for an enzyme).
[0077] A preferred detection method is so-called "real-time NASBA"
which allows continuous monitoring of the formation of the product
of the NASBA reaction over the course of the reaction. In a
preferred embodiment this may be achieved using a "molecular
beacons" probe comprising an HPV-specific sequence capable of
annealing to the NASBA product, a stem-duplex forming
oligonucleotide sequence and a pair of fluorescer/quencher
moieties, as known in the art and described herein. If the
molecular beacons probe is added to the reaction mixture prior to
amplification it may be possible to monitor the formation of the
NASBA product in real-time (Leone et al., Nucleic Acids Research,
1998, Vol 26, 2150-2155). Reagent kits and instrumentation for
performing real-time NASBA detection are available commercially
(e.g. NucliSens.TM. EasyQ system, from Organon Teknika).
[0078] In a further approach, the molecular beacons technology may
be incorporated into the primer 2 oligonucleotide allowing
real-time monitoring of the NASBA reaction without the need for a
separate hybridisation probe.
[0079] In a still further approach the products of the NASBA
reaction may be monitored using a generic labelled detection probe
which hybridises to a nucleotide sequence in the 5' terminus of the
primer 2 oligonucleotide. This is equivalent to the "NucliSens.TM."
detection system supplied by Organon Teknika. In this system
specificity for NASBA products derived from the target HPV mRNA may
be conferred by using HPV-specific capture probes comprising probe
oligonucleotides as described herein attached to a solid support
such as a magnetic microbead. Most preferably the generic labelled
detection probe is the ECL.TM. detection probe supplied by Organon
Teknika. NASBA amplicons are hybridized to the HPV-specific capture
probes and the generic ECL probe (via a complementary sequence on
primer 2). Following hybridization the bead/amplicon/ECL probe
complexes may be captured at the magnet electrode of an automatic
ECL reader (e.g. the NucliSens.TM. reader supplied by Organon
Teknika). Subsequently, a voltage pulse triggers the ECL.TM.
reaction.
[0080] The detection of HPV mRNA is also of clinical relevance in
cancers other than cervical carcinoma including, for example, head
and neck carcinoma, oral and tongue carcinoma, skin carcinoma, anal
and vaginal carcinoma. Detection of HPV mRNA may also be very
useful in the diagnosis of micrometastases in lymph nodes in the
lower part of the body. Hence, the invention also contemplates
screens for susceptibility to the above-listed cancers based on
screening for expression of HPV L1 and E6 transcripts.
[0081] In accordance with a further aspect of the invention there
is provided a kit for use in the detection of transcripts of the L1
and E6 genes of HPV, the kit comprising at least one primer-pair
suitable for use in amplification of a region of L1 transcripts
from at least HPV types 16, 18, 31 and 33, and preferably also HPV
45, and one or more primer-pairs which enable amplification of a
region of E6 transcripts from HPV types 16, 18, 31 and 33, and
preferably also HPV 45.
[0082] "Primer-pair" taken to mean are pair of primers which may be
used in combination to amplify a specific region of the L1 or E6
mRNA using any known nucleic acid technique. In preferred
embodiments the primer-pairs included in the kit will be suitable
for use in NASBA amplification or similar isothermal amplification
techniques.
[0083] The individual primers making up each primer-pair included
in the kit may be supplied separately (e.g. a separate container of
each primer) or, more preferably, may be supplied mixed in a single
container. Combinations of two or more primer-pairs may be supplied
ready-mixed in a single container within the kit. It may be
convenient to supply two or more primer-pairs in a single container
where the two or more amplification reactions are to be
"multiplexed", meaning performed simultaneously in a single
reaction vessel.
[0084] The primer-pair(s) suitable for use in amplification of a
region of E6 transcripts should enable amplification a region of E6
mRNA from at least the major cancer-associated HPV types 16, 18, 31
and 33, and preferably also HPV 45. There are several different
ways in which this can be achieved.
[0085] In one embodiment, the kit may contain separate primer-pairs
specific for each of HPV types 16, 18, 31 and 33, and preferably
also HPV 45. These primer-pairs may be supplied within the kit in
separate containers, or they may be supplied as mixtures of two or
more primer-pairs in a single container, for example to enable
multiplexing of the amplification reactions.
[0086] In a further embodiment, the kit may contain a single
primer-pair capable of amplifying a region of the E6 gene from HPV
types 16, 18, 31 and 33, and preferably also HPV 45, which thus
enables amplification of all four (preferably five) types in a
single amplification reaction. This could, for example, be achieved
with the use of a pair of degenerate primers or by selection of a
region of the E6 mRNA which is highly conserved across HPV
types.
[0087] The E6 primer-pair may correspond to any region of the E6
mRNA, an may enable amplification of all or part of the E6 open
reading frame and/or the E7 open reading frame.
[0088] The kit may further include primer-pairs suitable for use in
amplification of E6 mRNA from HPV types other than types 16, 18, 31
and 33, and preferably also HPV 45. For example, the kit may be
supplemented with E6 primers for detection of an HPV type which is
endemic in a particular geographical area or population.
[0089] The primer-pair(s) suitable for use in amplification of a
region of L1 transcripts should be capable of amplifying a region
of L1 mRNA from at least the major cancer-associated HPV types 16,
18, 31 and 33, and preferably also HPV 45, and will preferably be
suitable for use in amplification of a region of L1 mRNAs from
substantially all known HPV types. With the use of such primers it
is possible to test for active transcription of L1 mRNA from
multiple HPV types in a single amplification reaction.
[0090] It is possible to design primers capable of detecting L1
transcripts from multiple HPV types by selecting regions of the L1
transcript which are highly conserved.
[0091] In a further approach, specificity for multiple HPV types
may be achieved with the use of degenerate oligonucleotide primers
or complex mixtures of polynucleotides which exhibit minor sequence
variations, preferably corresponding to sites of sequence variation
between HPV genotypes. The rationale behind the use of such
degenerate primers or mixtures is that the mixture may contain at
least one primer-pair capable of detecting each HPV type.
[0092] In a still further approach specificity for multiple HPV
types may be achieved by incorporating into the primers one or more
inosine nucleotides, preferably at sites of sequence variation
between HPV genotypes.
[0093] The E6 and L1 primer-pairs may be supplied in separate
containers within the kit, or the L1 primer-pair(s) may be supplied
as a mixture with one or more E6 primer-pairs in a single
container.
[0094] The kits may further comprise one or more probes suitable
for use in detection of the products of amplification reactions
carried out using the primer-pairs included within the kit. The
probe(s) may be supplied as a separate reagent within the kit.
Alternatively, the probe(s) may be supplied as a mixture with one
or more primer-pairs.
[0095] The primers and probes included in the kit are preferably
single stranded DNA molecules. Non-natural synthetic
polynucleotides which retain the ability to base-pair with a
complementary nucleic acid molecule may also be used, including
synthetic oligonucleotides which incorporate modified bases and
synthetic oligonucleotides wherein the links between individual
nucleosides include bonds other than phosphodiester bonds. The
primers and probes may be produced according to techniques well
known in the art, such as by chemical synthesis using standard
apparatus and protocols for oligonucleotide synthesis.
[0096] The primers and probes will typically be isolated
single-stranded polynucleotides of no more than 100 bases in
length, more typically less than 55 bases in length. For the
avoidance of doubt it is hereby stated that the terms "primer" and
"probe" exclude naturally occurring full-length HPV genomes.
[0097] Several general types of oligonucleotide primers and probes
incorporating HPV-specific sequences may be included in the kit.
Typically, such primers and probes may comprise additional, non-HPV
sequences, for example sequences which are required for an
amplification reaction or which facilitate detection of the
products of the amplification reaction.
[0098] The first type of primers are primer 1 oligonucleotides
(also referred to herein as NASBA P1 primers), which are
oligonucleotides of generally approximately 50 bases in length,
containing an average of about 20 bases at the 3' end that are
complementary to a region of the target mRNA. Oligonucleotides
suitable for use as NASBA P1 primers are denoted "P1/PCR" in Table
1. P1 primer oligonucleotides have the general structure
X.sub.1-SEQ, wherein SEQ represents an HPV-specific sequence and
X.sub.1 is a sequence comprising a promoter that is recognized by a
specific RNA polymerase. Bacteriophage promoters, for example the
T7, T3 and SP6 promoters, are preferred for use in the
oligonucleotides of the invention, since they provide advantages of
high level transcription which is dependent only on binding of the
appropriate RNA polymerase. In a preferred embodiment, sequence
"X.sub.1" may comprise the sequence AATTCTAATACGACTCACTATAGGG (SEQ
ID No 171) or the sequence AATTCTAATACGACTCACTATAGGGAGAAGG (SEQ ID
No 172). These sequences contains a T7 promoter, including the
transcription initiation site for T7 RNA polymerase.
[0099] The HPV-specific sequences in the primers denoted in Table 1
as "P1/PCR" may also be adapted for use in standard PCR primers.
When these sequences are used as the basis of NASBA P1 primers they
have the general structure X.sub.1-SEQ, as defined above. The
promoter sequence X.sub.1 is essential in a NASBA P1 primer.
However, when the same sequences are used as the basis of standard
PCR primers it is not necessary to include X.sub.1.
[0100] A second type of primers are NASBA primer 2 oligonucleotides
(also referred to herein as NASBA P2 primers) which generally
comprise a sequence of approximately 20 bases substantially
identical to a region of the target mRNA. The oligonucleotide
sequences denoted in Table 1 as "P2/PCR" are suitable for use in
both NASBA P2 primers and standard PCR primers.
[0101] Oligonucleotides intended for use as NASBA P2 primers may,
in a particular but non-limiting embodiment, further comprise a
sequence of nucleotides at the 5' end which is unrelated to the
target mRNA but which is capable of hybridising to a generic
detection probe. The detection probe will preferably be labelled,
for example with a fluorescent, luminescent or enzymatic label. In
one embodiment the detection probe is labelled with a label that
permits detection using ECL.TM. technology, although it will be
appreciated that the invention is in no way limited to this
particular method of detection. In a preferred embodiment the 5'
end of the primer 2 oligonucleotides may comprise the sequence
GATGCAAGGTCGCATATGAG (SEQ ID No 170). This sequence is capable of
hybridising to a generic ECL.TM. probe commercially available from
Organon Teknika having the following structure:
TABLE-US-00001 Ru(bpy).sub.3.sup.2+-GAT GCA AGG TCG CAT ATG
AG-3'
[0102] In a different embodiment the primer 2 oligonucleotide may
incorporate "molecular beacons" technology, which is known in the
art and described, for example, in WO 95/13399 by Tyagi and Kramer,
Nature Biotechnology. 14: 303-308, 1996, to allow for real-time
monitoring of the NASBA reaction.
[0103] Target-specific probe oligonucleotides may also be included
within the kit. Probe oligonucleotides generally comprise a
sequence of approximately 20-25 bases substantially identical to a
region of the target mRNA, or the complement thereof. Example
HPV-specific oligonucleotide sequences which are suitable for use
as probes are denoted "PO" in Table 1. The probe oligonucleotides
may be used as target-specific hybridisation probes for detection
of the products of a NASBA or PCR reaction. In this connection the
probe oligonucleotides may be coupled to a solid support, such as
paramagnetic beads, to form a capture probe (see below). In a
preferred embodiment the 5' end of the probe oligonucleotide may be
labelled with biotin. The addition of a biotin label facilitates
attachment of the probe to a solid support via a
biotin/streptavidin or biotin/avidin linkage.
[0104] Target-specific probes enabling real-time detection of
amplification products may incorporate "molecular beacons"
technology which is known in the art and described, for example, by
Tyagi and Kramer, Nature Biotechnology. 14: 303-308, 1996 and in WO
95/13399. Example HPV-specific oligonucleotide sequences suitable
for use as molecular beacons probes are denoted "MB" in Table
1.
[0105] The term "molecular beacons probes" as used herein is taken
to mean molecules having the structure:
TABLE-US-00002 X.sub.2-arm.sub.1-target-arm.sub.2-X.sub.3
wherein "target" represents a target-specific sequence of
nucleotides, "X.sub.2" and "X.sub.3" represent a fluorescent moiety
and a quencher moiety capable of substantially or completely
quenching the fluorescence from the fluorescent moiety when the two
are held together in close proximity and "arm.sub.1" and
"arm.sub.2" represent complementary sequences capable of forming a
stem duplex.
[0106] Preferred combinations of "arm.sub.1" and "arm.sub.2"
sequences are as follows, however these are intended to be
illustrative rather than limiting to the invention:
TABLE-US-00003 cgcatg-SEQ-catgcg ccagct-SEQ-agctgg cacgc-SEQ-gcgtg
cgatcg-SEQ-cgatcg ccgtcg-SEQ-cgacgg cggacc-SEQ-ggtccg
ccgaagg-SEQ-ccttcgg cacgtcg-SEQ-cgacgtg cgcagc-SEQ-gctgcg
ccaagc-SEQ-gcttgg ccaagcg-SEQ-cgcttgg cccagc-SEQ-gctggg
ccaaagc-SEQ-gctttgg cctgc-SEQ-gcagg ccaccc-SEQ-gggtgg
ccaagcc-SEQ-ggcttgg ccagcg-SEQ-cgctgg cgcatg-SEQ-catgcg
[0107] The use of molecular beacons technology allows for real-time
monitoring of amplification reactions, for example NASBA
amplification (see Leone et al., Nucleic Acids Research., 1998,
vol: 26, pp 2150-2155). The molecular beacons probes generally
include complementary sequences flanking the HPV-specific sequence,
represented herein by the notation arm.sub.1 and arm.sub.2, which
are capable of hybridising to each other form a stem duplex
structure. The precise sequences of arm.sub.1 and arm.sub.2 are not
material to the invention, except for the requirement that these
sequences must be capable of forming a stem duplex when the probe
is not bound to a target HPV sequence.
[0108] Molecular beacons probes also include a fluorescent moiety
and a quencher moiety, the fluorescent and the quencher moieties
being represented herein by the notation X.sub.2 and X.sub.3. As
will be appreciated be the skilled reader, the fluorescer and
quencher moieties are selected such that the quencher moiety is
capable of substantially or completely quenching the fluorescence
from the fluorescent moiety when the two moieties are in close
proximity, e.g. when the probe is in the hairpin "closed"
conformation in the absence of the target sequence. Upon binding to
the target sequence, the fluorescent and quencher moieties are held
apart such that the fluorescence of the fluorescent moiety is no
longer quenched.
[0109] Many examples of suitable pairs of quencher/fluorescer
moieties which may be used in accordance with the invention are
known in the art (see WO 95/13399, Tyagi and Kramer, ibid). A broad
range of fluorophores in many different colours made be used,
including for example 5-(2'-aminoethyl)aminonaphthalene-1-sulphonic
acid (EDANS), fluorescein, FAM and Texas Red (see Tyagi, Bratu and
Kramer, 1998, Nature Biotechnology, 16, 49-53. The use of probes
labelled with different coloured fluorophores enables "multiplex"
detection of two or more different probes in a single reaction
vessel. A preferred quencher is
4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), a
non-fluorescent chromophore, which serves as a "universal" quencher
for a wide range of fluorophores. The fluorescer and quencher
moieties may be covalently attached to the probe in either
orientation, either with the fluorescer at or near the 5' end and
the quencher at or near the 3' end or vice versa. Protocols for the
synthesis of molecular beacon probes are known in the art. A
detailed protocol for synthesis is provided in a paper entitled
"Molecular Beacons: Hybridization Probes for Detection of Nucleic
Acids in Homogenous Solutions" by Sanjay Tyagi et al., Department
of Molecular Genetics, Public Health Research Institute, 455 First
Avenue, New York, N.Y. 10016, USA, which is available online via
the PHRI website (at phri.nyu.edu or molecular-beacons.org).
[0110] Suitable combinations of the NASBA P1 and NASBA P2 primers
may be used to drive a NASBA amplification reaction. In order to
drive a NASBA amplification reaction the primer 1 and primer 2
oligonucleotides must be capable of priming synthesis of a
double-stranded DNA from a target region of mRNA. For this to occur
the primer 1 and primer 2 oligonucleotides must comprise
target-specific sequences which are complementary to regions of the
sense and the antisense strand of the target mRNA,
respectively.
[0111] In the first phase of the NASBA amplification cycle, the
so-called "non-cyclic" phase, the primer 1 oligonucleotide anneals
to a complementary sequence in the target mRNA and its 3' end is
extended by the action of an RNA-dependent DNA polymerase (e.g.
reverse transcriptase) to form a first-strand cDNA synthesis. The
RNA strand of the resulting RNA:DNA hybrid is then digested, e.g.
by the action of RNaseH, to leave a single stranded DNA. The primer
2 oligonucleotide anneals to a complementary sequence towards the
3' end of this single stranded DNA and its 3' end is extended (by
the action of reverse transcriptase), forming a double stranded
DNA. RNA polymerase is then able to transcribe multiple RNA copies
from the now transcriptionally active promoter sequence within the
double-stranded DNA. This RNA transcript, which is antisense to the
original target mRNA, can act as a template for a further round of
NASBA reactions, with primer 2 annealing to the RNA and priming
synthesis of the first cDNA strand and primer 1 priming synthesis
of the second cDNA strand. The general principles of the NASBA
reaction are well known in the art (see Compton, J. Nature. 350:
91-92).
[0112] The target-specific probe oligonucleotides described herein
may also be attached to a solid support, such as magnetic
microbeads, and used as "capture probes" to immobilise the product
of the NASBA amplification reaction (a single stranded RNA). The
target-specific "molecular beacons" probes described herein may be
used for real-time monitoring of the NASBA reaction.
[0113] Kits according to the invention may also including a
positive control containing E6 and/or L1 mRNA from a known HPV
type. Suitable controls include, for example, nucleic acid extracts
prepared from cell lines infected with known HPV types (e.g. HeLa,
CaSki).
[0114] Kits further may contain internal control amplification
primers, e.g. primers specific for human U1A RNA.
[0115] Kits containing primers (and optionally probes) suitable for
use in NASBA amplification may further comprise a mixture of
enzymes required for the NASBA reaction, e.g. enzyme mixture
containing an RNA directed DNA polymerase (e.g. a reverse
transcriptase), a ribonuclease that hydrolyses the RNA strand of an
RNA-DNA hybrid without hydrolysing single or double stranded RNA or
DNA (e.g. RNaseH) and an RNA polymerase. The RNA polymerase should
be one which recognises the promoter sequence present in the 5'
terminal region of the NASBA P1 primers supplied in the reagent
kit. The kit may also comprise a supply of NASBA buffer containing
the ribonucleosides and deoxyribonucleosides required for RNA and
DNA synthesis. The composition of a standard NASBA reaction buffer
will be well known to those skilled in the art (see also Leone et
al., ibid).
TABLE-US-00004 TABLE 1 E6-specific sequences for inclusion in
NASBA/PCR primers and probes Primer/ SEQ probe HPV ID type Sequence
Type nt 1 P2/PCR CCACAGGAGCGACCCAGAAAGTTA 16 116 2 P1/PCR
X.sub.1-ACGGTTTGTTGTATTGCTGTTC 16 368 3 P2/PCR CCACAGGAGCGACCCAGAAA
16 116 4 P1/PCR X.sub.1-GGTTTGTTGTATTGCTGTTC 16 368 5 P1/PCR
X.sub.1-ATTCCCATCTCTATATACTA 16 258 6 P1/PCR
X.sub.1-TCACGTCGCAGTAACTGT 16 208 7 P1/PCR
X.sub.1-TTGCTTGCAGTACACACA 16 191 8 P1/PCR
X.sub.1-TGCAGTACACACATTCTA 16 186 9 P1/PCR
X.sub.1-GCAGTACACACATTCTAA 16 185 10 P2/PCR ACAGTTATGCACAGAGCT 16
142 11 P2/PCR ATATTAGAATGTGTGTAC 16 182 12 P2/PCR
TTAGAATGTGTGTACTGC 16 185 13 P2/PCR GAATGTGTGTACTGCAAG 16 188 14 PO
ACAGTTATGCACAGAGCT 16 142 15 PO ATATTAGAATGTGTGTAC 16 182 16 PO
TTAGAATGTGTGTACTGC 16 185 17 PO GAATGTGTGTACTGCAAG 16 188 18 PO
CTTTGCTTTTCGGGATTTATGC 16 235 19 PO TATGACTTTGCTTTTCGGGA 16 230 20
MB X.sub.2-arm.sub.1-TATGACTTTGCTTTTCGGGA- 16 230 arm.sub.2-X.sub.3
21 P2/PCR CAGAGGAGGAGGATGAAATAGTA 16 656 22 P1/PCR
X.sub.1-GCACAACCGAAGCGTAGAGTCACAC 16 741 23 PO
TGGACAAGCAGAACCGGACAGAGC 16 687 24 P2/PCR CAGAGGAGGAGGATGAAATAGA 16
656 25 P1/PCR X.sub.1-GCACAACCGAAGCGTAGAGTCA 16 741 26 PO
AGCAGAACCGGACAGAGCCCATTA 16 693 27 P2/PCR ACGATGAAATAGATGGAGTT 18
702 28 P1/PCR X.sub.1-CACGGACACACAAAGGACAG 18 869 28 PO
AGCCGAACCACAACGTCACA 18 748 30 P2/PCR GAAAACGATGAAATAGATGGAG 18 698
31 P1/PCR X.sub.1-ACACCACGGACACACAAAGGACAG 18 869 32 PO
GAACCACAACGTCACACAATG 18 752 33 MB
X.sub.2-arm.sub.1-GAACCACAACGTCACACAATG- 18 752 arm.sub.2-X.sub.3
34 P2/PCR TTCCGGTTGACCTTCTATGT 18 651 35 P1/PCR
X.sub.1-GGTCGTCTGCTGAGCTTTCT 18 817 36 P2/PCR GCAAGACATAGAAATAACCTG
18 179 37 P1/PCR X.sub.1-ACCCAGTGTTAGTTAGTT 18 379 38 PO
TGCAAGACAGTATTGGAACT 18 207 39 P2/PCR GGAAATACCCTACGATGAAC 31 164
40 P1/PCR X.sub.1-GGACACAACGGTCTTTGACA 31 423 41 PO
ATAGGGACGACACACCACACGGAG 31 268 42 P2/PCR GGAAATACCCTACGATGAACTA 31
164 43 P1/PCR X.sub.1-CTGGACACAACGGTCTTTGACA 31 423 44 PO
TAGGGACGACACACCACACGGA 31 269 45 P2/PCR ACTGACCTCCACTGTTATGA 31 617
46 P1/PCR X.sub.1-TATCTACTTGTGTGCTCTGT 31 766 47 PO
GACAAGCAGAACCGGACACATC 31 687 48 P2/PCR TGACCTCCACTGTTATGAGCAATT 31
619 49 P1/PCR X.sub.1-TGCGAATATCTACTTGTGTGCTCT 31 766 GT 50 PO
GGACAAGCAGAACCGGACACATCCAA 31 686 51 MB X.sub.2-arm.sub.1- 31 686
GGACAAGCAGAACCGGACACATCCAA- arm.sub.2-X.sub.3 52 P2/PCR
ACTGACCTCCACTGTTAT 31 617 53 P1/PCR X.sub.1-CACGATTCCAAATGAGCCCAT
31 809 54 P2/PCR TATCCTGAACCAACTGACCTAT 33 618 55 P1/PCR
X.sub.1-TTGACACATAAACGAACTG 33 763 56 PO CAGATGGACAAGCACAACC 33 694
57 P2/PCR TCCTGAACCAACTGACCTAT 33 620 58 P1/PCR
X.sub.1-CCCATAAGTAGTTGCTGTAT 33 807 59 PO GGACAAGCACAACCAGCCACAGC
33 699 60 MB X.sub.2-arm.sub.1- 33 699 GGACAAGCACAACCAGCCACAGC-
arm.sub.2-X.sub.3 61 P2/PCR GACCTTTGTGTCCTCAAGAA 33 431 62 P1/PCR
X.sub.1-AGGTCAGTTGGTTCAGGATA 33 618 63 PO AGAAACTGCACTGTGACGTGT 33
543 64 P2/PCR ATTACAGCGGAGTGAGGTAT 35 217 65 P1/PCR
X.sub.1-GTCTTTGCTTTTCAACTGGA 35 442 66 PO ATAGAGAAGGCCAGCCATAT 35
270 67 P2/PCR TCAGAGGAGGAGGAAGATACTA 35 655 68 P1/PCR
X.sub.1-GATTATGCTCTCTGTGAACA 35 844 69 P2/PCR CCCGAGGCAACTGACCTATA
35 610 70 P1/PCR X.sub.1-GTCAATGTGTGTGCTCTGTA 35 770 71 PO
GACAAGCAAAACCAGACACCTCCAA 35 692 72 PO GACAAGCAAAACCAGACACC 35 692
73 P2/PCR TTGTGTGAGGTGCTGGAAGAAT 52 144 74 P1/PCR
X.sub.1-CCCTCTCTTCTAATGTTT 52 358 75 PO GTGCCTACGCTTTTTATCTA 52 296
76 P2/PCR GTGCCTACGCTTTTTATCTA 52 296 77 P1/PCR
X.sub.1-GGGGTCTCCAACACTCTGAACA 52 507 78 PO TGCAAACAAGCGATTTCA 52
461 79 P2/PCR TCAGGCGTTGGAGACATC 58 157 80 P1/PCR
X.sub.1-AGCAATCGTAAGCACACT 58 301 81 P2/PCR TCTGTGCATGAAATCGAA 58
173 82 P1/PCR X.sub.1-AGCACACTTTACATACTG 58 291 83 PO
TGAAATGCGTTGAATGCA 58 192 84 PO TTGCAGCGATCTGAGGTATATG 58 218 85
P2/PCR TACACTGCTGGACAACAT B(11) 514 86 P1/PCR
X.sub.1-TCATCTTCTGAGCTGTCT B(11) 619 87 P2/PCR
TACACTGCTGGACAACATGCA B(11) 514 88 P1/PCR
X.sub.1-GTCACATCCACAGCAACAGGTCA B(11) 693 89 PO
GTAGGGTTACATTGCTATGA B(11) 590 90 PO GTAGGGTTACATTGCTATGAGC B(11)
590 91 P2/PCR TGACCTGTTGCTGTGGATGTGA B(11) 693 92 P1/PCR
X.sub.1-TACCTGAATCGTCCGCCAT B(11) 832 93 PO ATWGTGTGTCCCATCTGC
B(11) 794 94 P2/PCR CATGCCATAAATGTATAGA C(18 295 39 45) 95 P1/PCR
X.sub.1-CACCGCAGGCACCTTATTAA C(18 408 39 45 96 PO
AGAATTAGAGAATTAAGA C(18 324 39 45 97 P2/PCR GCAGACGACCACTACAGCAAA
39 210 98 P1/PCR X.sub.1-ACACCGAGTCCGAGTAATA 39 344 99 PO
ATAGGGACGGGGAACCACT 39 273 100 P2/PCR TATTACTCGGACTCGGTGT 39 344
101 P1/PCR X.sub.1-CTTGGGTTTCTCTTCGTGTTA 39 558 102 PO
GGACCACAAAACGGGAGGAC 39 531 103 P2/PCR GAAATAGATGAACCCGACCA 39 703
104 P1/PCR X.sub.1-GCACACCACGGACACACAAA 39 886 105 PO
TAGCCAGACGGGATGAACCACAGC 39 749 106 P2/PCR AACCATTGAACCCAGCAGAAA 45
430 107 P1/PCR X.sub.1-TCTTTCTTGCCGTGCCTGGTCA 45 527 108 PO
GTACCGAGGGCAGTGTAATA 45 500 109 P2/PCR AACCATTGAACCCAGCAGAAA 45 430
110 P1/PCR X.sub.1-TCTTTCTTGCCGTGCCTGGTCA 45 527 111 P2/PCR
GAAACCATTGAACCCAGCAGAAAA 45 428 112 P1/PCR
X.sub.1-TTGCTATACTTGTGTTTCCCTACG 45 558 113 PO GTACCGAGGGCAGTGTAATA
45 500 114 PO GGACAAACGAAGATTTCACA 45 467 115 P2/PCR
GTTGACCTGTTGTGTTACCAGCAAT 45 656 116 P1/PCR
X.sub.1-CACCACGGACACACAAAGGACAAG 45 868
117 P2/PCR CTGTTGACCTGTTGTGTTACGA 45 654 118 P1/PCR
X.sub.1-CCACGGACACACAAAGGACAAG 45 868 119 P2/PCR
GTTGACCTGTTGTGTTACGA 45 656 120 P1/PCR X.sub.1-ACGGACACACAAAGGACAAG
45 868 121 PO GAGTCAGAGGAGGAAAACGATG 45 686 122 PO
AGGAAAACGATGAAGCAGATGGAGT 45 696 123 PO ACAACTACCAGCCCGACGAGCCGAA
45 730 124 P2/PCR GGAGGAGGATGAAGTAGATA 51 658 125 P1/PCR
X.sub.1-GCCCATTAACATCTGCTGTA 51 807 126 P2/PCR
AGAGGAGGAGGATGAAGTAGATA 51 655 127 P1/PCR
X.sub.1-ACGGGCAAACCAGGCTTAGT 51 829 128 PO GCAGGTGTTCAAGTGTAGTA 51
747 129 PO TGGCAGTGGAAAGCAGTGGAGACA 51 771 130 P2/PCR
TTGGGGTGCTGGAGACAAACATCT 56 519 131 P1/PCR
X.sub.1-TTCATCCTCATCCTCATCCTCTGA 56 665 132 P2/PCR
TGGGGTGCTGGAGACAAACATC 56 520 133 P1/PCR
X.sub.1-CATCCTCATCCTCATCCTCTGA 56 665 134 P2/PCR
TTGGGGTGCTGGAGACAAACAT 56 519 135 P1/PCR
X.sub.1-CCACAAACTTACACTCACAACA 56 764 136 PO
AAAGTACCAACGCTGCAAGACGT 56 581 137 PO AGAACTAACACCTCAAACAGAAAT 56
610 138 PO AGTACCAACGCTGCAAGACGTT 56 583 139 P1/PCR
X.sub.1-TTGGACAGCTCAGAGGATGAGG 56 656 140 P2/PCR
GATTTTCCTTATGCAGTGTG 56 279 141 P1/PCR X.sub.1-GACATCTGTAGCACCTTATT
56 410 142 PO GACTATTCAGTGTATGGAGC 56 348 143 PO
CAACTGAYCTMYACTGTTATGA A (16 31 35) 144 MB X.sub.2-arm.sub.1- A (16
CAACTGAYCTMYACTGTTATGA- 31 35) arm.sub.2-X.sub.3 145 PO
GAAMCAACTGACCTAYWCTGCTAT A (33 52 58) 146 MB X.sub.2-arm.sub.1- A
(33 GAAMCAACTGACCTAYWCTGCTAT- 52 58) arm.sub.2-X.sub.3 147 PO
AAGACATTATTCAGACTC C (18 45 39) 148 MB
X.sub.2-arm.sub.1-AAGACATTATTCAGACTC- C (18 arm.sub.2-X.sub.3 45
39)
TABLE-US-00005 TABLE 2 L1-specific sequences for inclusion in
NASBA/PCR primers and probes SEQ Primer/probe ID type Sequence 149
P2/PCR AATGGCATTTGTTGGGGTAA 150 P1/PCR X.sub.1-TCATATTCCTCCCCATGTC
151 PO TTGTTACTGTTGTTGATACTAC 152 P2/PCR AATGGCATTTGTTGGSRHAA 153
P1/PCR X.sub.1-TCATATTCCTCMMCATGDC 154 PO TTGTTACTGTTGTTGATACYAC
155 PO TTGTTACTGTTGTTGATACCAC 156 P2/PCR AATGGCATTTGTTGGSIIAA 157
P2/PCR AATGGCATTTGTTGGIIHAA 158 P2/PCR AATGGCATTTGTTGGIRIAA 159
P2/PCR AATGGCATTTGTTGGGGTAA 160 P2/PCR AATGGCATTTGTTGGGGAAA 161
P2/PCR AATGGCATTTGTTGGCATAA 162 P2/PCR AATGGCATTTGTTGGGGCAA 163
P2/PCR AATGGCATTTGTTGGCACAA 164 P1/PCR X.sub.1-TCATATTCCTCMICATGIC
165 P1/PCR X.sub.1-TCATATTCCTCAACATGIC 166 P1/PCR
X.sub.1-TCATATTCCTCIICATGTC 167 P1/PCR X.sub.1-TCATATTCCTCIICATGGC
168 P1/PCR X.sub.1-TCATATTCCTCIICATGAC 3' 169 P1/PCR
X.sub.1-TCATATTCCTCIICATGCC 3'
[0116] Preferred primers suitable for use in detection of HPV L1
and E6 mRNA by NASBA are listed in the following tables. However,
these are merely illustrative and it is not intended that the scope
of the invention should be limited to these specific molecules.
[0117] In the following Tables the NASBA P2 primers (p2) include
the sequence GATGCAAGGTCGCATATGAG (SEQ ID No. 170) at the 5' end;
the NASBA P1 primers (p1) include the sequence
AATTCTAATACGACTCACTATAGGGAGAAGG (SEQ ID No. 172) at the 5' end.
Oligonucleotides suitable for use as probes are identified by "po".
The P2 primers generally contain HPV sequences from the postive
strand, whereas the p1 primers generally contain HPV sequences from
the negative strand. nt-refers to nucleotide position in the
relevant HPV genomic sequence.
TABLE-US-00006 TABLE 3 Preferred E6 NASBA primers and probes HPV
Primer name Sequence Type nt HAe6701p2
GATGCAAGGTCGCATATGAGCCACAGGAGCGACCC 16 116 (SEQ ID 173) AGAAAGTTA
HAe6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGACGG 16 368 (SEQ ID 174)
TTTGTTGTATTGCTGTTC HAe6702p2 GATGCAAGGTCGCATATGAGCCACAGGAGCGACCC 16
116 (SEQ ID 175) AGAAA HAe6702p1
AATTCTAATACGACTCACTATAGGGAGAAGGGGTT 16 368 (SEQ ID 176)
TGTTGTATTGCTGTTC HPV16p1 AATTCTAATACGACTCACTATAGGGAGAAGGATTC 16 258
(SEQ ID 177) CCATCTCTATATACTA HAe6702Ap1
AATTCTAATACGACTCACTATAGGGAGAAGGTCA 16 208 (SEQ ID 178)
CGTCGCAGTAACTGT HAe6702Bp1 AATTCTAATACGACTCACTATAGGGAGAAGGTTG 16
191 (SEQ ID 179) CTTGCAGTACACACA HAe6702Cp1
AATTCTAATACGACTCACTATAGGGAGAAGGTGC 16 186 (SEQ ID 180)
AGTACACACATTCTA HAe6702Dp1 AATTCTAATACGACTCACTATAGGGAGAAGGGCA 16
185 (SEQ ID 181) GTACACACATTCTAA H16e6702Ap2
GATGCAAGGTCGCATATGAGACAGTTATGCACAGA 16 142 (SEQ ID 182) GCT
H16e6702Bp2 GATGCAAGGTCGCATATGAGATATTAGAATGTGTG 16 182 (SEQ ID 183)
TAC H16e6702Cp2 GATGCAAGGTCGCATATGAGTTAGAATGTGTGTAC 16 185 (SEQ ID
184) TGC H16e6702Dp2 GATGCAAGGTCGCATATGAGGAATGTGTGTACTGC 16 188
(SEQ ID 185) AAG H16e6702Apo ACAGTTATGCACAGAGCT 16 142 (SEQ ID 10)
H16e6702Bpo ATATTAGAATGTGTGTAC 16 182 (SEQ ID 11) H16e6702Cpo
TTAGAATGTGTGTACTGC 16 185 (SEQ ID 12) H16e6702Dpo
GAATGTGTGTACTGCAAG 16 188 (SEQ ID 13) HAe6701po
CTTTGCTTTTCGGGATTTATGC 16 235 (SEQ ID 18) HAe6702po
TATGACTTTGCTTTTCGGGA 16 230 (SEQ ID 19) HAe6702mbl
X.sub.2-cgcatgTATGACTTTGCTTTTCGGGAcatgcg- 16 230 (SEQ ID 186)
X.sub.3 HAe6702mb2 X.sub.2-ccagctTATGACTTTGCTTTTCGGGAagctgg- 16 230
(SEQ ID 187) X.sub.3 HAe6702mb3 X.sub.2-cacgcTATGACTTTGCT
TTTCGGGAgcgtg-X.sub.3 16 230 (SEQ ID 188) H16e6702mb4
X.sub.2-cgatcgTATGACTTTGCTTTTCGGGAcgatcg- 16 230 (SEQ ID 189)
X.sub.3 HAe6703p2 GATGCAAGGTCGCATATGAGCAGAGGAGGAGGATG 16 656 (SEQ
ID 190) AAATAGTA HAe6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGGCAC 16
741 (SEQ ID 191) AACCGAAGCGTAGAGTCACAC HAe6703po
TGGACAAGCAGAACCGGACAGAGC 16 687 (SEQ ID 23) HAe6704p2
GATGCAAGGTCGCATATGAGCAGAGGAGGAGGATG 16 656 (SEQ ID 192) AAATAGA
HAe6704p1 AATTCTAATACGACTCACTATAGGGAGAAGGGCAC 16 741 (SEQ ID 193)
AACCGAAGCGTAGAGTCA HAe6704po AGCAGAACCGGACAGAGCCCATTA 16 693 (SEQ
ID 26) H18e6701p2 GATGCAAGGTCGCATATGAGACGATGAAATAGATG 18 702 (SEQ
ID 194) GAGTT H18e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGCACG 18 869
(SEQ ID 195) GACACACAAAGGACAG H18e6701po AGCCGAACCACAACGTCACA 18
748 (SEQ ID 29) H18e6702p2 GATGCAAGGTCGCATATGAGGAAAACGATGAAATA 18
698 (SEQ ID 196) GATGGAG H18e6702p1
AATTCTAATACGACTCACTATAGGGAGAAGGACAC 18 869 (SEQ ID 197)
CACGGACACACAAAGGACAG H18e6702po GAACCACAACGTCACACAATG 18 752 (SEQ
ID 32) H18e6702mb1 X.sub.2-cgcatgGAACCACAACGTCACACAATGcatgcg- 18
752 (SEQ ID 198) X.sub.3 H18e6702mb2
X.sub.2-ccgtcgGAACCACAACGTCACACAATGcgacgg- 18 752 (SEQ ID 199)
X.sub.3 H18e6702mb3 X.sub.2-cggaccGAACCACAACGTCACACAATGggtccg- 18
752 (SEQ ID 200) X.sub.3 H18e6702mb4
X.sub.2-cgatcgGAACCACAACGTCACACAATGcgatcg- 18 752 (SEQ ID 201)
x.sub.3 H18e6703p2 GATGCAAGGTCGCATATGAGTTCCGGTTGACCTTC 18 651 (SEQ
ID 202) TATGT H18e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGGGTC 18 817
(SEQ ID 203) GTCTGCTGAGCTTTCT H18e6704p2
GATGCAAGGTCGCATATGAGGCAAGACATAGAAAT 18 179 (SEQ ID 204) AACCTG
H18e6704p1 AATTCTAATACGACTCACTATAGGGAGAAGGACCC 18 379 (SEQ ID 205)
AGTGTTAGTTAGTT H18e6704po TGCAAGACAGTATTGGAACT 18 207 (SEQ ID 38)
H31e6701p2 GATGCAAGGTCGCATATGAGGGAAATACCCTACGA 31 164 (SEQ ID 206)
TGAAC H31e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGGGAC 31 423 (SEQ ID
207) ACAACGGTCTTTGACA H31e6701po ATAGGGACGACACACCACACGGAG 31 268
(SEQ ID 41) H31e6702p2 GATGCAAGGTCGCATATGAGGGAAATACCCTACGA 31 164
(SEQ ID 208) TGAACTA H31e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGCTGG
31 423 (SEQ ID 209) ACACAACGGTCTTTGACA H31e6702po
TAGGGACGACACACCACACGGA 31 269 (SEQ ID 44) H31e6703p2
GATGCAAGGTCGCATATGAGACTGACCTCCACTGT 31 617 (SEQ ID 210) TATGA
H31e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGTATC 31 766 (SEQ ID 211)
TACTTGTGTGCTCTGT H31e6703po GACAAGCAGAACCGGACACATC 31 687 (SEQ ID
47) H31e6704p2 GATGCAAGGTCGCATATGAGTGACCTCCACTGTTA 31 619 (SEQ ID
212) TGAGCAATT H31e6704p1 AATTCTAATACGACTCACTATAGGGAGAAGGTGCG 31
766 (SEQ ID 213) AATATCTACTTGTGTGCTCT GT H31e6704po
GGACAAGCAGAACCGGACACATCCAA 31 686 (SEQ ID 50) H31e6704mb1
X.sub.2-ccgaaggGGACAAGCAGAACCGGACACATCC 31 686 (SEQ ID 214)
AAccttcgg-X.sub.3 H31e6704mb2
X.sub.2-ccgtcgGGACAAGCAGAACCGGACACATCCA 31 686 (SEQ ID 215)
Acgacgg-X.sub.3 H31e6704mb3
X.sub.2-cacgtcgGGACAAGCAGAACCGGACACATCCAA 31 686 (SEQ ID 216)
cgacgtg-X.sub.3 H31e6704mb4
X.sub.2-cgcagoGGACAAGCAGAACCGGACACATCCAA 31 686 (SEQ ID 217)
gctgcg-X.sub.3 H31e6704mb5 X.sub.2-cgatcgGGACAAGCAGAACCGGACACATCCAA
31 686 (SEQ ID 218) cgatcg-X.sub.3 H31e6705p2
GATGCAAGGTCGCATATGAGACTGACCTCCACTGT 31 617 (SEQ ID 219) TAT
H31e6705p1 AATTCTAATACGACTCACTATAGGGAGAAGGCACG 31 809 (SEQ ID 220)
ATTCCAAATGAGCCCAT H33e6701p2 GATGCAAGGTCGCATATGAGTATCCTGAACCAACT 33
618 (SEQ ID 221) GACCTAT H33e6701p1
AATTCTAATACGACTCACTATAGGGAGAAGGTTGA 33 763 (SEQ ID 222)
CACATAAACGAACTG H33e6701po CAGATGGACAAGCACAACC 33 694 (SEQ ID 56)
H33e6703p2 GATGCAAGGTCGCATATGAGTCCTGAACCAACTGA 33 620 (SEQ ID 223)
CCTAT H33e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGCCCA 33 807 (SEQ ID
224) TAAGTAGTTGCTGTAT H33e6703po GGACAAGCACAACCAGCCACAGC 33 699
(SEQ ID 59) H33e6703mb1 X.sub.2-ccaagoGGACAAGCACAACCAGCCACAGCgct 33
699 (SEQ ID 225) tgg-X.sub.3 H33e6703mb2
X.sub.2-ccaagcgGGACAAGCACAACCAGCCACAGC 33 699 (SEQ ID 226)
cgcttgg-X.sub.3 H33e6703mb3
X.sub.2-cccagcGGACAAGCACAACCAGCCACAGCgct 33 699 (SEQ ID 227)
ggg-X.sub.3 H33e6703mb4 X.sub.2-ccaaagoGGACAAGCACAACCAGCCACAGCg 33
699 (SEQ ID 228) ctttgg-X.sub.3 H33e6703mb5
X.sub.2-cctgoGGACAAGCACAACCAGCCACAGCgcagg- 33 699 (SEQ ID 229)
X.sub.3 H33e6703mb6 X.sub.2-cgatcgGGACAAGCACAACCAGCCACAGCcga 33 699
(SEQ ID 230) tcg-X.sub.3 H33e6702p2
GATGCAAGGTCGCATATGAGGACCTTTGTGTCCTC 33 431 (SEQ ID 231) AAGAA
H33e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGAGGT 33 618 (SEQ ID 232)
CAGTTGGTTCAGGATA H33e6702po AGAAACTGCACTGTGACGTGT 33 543 (SEQ ID
63) H35e6701p2 GATGCAAGGTCGCATATGAGATTACAGCGGAGTGA 35 217 (SEQ ID
233) GGTAT H35e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGGTCT 35 442
(SEQ ID 234) TTGCTTTTCAACTGGA H35e5601po ATAGAGAAGGCCAGCCATAT 35
270 (SEQ ID 66) H35e6702p2 GATGCAAGGTCGCATATGAGTCAGAGGAGGAGGAA 35
655
(SEQ ID 235) GATACTA H35e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGGATT
35 844 (SEQ ID 236) ATGCTCTCTGTGAACA H35e6703p2
GATGCAAGGTCGCATATGAGCCCGAGGCAACTGAC 35 610 (SEQ ID 237) CTATA
H35e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGGTCA 35 770 (SEQ ID 238)
ATGTGTGTGCTCTGTA H35e6702po GACAAGCAAAACCAGACACCTCCAA 35 692 (SEQ
ID 71) H35e6703po GACAAGCAAAACCAGACACC 35 692 (SEQ ID 72)
H52e6701p2 GATGCAAGGTCGCATATGAGTTGTGTGAGGTGCTG 52 144 (SEQ ID 239)
GAAGAAT H52e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGCCCT 52 358 (SEQ
ID 240) CTCTTCTAATGTTT H52e6701po GTGCCTACGCTTTTTATCTA 52 296 (SEQ
ID 75) H52e6702p2 GATGCAAGGTCGCATATGAGGTGCCTACGCTTTTT 52 296 (SEQ
ID 241) ATCTA H52e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGGGGG 52 507
(SEQ ID 242) TCTCCAACACTCTGAACA H52e6702po TGCAAACAAGCGATTTCA 52
461 (SEQ ID 78) H58e6701p2 GATGCAAGGTCGCATATGAGTCAGGCGTTGGAGAC 58
157 (SEQ ID 243) ATC H58e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGAGCA
58 301 (SEQ ID 244) ATCGTAAGCACACT H58e6702p2
GATGCAAGGTCGCATATGAGTCTGTGCATGAAATC 58 173 (SEQ ID 245) GAA
H58e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGAGCA 58 291 (SEQ ID 246)
CACTTTACATACTG H58e6701po TGAAATGCGTTGAATGCA 58 192 (SEQ ID 83)
H58e6702po TTGCAGCGATCTGAGGTATATG 58 218 (SEQ ID 84) HBe6701p2
GATGCAAGGTCGCATATGAGTACACTGCTGGACAA B(11) 514 (SEQ ID 247) CAT
HBe6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGTCAT B(11) 619 (SEQ ID
248) CTTCTGAGCTGTCT HBe6702p2 GATGCAAGGTCGCATATGAGTACACTGCTGGACAA
B(11) 514 (SEQ ID 249) CATGCA HBe6702p1
AATTCTAATACGACTCACTATAGGGAGAAGGGTCA B(11) 693 (SEQ ID 250)
CATCCACAGCAACAGGTCA HBe6701po GTAGGGTTACATTGCTATGA B(11) 590 (SEQ
ID 89) HBe6702po GTAGGGTTACATTGCTATGAGC B(11) 590 (SEQ ID 90)
HBe6703p2 GATGCAAGGTCGCATATGAGTGACCTGTTGCTGTG B(11) 693 (SEQ ID
251) GATGTGA HBe6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGTACC B(11)
832 (SEQ ID 252) TGAATCGTCCGCCAT HBe6703po ATWGTGTGTCCCATCTGC B(11)
794 (SEQ ID 93) HCe6701p2 GATGCAAGGTCGCATATGAGCATGCCATAAATGTA C(18
295 (SEQ ID 253) TAGA 39 45) HCe6701p1
AATTCTAATACGACTCACTATAGGGAGAAGGCACC C (18 408 (SEQ ID 254)
GCAGGCACCTTATTAA 39 45 HCe6701po AGAATTAGAGAATTAAGA C(18 324 (SEQ
ID 96) 39 45 H39e6701p2 GATGCAAGGTCGCATATGAGGCAGACGACCACTAC 39 210
(SEQ ID 255) AGCAAA H39e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGACAC
39 344 (SEQ ID 256) CGAGTCCGAGTAATA H39e6701po ATAGGGACGGGGAACCACT
39 273 (SEQ ID 99) H39e6702p2 GATGCAAGGTCGCATATGAGTATTACTCGGACTCG
39 344 (SEQ ID 257) GTGT H39e6702p1
AATTCTAATACGACTCACTATAGGGAGAAGGCTTG 39 558 (SEQ ID 258)
GGTTTCTCTTCGTGTTA H39e6702po GGACCACAAAACGGGAGGAC 39 531 (SEQ ID
102) H39e6703p2 GATGCAAGGTCGCATATGAGGAAATAGATGAACCC 39 703 (SEQ ID
259) GACCA H39e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGGCAC 39 886
(SEQ ID 260) ACCACGGACACACAAA H39e6703po TAGCCAGACGGGATGAACCACAGC
39 749 (SEQ ID 105) HPV45p2 GATGCAAGGTCGCATATGAGAACCATTGAACCCAG 45
430 (SEQ ID 261) CAGAAA HPV45p1 AATTCTAATACGACTCACTATAGGGAGAAGGTCTT
45 527 (SEQ ID 262) TCTTGCCGTGCCTGGTCA HPV45po GTACCGAGGGCAGTGTAATA
45 500 (SEQ ID 108) H45e6701p2 GATGCAAGGTCGCATATGAGAACCATTGAACCCAG
45 430 (SEQ ID 263) CAGAAA H45e6701p1
AATTCTAATACGACTCACTATAGGGAGAAGGTCTT 45 527 (SEQ ID 264)
TCTTGCCGTGCCTGGTCA H45e6702p2 GATGCAAGGTCGCATATGAGGAAACCATTGAACCC
45 428 (SEQ ID 265) AGCAGAAAA H45e6702p1
AATTCTAATACGACTCACTATAGGGAGAAGGTTGC 45 558 (SEQ ID 266)
TATACTTGTGTTTCCCTACG H45e6701po GTACCGAGGGCAGTGTAATA 45 500 (SEQ ID
113) H45e6702po GGACAAACGAAGATTTCACA 45 467 (SEQ ID 114) H45e6703p2
GATGCAAGGTCGCATATGAGGTTGACCTGTTGTGT 45 656 (SEQ ID 267) TACCAGCAAT
H45e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGCACC 45 868 (SEQ ID 268)
ACGGACACACAAAGGACAAG H45e6704p2 GATGCAAGGTCGCATATGAGCTGTTGACCTGTTGT
45 654 (SEQ ID 269) GTTACGA H45e6704p1
AATTCTAATACGACTCACTATAGGGAGAAGGCCAC 45 868 (SEQ ID 270)
GGACACACAAAGGACAAG H45e6705p2 GATGCAAGGTCGCATATGAGGTTGACCTGTTGTGT
45 656 (SEQ ID 271) TACGA H45e6705p1
AATTCTAATACGACTCACTATAGGGAGAAGGACGG 45 868 (SEQ ID 272)
ACACACAAAGGACAAG H45e6703po GAGTCAGAGGAGGAAAACGATG 45 686 (SEQ ID
121) H45e6704po AGGAAAACGATGAAGCAGATGGAGT 45 696 (SEQ ID 122)
H45e6705po ACAACTACCAGCCCGACGAGCCGAA 45 730 (SEQ ID 123) H51e6701p2
GATGCAAGGTCGCATATGAGGGAGGAGGATGAAGT 51 658 (SEQ ID 273) AGATA
H51e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGGCCC 51 807 (SEQ ID 274)
ATTAACATCTGCTGTA H51e6702p2 GATGCAAGGTCGCATATGAGAGAGGAGGAGGATGA 51
655 (SEQ ID 275) AGTAGATA H51e6702p1
AATTCTAATACGACTCACTATAGGGAGAAGGACGG 51 829 (SEQ ID 276)
GCAAACCAGGCTTAGT H51e6701po GCAGGTGTTCAAGTGTAGTA 51 747 (SEQ ID
128) H51e6702po TGGCAGTGGAAAGCAGTGGAGACA 51 771 (SEQ ID 129)
H56e6701p2 GATGCAAGGTCGCATATGAGTTGGGGTGCTGGAGA 56 519 (SEQ ID 277)
CAAACATCT H56e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGTTCA 56 665
(SEQ ID 278) TCCTCATCCTCATCCTCTGA H56e6702p2
GATGCAAGGTCGCATATGAGTGGGGTGCTGGAGAC 56 520 (SEQ ID 279) AAACATC
H56e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGCATC 56 665 (SEQ ID 280)
CTCATCCTCATCCTCTGA H56e6703p2 GATGCAAGGTCGCATATGAGTTGGGGTGCTGGAGA
56 519 (SEQ ID 281) CAAACAT H56e6703p1
AATTCTAATACGACTCACTATAGGGAGAAGGCCAC 56 764 (SEQ ID 282)
AAACTTACACTCACAACA H56e6701po AAAGTACCAACGCTGCAAGACGT 56 581 (SEQ
ID 136) H56e6702po AGAACTAACACCTCAAACAGAAAT 56 610 (SEQ ID 137)
H56e6703po AGTACCAACGCTGCAAGACGTT 56 583 (SEQ ID 138) H56e6703po1
TTGGACAGCTCAGAGGATGAGG 56 656 (SEQ ID 139) H56e6704p2
GATGCAAGGTCGCATATGAGGATTTTCCTTATGCA 56 279 (SEQ ID 283) GTGTG
H56e6704p1 AATTCTAATACGACTCACTATAGGGAGAAGGGACA 56 410 (SEQ ID 284)
TCTGTAGCACCTTATT H56e6704po GACTATTCAGTGTATGGAGC 56 348 (SEQ ID
142) HPVAPO1A CAACTGAYCTMYACTGTTATGA A (16 (SEQ ID 143) 31 35)
HPVApo1Amb1 X.sub.2- A (16 (SEQ ID 285)
cgcatgCAACTGAYCTMYACTGTTATGAcatgcg- 31 35) X.sub.3 HPVApo1Amb2
X.sub.2-ccgtcgCAACTGAYCTMYACTGTTATGAcga A (16 (SEQ ID 286)
cgg-X.sub.3 31 35) HPVApo1Amb3
X.sub.2-ccacccCAACTGAYCTMYACTGTTATGAgg A (16 (SEQ ID 287)
gtgg-X.sub.3 31 35) HPVApo1Amb4
X.sub.2-cgatcgCAACTGAYCTMYACTGTTATGAcga A (16 (SEQ ID 288)
tcg-X.sub.3 31 35) HPVAPO4A GAAMCAACTGACCTAYWCTGCTAT A (33 (SEQ ID
145) 52 58) HPVAPO4Amb1 X.sub.2-ccaagcGAAMCAACTGACCTAYWCTGCTATgc A
(33 (SEQ ID 289) ttgg-X.sub.3 52 58) HPVAPO4Amb2
X.sub.2-ccaagccGAAMCAACTGACCTAYWCTGCTAT A (33 (SEQ ID 290)
ggcttgg-X.sub.3 52 58)
HPVAPO4Amb3 X.sub.2-ccaagcgGAAMCAACTGACCTAYWCTGCTA A (33 (SEQ ID
291) Tcgcttgg-X.sub.3 52 58) HPVAPO4Amb4
X.sub.2-ccagcgGAAMCAACTGACCTAYWCTGCTATcg A (33 (SEQ ID 292)
ctgg-X.sub.3 52 58) HPVAPO4Amb5
X.sub.2-cgatcgGAAMCAACTGACCTAYWCTGCTATcg A (33 (SEQ ID 293)
atcg-X.sub.3 52 58) HPVCPO4 AAGACATTATTCAGACTC C (18 (SEQ ID 147)
45 39) HPVCPO4Amb1 X.sub.2-ccaagcAAGACATTATTCAGACTCgcttgg-X.sub.3 C
(18 (SEQ ID 294) 45 39) HPVCPO4Amb2
X.sub.2-cgcatgAAGACATTATTCAGACTCcatgcg-X.sub.3 C (18 (SEQ ID 295)
45 39) HPVCPO4Amb3 X.sub.2-cccagcAAGACATTATTCAGACTCgctggg-X.sub.3 C
(18 (SEQ ID 296) 45 39) HPVCPO4Amb4
X.sub.2-cgatcgAAGACATTATTCAGACTCcgatcg-X.sub.3 C (18 (SEQ ID 297)
45 39)
[0118] Pairs of P1 and P2 primers having the same prefix (e.g.
HAe6701p1 and HAe6701p2) are intended to be used in combination.
However, other combinations may also be used, as summarised below
for HPV types 16, 18, 31, 33 and 45.
[0119] Suitable primer-pairs for amplification of HPV 16 E6 mRNA
are as follows:
HAe6701p2 or HAe6702p2 (both nt 116) with HAe6701p1 or HAe6702p1
(both nt 368). HAe6701p2 or HAe6702p2 (both nt 116) with HPV16p1
(nt 258). H16e6702Ap2 (nt 142), H16e6702 Bp2 (nt 182), H16e6702 Cp2
(nt 185) or H16e6702Dp2 (nt 188) with HAe6701p1 or HAe6702p1 (both
nt 368). HAe6701p2 or HAe6702p2 (both nt 116) with HAe6702Ap1 (nt
208), HAe6702 Bp1 (nt 191), HAe6702 Cp1 (nt 186) or HAe6702Dp1
(185). These combinations are suitable for amplification of all E6
splice variants. HAe6703p2 or HAe6704p2 (both nt 656) with
HAe6703p1 or HAe6704p1 (both nt 741). These combinations are
suitable for amplification of all transcripts containing the E7
coding region (at least up to nt 741).
[0120] The following primer-pairs are preferred for amplification
of HPV 18 E6 mRNA:
H18e6701p2 (nt 702) or H18e6702p2 (nt 698) with H18e6701p1 or
H18e6702p1 (both nt 869). H18e6703p2 (nt 651) with H18e6703p1 (nt
817). H18e6704p2 (nt 179) with H18e6704p1 (nt 379).
[0121] The following primer-pairs are preferred for amplification
of HPV 31 E6 mRNA:
H31e6701p2 or H31e6702p2 (both nt 164) with H31e6701p1 or
H31e6702p1 (both nt 423). H31e6703p2 (nt 617), H31e6704p2 (nt 619)
or H31e6705p2 (nt 617) with H31e6703p1 (nt 766), H31e6704p1 (766)
or H31e6705p1 (nt 809).
[0122] The following primer-pairs are preferred for amplification
of HPV 33 E6 mRNA:
H33e6701p2 (nt 618) or H33e6703p2 (nt 620) with H33e6701p1 (nt 763)
or H33e6703p1 (nt 807). H33e6702p2 (nt 431) with H33e6702p1 (nt
618).
[0123] The following primer pair is preferred for amplification of
HPV 45:
HPV45p2 (nt 430) with HPV45p1 (nt 527)
TABLE-US-00007 TABLE 4-E6 PCR primers HPV Primer name Sequence type
nt HAe6701PCR2 (SEQ ID 1) CCACAGGAGCGACCCAGAAAGTTA 16 116
HAe6701PCR1 (SEQ ID 2) ACGGTTTGTTGTATTGCTGTTC 16 368 HAe6702PCR2
(SEQ ID 3) CCACAGGAGCGACCCAGAAA 16 116 HAe6702PCR1 (SEQ ID 4)
GGTTTGTTGTATTGCTGTTC 16 368 HAe6703PCR2 (SEQ ID 21)
CAGAGGAGGAGGATGAAATAGTA 16 656 HAe6703PCR1 (SEQ ID 22)
GCACAACCGAAGCGTAGAGTCACAC 16 741 HAe6704PCR2 (SEQ ID 24)
CAGAGGAGGAGGATGAAATAGA 16 656 HAe6704PCR1 (SEQ ID 25)
GCACAACCGAAGCGTAGAGTCA 16 741 H18e6701PCR2 (SEQ ID 27)
ACGATGAAATAGATGGAGTT 18 702 H18e6701PCR1 (SEQ ID 28)
CACGGACACACAAAGGACAG 18 869 H18e6702PCR2 (SEQ ID 30)
GAAAACGATGAAATAGATGGAG 18 698 H18e6702PCR1 (SEQ ID 31)
ACACCACGGACACACAAAGGACAG 18 869 H18e6703PCR2 (SEQ ID 34)
TTCCGGTTGACCTTCTATGT 18 651 H18e6703PCR1 (SEQ ID 35)
GGTCGTCTGCTGAGCTTTCT 18 817 H18e6704PCR2 (SEQ ID 36)
GCAAGACATAGAAATAACCTG 18 179 H18e6704PCR1 (SEQ ID 37)
ACCCAGTGTTAGTTAGTT 18 379 H31e6701PCR2 (SEQ ID 39)
GGAAATACCCTACGATGAAC 31 164 H31e6701PCR1 (SEQ ID 40)
GGACACAACGGTCTTTGACA 31 423 H31e6702PCR2 (SEQ ID 42)
GGAAATACCCTACGATGAACTA 31 164 H31e6702PCR1 (SEQ ID 43)
CTGGACACAACGGTCTTTGACA 31 423 H31e6703PCR2 (SEQ ID 45)
ACTGACCTCCACTGTTATGA 31 617 H31e6703PCR1 (SEQ ID 46)
TATCTACTTGTGTGCTCTGT 31 766 H31e6704PCR2 (SEQ ID 48)
TGACCTCCACTGTTATGAGCAATT 31 619 H31e6704PCR1 (SEQ ID 49)
TGCGAATATCTACTTGTGTGCTCTGT 31 766 H31e6705PCR2 (SEQ ID 52)
ACTGACCTCCACTGTTAT 31 617 H31e6705PCR1 (SEQ ID 53)
CACGATTCCAAATGAGCCCAT 31 809 H33e6701PCR2 (SEQ ID 54)
TATCCTGAACCAACTGACCTAT 33 618 H33e6701PCR1 (SEQ ID 55)
TTGACACATAAACGAACTG 33 763 H33e6703PCR2 (SEQ ID 57)
TCCTGAACCAACTGACCTAT 33 620 H33e6703PCR1 (SEQ ID 58)
CCCATAAGTAGTTGCTGTAT 33 807 H33e6702PCR2 (SEQ ID 61)
GACCTTTGTGTCCTCAAGAA 33 431 H33e6702PCR1 (SEQ ID 62)
AGGTCAGTTGGTTCAGGATA 33 618 H35e6701PCR2 (SEQ ID 64)
ATTACAGCGGAGTGAGGTAT 35 217 H35e6701PCR1 (SEQ ID 65)
GTCTTTGCTTTTCAACTGGA 35 442 H35e6702PCR2 (SEQ ID 67)
TCAGAGGAGGAGGAAGATACTA 35 655 H35e6702PCR1 (SEQ ID 68)
GATTATGCTCTCTGTGAACA 35 844 H35e6703PCR2 (SEQ ID 69)
CCCGAGGCAACTGACCTATA 35 610 H35e6703PCR1 (SEQ ID 70)
GTCAATGTGTGTGCTCTGTA 35 770 H52e6701PCR2 (SEQ ID 73)
TTGTGTGAGGTGCTGGAAGAAT 52 144 H52e6701PCR1 (SEQ ID 74)
CCCTCTCTTCTAATGTTT 52 358 H52e6702PCR2 (SEQ ID 75)
GTGCCTACGCTTTTTATCTA 52 296 H52e6702PCR1 (SEQ ID 77)
GGGGTCTCCAACACTCTGAACA 52 507 H58e6701PCR2 (SEQ ID 79)
TCAGGCGTTGGAGACATC 58 157 H58e6701PCR1 (SEQ ID 80)
AGCAATCGTAAGCACACT 58 301 H58e6702PCR2 (SEQ ID 81)
TCTGTGCATGAAATCGAA 58 173 H58e6702PCR1 (SEQ ID 82)
AGCACACTTTACATACTG 58 291 HBe6701PCR2 (SEQ ID 85)
TACACTGCTGGACAACAT B(11) 514 HBe6701PCR1 (SEQ ID 86)
TCATCTTCTGAGCTGTCT B(11) 619 HBe6702PCR2 (SEQ ID 87)
TACACTGCTGGACAACATGCA B(11) 514 HBe6702PCR1 (SEQ ID 88)
GTCACATCCACAGCAACAGGTCA B(11) 693 HBe6703PCR2 (SEQ ID 91)
TGACCTGTTGCTGTGGATGTGA B(11) 693 HBe6703PCR1 (SEQ ID 92)
TACCTGAATCGTCCGCCAT B(11) 832 HCe6701PCR2 (SEQ ID 94)
CATGCCATAAATGTATAGA C (18 295 39 45 HCe6701PCR1 (SEQ ID 95)
CACCGCAGGCACCTTATTAA C (18 408 39 45 H39e6701PCR2 (SEQ ID 97)
GCAGACGACCACTACAGCAAA 39 210 H39e6701PCR1 (SEQ ID 98)
ACACCGAGTCCGAGTAATA 39 344 H39e6702PCR2 (SEQ ID 100)
TATTACTCGGACTCGGTGT 39 344 H39e6702PCR1 (SEQ ID 101)
CTTGGGTTTCTCTTCGTGTTA 39 558 H39e6703PCR2 (SEQ ID 103)
GAAATAGATGAACCCGACCA 39 703 H39e6703PCR1 (SEQ ID 104)
GCACACCACGGACACACAAA 39 886 H45e6701PCR2 (SEQ ID 106)
AACCATTGAACCCAGCAGAAA 45 430 H45e6701PCR1 (SEQ ID 107)
TCTTTCTTGCCGTGCCTGGTCA 45 527 H45e6702PCR2 (SEQ ID 111)
GAAACCATTGAACCCAGCAGAAAA 45 428 H45e6702PCR1 (SEQ ID 112)
TTGCTATACTTGTGTTTCCCTACG 45 558 H45e6703PCR2 (SEQ ID 115)
GTTGACCTGTTGTGTTACCAGCAAT 45 656 H45e6703PCR1 (SEQ ID 116)
CACCACGGACACACAAAGGACAAG 45 868 H45e6704PCR2 (SEQ ID 117)
CTGTTGACCTGTTGTGTTACGA 45 654 H45e6704PCR1 (SEQ ID 118)
CCACGGACACACAAAGGACAAG 45 868 H45e6705PCR2 (SEQ ID 119)
GTTGACCTGTTGTGTTACGA 45 656 H45e6705PCR1 (SEQ ID 120)
ACGGACACACAAAGGACAAG 45 868 H51e6701PCR2 (SEQ ID 124)
GGAGGAGGATGAAGTAGATA 51 658 H51e6701PCR1 (SEQ ID 125)
GCCCATTAACATCTGCTGTA 51 807 H51e6702PCR2 (SEQ ID 126)
AGAGGAGGAGGATGAAGTAGATA 51 655 H51e6702PCR1 (SEQ ID 127)
ACGGGCAAACCAGGCTTAGT 51 829 H56e6701PCR2 (SEQ ID 130)
TTGGGGTGCTGGAGACAAACATCT 56 519 H56e6701PCR1 (SEQ ID 131)
TTCATCCTCATCCTCATCCTCTGA 56 665 H56e6702PCR2 (SEQ ID 132)
TGGGGTGCTGGAGACAAACATC 56 520 H56e6702PCR1 (SEQ ID 133)
CATCCTCATCCTCATCCTCTGA 56 665 H56e6703PCR2 (SEQ ID 134)
TTGGGGTGCTGGAGACAAACAT 56 519 H56e6703PCR1 (SEQ ID 135)
CCACAAACTTACACTCACAACA 56 764 H56e6704PCR2 (SEQ ID 140)
GATTTTCCTTATGCAGTGTG 56 279 H56e6704PCR1 (SEQ ID 141)
GACATCTGTAGCACCTTATT 56 410
[0124] Preferred PCR primer-pairs for HPV types 16, 18, 31 and 33
are analogous to the NASBA primer-pairs.
TABLE-US-00008 TABLE 5 Preferred L1 NASBA primers and probes Primer
name Sequence Onc2A2 5' GATGCAAGGTCGCATATGAGAATGGCATTTGTTGGGGTAA 3'
(SEQ ID 298) Onc2A1 5'
AATTCTAATACGACTCACTATAGGGAGAAGGTCATATTCCTCCCCATGTC 3' (SEQ ID 299)
Onc2PoA 5' TTGTTACTGTTGTTGATACTAC 3' (SEQ ID 151) Onc2B2 5'
GATGCAAGGTCGCATATGAGAATGGCATTTGTTGGSRHAA 3' (SEQ ID 300) Onc2B1 5'
AATTCTAATACGACTCACTATAGGGAGAAGGTCATATTCCTCMMCATGDC 3' (SEQ ID 301)
Onc2PoB 5' TTGTTACTGTTGTTGATACYAC 3' (SEQ ID 154) Onc2PoC 5'
TTGTTACTGTTGTTGATACCAC 3' (SEQ ID 155) Onc2C2 5'
GATGCAAGGTCGCATATGAGAATGGCATTTGTTGGSIIAA 3' (SEQ ID 302) Onc2D2 5'
GATGCAAGGTCGCATATGAGAATGGCATTTGTTGGIIHAA 3' (SEQ ID 303) Onc2E2 5'
GATGCAAGGTCGCATATGAGAATGGCATTTGTTGGIRIAA 3' (SEQ ID 304) Onc2F2 5'
GATGCAAGGTCGCATATGAGAATGGCATTTGTTGGGGTAA 3' (SEQ ID 305) Onc2G2 5'
GATGCAAGGTCGCATATGAGAATGGCATTTGTTGGGGAAA 3' (SEQ ID 306) Onc2H2 5'
GATGCAAGGTCGCATATGAGAATGGCATTTGTTGGCATAA 3' (SEQ ID 307) Onc2I2 5'
GATGCAAGGTCGCATATGAGAATGGCATTTGTTGGGGCAA 3' (SEQ ID 308) Onc2J2 5'
GATGCAAGGTCGCATATGAGAATGGCATTTGTTGGCACAA 3' (SEQ ID 309) Onc2K1 5'
AATTCTAATACGACTCACTATAGGGAGAAGGTCATATTCCTCMICATGIC 3' (SEQ ID 310)
Onc2L1 5' AATTCTAATACGACTCACTATAGGGAGAAGGTCATATTCCTCAACATGIC 3'
(SEQ ID 311) Onc2M1 5'
AATTCTAATACGACTCACTATAGGGAGAAGGTCATATTCCTCIICATGTC 3' (SEQ ID 312)
Onc2N1 5' AATTCTAATACGACTCACTATAGGGAGAAGGTCATATTCCTCIICATGGC 3'
(SEQ ID 313) Onc201 5'
AATTCTAATACGACTCACTATAGGGAGAAGGTCATATTCCTCIICATGAC 3' (SEQ ID 314)
Onc2P1 5' AATTCTAATACGACTCACTATAGGGAGAAGGTCATATTCCTCIICATGCC 3'
(SEQ ID 315)
TABLE-US-00009 TABLE 6 Preferred L1 PCR primers Primer name
Sequence Onc2A1-PCR 5' AATGGCATTTGTTGGGGTAA 3' (SEQ ID 149)
Onc2A2-PCR 5' TCATATTCCTCCCCATGTC 3' (SEQ ID 150) Onc2B1-PCR 5'
AATGGCATTTGTTGGSRHAA 3' (SEQ ID 152) Onc2B2-PCR 5'
TCATATTCCTCMMCATGDC 3' (SEQ ID 153) Onc2C1-PCR 5'
AATGGCATTTGTTGGSIIAA 3' (SEQ ID 156) Onc2D1-PCR 5'
AATGGCATTTGTTGGIIHAA 3' (SEQ ID 157) Onc2E1-PCR 5'
AATGGCATTTGTTGGIRIAA 3' (SEQ ID 158) Onc2F1-PCR 5'
AATGGCATTTGTTGGGGTAA 3' (SEQ ID 159) Onc2G1-PCR 5'
AATGGCATTTGTTGGGGAAA 3' (SEQ ID 160) Onc2H1-PCR 5'
AATGGCATTTGTTGGCATAA 3' (SEQ ID 161) Onc2I1-PCR 5'
AATGGCATTTGTTGGGGCAA 3' (SEQ ID 162) Onc2J1-PCR 5'
AATGGCATTTGTTGGCACAA 3' (SEQ ID 163) Onc2K2-PCR 5'
TCATATTCCTCMICATGIC 3' (SEQ ID 164) Onc2L2-PCR 5'
TCATATTCCTCAACATGIC 3' (SEQ ID 165) Onc2M2-PCR 5'
TCATATTCCTCIICATGTC 3' (SEQ ID 166) Onc2N2-PCR 5'
TCATATTCCTCIICATGGC 3' (SEQ ID 167) Onc2O2-PCR 5'
TCATATTCCTCIICATGAC 3' (SEQ ID 168) Onc2P2-PCR 5'
TCATATTCCTCIICATGCC 3' (SEQ ID 169)
[0125] The HPV-specific sequences in SEQ ID NOs:149 and 150
(primers Onc2A2/Onc2A1-PCR and Onc2A1/Onc2A2-PCR) are identical to
fragments of the HPV type 16 genomic sequence from position
6596-6615 (SEQ ID NO:149; Onc2A2/Onc2A1-PCR), and from position
6729 to 6747(SEQ ID NO:150; Onc2A1/Onc2A2-PCR).
[0126] The HPV-specific sequences SEQ ID NOs:152 and 153
(Onc2B2/Onc2B1-PCR and Onc2B1/Onc2B2-PCR) are variants of the above
sequences, respectively, including several degenerate bases.
Representations of the sequences of degenerate oligonucleotide
molecules provided herein use the standard IUB code for mixed base
sites: N=G,A,T,C; V=G,A,C; B=G,T,C; H=A,T,C; D=G,A,T; K=G,T; S=G,C;
W=A,T; M=A,C; Y=C,T; R=A,G.
[0127] It is also possible to use variants of the HPV-specific
sequences SEQ ID NO:152 (Onc2B2/Onc2B1-PCR) and SEQ ID NO:153
(Onc2B 1/Onc2B2-PCR) wherein any two of nucleotides "SRH" towards
the 3' end of the sequence are replaced with inosine (I), as
follows:
TABLE-US-00010 5' AATGGCATTTGTTGGIIHAA 3' (SEQ ID 157) 5'
AATGGCATTTGTTGGSIIAA 3' (SEQ ID 156) 5' AATGGCATTTGTTGGIRIAA 3'
(SEQ ID 158)
[0128] The HPV-specific sequences SEQ ID NOs: 156-163 (present in
primers Onc2C2, Onc2D2, Onc2E2, Onc2F2, Onc2G2, Onc2H2, Onc212,
Onc2J2, Onc2C1-PCR, Onc2D1-PCR, Onc2E1-PCR, Onc2F1-PCR, Onc2G1-PCR,
Onc2H1-PCR, Onc2I1-PCR and Onc2J1-PCR) are variants based on the
HPV-specific sequence SEQ ID NO:152 (Onc2B2/Onc2B1-PCR), whereas
the HPV-specific sequences SEQ ID NOs: 164-169 (present in primers
Onc2K1, Onc2L1, Onc2M1, Onc2N1, Onc2O1, Onc2P1, Onc2K2-PCR,
Onc2L2-PCR, Onc2M2-PCR, Onc2N2-PCR, Onc2O2-PCR and Onc2P2-PCR are
variants based on the HPV-specific sequence SEQ ID NO:153
(Onc2B1/Onc2B2-PCR). These variants include degenerate bases and
also inosine (I) residues. This sequence variation enables
oligonucleotides incorporating the variant sequences to bind to
multiple HPV types. Inosine bases do not interfere with
hybridization and so may be included at sites of variation between
HPV types in order to construct a "consensus" primer able to bind
to multiple HPV types.
[0129] Any one or more of primers Onc2A2, Onc2B2, Onc2C2, Onc2D2,
Onc2E2, Onc2F2, Onc2G2, Onc2H2, Onc2I2 and Onc2J2, may be used in
combination with any one or more of primers Onc2A1, Onc2B1, Onc2K1,
Onc2L1, Onc2M1, Onc2N1, Onc2O1 and Onc2P1, for NASBA amplification
of HPV L1 mRNA.
[0130] Any one or more of primers Onc2A1-PCR, Onc2B1-PCR,
Onc2C1-PCR, Onc2D1-PCR, Onc2E1-PCR, Onc2F1-PCR, Onc2G1-PCR,
Onc2H1-PCR, Onc2I1-PCR and Onc2J1-PCR, may be used in combination
with any one or more of primers Onc2A2-PCR, Onc2B2-PCR, Onc2K2-PCR,
Onc2L2-PCR, Onc2M2-PCR, Onc2N2-PCR, Onc2O2-PCR and Onc2P2-PCR for
PCR amplification of HPV L1 mRNA.
[0131] The invention will be further understood with reference to
the following experimental examples and figures in which:
[0132] FIG. 1A shows the results of a single reaction real-time
NASBA assay using a FAM molecular beacon for HPV 16 on a patient
sample while FIG. 1B shows multiplexed real-time NASBA assay using
the FAM molecular beacon of FIG. 1A and a molecular beacon labeled
with Texas red for UTA,
[0133] FIG. 2A shows single reaction real-time NASBA with FAM
molecular beacon for HPV 18 on a patient sample while FIG. 2B shows
a multiplexed version with a Texas red labeled molecular beacon for
HPV 18 and a FAM labeled beacon for HPV 33,
[0134] FIG. 3A shows single reaction real-time NASBA with HPV 31
FAM labeled molecular beacon while FIG. 3B shows multiplexed
version including HPV 45 Texas red labeled molecular beacon,
[0135] FIG. 4A shows single reaction real-time NASBA with HPV 33
FAM labeled molecular beacon while FIG. 4B is a multiplexed version
including a Texas red labeled HPV 18 molecular beacon,
[0136] FIG. 5A shows single reaction real-time NASBA with HPV45 FAM
labeled molecular beacon while FIG. 5B shows the multiplexed
version including HPV 45 Texas red labeled molecular beacon and a
FAM labeled HPV 31 molecular beacon, and
[0137] FIG. 6 shows HPV detected by PreTect HPV-Proofer and PCR
compared to cytology or histology.
EXAMPLE 1
Detection of HPV mRNA by NASBA-Based Nucleic Acid Amplification and
Real-Time Detection
Collection and Preparation of Clinical Samples
[0138] Pap smears and HPV samples were collected from 5970 women in
the cervical screening program in Oslo, Norway. Samples intended
for RNA/DNA extraction were treated as follows:
[0139] Cervical samples were collected from each women attending
the cervical screening program using a cytobrush (Rovers Medical
Devices, The Netherlands). The cytobrush was then immersed in 9 ml
lysis buffer (5M Guanidine thiocyanate). Since RNA is best
protected in the 5M guanidine thiocyanate at -70.degree. C. only 1
ml of the total volume of sample was used for each extraction
round. The samples in lysis buffer were stored at -20.degree. C.
for no more than one week, then at -70.degree. C. until isolation
of DNA/RNA.
[0140] RNA and DNA were automatically isolated from 5300 women in
the first round of extraction, using 1 ml from the total sample of
9 ml in lysis buffer. RNA and DNA were extracted according to the
"Booms" isolation method from Organon Teknika (Organon Teknika B.
V., Boselind 15, P.O. Box 84, 5280 AB Baxtel, The Netherlands; now
Biomerieux, 69280 Marcy l'Etoile, France) using the Nuclisens.TM.
extractor following the protocol for automated extraction.
Cell Lines
[0141] DNA and RNA from HeLa (HPV 18), SiHa (HPV 16) and CaSki (HPV
16) cell lines were used as positive controls for the PCR and NASBA
reactions. These cells were also used as sample material in the
sensitivity study (Example 2). SiHa cells have integrated 1-2
copies of HPV 16 per cell, whilst CaSki cells have between 60-600
copies of HPV 16, both integrated and in the episomal state. HeLa
cells have approximately 10-50 copies of HPV 18 per cell.
HPV Detection and Typing by PCR
[0142] Isolated DNA from cervical scrapes was subjected to PCR
using the consensus GPS+/6+ primers (EP-B-0 517 704). The PCR was
carried out in 50 .mu.l reaction volume containing 75 mM Tris-HCl
(pH 8.8 at 25.degree. C.), 20 mM (NH.sub.4).sub.2SO.sub.4, 0.01%
Tween 20.TM., 200 mM each of dNTP, 1.5 mM MgCl.sub.2, 1 U
recombinant Taq DNA Polymerase (MBI Fermentas), 3 .mu.l DNA sample
and 50 pmol of each GP5+ and GP6+ primers. A 2 minutes denaturation
step at 94.degree. C. was followed by 40 cycles of amplification
with a PCR processor (Primus 96, HPL block, MWG, Germany). Each
cycle included a denaturation step at 1 minutes, a primer annealing
step at 40.degree. C. for 2 minutes and a chain elongation step at
72.degree. C. for 1.5 minutes. The final elongation step was
prolonged by 4 minutes to ensure a complete extension of the
amplified DNA.
[0143] The GP5+/6+ positive samples were subjected to HPV type 16,
31 and 33 PCR protocols as follows:
HPV 16, 31 and 33: The PCR was carried out in 50 .mu.l containing
75 mM Tris-HCl (pH 8.8 at 25.degree. C.), 200 mM each of dNTP, 1.5
mM MgCl.sub.2, 2.5 U recombinant Taq DNA Polymerase (MBI
Fermentas), 3 .mu.l DNA sample and 25 pmol of each primers. A 2
minutes denaturation step at 94.degree. C. was followed by 35
cycles of amplification with a PCR processor (Primus 96, HPL block,
MWG, Germany). Each cycle included a denaturation step at 30 sec, a
primer annealing step at 57.degree. C. for 30 sec and a chain
elongation step at 72.degree. C. for 1 minutes. The final
elongation step was prolonged by 10 minutes to ensure a complete
extension of the amplified DNA. The protocol for HPV 33 had a
primer annealing step at 52.degree. C. HPV 18 protocol: Primers
were designed to identify HPV type 18. The PCR was carried out in
50 .mu.l containing 75 mM Tris-HCl (pH 8.8 at 25.degree. C.), 20 mM
(NH.sub.4).sub.2SO.sub.4, 0.01% Tween 20, 200 mM each of dNTP, 2.0
mM MgCl.sub.2, 2.5 U recombinant Taq DNA Polymerase (MBI
Fermentas), 3 .mu.l DNA sample and 25 pmol of each primer. A 2
minutes denaturation step at 94.degree. C. was followed by 35
cycles of amplification in a PCR processor (Primus 96, HPL block,
MWG, Germany). Each cycle included a denaturation step at 30 sec, a
primer annealing step at 57.degree. C. for 30 sec and a chain
elongation step at 72.degree. C. for 1 minutes. The final
elongation step was prolonged by 10 minutes to ensure a complete
extension of the amplified DNA.
[0144] A primer set directed against the human-globin gene was used
as a control of the DNA quality (Operating procedure, University
Hospital Vrije Universiteit, Amsterdam, The Netherlands). The PCR
was carried out in 50 .mu.l containing 75 mM Tris-HCl (pH 8.8 at
25.degree. C.), 200 mM each of dNTP, 1.5 mM MgCl.sub.2, 1 U
Recombinant Taq DNA Polymerase (MBI Fermentas), 3 .mu.l DNA sample
and 25 pmol of each primer. A 2 minutes denaturation step at
94.degree. C. was followed by 35 cycles of amplification with a PCR
processor (Primus 96, HPL block, MWG, Germany). Each cycle included
a denaturation step at 94.degree. C. for 1 minute, a primer
annealing step at 55.degree. C. for 11/2 minutes and a chain
elongation step at 72.degree. C. for 2 minutes. The final
elongation step was prolonged by 4 minutes to ensure a complete
extension of the amplified DNA. HeLa was used as positive controls
for HPV 18, while SiHa or CaSki were used as positive control for
HPV 16. Water was used as negative control.
Primers Used for HPV PCR:
TABLE-US-00011 [0145] Length Type Primer Primer sequence (SEQ ID
No.) Position (bp) HPV16 Pr1 5' TCA AAA GCC ACT GTG TCC TGA 3'
(318) 421-440 119 Pr2 5' CGT GTT CTT GAT GAT CTG CAA 3' (319)
521-540 HPV18 Pr1 (5' TTC CGG TTG ACC TTC TAT GT 3') (320) 651-670
186 Pr2 (5' GGT CGT CTG CTG AGC TTT CT 3') (321) 817-836 HPV31 Pr1
5' CTA CAG TAA GCA TTG TGC TAT GC 3' (322) 3835-3875 153 Pr2 5' ACG
TAA TGG AGA GGT TGC AAT AAC CC 3' (323) 3963-3988 HPV33 Pr1 5' AAC
GCC ATG AGA GGA CAC AAG 3' (324) 567-587 211 Pr2 5' ACA CAT AAA CGA
ACT GTG TGT 3' (346) 758-778 Gp+ Gp5+ 5' TTT GTT ACT GTG GTA GAT
ACT AC 3' (338) 6624-6649 150 Gp6+ 5' GAA AAA TAA ACT GTA AAT CAT
ATT C (339) 6719-6746 BGPCO3 Pr1 5' ACA CAA CTG TGT TCA CTA GC
(340) BGPCO5 Pr2 5' GAA ACC CAA GAG TCT TCT CT (341)
[0146] Visualization of the PCR products was done on a DNA 500 chip
(Agilent Technologies, USA) according to their manual. The DNA chip
uses micro scale gel electrophoresis with an optimal detection
limit of 0.5-50 ng/ml. The results were interpreted using the
Bioanalyzer 2100 software (Agilent Technologies, USA).
[0147] The following table confirms primers used for HPV PCR in
patient samples and indicates additional PCR primers useful for HPV
35, 39, 45, 51, 52, 58 and HPV 6/11.
PCR Primers for Detection of HPV.
TABLE-US-00012 [0148] Length Type Primer Primer sequence (SEQ ID
No.) Position (bp) HPV 6/11 Pr1 5' TAC ACT GCT GGA CAA CAT 3' (316)
514-531 123 Pr2 5' TCA TCT TCT GAG CTG TCT 3' (317) 619-636 HPV16
Pr1 5' TCA AAA GCC ACT GTG TCC TGA 3' (318) 421-441 120 Pr2 5' CGT
GTT CTT GAT GAT CTG CAA 3' (319) 520-540 HPV18 Pr1 5' TTC CGG TTG
ACC TTC TAT GT 3' (320) 651-670 186 Pr2 5' GGT CGT CTG CTG AGC TTT
CT 3' (321) 817-836 HPV31 Pr1 5' CTA CAG TAA GCA TTG TGC TAT GC 3'
(322) 3835-3857 155 Pr2 5' ACG TAA TGG AGA GGT TGC AAT AAC CC 3'
(323) 3964-3989 HPV33 Pr1 5' AAC GCC ATG AGA GGA CAC AAG 3' (324)
567-587 212 Pr2 5' ACA CAT AAA CGA ACT GTG GTG 3' (325) 758-778 HPV
35 Pr1 5' CCC GAG GCA ACT GAC CTA TA 3' (326) 610-629 231 Pr2 5'
GGG GCA CAC TAT TCC AA ATG 3' (327) 821-840 HPV 39 Pr1 5' GCA GAC
GAC CAC TAC AGC AAA 3' (328) 210-230 153 Pr2 5' ACA CCG AGT CCG AGT
AAT A 3' (329) 344-362 HPV 45 Pr1 5' GAA ACC ATT GAA CCC AGC AGA
AAA 3' (330) 428-451 154 Pr2 5' TTG CTA TAC TTG TGT TTC CCT ACG 3'
(331) 558-581 HPV 51 Pr1 5' GGA GGA GGA TGA AGT AGA TA 3' (332)
658-677 169 Pr2 5' GCC CAT TAA CAT CTG CTG TA 3' (333) 807-826 HPV
52 Pr1 5' GTG CCT ACG CTT TTT ATC TA 3' (334) 296-315 233 Pr2 5'
GGG GTC TCC AAC ACT CTG AAC A 3' (335) 507-528 HPV 58 Pr1 5' TCA
GGC GTT GGA GAC ATC 3' (336) 157-174 162 Pr2 5' AGC AAT CGT AAG CAC
ACT 3' (337) 301-318 Gp+ Gp5+ 5' TTT GTT ACT GTG GTA GAT ACT AC 3'
(338) 150 Gp6+ 5' GAA AAA TAA ACT GTA AAT CAT ATT C (339) BGPCO3
Pr1 5' ACA CAA CTG TGT TCA CTA GC (340) BGPCO5 Pr2 5' GAA ACC CAA
GAG TCT TCT CT (341)
NASBA RNA Amplification
[0149] Precautions for avoiding contamination:
[0150] 1. Perform nucleic acid release, isolation and
amplification/detection in separate laboratory areas.
[0151] 2. Store and prepare reagents for nucleic acid release,
isolation and amplification/detection at the laboratory areas where
nucleic acid release, isolation and amplification/detection are to
be performed, respectively.
[0152] 3. Keep all tubes and vials closed when not in use.
[0153] 4. Pipettes and other equipment that have been used in one
laboratory area must not be used in the other areas.
[0154] 5. Use a fresh pipette or pipette tip for each pipetting
action.
[0155] 6. Use pipettes with aerosol resistant tips for fluids
possibly containing nucleic acid. Pipetting of solutions must
always be performed out of or into an isolated tube that is opened
and closed exclusively for this action. All other tubes and vials
should be kept closed and separated from the one handled.
[0156] 7. Use disposable gloves when working with clinical material
possibly containing target-RNA or amplified material. If possible,
change gloves after each pipetting step in the test procedure,
especially after contact with possibly contaminated material.
[0157] 8. Collect used disposable material in a container. Close
and remove container after each test run.
[0158] 9. Soak tube racks used during nucleic acid isolation or
amplification/detection in a detergent (e.g. Merck Extran MA01
alkaline) for at least one hour after each test run.
[0159] The following procedure was carried out using reagents from
the Nuclisens.TM. Basic Kit, supplied by Organon Teknika.
[0160] Procedure for n=10 samples:--
[0161] 1. Prepare Enzyme Solution.
[0162] Add 55 .mu.l of enzyme diluent (from Nuclisens.TM. Basic
Kit; contains sorbitol in aqueous solution) to each of 3
lyophilized enzyme spheres (from Nuclisens.TM. Basic Kit; contains
AMV-RT, RNase H, T7 RNA polymerase and BSA). Leave this enzyme
solution at least for 20 minutes at room temperature. Gather the
enzyme solutions in one tube, mix well by flicking the tube with
your finger, spin down briefly and use within 1 hour. Final
concentrations in the enzyme mix are 375 mM sorbitol, 2.5 .mu.g
BSA, 0.08 U RNase H, 32 U T7 RNA polymerase and 6.4 U AMV-reverse
transcriptase.
[0163] 2. Prepare Reagent Sphere/KCl Solution.
[0164] For 10 samples: add 80 .mu.l reagent sphere diluent (from
Nuclisens.TM. Basic Kit; contains Tris/HCl (pH 8.5), 45% DMSO) to
the lyophilized reagent sphere (from Nuclisens.TM. Basic Kit;
contains nucleotides, dithiotreitol and MgCl.sub.2) and immediately
vortex well. Do this with 3 reagent spheres and mix the solutions
in one tube.
[0165] Add 3 .mu.l NASBA water (from Nuclisens.TM. Basic Kit) to
the reconstituted reagent sphere solution and mix well.
[0166] Add 56 .mu.l of KCl stock solution (from Nuclisens.TM. Basic
Kit) and mix well. Use of this KCl/water mixture will result in
NASBA reactions with a final KCl concentration of 70 mM. Final
concentrations in the reagent/KCl solution are 1 mM of each dNTP, 2
mM of ATP, UTP and CTP, 1.5 mM GTP, and 0.5 mM ITP, 0.5 mM
dithiotreitol, 70 mM KCl, 12 mM MgCl.sub.2, 40 mM Tris-HCl (pH
8.5).
[0167] 3. Prepare Primer/Probe Solution Containing Target-Specific
Primers and Molecular Beacon Probe.
[0168] For each target reaction transfer 91 .mu.l of the reagent
sphere/KCl solution (prepared in step 2) into a fresh tube. Add 25
.mu.l of primers/molecular beacon probe solution (to give final
concentration of .about.0.1-0.5 .mu.M each of the sense and
antisense primers and .about.15-70 pmol molecular beacon probe per
reaction). Mix well by vortexing. Do not centrifuge.
[0169] In case less than 10 target RNA amplifications are being
performed refer to the table below for the appropriate amounts of
reagent sphere solution, KCl/water solution and primers to be used.
Primer solutions should be used within 30 minutes after
preparation.
TABLE-US-00013 Reagent sphere Reactions (n) solution (.mu.l)
KCl/water (.mu.l) Primer mix (.mu.l) 10 80 30 10 9 72 27 9 8 64 24
8 7 56 21 7 6 48 18 6 4 32 12 4 3 24 9 3 2 16 6 2 1 8 3 1
[0170] 4. Addition of Samples
[0171] For each target RNA reaction:
[0172] In a 96 well microtiter plate pipette 10 .mu.l of the
primer/probe solution (prepared in step 3) into each of 10 wells.
Add 5 .mu.l nucleic acid extract to each well. Incubate the
microtiter plate for 4 minutes at 65.+-.1.degree. C. Cool to at
41.+-.0.5.degree. C. for 4 minutes. Then to each well add 5 .mu.l
enzyme solution. Immediately place the microtiter plate in a
fluorescent detection instrument (e.g. NucliSens.TM. EasyQ
Analyzer) and start the amplification.
Results from Clinical Study
[0173] Table 7 shows the distribution of real-time NASBA HPV
positive (L1 and/or E6 expression) and PCR HPV positive cases
related to cytology results. PCR amplification was carried out as
described by Karlsen et al., J Clin Microbiol. 34: 2095-2100, 1996.
The figures for expected histology are based on average results
from similar study on CIN III lesions (Clavel et al., Br J Cancer,
84: 1616-1623, 2001). The results from several example cases are
listed in Table 8.
TABLE-US-00014 TABLE 7 Normal Benign Condyloma CIN III Cytology
4474 66 16 15 PCR 9.0% 44.6% 87.5% 73.3% Real-time 1% 24.6% 37.5%
73.3% NASBA Expected 0.2% 5-15% 15-20% 71% Histology
TABLE-US-00015 TABLE 8 Internal No. Cytology PCR L1 NASBA E6 NASBA
84 Neg Neg Neg 31 289 Neg 31 Pos 31 926 Neg Neg Pos 16 743 Benign
Neg Neg 33 1512 Benign 16 Pos 16 3437 Benign Neg Neg 18 3696 Benign
16 Pos Neg 2043 Condyloma 16, 51 Pos 16 3873 Condyloma 16, 51 Pos
16 3634 CIN II 33 Neg 33 4276 CIN III Neg Neg 18 4767 CIN III 18
Neg 18 1482 CIN III Neg Pos 16 5217 CIN III 31 Neg 31 4696 CIN III
Neg Neg Neg
EXAMPLE 2
Sensitivity of Real-Time NASBA on Control Cell Lines
[0174] Cervical cancer cell lines, CaSki, SiHa and HeLa were
diluted in lysis buffer either before automated extraction of
nucleic acids using the Boom's extraction method from Organon
Teknika/bioMerieux (parallels 1 and 3), or after nucleic acid
extraction (parallel 2). Real-time NASBA was performed using
molecular beacons probes labelled with Texas red (16, L1 and 18) or
FAM (U1A, 33 and 31) following the protocol described above.
TABLE-US-00016 TABLE 9 CaSki CaSki HeLa Primer sets and probes 16
16 16 33 33 33 18 31 18 31 18 31 E6 U1 E6 U1 E6 U1 L1 E6 L1 E6 L1
E6 E6 E6 E6 E6 E6 E6 Number of Parallels Cells 1 1 2 2 3 3 1 1 2 2
3 3 1 1 2 2 3 3 100 000 + + + + + + + - + - + - + - + - + - 10 000
+ + + + + + + - + - + - + - + - + - 1 000 + + + + + + + - + - + - +
- + - + - 100 + + + + + + + - + - + - + - + - + - 10 + + + + + + -
- - - + - + - + - + - 1 - - + - + - - - - - - - + - + - + -
10.sup.-1 - - - - - - - - - - - - - - - - - -
[0175] Thus, it is possible to detect HPV E6 mRNA in less than 1
cell using real-time NASBA.
[0176] Real-time NASBA was tested both as a multiplex assay and as
single reactions. The results from the following sensitivity study
are based on parallel runs of CaSki, SiHa and HeLa cell lines, and
on three parallel runs on synthetic DNA oligos for HPV type 16, 18,
31 and 33. The definition of the detection limit is that both of
the samples in the parallel are positive. The number in the
brackets (x) indicates that the specified amount of cells also have
been detected in some runs. Sensitivity is defined as the amount of
cells necessary for detection of HPV in two parallel runs. The HPV
types are determined from PCR and the specificity is based on NASBA
compared to PCR.
Sensitivity
[0177] PCR: the HPV consensus PCR using Gp5+/6+ detected only down
to 10.sup.4 SiHa and HeLa cells, and down to 10.sup.3 CaSki cells.
However, the type specific PCR primer-sets were more sensitive,
detecting 10.sup.3 (10.sup.2) SiHa cells and 0.1 CaSki cells for
HPV 16 type specific PCR primer-set, while the HPV 18 type specific
PCR primer-set detected 10.sup.2 HeLa cells.
[0178] Real-time NASBA: Real-time NASBA with primers specific for
U1A, detected 10(1) SiHa and CaSki cells and 1 HeLa cell in the
reaction mixture. For the HPV 16 specific primers, the lower
detection limit was (10) (10.sup.2, 1) SiHa cells and 10 (1) CaSki
cells and for the HPV 18 specific primers the detection limit was 1
(0.1) HeLa cell. The universal L1 primers detected 10 CaSki cells.
HeLa cells and SiHa cells were not detected with the universal L1
primers.
[0179] Real-time multiplex NASBA with the U1A specific primers, had
a lower detection limit of 10.sup.2(10) SiHa cells and 10(1) CaSki
cells when combined with the HPV 16 specific primers, which had a
lower detection limit for 10(1) SiHa and 10(1) CaSki cells. The L1
specific primers in combination with the HPV 33 specific primers
detected 10.sup.3(102) CaSki cells. There was no competing HPV 33
sample in the reaction. For the HPV 18 specific primers, the lower
detection limit was 1 (0.1) HeLa cell when combined with the HPV 31
specific primers. There was no competing HPV 31 sample in the
reaction. Sensitivity of the HPV 31 and HPV 33 specific primers
were not tested, due to lack of cell lines harbouring these HPV
types. They were tested against samples containing HPV 31 and HPV
33, but the amount of cells and the copy number of HPV 31 and HPV
33 in these cells were unknown and most probably varied in
different samples.
TABLE-US-00017 TABLE 10 sensitivity of real-time NASBA compared to
PCR NASBA PCR Primer SiHa CaSki HeLa SiHa CaSki HeLa GP5+/6+ -- --
-- 10.sup.4 10.sup.3 10.sup.3 L1 -- 10.sup.3 (10.sup.2) -- -- -- --
U1A 10.sup.2 (10) 10 (1) -- -- -- -- HPV 16 10 (1) 10 -- 10.sup.3
(10.sup.2) 0.1 -- HPV 18 -- -- 1 (0, 1) -- -- 10.sup.2
[0180] Real-time NASBA was performed on samples from women admitted
to Ostfold Central Hospital for treatment of CIN in the period of
1999-2001 (see example 3). Molecular beacon probes labeled with FAM
or Texas red were used together with the nucleic acid extraction
and NASBA protocols described above. The results are shown in FIGS.
1A and 1B (HPV 16--patient sample 205), FIGS. 2A and 2B (HPV
18--patient sample 146), FIGS. 3A and 3B (HPV 31--patient sample
236), FIGS. 4A and 4B (HPV 33--patient sample 218) and FIGS. 5A and
5B (HPV 45--patient sample 343). In each case, the "A" figure is a
single reaction while the "B" figure is the multiplex assay.
[0181] Specificity: Cross reactivity of Real-time NASBA. Real-time
NASBA primer combinations were tested against 490 cervical samples
from the Oslo study positive with PCR for HPV 6/11, 16, 18, 31, 33,
35, 39, 45, 51, 52, 58 or HPV X to check for cross reactivity
between HPV types using NASBA. All samples have been typed by
consensus PCR and type specific PCR for the respective HPV types,
except for HPV type 39(2), 52(1) and 58(2). These samples are added
to test against PreTect HPV-Proofer. HPV X are positive for
consensus Gp5+/6+ PCR but negative for HPV6/11, 16, 18, 31, 33, 35,
45 and 51 by type specific PCR. Results are shown in table 14. No
cross-reactivity was shown. Sequence confirmation of a selected
number of cases from table 14 is shown in table 14a.
[0182] PCR: a total of 773 cervical samples were tested with PCR
and the PreTect HPV-Proofer (Real time multiplex NASBA), and a
total of 24.6% (190/773) samples were positive with the Gp5+/6+
consensus PCR primers. 74.1% (83/112) were typed to be HPV 16, 13%
(15/112) HPV 18, 17% (19/112) HPV 31 and 12% (13/112) HPV 33
including multiple HPV infections. A total of 103 samples had
single or multiple HPV infections, and 91.3% (94/103) had only a
single HPV infection. Double HPV infections occurred in 8.7%
(9/103) of the samples. All samples were first tested with the
consensus Gp5+/6+ PCR primers. The HPV PCR negative samples from
the consensus Gp5+/6+ were then tested with .beta.-globin control
primers for a verification of intact DNA. The HPV PCR positive
samples were not subjected to this DNA control. The HPV negative
samples in this study were all positive with .beta.-globin control
PCR primers. Only DNA samples positive with Gp5+/6+ PCR were
subjected to HPV type specific PCR. HPV types of interest were HPV
16, 18, 31 and 33.
[0183] Real-time multiplex NASBA: For the real-time NASBA
reactions, the primers and probes for the U1A gene product were
used as a performance control for intact RNA. Samples negative for
U1A were rejected.
[0184] A total of 14.2% (110/773) of the samples were positive with
at least one of the HPV type-specific NASBA primers including
samples showing multiple HPV infections. From these samples 54.5%
(60/110) were positive with HPV 16 NASBA primers, 13.6% (15/110)
with HPV 18 primers, 21.8% (24/110) with HPV 31 primers and 13.6%
(15/110) with HPV 33 primers. A total of 45 samples were positive
with the L1 consensus primers and usually together with HPV 16
E6/E7 oncogene expression 82.2% (37/45). The consensus L1 was
detected in 2.2% (1/45) together with either HPV 18, 31 and 33
respectively. L1 was also detected alone in 8.9% (4/45) cases, and
they all were PCR positive with Gp5+/6+ primers. A total of 108
samples had single or multiple HPV infections, and 98.1% (106/108)
had only a single HPV infections. Double mRNA expression occurred
in 1.9% (2/108) of the samples.
[0185] Real-time multiplex NASBA compared to PCR: a total of 87
samples showed presence of HPV 16 DNA or RNA with HPV 16 PCR or
PreTect HPV-Proofer. 64.4% (56/87) were determined to be positive
for HPV 16 with both PCR and real-time NASBA. 39.1% (34/87) were
only positive with PCR and 3.4% (3/87) were positive only with
real-time NASBA. For HPV 18, a total of 20 samples showed presence
of HPV 18 DNA or RNA with either PCR or real-time NASBA. From these
20 samples, 50% (10/20) were positive with both tests, and 35%
(7/20) were only positive with PCR and 15% (3/20) were only
positive with real-time NASBA. A total of 27 samples showed
presence of HPV 31 DNA or RNA with either PCR or real-time NASBA.
Out of these 27 samples, 59.3% (16/27) were positive with both
tests and 11.1% (3/27) were positive only with the PCR test and
18.5% (5/27) were only positive with the real-time NASBA test. For
HPV 33, a total of 18 samples showed presence of HPV DNA or RNA
with either PCR or PreTect-HPV Proofer and 55.6% (10/18) of the
samples were tested positive with both tests. 16.7% (3/18) were
only positive with PCR and 22.2% (4/18) were only positive with
real-time NASBA.
TABLE-US-00018 TABLE 11 statistical distribution of HPV in samples
with PCR and real-time NASBA PCR % NASBA % Total samples 773 773
Total positive 190 24.6 110 14.2 samples HPV16 83 74.1 60 54.5 HPV
18 15 13 15 13.6 HPV 31 19 17 24 21.8 HPV33 13 12 15 13.6 HPV X 78
69.6 -- --
TABLE-US-00019 TABLE 12 correspondence between PCR and real-time
NASBA Both Only % % Only % Total tests % PCR + (PCR) (Total) NASBA
% NASBA (total) HPV 16 87 56 64.4 34 41.0 39.1 3 5 3.4 HPV 18 20 10
50.0 7 46.7 35.0 3 20 15.0 HPV 31 27 16 59.3 3 15.8 11.1 5 20.8
18.5 HPV 33 18 10 55.6 3 23.1 16.7 4 26.7 22.2
TABLE-US-00020 TABLE 13 Real-time NASBA results for L1 Total % L1
(NASBA) 45 100 L1 + HPV 16 37 82.2 L1 alone 4 8.9 L1 + HPV 18 1 2.2
L1 + HPV 31 1 2.2 L1 + HPV 33 1 2.2
TABLE-US-00021 TABLE 14 Genetic specificity of real-time multiplex
NASBA compared to PCR NASBA HPV (PCR) Total Primers 6/11 16 18 31
33 35 39 45 51 52 58 X Number 16 2 28 1 0 1 1 0 0 5 0 0 2 18 1 1 18
0 1 0 0 0 1 0 0 1 31 1 0 1 13 1 5 0 1 1 0 0 0 33 1 2 0 2 12 2 0 1 2
0 0 1 45 2 1 1 1 1 1 0 17 1 0 0 1 Sum 43 71 36 32 25 23 2 23 31 1 2
201 490 tested
TABLE-US-00022 TABLE 14a DNA sequencing from Gp5+ PCR primers (not
to be included in the article) HPV type by PreTect HPV- HPV type by
Sequencing Int No HPV type by PCR Proofer (BLAST) 1272 16 16 16 132
35 35 2655 58 58 2924 33 33 33 2942 18 18 18 2987 16 16 16 3016 33
33 33 3041 35 35 3393 35 35 3873 16 16 16 4767 18 18 18 5707 18 18
18 845 X 39
Discussion
[0186] Sensitivity of real-time NASBA was generally better than the
sensitivity of PCR. The general sensitivity of real-time NASBA for
all the markers were between 1 and 10.sup.2 cells, which is
considerable better than for the PCR reaction with a sensitivity
range from 10.sup.2 to 10.sup.4. As expected, the sensitivity of
the specific primers and probes were better than the sensitivity of
the universal primers and probes. Real-time NASBA was just as
sensitive or more sensitive than real-time multiplex NASBA.
[0187] Real-time NASBA primers and molecular beacon probe directed
towards U1A (a human house keeping gene) were used as a performance
control of the sample material in the real-time NASBA reaction to
ensure that the RNA in the sample material was intact. A positive
signal from this reaction was necessary for a validation of the
real-time NASBA reaction.
[0188] The sensitivity of the universal real-time NASBA with L1
(the major capsid protein of HPV) was much better than for the
universal Gp5+/6+ PCR, also directed against L1, with a sensitivity
of 10 cells compared to 10.sup.3 (10.sup.2) CaSki cells. These two
primer sets (PCR and NASBA) have their targets in the same region
of the conserved L1 gene of different HPV types. The differences in
sensitivity may be due to the fact that there is usually one copy
of each gene per cell, while the copy number of mRNA may be several
hundreds. The real-time NASBA L1 primers did not detect SiHa or
HeLa cells as the Gp5+/6+ PCR primers did, indicating lack of L1
expression in these cell lines. Gp5+/6+ PCR primers detected
10.sup.4 SiHa or HeLa cells. Considering the amount of HPV copies
in each cell, it makes sense that the CaSki cells were detected in
1/10 the amount of cells from SiHa and HeLa since CaSki cells have
60-600 HPV copies per cell, both integrated and episomal, while
SiHa cells have 1-2 HPV copies integrated per cell and HeLa cells
have 10-50 HPV copies integrated per cell. The L1 primer set
detected only CaSki cells, with both integrated and episomal forms
of HPV, and not in SiHa or HeLa cells, with only integrated forms
of HPV. This might indicate that the L1 gene is only expressed in
episomal states of HPV infection, and therefore L1 may be a
valuable marker for integration and persistence of HPV
infection.
[0189] The HPV type-specific NASBA primers are directed against the
full length E6/E7 transcript, which are expressed in large amount
in cancer cells due to lack of E2 gene product. The real-time NASBA
16 type specific primers detected 10(1) SiHa cells and 10(1) CaSki
cells compared to HPV 16 PCR primers that detected
10.sup.3(10.sup.2) SiHa cells. The explanation for this might be
the different amount of HPV copies in each cell line. The CaSki
cells have both integrated and episomal forms of HPV, while SiHa
has only integrated forms of HPV. This may be due to high
expression of mRNA from the E6/E7 genes. For detection of CaSki
cells, the detection limit for the NASBA HPV 16 primers were 10(1)
CaSki cells compared to 0.1 CaSki cells for the HPV 16 PCR primers.
This is peculiar, but an explanation may be that the CaSki cells
contain from 60-600 copies of HPV 16 DNA, so that it is possible to
detect 0.1 CaSki cells with 6-60 HPV 16 DNA copies. The lower
sensitivity of real-time NASBA compared to PCR may indicate that
the expression of E6/E7 in the CaSki cells is moderate/low.
Degradation of the unstable mRNA may also be an explanation. The
amount of HPV copies in the CaSki cells may be in the order of
60-600 times more than in the SiHa cells, which is shown by the
more sensitive detection of CaSki cells.
[0190] The type specific HPV 18 PCR primers detected 10.sup.2 HeLa
cells. This is a magnitude of 100 better than the HPV consensus
Gp5+/6+ primers and states that specific primers are generally more
sensitive than consensus primers. The sensitivity of the type
specific HPV 18 NASBA primers was 1 (0,1) HeLa cells, indicating
high expression of E6/E7 in HeLa cells.
[0191] The sensitivity of U1A NASBA primers was 10 SiHa or CaSki.
The target for the U1A primer set is a human housekeeping gene that
is expressed in every human cell.
[0192] The sensitivity of PCR and NASBA varies for different primer
sets and sample material, and generally type specific primers are
more sensitive than consensus primers due to base pair mismatch in
consensus primer sets. The annealing temperature for the primers in
the PCR reaction can be optimised, giving optimal reaction
condition for the primers. In contrast to the annealing temperature
in PCR, the annealing temperature for the NASBA primers must be
fixed at 41.degree. C. This lack of temperature flexibility may
make the NASBA primers less sensitive and specific than the PCR
primers.
[0193] PCR amplifies double stranded DNA and the target is usually
present as one copy per cell and this makes it vulnerable to the
number of cells in the sample material. The target for the NASBA
reaction is RNA, and mRNA may be present as multiple copies per
cell, depending on the expression of the genes. By choosing a gene
that is highly expressed, the mRNA copy number may be several
hundred per cell and therefore easier to detect.
[0194] dsDNA is relatively stable in the cell and the material
stays intact for a long time. In contrast to dsDNA, mRNA is
generally not very stable and degradation of mRNA is rapid
depending on the cell. There is no detected DNase or RNase activity
in the lysis buffer so both dsDNA and ssRNA should be stable.
Autocatalytic activity may degrade both DNA and RNA. The DNA/RNA
from the cervix sample should stay intact, when stored in the lysis
buffer, for 24 hours at 15-30.degree. C., 7 days at 2-8.degree. C.
or at -70.degree. C. for long term storage.
[0195] A limitation in the real-time NASBA reaction is the
concentration of the molecular beacon probes. The amount of
products will exceed the concentration of the molecular beacon
probes and therefore it will not be detected because a high
molecular beacon probe concentration will make the reaction mixture
more complex and inhibit the amplification reaction.
[0196] Nucleotides may also be a limitation to the final amount of
the amplification product, both in the PCR and in the NASBA
reaction. The final concentration of the amplified product may in
itself inhibit further amplification because of the amount of
product and the complexity of the reaction mixture. During a NASBA
reaction in the presence of molecular beacons, the probe might
compete with the amplification by hybridising to the template,
making it unavailable for following RNA synthesis. In this way, RNA
is subtracted as substrate for the reverse transcription steps and
further RNA synthesis by T7 RNA polymerase. This competition is not
significant with low amounts of molecular beacon, and with a high
amount of molecular beacon this inhibition can be overcome by a
higher number of copies of input RNA.
[0197] The linear relationship between the amount of input RNA and
the time-to-positive signal was tested in a ten-fold serial
dilutions of different HPV cell lines. There was a clear indication
that a positive signal was dependent on the amount of input RNA and
time. The multiplex reaction needed more time than the single
reaction to show a positive signal. This might be due to
competition in the more complex mixture in the multiplex reaction
vessel and also to the fact that the multiplex reaction has a
different and lower concentration of primer and probe. The
relationship between amount of target RNA and time to positive
signal opens up for a real-time multiplex quantitative
amplification reaction with internal RNA standards in each reaction
vessel.
[0198] Real-time NASBA: single vs. multiplex. Real-time NASBA was
generally more sensitive than real-time multiplex NASBA. This was
as expected because of competition between primers and probes in
the multiplex reaction. The final concentration of primers and
molecular beacon probes were optimised in the multiplex reaction so
that for at least one of the primer and probe sets the
concentration were lower than in the single reaction. From this it
follows that with a lower concentration of primers, the less
sensitive the reaction, or at least the less rapid the reaction. It
will take longer time to reach the exponential stage of the
amplification reaction and therefore longer time to detect the
products. The concentration of the primers will not be a limitation
to the final concentration of the product in the NASBA reaction
because the double stranded DNA created from the primers will
continue to serve as a template for the RNA polymerase over and
over again in a loop. The sensitivity of multiplex real-time NASBA
was the same for HPV 16 and HPV 18 compared to single real-time
NASBA, but the sensitivity for L1 decreased drastically from a
detection limit of 10(1) in the single reaction to
10.sup.3(10.sup.2) CaSki cells in the multiplex reaction. For U1A
NASBA primers, the sensitivity decreased from 10(1) to 10.sup.2(10)
SiHa cells, while the detection limit remained the same for the
CaSki cells. This decrease in detection limit may be to more
complex competition of primers and molecular beacon probes in the
multiplex reaction. The final concentration of primers and
molecular beacon probes may not be the best and the different
primers and molecular beacon probes in the multiplex reaction may
interfere with each other. The U1A NASBA primers detected 1 HeLa
cell. One might expect the same detection level in all the cell
lines, but the sensitivity of HeLa cells were 1/10 of the detection
level of SiHa and CaSki cells. These cell lines are cancer cells
and they might have different impact on the cells so that the
expression of U1A is different. The differences may also be due to
different amount of cells in each reaction, because of counting
errors during harvesting of the cells.
[0199] Real-time NASBA showed no cross reactivity between HPV 16,
18, 31 and 33 or with HPV 6/11, 35, 39, 45, 51, 52, 58 or HPV
X.
[0200] The specificity of the PCR reaction may be better than the
specificity of the real-time NASBA reaction because the NASBA
reaction is an isothermal reaction at 41.degree. C. with no
possibilities to change the annealing temperature of the primers.
The primers are basically designed the same way as for the PCR
primers. In a PCR reaction, you have the possibility to change the
annealing temperature, in contrast to the NASBA reaction, and
therefore choose an annealing temperature that is optimal for the
two primers. This makes the annealing of the primers more specific.
The PCR results where visualized with gel electrophoresis. But the
molecular beacon probes in the real-time NASBA reaction is an
additional parameter compared to PCR and therefore may give the
overall NASBA reaction a better specificity. It is also easier to
find two different regions on the DNA sequence for primer annealing
because there is much greater flexibility in the length of the PCR
product, than for the NASBA product, which should be less than 250
bp. It is important for the specificity of the NASBA reaction to
choose a unique area that is not conserved among the different HPV
types. A couple of base pair mismatches may still give an
amplification or hybridisation of the target.
[0201] Detection of CaSki (integrated and episomal state) cells
with the universal L1 NASBA primers and not SiHa or HeLa (both
integrated) may give an indication that integrated HPV doesn't show
any L1 expression, while HPV in the episomal state may have L1
expression.
[0202] In summary, an identification assay has been developed for
HPV type 16, 18, 31 and 33 that can accurately identify the
oncogenic E6/E7 expression of these HPV types. The assay can also
identify the expression of the major capsid protein, L1.
EXAMPLE 3
Further Clinical Study in 190 Patients
Patients/Clinical Samples
[0203] Biopsies from 190 women admitted to Ostfold central-hospital
for treatment of CIN in the period 1999-2001. The mean age of the
190 women included in the study was 37.4 years (range 22-74 years).
Biopsies were frozen in -80.degree. C. immediately after
collection.
Cytological Examination of Samples
[0204] The routine cytological reports were used to record
cytological findings. No attempt was made to re-evaluate the
slides. Each one of them indicated a CIN II-III condition, i.e. a
high grade dysplasia or HSIL, which was the basis for hospital
admittance, colposcopy and biopsy.
Histological Examination of Samples
[0205] A biopsy, here termed biopsy 1, was taken after a high-grade
cytology report. If it confirmed a high-grade lesion (CIN II or
III), the patient was again admitted to hospital, this time for
colposcopically guided conization. Before the conization, but after
local anesthesia was applied, a second biopsy (biopsy 2) was taken
from an area of portio where a dysplasia was most likely to be
localised, judged from the gross findings. This biopsy (2.times.2
mm) was frozen within 2 minutes in a -80.degree. C. freezer.
[0206] Biopsy 2 was split in two when frozen and half was used for
DNA/RNA extraction. The other half was fixed in 10% buffered
formaldehyde and processed for histopathological examination. Some
lesions were not correctly oriented in the paraffin block and had
to be reoriented or serial sectioned in order to show the relevant
surface epithelium. Consequently, it cannot be guaranteed that
exactly the same tissue was used for the extraction and for the
histopathological evaluation. The cone specimen, finally, was
evaluated by the local pathologist, who in all cases could confirm
the presence of dysplasia. It was not always the same grade as in
the original biopsy, and, in many cases, not the same as in biopsy
2.
Extraction of Nucleic Acids
[0207] Nucleic acids were isolated using the automated Nuclisens
Extractor as previously described (Boom et al., 1990). Each biopsy
was cut in two pieces, one intended for histological examination
and the other half for RNA analysis. The material intended for RNA
analysis was divided into smaller pieces while kept on dry ice
(-80.degree. C.) and put into 1 ml of lysisbuffer (as above)
followed by 20 seconds of homogenisation using disposable pestles.
100 ml of the sample was further diluted 10 fold in lysisbuffer and
100 ml was then extracted for DNA/RNA. The extracted DNA/RNA was
eluted with .about.40 ml of elution buffer (Organon Teknika) and
stored at -70.degree. C.
[0208] All molecular beacon probes used in this study employ the
fluorophore FAM (6-carboxyfluorescein) at the 5' end of the
structure. This was bound to a variable stem-loop sequence coupled
to the universal quencher 4-(4' dimethylaminophenylazo)benzoic acid
(DABCYL) at the 3' end. The probes were delivered by Eurogentec,
Belgium. Final concentration of MBs used in the reaction was 2.5
mM. For the real-time NASBA we made use of the NucliSens Basic Kit
(Organon Teknika, Netherlands), intended for the development of
user-defined RNA amplification assays. The NASBA amplification was
carried out in a volume of 20 .mu.l. The primer-sets and probes
were directed against full-length E6/E7 mRNA for the high-risk HPV
16, 18, 31, and 33. As performance control, to avoid false negative
results due to degradation of nucleic acid, we used a primer set
and probe directed against the human U1 small nuclear
ribonucleoprotein (snRNP) specific A protein (U1A mRNA) (Nelissen
et al., 1991). All samples were run in duplicate on separate
machines (microplate readers for measuring fluorescence and
absorbance, Bio-tek FL-600 FA from MWG). mRNA isolated from
CaSki/SiHa or HeLa cells served as positive controls for HPV 16 and
HPV 18 transcripts, respectively. Negative controls, included for
every 7 reaction, consisted of a reaction containing all reagents
except mRNA.
HPV DNA Analysis; Polymerase Chain Reaction
[0209] The same extracts and amounts as used in the NASBA reaction
were used for PCR. The L1 consensus primers Gp5+/Gp6+ were used to
detect all samples containing HPV DNA. The PCR amplification was
carried out as described above. The first DNA denaturation was done
for 2 minutes at 94.degree. C., then 40 cycles of PCR were run:
denaturation 1 minute at 94.degree. C., annealing for 2 minutes at
40.degree. C., extension for 1.5 minutes at 72.degree. C., followed
by a final extension for 4 minutes at 72.degree. C. Typing of HPV
was performed by using PCR type-specific primers against HPV 16,
18, 31, and 33 (6/11, 35, 45, 51, 52, 58), as described above.
Results
[0210] Originally 190 patients were biopsied after being given the
diagnosis CIN I, CINII, or CIN III by cytology. A high-grade lesion
was confirmed by histologically examination, 150 samples diagnosed
as CIN III (78.9%). Biopsy 2, taken before conization, was used for
RNA analysis. However, histological examination of this biopsy
diagnosed only 53 samples of the originally 150 as CIN III [54 were
given no diagnosis, 24 diagnosed as CIN II, 18 as CIN I, and 4 as
HPV/condylom]. The number of CIN II samples increased from 16
(8.4%) to 30 (15.8%) [by Histology 124 diagnosed as CIN III, 4 as
CIN II, 1 as carcinom, and 1 as CIN I. 12 CIN II cases from
Histology I were given a lower diagnosis in Histology II]. The
degree of CIN I increased from 6 samples (3.2%) to 32 samples
(16.8%). The 2 squamous cell carcinomas were in Histology II
diagnosed as CIN III, the adenocarcinom as CIN II. In 71 samples
(38.4%) high-grade lesions were not detected.
[0211] HPV oncogenic RNA was detected in 69 (36%) of the 190
patients. Of the 53 samples (28%) diagnosed as CIN III in Histology
II, we found 40 (76%) cases showing HPV 16, 18, 31, or 33 oncogenic
expression. In addition, we found oncogenic expression in 9 of 30
cases (30%) of CIN II, in 4 of 32 cases (13%) of CIN I, in 14 of 71
cases (20%) not showing cell abnormalities, and in 2 of 4 (50%)
samples diagnosed as HPV/condyloma.
[0212] HPV 16 RNA was found in 42 of the 190 patients, HPV 18 was
found in 7 (3.7%), HPV 31 in 15 (7.9%), and HPV 33 in 8 (4.2%). One
patient had mixed infection with HPV 16 and HPV 18, and one with
HPV 16 and HPV 31.
[0213] Using the consensus Gp5+/Gp6+ primers directed against the
L1 gene, encoding the major capsid protein, PCR detected HPV in 81
of the 190 cervical biopsies (43%). Of the 119 cases given a
diagnosis in the second histological examination (115 diagnosed as
CIN, 4 as HPV/condyloma) 63 were found to contain HPV DNA. The
additional 18 cases detected were not given any histological
diagnosis. 20 of the 81 cases were not detected by NASBA; 7 out of
these were given the diagnosis CIN III, 2 were diagnosed as CIN II,
4 diagnosed as CIN I, and 7 given no diagnosis.
[0214] Type-specific PCR detected 85 cases containing HPV; 66
having HPV 16, 10 HPV 18, 14 HPV 31, 7 HPV 33. 12 cases had
multiple infection: 3 with HPV 16+18; 4 with HPV 16+33, 5 with HPV
16+31. 20 no diagnosis.
EXAMPLE 4
HPV Detected by PreTect HPV-Proofer and PCR Compared to Cytology
and Histology
[0215] Normal and ASCUS samples (including borderline smears) were
determined by cytology. All samples were tested with consensus PCR
and PreTect HPV-Proofer but only the consensus positive samples
were typed by PCR. The CIN 3 and cancer samples were determined by
histology and all the samples were tested with all three methods.
The results are shown in FIG. 6. Concordance between real-time
multiplex NASBA and PCR compared to cytology or histology is shown
in Table 15 below.
TABLE-US-00023 TABLE 15 Concordance between real-time multiplex
NASBA and PCR compared to cytology or histology Cytology/Histology
Concordance.sup.a (Number) Concordance.sup.b (Number) Normal 98.2%
(4043) 42.8% (138) ASCUS.sup.c 94.5% (55) 78.6% (14) CIN 3 94.3%
(53) 93.2% (44) Cancer 99.0% (196) 98.8% (170) Only samples
positive by Gp5+/6+ PCR have been typed. .sup.aIncluding PCR and
real-time multiplex NASBA positive and negative samples.
.sup.bIncluding only PCR and/or real-time multiplex NASBA positive
samples. .sup.cASCUS excluding borderline smears.
EXAMPLE 5
[0216] The invention provides a kit for detection of mRNA
transcripts from the E6 gene(s) of HPV the kit comprising one or
more of, two or more of and preferably all of the following primer
pairs and accompanying identification probes.
TABLE-US-00024 HPV 16:HPV16.txt 7905 b.p HPV16P2: p2:116 (20) (SEQ
ID No. 175) GATGCAAGGTCGCATATGAGCCACAGGAGCGACCCAGAAA 16 pl (no7)
(SEQ ID No. 177) AATTCTAATACGACTCACTATAGGGAGAAGG ATT CCC ATC TCT
ATA TAC TA (51 baser) HPV16PO2: po:230 (20) H16e6702po (SEQ ID No.
19) TATGACTTTGCTTTTCGGGA HPV 18:HPV18.txt 7857 b.p HPV18P2: p2:698
(22) H18e6702p2 (SEQ ID No. 196)
GATGCAAGGTCGCATATGAGGAAAACGATGAAATAGATGGAG HPV18P4: p1:817 (20)
H18e6703p1 (SEQ ID No. 203)
AATTCTAATACGACTCACTATAGGGAGAAGGGGTCGTCTGCTGAGCTTTCT (Multiplex)
HPV18PO2: po:752 (21) H18e6702po (SEQ ID No. 32)
GAACCACAACGTCACACAATG HPV 31:HPV31.txt 7912 b.p HPV31P3: p2:617
(20) H31e6703p2 (SEQ ID No. 210)
GATGCAAGGTCGCATATGAGACTGACCTCCACTGTTATGA p1:766 (20) H31e6703p1
(SEQ ID No. 211)
AATTCTAATACGACTCACTATAGGGAGAAGGTATCTACTTGTGTGCTCTGT HPV31PO4:
po:686 (26) H31e6704po (SEQ ID No. 50) GGACAAGCAGAACCGGACACATCCAA
HPV 33:HPV33.txt 7909 b.p HPV33P1: p2:618 (22) H33e6701p2 (SEQ ID
No. 221) GATGCAAGGTCGCATATGAGTATCCTGAACCAACTGACCTAT p1:763 (19)
H33e6701p1 (SEQ ID No. 222)
AATTCTAATACGACTCACTATAGGGAGAAGGTTGACACATAAACGAACTG HPV33PO3: po:699
(23) H33e6703po (SEQ ID No. 59) GGACAAGCACAACCAGCCACAGC
[0217] As alternative to the probes shown above the kit may
optionally include one or more of the following molecular beacon
probes:
TABLE-US-00025 Molecular Beacon Probes: H16e6702mb2-FAM (SEQ ID No.
187) ccagctTATGACTTTGCTTTTCGGGAagctgg H18e6702mb1-TxR (SEQ ID No.
198) cgcatgGAACCACAACGTCACACAATGcatgcg H31e6704mb2-FAM ((SEQ ID No.
215) ccgtcgGGACAAGCAGAACCGGACACATCCAAcgacgg H33e6703mb1-FAM (SEQ ID
No. 225) ccaagcGGACAAGCACAACCAGCCACAGCgcttgg
[0218] Preferably the kit of the invention also includes the
following primer pair and probe: HPV45: HPV45.txt 7858 bp
(X74479)
TABLE-US-00026 HPV45P1: p2:430 (21): H45e6701p2 (SEQ ID No. 261)
GATGCAAGGTCGCATATGAGAACCATTGAACCCAGCAGAAA p1:527 (22): H45e6701p1
(SEQ ID No. 262) AATTCTAATACGACTCACTATAGGGAGAAGGTCTTTCTTGCCGT
GCCTGGTCA HPV45PO1: po:500 (20): H45e6701po (SEQ ID No. 108)
GTACCGAGGGCAGTGTAATA
[0219] The HPV 45 probe above may be replaced by an HPV molecular
beacon probe as follows:
TABLE-US-00027 H45e6701mb1 (SEQ ID No. 342)
cgatcgGTACCGAGGGCAGTGTAATAcgatcg
[0220] In addition the kit may include one or more of the following
primer pairs and accompanying identification probes depending on
the geographical area of use of the kit.
TABLE-US-00028 HPV52: HPV52.txt 7942 bp (X74481) HPV52P1: p2:144
(22): H52e6701p2 (SEQ ID No. 239)
GATGCAAGGTCGCATATGAGTTGTGTGAGGTGCTGGAAGAAT p1:358 (18): H52e6701p1
(SEQ ID No. 240) AATTCTAATACGACTCACTATAGGGAGAAGGCCCTCTCTT CTAATGTTT
HPV52PO1: Po:296 (20): H52e6701po (SEQ ID No. 334)
GTGCCTACGCTTTTTATCTA HPV58 HPV58.txt 7824 bp (D90400) HPV58P2:
p2:173 (18): H58e6702p2 (SEQ ID No. 245)
GATGCAAGGTCGCATATGAGTCTGTGCATGAAATCGAA p1:291 (18): H58e6702p1 (SEQ
ID No. 246) AATTCTAATACGACTCACTATAGGGAGAAGGAGCACACTTT ACATACTG
HPV58PO2: po:218 (22): H58e6702po (SEQ ID No. 84)
TTGCAGCGATCTGAGGTATATG HPV51 HPV51.txt 7808 bp (M62877) HPV51PA/P:
p2:655 (23): H51e6702p2 (SEQ ID No. 275) GATGCAAGGTCGCATATGAG AGA
GGA GGA GGA TGA AGT AGA TA p1:807 (20): H51e6701p1 (SEQ ID No. 274)
AATTCTAATACGACTCACTATAGGGAGAAGG GCC CAT TAA CAT CTG CTG TA
HPV51POA: po:771 (24): H51e67o2po (SEQ ID No. 129) TGG CAG TGG AAA
GCA GTG GAG ACA
[0221] The probes shown above may be replaced in the kit by the
following molecular beacon probes:
TABLE-US-00029 H52e6701mb1 (SEQ ID No. 343)
cgatcgGTGCCTACGCTTTTTATCTAcgatcg H58e6702mb1 (SEQ ID No. 344)
ccgtcgTTGCAGCGATCTGAGGTATATGcgacgg H51e6702mb1 (SEQ ID No. 345)
cgatcgTGG CAG TGG AAA GCA GTG GAG ACAcgatcg
Sequence CWU 1
1
346124DNAArtificial sequence/note="HPV Primer" 1ccacaggagc
gacccagaaa gtta 24222DNAArtificial sequence/note="HPV Primer"
2acggtttgtt gtattgctgt tc 22320DNAArtificial sequence/note="HPV
Primer" 3ccacaggagc gacccagaaa 20420DNAArtificial
sequence/note="HPV Primer" 4ggtttgttgt attgctgttc
20520DNAArtificial sequence/note="HPV Primer" 5attcccatct
ctatatacta 20618DNAArtificial sequence/note="HPV Primer"
6tcacgtcgca gtaactgt 18718DNAArtificial sequence/note="HPV Primer"
7ttgcttgcag tacacaca 18818DNAArtificial sequence/note="HPV Primer"
8tgcagtacac acattcta 18918DNAArtificial sequence/note="HPV Primer"
9gcagtacaca cattctaa 181018DNAArtificial sequence/note="HPV Primer"
10acagttatgc acagagct 181118DNAArtificial sequence/note="HPV
Primer" 11atattagaat gtgtgtac 181218DNAArtificial
sequence/note="HPV Primer" 12ttagaatgtg tgtactgc
181318DNAArtificial sequence/note="HPV Primer" 13gaatgtgtgt
actgcaag 181418DNAArtificial sequence/note="HPV probe" 14acagttatgc
acagagct 181518DNAArtificial sequence/note="HPV probe" 15atattagaat
gtgtgtac 181618DNAArtificial sequence/note="HPV probe" 16ttagaatgtg
tgtactgc 181718DNAArtificial sequence/note="HPV probe" 17gaatgtgtgt
actgcaag 181822DNAArtificial sequence/note="HPV probe" 18ctttgctttt
cgggatttat gc 221920DNAArtificial sequence/note="HPV probe"
19tatgactttg cttttcggga 202020DNAArtificial sequence/note="HPV
probe" 20tatgactttg cttttcggga 202123DNAArtificial
sequence/note="HPV Primer" 21cagaggagga ggatgaaata gta
232225DNAArtificial sequence/note="HPV Primer" 22gcacaaccga
agcgtagagt cacac 252324DNAArtificial sequence/note="HPV probe"
23tggacaagca gaaccggaca gagc 242422DNAArtificial sequence/note="HPV
Primer" 24cagaggagga ggatgaaata ga 222522DNAArtificial
sequence/note="HPV Primer" 25gcacaaccga agcgtagagt ca
222624DNAArtificial sequence/note="HPV probe" 26agcagaaccg
gacagagccc atta 242720DNAArtificial sequence/note="HPV Primer"
27acgatgaaat agatggagtt 202820DNAArtificial sequence/note="HPV
Primer" 28cacggacaca caaaggacag 202920DNAArtificial
sequence/note="HPV probe" 29agccgaacca caacgtcaca
203022DNAArtificial sequence/note="HPV Primer" 30gaaaacgatg
aaatagatgg ag 223124DNAArtificial sequence/note="HPV Primer"
31acaccacgga cacacaaagg acag 243221DNAArtificial sequence/note="HPV
probe" 32gaaccacaac gtcacacaat g 213321DNAArtificial
sequence/note="HPV probe" 33gaaccacaac gtcacacaat g
213420DNAArtificial sequence/note="HPV Primer" 34ttccggttga
ccttctatgt 203520DNAArtificial sequence/note="HPV Primer"
35ggtcgtctgc tgagctttct 203621DNAArtificial sequence/note="HPV
Primer" 36gcaagacata gaaataacct g 213718DNAArtificial
sequence/note="HPV Primer" 37acccagtgtt agttagtt
183820DNAArtificial sequence/note="HPV probe" 38tgcaagacag
tattggaact 203920DNAArtificial sequence/note="HPV Primer"
39ggaaataccc tacgatgaac 204020DNAArtificial sequence/note="HPV
Primer" 40ggacacaacg gtctttgaca 204124DNAArtificial
sequence/note="HPV probe" 41atagggacga cacaccacac ggag
244222DNAArtificial sequence/note="HPV Primer" 42ggaaataccc
tacgatgaac ta 224322DNAArtificial sequence/note="HPV Primer"
43ctggacacaa cggtctttga ca 224422DNAArtificial sequence/note="HPV
probe" 44tagggacgac acaccacacg ga 224520DNAArtificial
sequence/note="HPV Primer" 45actgacctcc actgttatga
204620DNAArtificial sequence/note="HPV Primer" 46tatctacttg
tgtgctctgt 204722DNAArtificial sequence/note="HPV probe"
47gacaagcaga accggacaca tc 224824DNAArtificial sequence/note="HPV
Primer" 48tgacctccac tgttatgagc aatt 244926DNAArtificial
sequence/note="HPV Primer" 49tgcgaatatc tacttgtgtg ctctgt
265026DNAArtificial sequence/note="HPV probe" 50ggacaagcag
aaccggacac atccaa 265126DNAArtificial sequence/note="HPV probe"
51ggacaagcag aaccggacac atccaa 265218DNAArtificial
sequence/note="HPV Primer" 52actgacctcc actgttat
185321DNAArtificial sequence/note="HPV Primer" 53cacgattcca
aatgagccca t 215422DNAArtificial sequence/note="HPV Primer"
54tatcctgaac caactgacct at 225519DNAArtificial sequence/note="HPV
Primer" 55ttgacacata aacgaactg 195619DNAArtificial
sequence/note="HPV probe" 56cagatggaca agcacaacc
195720DNAArtificial sequence/note="HPV Primer" 57tcctgaacca
actgacctat 205820DNAArtificial sequence/note="HPV Primer"
58cccataagta gttgctgtat 205923DNAArtificial sequence/note="HPV
probe" 59ggacaagcac aaccagccac agc 236023DNAArtificial
sequence/note="HPV probe" 60ggacaagcac aaccagccac agc
236120DNAArtificial sequence/note="HPV Primer" 61gacctttgtg
tcctcaagaa 206220DNAArtificial sequence/note="HPV Primer"
62aggtcagttg gttcaggata 206321DNAArtificial sequence/note="HPV
probe" 63agaaactgca ctgtgacgtg t 216420DNAArtificial
sequence/note="HPV Primer" 64attacagcgg agtgaggtat
206520DNAArtificial sequence/note="HPV Primer" 65gtctttgctt
ttcaactgga 206620DNAArtificial sequence/note="HPV probe"
66atagagaagg ccagccatat 206722DNAArtificial sequence/note="HPV
Primer" 67tcagaggagg aggaagatac ta 226820DNAArtificial
sequence/note="HPV Primer" 68gattatgctc tctgtgaaca
206920DNAArtificial sequence/note="HPV Primer" 69cccgaggcaa
ctgacctata 207020DNAArtificial sequence/note="HPV Primer"
70gtcaatgtgt gtgctctgta 207125DNAArtificial sequence/note="HPV
probe" 71gacaagcaaa accagacacc tccaa 257220DNAArtificial
sequence/note="HPV probe" 72gacaagcaaa accagacacc
207322DNAArtificial sequence/note="HPV Primer" 73ttgtgtgagg
tgctggaaga at 227418DNAArtificial sequence/note="HPV Primer"
74ccctctcttc taatgttt 187520DNAArtificial sequence/note="HPV probe"
75gtgcctacgc tttttatcta 207620DNAArtificial sequence/note="HPV
Primer" 76gtgcctacgc tttttatcta 207722DNAArtificial
sequence/note="HPV Primer" 77ggggtctcca acactctgaa ca
227818DNAArtificial sequence/note="HPV probe" 78tgcaaacaag cgatttca
187918DNAArtificial sequence/note="HPV Primer" 79tcaggcgttg
gagacatc 188018DNAArtificial sequence/note="HPV Primer"
80agcaatcgta agcacact 188118DNAArtificial sequence/note="HPV
Primer" 81tctgtgcatg aaatcgaa 188218DNAArtificial
sequence/note="HPV Primer" 82agcacacttt acatactg
188318DNAArtificial sequence/note="HPV probe" 83tgaaatgcgt tgaatgca
188422DNAArtificial sequence/note="HPV probe" 84ttgcagcgat
ctgaggtata tg 228518DNAArtificial sequence/note="HPV Primer"
85tacactgctg gacaacat 188618DNAArtificial sequence/note="HPV
Primer" 86tcatcttctg agctgtct 188721DNAArtificial
sequence/note="HPV Primer" 87tacactgctg gacaacatgc a
218823DNAArtificial sequence/note="HPV Primer" 88gtcacatcca
cagcaacagg tca 238920DNAArtificial sequence/note="HPV probe"
89gtagggttac attgctatga 209022DNAArtificial sequence/note="HPV
probe" 90gtagggttac attgctatga gc 229122DNAArtificial
sequence/note="HPV Primer" 91tgacctgttg ctgtggatgt ga
229219DNAArtificial sequence/note="HPV Primer" 92tacctgaatc
gtccgccat 199318DNAArtificial sequence/note="HPV probe"
93atwgtgtgtc ccatctgc 189419DNAArtificial sequence/note="HPV
Primer" 94catgccataa atgtataga 199520DNAArtificial
sequence/note="HPV Primer" 95caccgcaggc accttattaa
209618DNAArtificial sequence/note="HPV probe" 96agaattagag aattaaga
189721DNAArtificial sequence/note="HPV Primer" 97gcagacgacc
actacagcaa a 219819DNAArtificial sequence/note="HPV Primer"
98acaccgagtc cgagtaata 199919DNAArtificial sequence/note="HPV
probe" 99atagggacgg ggaaccact 1910019DNAArtificial
sequence/note="HPV Primer" 100tattactcgg actcggtgt
1910121DNAArtificial sequence/note="HPV Primer" 101cttgggtttc
tcttcgtgtt a 2110220DNAArtificial sequence/note="HPV probe"
102ggaccacaaa acgggaggac 2010320DNAArtificial sequence/note="HPV
Primer" 103gaaatagatg aacccgacca 2010420DNAArtificial
sequence/note="HPV Primer" 104gcacaccacg gacacacaaa
2010524DNAArtificial sequence/note="HPV probe" 105tagccagacg
ggatgaacca cagc 2410621DNAArtificial sequence/note="HPV Primer"
106aaccattgaa cccagcagaa a 2110722DNAArtificial sequence/note="HPV
Primer" 107tctttcttgc cgtgcctggt ca 2210820DNAArtificial
sequence/note="HPV probe" 108gtaccgaggg cagtgtaata
2010921DNAArtificial sequence/note="HPV Primer" 109aaccattgaa
cccagcagaa a 2111022DNAArtificial sequence/note="HPV Primer"
110tctttcttgc cgtgcctggt ca 2211124DNAArtificial sequence/note="HPV
Primer" 111gaaaccattg aacccagcag aaaa 2411224DNAArtificial
sequence/note="HPV Primer" 112ttgctatact tgtgtttccc tacg
2411320DNAArtificial sequence/note="HPV probe" 113gtaccgaggg
cagtgtaata 2011420DNAArtificial sequence/note="HPV probe"
114ggacaaacga agatttcaca 2011525DNAArtificial sequence/note="HPV
Primer" 115gttgacctgt tgtgttacca gcaat 2511624DNAArtificial
sequence/note="HPV Primer" 116caccacggac acacaaagga caag
2411722DNAArtificial sequence/note="HPV Primer" 117ctgttgacct
gttgtgttac ga 2211822DNAArtificial sequence/note="HPV Primer"
118ccacggacac acaaaggaca ag 2211920DNAArtificial sequence/note="HPV
Primer" 119gttgacctgt tgtgttacga 2012020DNAArtificial
sequence/note="HPV Primer" 120acggacacac aaaggacaag
2012122DNAArtificial sequence/note="HPV probe" 121gagtcagagg
aggaaaacga tg 2212225DNAArtificial sequence/note="HPV probe"
122aggaaaacga tgaagcagat ggagt 2512325DNAArtificial
sequence/note="HPV probe" 123acaactacca gcccgacgag ccgaa
2512420DNAArtificial sequence/note="HPV Primer" 124ggaggaggat
gaagtagata 2012520DNAArtificial sequence/note="HPV Primer"
125gcccattaac atctgctgta 2012623DNAArtificial sequence/note="HPV
Primer" 126agaggaggag gatgaagtag ata 2312720DNAArtificial
sequence/note="HPV Primer" 127acgggcaaac caggcttagt
2012820DNAArtificial sequence/note="HPV probe" 128gcaggtgttc
aagtgtagta 2012924DNAArtificial sequence/note="HPV probe"
129tggcagtgga aagcagtgga gaca 2413024DNAArtificial
sequence/note="HPV Primer" 130ttggggtgct ggagacaaac atct
2413124DNAArtificial sequence/note="HPV Primer" 131ttcatcctca
tcctcatcct ctga 2413222DNAArtificial sequence/note="HPV Primer"
132tggggtgctg gagacaaaca tc 2213322DNAArtificial sequence/note="HPV
Primer" 133catcctcatc ctcatcctct ga 2213422DNAArtificial
sequence/note="HPV Primer" 134ttggggtgct ggagacaaac at
2213522DNAArtificial sequence/note="HPV Primer" 135ccacaaactt
acactcacaa ca 2213623DNAArtificial sequence/note="HPV probe"
136aaagtaccaa cgctgcaaga cgt 2313724DNAArtificial
sequence/note="HPV probe" 137agaactaaca cctcaaacag aaat
2413822DNAArtificial sequence/note="HPV probe" 138agtaccaacg
ctgcaagacg tt 2213922DNAArtificial sequence/note="HPV Primer"
139ttggacagct cagaggatga gg 2214020DNAArtificial sequence/note="HPV
Primer" 140gattttcctt atgcagtgtg 2014120DNAArtificial
sequence/note="HPV Primer" 141gacatctgta gcaccttatt
2014220DNAArtificial sequence/note="HPV probe" 142gactattcag
tgtatggagc 2014322DNAArtificial sequence/note="HPV probe"
143caactgayct myactgttat ga 2214422DNAArtificial sequence/note="HPV
probe" 144caactgayct myactgttat ga 2214524DNAArtificial
sequence/note="HPV probe" 145gaamcaactg acctaywctg ctat
2414624DNAArtificial sequence/note="HPV probe" 146gaamcaactg
acctaywctg ctat 2414718DNAArtificial sequence/note="HPV probe"
147aagacattat tcagactc 1814818DNAArtificial sequence/note="HPV
probe" 148aagacattat tcagactc 1814920DNAArtificial
sequence/note="HPV Primer" 149aatggcattt gttggggtaa
2015019DNAArtificial sequence/note="HPV Primer" 150tcatattcct
ccccatgtc 1915122DNAArtificial sequence/note="HPV probe"
151ttgttactgt tgttgatact
ac 2215220DNAArtificial sequence/note="HPV Primer" 152aatggcattt
gttggsrhaa 2015319DNAArtificial sequence/note="HPV Primer"
153tcatattcct cmmcatgdc 1915422DNAArtificial sequence/note="HPV
probe" 154ttgttactgt tgttgatacy ac 2215522DNAArtificial
sequence/note="HPV probe" 155ttgttactgt tgttgatacc ac
2215620DNAArtificial sequence/note="HPV Primer" 156aatggcattt
gttggsnnaa 2015720DNAArtificial sequence/note="HPV Primer"
157aatggcattt gttggnnhaa 2015820DNAArtificial sequence/note="HPV
Primer" 158aatggcattt gttggnrnaa 2015920DNAArtificial
sequence/note="HPV Primer" 159aatggcattt gttggggtaa
2016020DNAArtificial sequence/note="HPV Primer" 160aatggcattt
gttggggaaa 2016120DNAArtificial sequence/note="HPV Primer"
161aatggcattt gttggcataa 2016220DNAArtificial sequence/note="HPV
Primer" 162aatggcattt gttggggcaa 2016320DNAArtificial
sequence/note="HPV Primer" 163aatggcattt gttggcacaa
2016419DNAArtificial sequence/note="HPV Primer" 164tcatattcct
cmncatgnc 1916519DNAArtificial sequence/note="HPV Primer"
165tcatattcct caacatgnc 1916619DNAArtificial sequence/note="HPV
Primer" 166tcatattcct cnncatgtc 1916719DNAArtificial
sequence/note="HPV Primer" 167tcatattcct cnncatggc
1916819DNAArtificial sequence/note="HPV Primer" 168tcatattcct
cnncatgac 1916919DNAArtificial sequence/note="HPV Primer"
169tcatattcct cnncatgcc 1917020DNAArtificial sequenceSequence for
incorporation into NASBA primers to enable hybridisation with
universal ECL detection probe 170gatgcaaggt cgcatatgag
2017125DNAArtificial sequencePromoter sequence for incorporation
into NASBA P1 primer oligonucleotides 171aattctaata cgactcacta
taggg 2517231DNAArtificial sequencePromoter sequence for
incorporation into NASBA P1 primer oligonucleotides 172aattctaata
cgactcacta tagggagaag g 3117344DNAArtificial sequence/note="HPV
NASBA Primer" 173gatgcaaggt cgcatatgag ccacaggagc gacccagaaa gtta
4417453DNAArtificial sequence/note="HPV NASBA Primer" 174aattctaata
cgactcacta tagggagaag gacggtttgt tgtattgctg ttc
5317540DNAArtificial sequence/note="HPV NASBA Primer" 175gatgcaaggt
cgcatatgag ccacaggagc gacccagaaa 4017651DNAArtificial
sequence/note="HPV NASBA Primer" 176aattctaata cgactcacta
tagggagaag gggtttgttg tattgctgtt c 5117751DNAArtificial
sequence/note="HPV NASBA Primer" 177aattctaata cgactcacta
tagggagaag gattcccatc tctatatact a 5117849DNAArtificial
sequence/note="HPV NASBA Primer" 178aattctaata cgactcacta
tagggagaag gtcacgtcgc agtaactgt 4917949DNAArtificial
sequence/note="HPV NASBA Primer" 179aattctaata cgactcacta
tagggagaag gttgcttgca gtacacaca 4918049DNAArtificial
sequence/note="HPV NASBA Primer" 180aattctaata cgactcacta
tagggagaag gtgcagtaca cacattcta 4918149DNAArtificial
sequence/note="HPV NASBA Primer" 181aattctaata cgactcacta
tagggagaag ggcagtacac acattctaa 4918238DNAArtificial
sequence/note="HPV NASBA Primer" 182gatgcaaggt cgcatatgag
acagttatgc acagagct 3818338DNAArtificial sequence/note="HPV NASBA
Primer" 183gatgcaaggt cgcatatgag atattagaat gtgtgtac
3818438DNAArtificial sequence/note="HPV NASBA Primer" 184gatgcaaggt
cgcatatgag ttagaatgtg tgtactgc 3818538DNAArtificial
sequence/note="HPV NASBA Primer" 185gatgcaaggt cgcatatgag
gaatgtgtgt actgcaag 3818632DNAArtificial sequence/note="HPV
molecular beacon probe" 186cgcatgtatg actttgcttt tcgggacatg cg
3218732DNAArtificial sequence/note="HPV molecular beacon probe"
187ccagcttatg actttgcttt tcgggaagct gg 3218830DNAArtificial
sequence/note="HPV molecular beacon probe" 188cacgctatga ctttgctttt
cgggagcgtg 3018932DNAArtificial sequence/note="HPV molecular beacon
probe" 189cgatcgtatg actttgcttt tcgggacgat cg 3219043DNAArtificial
sequence/note="HPV NASBA Primer" 190gatgcaaggt cgcatatgag
cagaggagga ggatgaaata gta 4319156DNAArtificial sequence/note="HPV
NASBA Primer" 191aattctaata cgactcacta tagggagaag ggcacaaccg
aagcgtagag tcacac 5619242DNAArtificial sequence/note="HPV NASBA
Primer" 192gatgcaaggt cgcatatgag cagaggagga ggatgaaata ga
4219353DNAArtificial sequence/note="HPV NASBA Primer" 193aattctaata
cgactcacta tagggagaag ggcacaaccg aagcgtagag tca
5319440DNAArtificial sequence/note="HPV NASBA Primer" 194gatgcaaggt
cgcatatgag acgatgaaat agatggagtt 4019551DNAArtificial
sequence/note="HPV NASBA Primer" 195aattctaata cgactcacta
tagggagaag gcacggacac acaaaggaca g 5119642DNAArtificial
sequence/note="HPV NASBA Primer" 196gatgcaaggt cgcatatgag
gaaaacgatg aaatagatgg ag 4219755DNAArtificial sequence/note="HPV
NASBA Primer" 197aattctaata cgactcacta tagggagaag gacaccacgg
acacacaaag gacag 5519833DNAArtificial sequence/note="HPV molecular
beacon probe" 198cgcatggaac cacaacgtca cacaatgcat gcg
3319933DNAArtificial sequence/note="HPV molecular beacon probe"
199ccgtcggaac cacaacgtca cacaatgcga cgg 3320033DNAArtificial
sequence/note="HPV molecular beacon probe" 200cggaccgaac cacaacgtca
cacaatgggt ccg 3320133DNAArtificial sequence/note="HPV molecular
beacon probe" 201cgatcggaac cacaacgtca cacaatgcga tcg
3320240DNAArtificial sequence/note="HPV NASBA Primer" 202gatgcaaggt
cgcatatgag ttccggttga ccttctatgt 4020351DNAArtificial
sequence/note="HPV NASBA Primer" 203aattctaata cgactcacta
tagggagaag gggtcgtctg ctgagctttc t 5120441DNAArtificial
sequence/note="HPV NASBA Primer" 204gatgcaaggt cgcatatgag
gcaagacata gaaataacct g 4120549DNAArtificial sequence/note="HPV
NASBA Primer" 205aattctaata cgactcacta tagggagaag gacccagtgt
tagttagtt 4920640DNAArtificial sequence/note="HPV NASBA Primer"
206gatgcaaggt cgcatatgag ggaaataccc tacgatgaac 4020751DNAArtificial
sequence/note="HPV NASBA Primer" 207aattctaata cgactcacta
tagggagaag gggacacaac ggtctttgac a 5120842DNAArtificial
sequence/note="HPV NASBA Primer" 208gatgcaaggt cgcatatgag
ggaaataccc tacgatgaac ta 4220953DNAArtificial sequence/note="HPV
NASBA Primer" 209aattctaata cgactcacta tagggagaag gctggacaca
acggtctttg aca 5321040DNAArtificial sequence/note="HPV NASBA
Primer" 210gatgcaaggt cgcatatgag actgacctcc actgttatga
4021151DNAArtificial sequence/note="HPV NASBA Primer" 211aattctaata
cgactcacta tagggagaag gtatctactt gtgtgctctg t 5121244DNAArtificial
sequence/note="HPV NASBA Primer" 212gatgcaaggt cgcatatgag
tgacctccac tgttatgagc aatt 4421357DNAArtificial sequence/note="HPV
NASBA Primer" 213aattctaata cgactcacta tagggagaag gtgcgaatat
ctacttgtgt gctctgt 5721440DNAArtificial sequence/note="HPV
molecular beacon probe" 214ccgaagggga caagcagaac cggacacatc
caaccttcgg 4021538DNAArtificial sequence/note="HPV molecular beacon
probe" 215ccgtcgggac aagcagaacc ggacacatcc aacgacgg
3821640DNAArtificial sequence/note="HPV molecular beacon probe"
216cacgtcggga caagcagaac cggacacatc caacgacgtg 4021738DNAArtificial
sequence/note="HPV molecular beacon probe" 217cgcagcggac aagcagaacc
ggacacatcc aagctgcg 3821838DNAArtificial sequence/note="HPV
molecular beacon probe" 218cgatcgggac aagcagaacc ggacacatcc
aacgatcg 3821938DNAArtificial sequence/note="HPV NASBA Primer"
219gatgcaaggt cgcatatgag actgacctcc actgttat 3822052DNAArtificial
sequence/note="HPV NASBA Primer" 220aattctaata cgactcacta
tagggagaag gcacgattcc aaatgagccc at 5222142DNAArtificial
sequence/note="HPV NASBA Primer" 221gatgcaaggt cgcatatgag
tatcctgaac caactgacct at 4222250DNAArtificial sequence/note="HPV
NASBA Primer" 222aattctaata cgactcacta tagggagaag gttgacacat
aaacgaactg 5022340DNAArtificial sequence/note="HPV NASBA Primer"
223gatgcaaggt cgcatatgag tcctgaacca actgacctat 4022451DNAArtificial
sequence/note="HPV NASBA Primer" 224aattctaata cgactcacta
tagggagaag gcccataagt agttgctgta t 5122535DNAArtificial
sequence/note="HPV molecular beacon probe" 225ccaagcggac aagcacaacc
agccacagcg cttgg 3522637DNAArtificial sequence/note="HPV molecular
beacon probe" 226ccaagcggga caagcacaac cagccacagc cgcttgg
3722735DNAArtificial sequence/note="HPV molecular beacon probe"
227cccagcggac aagcacaacc agccacagcg ctggg 3522837DNAArtificial
sequence/note="HPV molecular beacon probe" 228ccaaagcgga caagcacaac
cagccacagc gctttgg 3722933DNAArtificial sequence/note="HPV
molecular beacon probe" 229cctgcggaca agcacaacca gccacagcgc agg
3323035DNAArtificial sequence/note="HPV molecular beacon probe"
230cgatcgggac aagcacaacc agccacagcc gatcg 3523140DNAArtificial
sequence/note="HPV NASBA Primer" 231gatgcaaggt cgcatatgag
gacctttgtg tcctcaagaa 4023251DNAArtificial sequence/note="HPV NASBA
Primer" 232aattctaata cgactcacta tagggagaag gaggtcagtt ggttcaggat a
5123340DNAArtificial sequence/note="HPV NASBA Primer" 233gatgcaaggt
cgcatatgag attacagcgg agtgaggtat 4023451DNAArtificial
sequence/note="HPV NASBA Primer" 234aattctaata cgactcacta
tagggagaag ggtctttgct tttcaactgg a 5123542DNAArtificial
sequence/note="HPV NASBA Primer" 235gatgcaaggt cgcatatgag
tcagaggagg aggaagatac ta 4223651DNAArtificial sequence/note="HPV
NASBA Primer" 236aattctaata cgactcacta tagggagaag ggattatgct
ctctgtgaac a 5123740DNAArtificial sequence/note="HPV NASBA Primer"
237gatgcaaggt cgcatatgag cccgaggcaa ctgacctata 4023851DNAArtificial
sequence/note="HPV NASBA Primer" 238aattctaata cgactcacta
tagggagaag ggtcaatgtg tgtgctctgt a 5123942DNAArtificial
sequence/note="HPV NASBA Primer" 239gatgcaaggt cgcatatgag
ttgtgtgagg tgctggaaga at 4224049DNAArtificial sequence/note="HPV
NASBA Primer" 240aattctaata cgactcacta tagggagaag gccctctctt
ctaatgttt 4924140DNAArtificial sequence/note="HPV NASBA Primer"
241gatgcaaggt cgcatatgag gtgcctacgc tttttatcta 4024253DNAArtificial
sequence/note="HPV NASBA Primer" 242aattctaata cgactcacta
tagggagaag gggggtctcc aacactctga aca 5324338DNAArtificial
sequence/note="HPV NASBA Primer" 243gatgcaaggt cgcatatgag
tcaggcgttg gagacatc 3824449DNAArtificial sequence/note="HPV NASBA
Primer" 244aattctaata cgactcacta tagggagaag gagcaatcgt aagcacact
4924538DNAArtificial sequence/note="HPV NASBA Primer" 245gatgcaaggt
cgcatatgag tctgtgcatg aaatcgaa 3824649DNAArtificial
sequence/note="HPV NASBA Primer" 246aattctaata cgactcacta
tagggagaag gagcacactt tacatactg 4924738DNAArtificial
sequence/note="HPV NASBA Primer" 247gatgcaaggt cgcatatgag
tacactgctg gacaacat 3824849DNAArtificial sequence/note="HPV NASBA
Primer" 248aattctaata cgactcacta tagggagaag gtcatcttct gagctgtct
4924941DNAArtificial sequence/note="HPV NASBA Primer" 249gatgcaaggt
cgcatatgag tacactgctg gacaacatgc a 4125054DNAArtificial
sequence/note="HPV NASBA Primer" 250aattctaata cgactcacta
tagggagaag ggtcacatcc acagcaacag gtca 5425142DNAArtificial
sequence/note="HPV NASBA Primer" 251gatgcaaggt cgcatatgag
tgacctgttg ctgtggatgt ga 4225250DNAArtificial sequence/note="HPV
NASBA Primer" 252aattctaata cgactcacta tagggagaag gtacctgaat
cgtccgccat 5025339DNAArtificial sequence/note="HPV NASBA Primer"
253gatgcaaggt cgcatatgag catgccataa atgtataga 3925451DNAArtificial
sequence/note="HPV NASBA Primer" 254aattctaata cgactcacta
tagggagaag gcaccgcagg caccttatta a 5125541DNAArtificial
sequence/note="HPV NASBA Primer" 255gatgcaaggt cgcatatgag
gcagacgacc actacagcaa a 4125650DNAArtificial sequence/note="HPV
NASBA Primer" 256aattctaata cgactcacta tagggagaag gacaccgagt
ccgagtaata 5025739DNAArtificial sequence/note="HPV NASBA Primer"
257gatgcaaggt cgcatatgag tattactcgg actcggtgt 3925852DNAArtificial
sequence/note="HPV NASBA Primer" 258aattctaata cgactcacta
tagggagaag gcttgggttt ctcttcgtgt ta 5225940DNAArtificial
sequence/note="HPV NASBA Primer" 259gatgcaaggt cgcatatgag
gaaatagatg aacccgacca 4026051DNAArtificial sequence/note="HPV NASBA
Primer" 260aattctaata cgactcacta tagggagaag ggcacaccac ggacacacaa a
5126141DNAArtificial sequence/note="HPV NASBA Primer" 261gatgcaaggt
cgcatatgag aaccattgaa cccagcagaa a 4126253DNAArtificial
sequence/note="HPV NASBA Primer" 262aattctaata cgactcacta
tagggagaag gtctttcttg ccgtgcctgg tca 5326341DNAArtificial
sequence/note="HPV NASBA Primer" 263gatgcaaggt cgcatatgag
aaccattgaa cccagcagaa a 4126453DNAArtificial sequence/note="HPV
NASBA Primer" 264aattctaata cgactcacta tagggagaag gtctttcttg
ccgtgcctgg tca 5326544DNAArtificial sequence/note="HPV NASBA
Primer" 265gatgcaaggt cgcatatgag gaaaccattg aacccagcag aaaa
4426655DNAArtificial sequence/note="HPV NASBA Primer" 266aattctaata
cgactcacta tagggagaag gttgctatac ttgtgtttcc ctacg
5526745DNAArtificial sequence/note="HPV NASBA Primer" 267gatgcaaggt
cgcatatgag gttgacctgt tgtgttacca gcaat 4526855DNAArtificial
sequence/note="HPV NASBA Primer" 268aattctaata cgactcacta
tagggagaag gcaccacgga cacacaaagg acaag 5526942DNAArtificial
sequence/note="HPV NASBA Primer" 269gatgcaaggt cgcatatgag
ctgttgacct gttgtgttac ga 4227053DNAArtificial sequence/note="HPV
NASBA Primer" 270aattctaata cgactcacta tagggagaag gccacggaca
cacaaaggac aag 5327140DNAArtificial sequence/note="HPV NASBA
Primer" 271gatgcaaggt cgcatatgag gttgacctgt tgtgttacga
4027251DNAArtificial sequence/note="HPV NASBA Primer" 272aattctaata
cgactcacta tagggagaag gacggacaca caaaggacaa g 5127340DNAArtificial
sequence/note="HPV NASBA Primer" 273gatgcaaggt cgcatatgag
ggaggaggat gaagtagata 4027451DNAArtificial sequence/note="HPV NASBA
Primer" 274aattctaata cgactcacta tagggagaag ggcccattaa catctgctgt a
5127543DNAArtificial sequence/note="HPV NASBA Primer" 275gatgcaaggt
cgcatatgag agaggaggag gatgaagtag ata 4327651DNAArtificial
sequence/note="HPV NASBA Primer" 276aattctaata cgactcacta
tagggagaag gacgggcaaa ccaggcttag t 5127744DNAArtificial
sequence/note="HPV NASBA Primer" 277gatgcaaggt cgcatatgag
ttggggtgct ggagacaaac atct 4427855DNAArtificial sequence/note="HPV
NASBA Primer" 278aattctaata cgactcacta tagggagaag gttcatcctc
atcctcatcc tctga
5527942DNAArtificial sequence/note="HPV NASBA Primer" 279gatgcaaggt
cgcatatgag tggggtgctg gagacaaaca tc 4228053DNAArtificial
sequence/note="HPV NASBA Primer" 280aattctaata cgactcacta
tagggagaag gcatcctcat cctcatcctc tga 5328142DNAArtificial
sequence/note="HPV NASBA Primer" 281gatgcaaggt cgcatatgag
ttggggtgct ggagacaaac at 4228253DNAArtificial sequence/note="HPV
NASBA Primer" 282aattctaata cgactcacta tagggagaag gccacaaact
tacactcaca aca 5328340DNAArtificial sequence/note="HPV NASBA
Primer" 283gatgcaaggt cgcatatgag gattttcctt atgcagtgtg
4028451DNAArtificial sequence/note="HPV NASBA Primer" 284aattctaata
cgactcacta tagggagaag ggacatctgt agcaccttat t 5128534DNAArtificial
sequence/note="HPV molecular beacon probe" 285cgcatgcaac tgayctmyac
tgttatgaca tgcg 3428634DNAArtificial sequence/note="HPV molecular
beacon probe" 286ccgtcgcaac tgayctmyac tgttatgacg acgg
3428734DNAArtificial sequence/note="HPV molecular beacon probe"
287ccaccccaac tgayctmyac tgttatgagg gtgg 3428834DNAArtificial
sequence/note="HPV molecular beacon probe" 288cgatcgcaac tgayctmyac
tgttatgacg atcg 3428936DNAArtificial sequence/note="HPV molecular
beacon probe" 289ccaagcgaam caactgacct aywctgctat gcttgg
3629038DNAArtificial sequence/note="HPV molecular beacon probe"
290ccaagccgaa mcaactgacc taywctgcta tggcttgg 3829138DNAArtificial
sequence/note="HPV molecular beacon probe" 291ccaagcggaa mcaactgacc
taywctgcta tcgcttgg 3829236DNAArtificial sequence/note="HPV
molecular beacon probe" 292ccagcggaam caactgacct aywctgctat cgctgg
3629336DNAArtificial sequence/note="HPV molecular beacon probe"
293cgatcggaam caactgacct aywctgctat cgatcg 3629430DNAArtificial
sequence/note="HPV molecular beacon probe" 294ccaagcaaga cattattcag
actcgcttgg 3029530DNAArtificial sequence/note="HPV molecular beacon
probe" 295cgcatgaaga cattattcag actccatgcg 3029630DNAArtificial
sequence/note="HPV molecular beacon probe" 296cccagcaaga cattattcag
actcgctggg 3029730DNAArtificial sequence/note="HPV molecular beacon
probe" 297cgatcgaaga cattattcag actccgatcg 3029840DNAArtificial
sequence/note="HPV NASBA Primer" 298gatgcaaggt cgcatatgag
aatggcattt gttggggtaa 4029950DNAArtificial sequence/note="HPV NASBA
Primer" 299aattctaata cgactcacta tagggagaag gtcatattcc tccccatgtc
5030040DNAArtificial sequence/note="HPV NASBA Primer" 300gatgcaaggt
cgcatatgag aatggcattt gttggsrhaa 4030150DNAArtificial
sequence/note="HPV NASBA Primer" 301aattctaata cgactcacta
tagggagaag gtcatattcc tcmmcatgdc 5030240DNAArtificial
sequence/note="HPV NASBA Primer" 302gatgcaaggt cgcatatgag
aatggcattt gttggsnnaa 4030340DNAArtificial sequence/note="HPV NASBA
Primer" 303gatgcaaggt cgcatatgag aatggcattt gttggnnhaa
4030440DNAArtificial sequence/note="HPV NASBA Primer" 304gatgcaaggt
cgcatatgag aatggcattt gttggnrnaa 4030540DNAArtificial
sequence/note="HPV NASBA Primer" 305gatgcaaggt cgcatatgag
aatggcattt gttggggtaa 4030640DNAArtificial sequence/note="HPV NASBA
Primer" 306gatgcaaggt cgcatatgag aatggcattt gttggggaaa
4030740DNAArtificial sequence/note="HPV NASBA Primer" 307gatgcaaggt
cgcatatgag aatggcattt gttggcataa 4030840DNAArtificial
sequence/note="HPV NASBA Primer" 308gatgcaaggt cgcatatgag
aatggcattt gttggggcaa 4030940DNAArtificial sequence/note="HPV NASBA
Primer" 309gatgcaaggt cgcatatgag aatggcattt gttggcacaa
4031050DNAArtificial sequence/note="HPV NASBA Primer" 310aattctaata
cgactcacta tagggagaag gtcatattcc tcmncatgnc 5031150DNAArtificial
sequence/note="HPV NASBA Primer" 311aattctaata cgactcacta
tagggagaag gtcatattcc tcaacatgnc 5031250DNAArtificial
sequence/note="HPV NASBA Primer" 312aattctaata cgactcacta
tagggagaag gtcatattcc tcnncatgtc 5031350DNAArtificial
sequence/note="HPV NASBA Primer" 313aattctaata cgactcacta
tagggagaag gtcatattcc tcnncatggc 5031450DNAArtificial
sequence/note="HPV NASBA Primer" 314aattctaata cgactcacta
tagggagaag gtcatattcc tcnncatgac 5031550DNAArtificial
sequence/note="HPV NASBA Primer" 315aattctaata cgactcacta
tagggagaag gtcatattcc tcnncatgcc 5031618DNAArtificial
sequenceprimer 316tacactgctg gacaacat 1831718DNAArtificial
sequenceprimer 317tcatcttctg agctgtct 1831821DNAArtificial
sequenceprimer 318tcaaaagcca ctgtgtcctg a 2131921DNAArtificial
sequenceprimer 319cgtgttcttg atgatctgca a 2132020DNAArtificial
sequenceprimer 320ttccggttga ccttctatgt 2032120DNAArtificial
sequenceprimer 321ggtcgtctgc tgagctttct 2032223DNAArtificial
sequenceprimer 322ctacagtaag cattgtgcta tgc 2332326DNAArtificial
sequenceprimer 323acgtaatgga gaggttgcaa taaccc 2632421DNAArtificial
sequenceprimer 324aacgccatga gaggacacaa g 2132521DNAArtificial
sequenceprimer 325acacataaac gaactgtggt g 2132620DNAArtificial
sequenceprimer 326cccgaggcaa ctgacctata 2032720DNAArtificial
sequenceprimer 327ggggcacact attccaaatg 2032821DNAArtificial
sequenceprimer 328gcagacgacc actacagcaa a 2132919DNAArtificial
sequenceprimer 329acaccgagtc cgagtaata 1933024DNAArtificial
sequenceprimer 330gaaaccattg aacccagcag aaaa 2433124DNAArtificial
sequenceprimer 331ttgctatact tgtgtttccc tacg 2433220DNAArtificial
sequenceprimer 332ggaggaggat gaagtagata 2033320DNAArtificial
sequenceprimer 333gcccattaac atctgctgta 2033420DNAArtificial
sequenceprimer 334gtgcctacgc tttttatcta 2033522DNAArtificial
sequenceprimer 335ggggtctcca acactctgaa ca 2233618DNAArtificial
sequenceprimer 336tcaggcgttg gagacatc 1833718DNAArtificial
sequenceprimer 337agcaatcgta agcacact 1833823DNAArtificial
sequenceprimer 338tttgttactg tggtagatac tac 2333925DNAArtificial
sequenceprimer 339gaaaaataaa ctgtaaatca tattc 2534020DNAArtificial
sequenceprimer 340acacaactgt gttcactagc 2034120DNAArtificial
sequenceprimer 341gaaacccaag agtcttctct 2034232DNAArtificial
sequence/note="HPV molecular beacon probe" 342cgatcggtac cgagggcagt
gtaatacgat cg 3234332DNAArtificial sequence/note="HPV molecular
beacon probe" 343cgatcggtgc ctacgctttt tatctacgat cg
3234434DNAArtificial sequence/note="HPV molecular beacon probe"
344ccgtcgttgc agcgatctga ggtatatgcg acgg 3434536DNAArtificial
sequence/note="HPV molecular beacon probe" 345cgatcgtggc agtggaaagc
agtggagaca cgatcg 3634621DNAArtificial sequenceprimer 346acacataaac
gaactgtgtg t 21
* * * * *