U.S. patent application number 10/241780 was filed with the patent office on 2003-09-04 for detection and identification of human papillomavirus by pcr and type-specific reverse hybridization.
This patent application is currently assigned to INNOGENETICS S.A.. Invention is credited to Kleter, Bernhard, Quint, Wim, Ter Schegget, Jan, Van Doorn, Leen-Jan.
Application Number | 20030165821 10/241780 |
Document ID | / |
Family ID | 8231037 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165821 |
Kind Code |
A1 |
Van Doorn, Leen-Jan ; et
al. |
September 4, 2003 |
Detection and identification of human papillomavirus by PCR and
type-specific reverse hybridization
Abstract
A method for detection and/or identification of HPV present in a
biological sample comprising amplification of HPV polynucleic acids
and of hybridization of said amplified polynucleic acids to a
number of probes whereby a short fragment of the L1 gene of HPV is
amplified after which, the amplimers are contacted with probes that
specifically hybridize to the said short fragment of the L1 gene of
at least one HPV type and a diagnostic kit to perform said method
and primers and probes used in the said method.
Inventors: |
Van Doorn, Leen-Jan;
(Ridderkerk, NL) ; Quint, Wim; (Nootdorp, NL)
; Kleter, Bernhard; (Delft, NL) ; Ter Schegget,
Jan; (Amsterdam, NL) |
Correspondence
Address: |
Charles A. Muserlian
c/o Bierman, Muserlian and Lucas
600 Third Avenue
New York
NY
10016
US
|
Assignee: |
INNOGENETICS S.A.
|
Family ID: |
8231037 |
Appl. No.: |
10/241780 |
Filed: |
September 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10241780 |
Sep 11, 2002 |
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09527030 |
Mar 16, 2000 |
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6482588 |
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09527030 |
Mar 16, 2000 |
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PCT/EP98/05829 |
Sep 14, 1998 |
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Current U.S.
Class: |
435/5 ; 435/6.14;
435/91.2 |
Current CPC
Class: |
C12Q 1/708 20130101;
C12Q 1/708 20130101; C12Q 2531/113 20130101 |
Class at
Publication: |
435/5 ; 435/6;
435/91.2 |
International
Class: |
C12Q 001/70; C12Q
001/68; C12P 019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 16, 1997 |
EP |
97870136.5 |
Claims
1. Method for detection and/or identification of HPV present in a
biological sample, comprising the following steps: (i)
amplification of a polynucleic acid fragment of HPV by use of: a
5'-primer specifically hybridizing to the A region or B region of
the genome of at least one HPV type, said A region or B region
being indicated in FIG. 1, and, a 3'-primer specifically
hybridizing to the C region of the genome of at least one HPV type,
said C region being indicated in FIG. 1; (ii) hybridizing the
amplified fragments from step (i) with at least one probe capable
of specific hybridization with the D region of at least one HPV
type, said D region being indicated in FIG. 1.
2. Method according to claim 1, characterized further in that: the
3'-end of said 5'-primer specifically hybridizing to the A region
of the genome of at least one HPV type, is situated at position
6572 of the genome of HPV 16, or at the corresponding position of
any other HPV genome, as indicated in FIG. 1, and/or, the 3'-end of
said 5'-primer specifically hybridizing to the B region of the
genome of at least one HPV type, is situated at position 6601 of
the genome of HPV 16, or at the corresponding position of any other
HPV genome, as indicated in FIG. 1, and/or, the 3'-end of said
3'-primer specifically hybridizing to the C region of the genome of
at least one HPV type, is situated at position 6624 of the genome
of HPV 16, or at the corresponding position of any other HPV
genome, as indicated in FIG. 1.
3. A method according to claim 2, characterized further in that:
said 5'-primer specifically hybridizing to the A region is chosen
from the following list: SGP3 (SEQ ID NO 2), SGP3A (SEQ ID NO 3),
SGP3B (SEQ ID NO 4), SGP3C (SEQ ID NO 10), SGP3D (SEQ ID NO 11),
SGP3E (SEQ ID NO 12), SGP3F (SEQ ID NO 13), SGP3G (SEQ ID NO 14),
and/or, said 5-primer specifically hybridizing to the b region is
chosen from the following list: SGP1 (SEQ ID NO 6), SGP1A (SEQ ID
NO 15), SGP1B (SEQ ID NO16), SGP1C (SEQ ID NO 17), SGP1D (SEQ ID NO
18), and/or, said 3'-primer specifically hybridizing to the C
region is chosen from the following list: SGP2 (SEQ ID NO 9), SGP2A
(SEQ ID NO 8), SGP2B (SEQ ID NO 19), SGP2C (SEQ ID NO 20), SGP2D
(SEQ ID NO 21), SGP2E (SEQ ID NO 22), SGP2F (SEQ ID NO 23), SGP2H
(SEQ ID NO 98), SGP2I (SEQ ID NO 154), SGP2J (SEQ ID NO 155), SGP2K
(SEQ ID NO 156), SGP2L (SEQ ID NO 157), SGP2M (SEQ ID NO 158),
SGP2N (SEQ ID NO 159), SGP2P (SEQ ID NO 160).
4. Method according to any of claims 1 to 3, characterized further
in that said probe mentioned in step (ii) is capable of specific
hybridization with the D region of the genome of only one HPV type,
and thus enables specific identification of this HPV type, when
this type is present in a biological sample.
5. Method according to any of claims 1 to 3, characterized further
in that said probe mentioned in step (ii) is capable of specific
hybridization with the D region of more than one HPV type, and thus
enables detection of any of said more than one HPV type, when any
of said types is present in a biological sample.
6. Method according to claim 4, characterized further in that said
probe capable of specific hybridization with the D region of the
genome of only one HPV type, more particularly hybridizes to the E
region, with said E region being a subregion of the D region, as
indicated in FIG. 1.
7. Method according to claim 4, characterized further in that said
probe capable of specific hybridization with the D region of the
genome of only one HPV type, more particularly specifically
hybridizes to the 22 bp region situated between the B region and
the C region, as indicated in FIG. 1.
8. Method according to claim 7, characterized further in that said
probe specifically hybridizing to said 22 bp region of only one HPV
type is chosen from the following list: HPV6 Pr1, HPV6 Pr2, HPV6
Pr3, HPV6 Pr4, HPV6 Pr5, HPV11 Pr1, HPV11 Pr2, HPV11 Pr3, HPV11
Pr4, HPV11 Pr5, HPV16 Pr1, HPV16 Pr2, HPV16 Pr3, HPV16 Pr4, HPV16
Pr5, HPV18 Pr1, HPV18 Pr2, HPV18 Pr3, HPV18 Pr4, HPV18 Pr5, HPV31
Pr1, HPV31 Pr2, HPV31 Pr3, HPV31 Pr4, HPV31 Pr5, HPV31 Pr21, HPV31
Pr22, HPV31 Pr23, HPV31 Pr24, HPV31 Pr25, HPV31 Pr26, HPV31 Pr31,
HPV31 Pr32, HPV33 Pr1, HPV33 Pr2, HPV33 Pr3, HPV33 Pr4, HPV33 Pr5,
HPV33 Pr21, HPV33 Pr22, HPV33 Pr23, HPV33 Pr24, HPV33 Pr25, HPV33
Pr26, HPV40 Pr1, HPV45 Pr1 (=SGPP68), HPV45 Pr2, HPV45 Pr3, HPV45
Pr4, HPV45 Pr5, HPV45 Pr11, HPV45 Pr12, HPV45 Pr13, HPV52 Pr1,
HPV52 Pr2, HPV52 Pr3, HPV52 Pr4, HPV52 Pr5, HPV52 Pr6, HPV56 Pr1,
HPV56 Pr2, HPV56 Pr3, HPV56 Pr11, HPV56 Pr12, HPV58 Pr1, HPV58 Pr2,
HPV58 Pr3, HPV58 Pr4 (SEQ ID NOs 24 to 91), and, SGPP35, SGPP39,
SGPP51 (=HPV51 Pr1), SGPP54, SGPP59, SGPP66, SGPP70 (=HPV70 Pr1),
SGPP13, SGPP34, SGPP42, SGPP43, SGPP44, SGPP53, SGPP55, SGPP69,
SGPP61, SGPP62, SGPP64, SGPP67, SGPP74 (=HPV74 Pr13), MM4 (=HPVM4
Pr11), MM7, MM8 (SEQ ID NOs 92 to 115), and, HPV18b Pr1, HPV18b
Pr2, HPV31 Vs40-1, HPV31 Vs40-2, HPV31 Vs40-3, HPV34 Pr1, HPV35
Pr1, HPV35 Pr2, HPV35 Pr3, HPV39 Pr1, HPV42 Pr1, HPV42 Pr2, HPV43
Pr1, HPV43 Pr2, HPV43 Pr3, HPV44 Pr1, HPV44 Pr2, HPV44 Pr3, HPV44
Pr4, HPV51 Pr2, HPV53 Pr1, HPV54 Pr1, HPV54 Pr11, HPV54 Pr11as,
HPV54 Pr12, HPV55 Pr1, HPV55 Pr11, HPV55 Pr12, HPV55 Pr13, HPV56
Vs74-1, HPV59 Pr1, HPV59 Pr11, HPV59 Pr12, HPV59 Pr13, HPV66 Pr1,
HPV67 Pr1, HPV67 Pr11, HPV67 Pr12, HPV67 Pr13, HPV67 Pr21, HPV67
Pr22, HPV67 Pr23, HPV68 Pr1, HPV68 Pr2, HPV68 Pr3, HPV68 Vs45-1,
HPV68 Vs45-2, HPV70 Pr1, HPV70 Pr12, HPV70 Pr13, HPV74 Pr1, HPV74
Pr11, HPV74 Pr12, HPV74 Pr2, HPV74 Pr3, HPVM4 Pr1, HPVM4 Pr12,
HPVM4 Pr21, HPVM4 Pr22 (SEQ ID NOs 161 to 219).
9. Method according to claim 5, characterized further in that said
probe capable of specific hybridization with the D region of the
genome of more than one HPV type, more particularly hybridizes to
the E region, with said E region being a subregion of the D region,
as indicated in FIG. 1.
10. Method according to claim 9, characterized further in that said
probe specifically hybridizing to said E region of more than one
HPV type, is chosen from the following list: HPVuni1, HPVuni2,
HPVuni3, HPVuni4, HPVuni5, HPVuni6, HPVuni7, HPVuni2L2, HPVuni2L3,
HPVuni2L4, HPVuni2L5, HPVuni2L6, HPVuni2L7, HPVuni4L1, HPVuni4L2,
HPVuni4L3, HPVuni4L4, HPVuni4L5, HPVuni4L6 (SEQ ID NOs 116 to 134),
and, HPVuni1A, HPVuni1B, HPVuni1C, HPVuni2A, HPVuni3A (SEQ ID NOs
220 to 224), and, HPV G1, HPV G1A1, HPV G1A2, HPV G1A3, HPV G1A4,
HPV G2, HPV G3, HPV G4, HPV G5, HPV G6, HPV R1, HPV R10, HPV R11,
HPV R2, HPV R3, HPV R4, HPV R5, HPV R6, HPV R7, HPV R8, HPV R9 (SEQ
ID NOs 225 to 245).
11. A primer as defined in any of claims 1 to 3, for use in the
detection and/or identification of HPV present in a biological
sample.
12. A primer combination consisting of a 5'-primer as defined in
any of claims 1 to 3 and of a 3'-primer as defined in any of claims
1 to 3, for use in the detection and/or identification of HPV
present in a biological sample.
13. A probe as defined in any of claims 1 and 4 to 10, for use in
the detection and/or identification of HPV present in a biological
sample.
14. A diagnostic kit for detection and/or identification of HPV,
possibly present in a biological sample, comprising the following
components: (i) at least one suitable primer, with said primers
being defined in any of claims 1 to 3; (ii) at least one suitable
probe, with said probes being defined in any of claims 1 and 4 to
10.
15. An isolated HPV polynucleic acid, defined by SEQ ID NO 135 to
153, or any fragment thereof, that can be used as a primer or as a
probe in a method for detection and/or identification of HPV
present in a sample.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of detection and
identification of Human Papillomavirus (HPV) infections in clinical
samples.
BACKGROUND OF THE INVENTION
[0002] Cervical cancer is the second most common malignancy in
women, following breast cancer. Carcinoma of the cervix is unique
in that it is the first major solid tumor in which HPV DNA is found
in virtually all cases and in precursor lesions worldwide.
[0003] Nowadays, 74 HPV genotypes have been characterized and are
numbered in chronological order of isolation. HPV is
epitheliotropic and infects only the skin (cutaneous types) or the
mucosa of the respiratory and anogenital tract (mucosal types).
Thirty-six of the 74 HPV types are known to infect the uterine
cervix. Based on the induced benign, premalignant or malignant
lesions, HPV is divided into low-risk (e.g., HPV types 6, 11, 42,
43 and 44) and high-risk types (e.g., types 16, 18, 31, 33 and 45),
respectively. The high-risk types account for more than 80% of all
invasive cervical cancers. Consequently, detection and
identification of HPV types is very important. The high-risk types
are more consistently found in high grade SIL (Squamous
Intraepithelial Lesion) and carcinoma in-situ than low-risk types
which are mainly found in low grade SIL. This epidemiological
observation is supported by molecular findings. For instance, the
E6 and E7 proteins from low-risk types 6 and 11 bind p53 and pRB
too weakly to immortalize keratinocytes in vitro or to induce
malignant transformation in vivo (Woodworth et al., 1990). The
circular ds-DNA genome of low-risk HPV types remains episomal
whereas the genome of high-risk HPV types is able to integrate into
the human genome.
[0004] Screening for malignant and premalignant disorders of the
cervix is usually performed according to the Papanicoloau (PAP)
system. The cervical smears are examined by light microscopy and
the specimens containing morphologically abnormal cells are
classified into PAP I to V, at a scale of increasing severity of
the lesion. This cytomorphological method is an indirect method and
measures the possible outcome of an HPV infection. Therefore, HPV
DNA detection and typing is of importance in secondary screening in
order to select patients for monitoring (follow-up) and treatment.
This means that cervical smears classified as PAP II (a typical
squamous metaplasia) or higher classes should be analyzed for
low-risk and high-risk HPV types. Follow-up studies have shown that
only high-risk HPV types are involved in the progression from
cytologically normal cervix cells to high grade SIL (Remminck et
al., 1995). These results indicate that the presence of high-risk
HPV types is a prognostic marker for development and detection of
cervical cancer.
[0005] Detection of HPV Infections
[0006] Diagnosis of HPV by culture is not possible. Also diagnosis
by detection of HPV anti-bodies appears to be hampered by
insufficient sensitivity and specificity. Direct methods to
diagnose an HPV infection are mainly based on detection of the
viral DNA genome by different formats of DNA/DNA hybridization with
or without prior amplification of HPV DNA. The polymerase chain
reaction (PCR) is a method that is highly efficient for
amplification of minute amounts of target DNA. Nowadays, mainly
three different primer pairs are used for universal amplification
of HPV DNA Two of these primer pairs, MY11/MY09 and GP5/GP6, are
directed to conserved regions among diffent HPV types in the L1
region (Manos et al., 1989; Van den Brule et al., 1990). The other
primer pair, CPI/CPIIg, is directed to conserved regions in the E1
region (Tieben et al., 1993).
[0007] Typing of HPV Isolates
[0008] There are several methods to identify the various HPV
types.
[0009] 1. HPV DNA can be typed by PCR primers that recognize only
one specific type. This method is known as type-specific PCR. Such
methods have been described for HPV types 6, 11, 16, 18, 31 and 33
(Claas et al., 1989; Cornelissen et al., 1989; Falcinelli et al.,
1992; Van den Brule et al., 1990; Young et al., 1989). The primers
are aimed at the E5, L1, E6, L1, E2 and E1 regions of the HPV
genome for types 6, 11, 16, 18, 31 and 33, respectively (Baay et
al., 1996). The synthesized amplimer sizes vary from 217 bp to 514
bp.
[0010] 2. Another method is general amplification of a genomic part
from all HPV types followed by hybridization with two cocktails of
type-specific probes differentiating between the oncogenic and
non-oncogenic groups, respectively. A similar typing method has
been described without prior amplification of HPV DNA. In the
Hybrid capture assay (Hybrid Capture Sharp Assay; Digene, Silver
Springs, Md.), each sample is tested for a group of "high-risk" HPV
types (16, 18, 31, 33, 35, 45, 51, 52 and 56) and for another group
of "low-risk" HPV types (6, 11, 42, 43 and 44) (Cox et al.,
1995).
[0011] At present, classification of human papillomaviruses can be
performed for instance by sequence analysis of a 450 bp PCR
fragment synthesized by the primers MY 11/MY09 in the L1 region
(Chan et al., 1995) or by the primers CPI and CPIIg in the E1
region (Tieben et al., 1993). Phylogenetic analysis of these
sequences allows classification of the different HPV types. By
definition, if the sequence differences between two HPV isolates is
higher than 10% they are classified as different types.
Consequently, if the sequence differs more than 10% from any known
HPV type it is classified as a novel HPV genotype. HPV isolates
that differ between 2-10% are classified as different subtypes.
Finally, if the sequence variation is below 2%, the 2 isolates are
classified within the same subtype as different variants.
AIMS OF THE INVENTION
[0012] It is an aim of the present invention to provide a rapid and
reliable method for detection and/or identification of HPV,
possibly present in a biological sample.
[0013] It is more particularly an aim of the present invention to
provide a method for detection and/or identification of HPV
comprising amplification of a polynucleic acid fragment of HPV and
subsequent hybridization of this fragment to suitable probes.
[0014] It is also an aim of the present invention to provide a
number of oligonucleotide primers and probes enabling said method
of detection and/or amplification of HPV.
[0015] It is also an aim of the present invention to provide new
HPV sequences.
[0016] It is furthermore an aim of the present invention to provide
protocols according to which said amplification and hybridization
steps can be performed. One format for the hybridization step is,
for instance, the reverse hybridization format, and more
particularly the LiPA technique.
[0017] It is also an aim of the present invention to compose
diagnostic kits comprising said primers and probes, permitting the
rapid and reliable detection and/or identification of HPV possibly
present in a biological sample.
[0018] All the aims of the present invention are met by the
following specific embodiments.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention provides a method for detection and/or
identification of HPV, possibly present in a biological sample,
comprising the following steps:
[0020] (i) amplification of a polynucleic acid fragment of HPV by
use of:
[0021] a 5'-primer specifically hybridizing to the A region or B
region of the genome of at least one HPV type, said A region and B
region being indicated in FIG. 1, and
[0022] a 3'-primer specifically hybridizing to the C region of the
genome of at least one HPV type, said C region being indicated in
FIG. 1;
[0023] (ii) hybridizing the amplified fragments from step (i) with
at least one probe capable of specific hybridization with the D
region of at least one HPV type, said D region being indicated in
FIG. 1.
[0024] According to one preferred embodiment of the present
invention, said probe mentioned in step (ii) is capable of specific
hybridization with the D region of the genome of only one HPV type,
and thus enables specific identification of this HPV type, when
this type is present in a biological sample.
[0025] According to another preferred embodiment of the present
invention, said probe mentioned in step (ii) is capable of specific
hybridization with the D region of more than one HPV type, and thus
enables detection of any of said more than one HPV type, when any
of said types is present in a biological sample.
[0026] According to another preferred embodiment of the present
invention, the 3'-end of said 5'-primer specifically hybridizing to
the A region of the genome of at least one HPV type, is situated at
position 6572 of the genome of HPV 16, or at the corresponding
position of any other HPV genome, as indicated in FIG. 1.
[0027] According to another preferred embodiment of the present
invention, the 3'-end of said 5'-primer specifically hybridizing to
the B region of the genome of at least one HPV type, is situated at
position 6601 of the genome of HPV 16, or at the corresponding
position of any other HPV genome, as indicated in FIG. 1.
[0028] According to another preferred embodiment of the present
invention, the 3'-end of said 3'-primer specifically hybridizing to
the C region of the genome of at least one HPV type, is situated at
position 6624 of the genome of HPV 16, or at the corresponding
position of any other HPV genome, as indicated in FIG. 1.
[0029] According to another preferred embodiment of the present
invention, said probe capable of specific hybridization with the D
region of the genome of only one HPV type, more particularly
specifically hybridizes to the E region, with said E region being a
subregion of the D region, as indicated in FIG. 1.
[0030] According to another preferred embodiment of the present
invention, said probe capable of specific hybridization with the D
region of the genome of only one HPV type, more particularly
specifically hybridizes to the 22 bp region situated between the B
region and the C region, as indicated in FIG. 1.
[0031] According to another preferred embodiment, said 5'-primer
specifically hybridizing to the A region of the genome of at least
one HPV type, is chosen from the following list:
[0032] SGP3, SGP3A, SGP3B, SGP3C, SGP3D, SGP3E, SGP3F, SGP3G.
[0033] The sequences of said primers are shown in table 1 and in
table 4.
[0034] According to another preferred embodiment, said 5'-primer
specifically hybridizing to the B region of the genome of at least
one HPV type, is chosen from the following list:
[0035] SGP1, SGP1A, SGP1B, SGP1C, SGP1D.
[0036] The sequences of said primers are shown in table 1, in table
4 and in table 11.
[0037] According to another preferred embodiment, said 3'-primer
specifically hybridizing to the C region of the genome of at least
one HPV type, is chosen from the following list:
[0038] SGP2, SGP2A, SGP2B, SGP2C, SGP2D, SGP2E, SGP2F, SGP2H,
SGP2I, SGP2J, SGP2K, SGP2L, SGP2M, SGP2N, SGP2P.
[0039] The sequences of said primers are shown in table 1, in table
4 and in table 11.
[0040] According to another preferred embodiment, said probe
capable of specific hybridization with the aforementioned 22 bp
region of only one HPV type, is chosen from the following list:
[0041] HPV6 Pr1, HPV6 Pr2, HPV6 Pr3, HPV6 Pr4, HPV6 Pr5, HPV11 Pr1,
HPV11 Pr2, HPV11 Pr3, HPV11 Pr4, HPV11 Pr5, HPV16 Pr1, HPV16 Pr2,
HPV16 Pr3, HPV16 Pr4, HPV16 Pr5, HPV18 Pr1, HPV18 Pr2, HPV18 Pr3,
HPV18 Pr4, HPV18 Pr5, HPV31 Pr1, HPV31 Pr2, HPV31 Pr3, HPV31 Pr4,
HPV31 Pr5, HPV31 Pr21, HPV31 Pr22, HPV31 Pr23, HPV31 Pr24, HPV31
Pr25, HPV31 Pr26, HPV31 Pr31, HPV31 Pr32, HPV33 Pr1, HPV33 Pr2,
HPV33 Pr3, HPV33 Pr4, HPV33 Pr5, HPV33 Pr21, HPV33 Pr22, HPV33
Pr23, HPV33 Pr24, HPV33 Pr25, HPV33 Pr26, HPV40 Pr1, HPV45 Pr1
(=SGPP68), HPV45 Pr2, HPV45 Pr3, HPV45 Pr4, HPV45 Pr5, HPV45 Pr11,
HPV45 Pr12, HPV45 Pr13, HPV52 Pr1, HPV52 Pr2, HPV52 Pr3, HPV52 Pr4,
HPV52 Pr5, HPV52 Pr6, HPV56 Pr1, HPV56 Pr2, HPV56 Pr3, HPV56 Pr11,
HPV56 Pr12, HPV58 Pr1, HPV58 Pr9, HPV58 Pr3, HPV58 Pr4, SGPP35,
SGPP39, SGPP51 (=HPV51 Pr1), SGPP54, SGPP59, SGPP66, SGPP70 (=HPV70
Pr11), SGPP13, SGPP34, SGPP42, SGPP43, SGPP44, SGPP53, SGPP55,
SGPP69, SGPP61,
[0042] SGPP62, SGPP64, SGPP67, SGPP74 (=HPV74 Pr13), MM4 (=HPVM4
Pr11), MM7, MM8, HPV18b Pr1, HPV18b Pr2, HPV31 Vs40-1, HPV31
Vs40-2, HPV31 Vs40-3, HPV34 Pr1, HPV35 Pr1, HPV35 Pr2, HPV35 Pr3,
HPV39 Pr1, HPV42 Pr1, HPV42 Pr2, HPV43 Pr1, HPV43 Pr2, HPV43 Pr3,
HPV44 Pr1, HPV44 Pr2, HPV44 Pr3, HPV44 Pr4, HPV45 Pr5, HPV51 Pr2,
HPV53 Pr1, HPV54 Pr1, HPV54 Pr11, HPV54 Pr11as, HPV54-Pr12, HPV55
Pr1, HPV55 Pr11, HPV55 Pr12, HPV55 Pr13, HPV56 Vs74-1, HPV59 Pr1,
HPV59 Pr11, HPV59 Pr12, HPV59 Pr13, HPV66 Pr1, HPV67 Pr1, HPV
67Pr11, HPV67 Pr12, HPV67 Pr13, HPV67 Pr21, HPV67 Pr22, HPV67 Pr23,
HPV68 Pr1, HPV68 Pr2, HPV68 Pr3, HPV68 Vs45-1, HPV68 Vs45-2, HPV70
Pr1, HPV70 Pr12, HPV70 Pr13, HPV74 Pr1, HPV74 Pr11, HPV74 Pr12,
HPV74 Pr2, HPV74 Pr3, HPVM4 Pr1, HPVM4 Pr12, HPVM4 Pr21, HPVM4
Pr22.
[0043] The sequences of said probes are shown in table 7 and table
12.
[0044] It is to be understood that combinations of the
aforementioned embodiments are also preferred embodiments, for
instance a method characterized in that said 5'-primer specifically
hybridizing to the A region is chosen from the aforementioned
respective list and that said 3'-primer specifically hybridizing to
the C region is chosen from the aforementioned respective list.
[0045] It is an important feature of the present invention that the
amplified polynucleic acid fragments of HPV fall within a short
region of the L1 gene, a region that presents a high degree of
sequence variability. Said region is denoted D region and for any
HPV type consists of the region corresponding in a sequence
alignment to the region from position 6553 to position 6646 of the
genome of HPV 16, with the numbering being according to isolate
PPH16, with Genbank accession number K02718. The advantage of
amplifying a short fragment is that higher sensitivity can be
obtained, i.e. a lower number of copies of HPV polynucleic acids
can be detected and/or identified. The aforementioned primers may
be used to amplify a fragment of approximately 65 bp (by use of
5'-primers specifically hybridizing to the B region and 3'-primers
specifically hybridizing to the C region) or a fragment of
approximately 94 bp (by use of 5'-primers specifically hybridizing
to the A region and 3'-primers specifically hybridizing to the C
region). However, it is obvious to one skilled in the art that
other primers may be used in order to amplify other fragments
within or overlapping with said D region. Preferred primers are
shown in table 1 and in table 4. These primers permit amplification
of polynucleic acid fragments of a large group of HPV types, but it
may be desirable for some purposes to chose primers that
selectively amplify a smaller group of HPV types.
[0046] The different types of HPV in a sample can be identified by
hybridization of polynucleic acids of said types of HPV to at least
one, preferably at least two, more preferably at least three, even
more preferably at least four and most preferably at least five
oligonucleotide probes. These probes may be designed to
specifically hybridize to the D region of only one HPV genome, said
D region being indicated in FIG. 1. Tables 7 and 12 contain a list
of preferred probes specifically hybridizing to the 22 bp region
within said D region, situated between the B region and the C
region. These probes may be used together under the same conditions
of hybridization and washing, for instance in a LiPA format (see
below). Probes that have been optimized to work together in a LiPA
format are for instance the combination of HPV6 Pr1, HPV11 Pr1,
HPV16 Pr1, HPV18 Pr1, HPV31 Pr25, HPV31 Pr31, HPV31 Pr32, HPV33
Pr21, HPV33 Pr25, HPV40 Pr1, HPV45 Pr11, HPV45 Pr12, HPV45 Pr13
HPV52 Pr5, HPV52 Pr6, HPV56 Pr11, HPV56 Pr12, HPV58 Pr2, HPV58 Pr3
and HPV58 Pr4 (see example 4), the combination of HPV6 Pr1, HPV11
Pr5, HPV16 Pr1, HPV18 Pr1, HPV18b Pr2, HPV31 Pr31, c31-3, HPV33
Pr21, HPV34 Pr1, HPV35 Pr1, HPV39 Pr1, HPV40 Pr1, HPV42 Pr1, HPV43
Pr3, HPV44 Pr1, HPV45 Pr11, HPV51 Pr2, HPV52 Pr5, HPV53 Pr1, HPV56
Pr12, c56-1, HPV58 Pr2, HPV59 Pr12, HPV66 Pr1, HPV68 Pr1, c68-1,
HPV70 Pr12 and HPV74 Pr1, or the combination outlined in example 7.
Probes specifically hybridizing to said 22 bp region should permit
discrimination of all genital low-risk types including HPV types 6,
11, 34, 40, 42-44, 53, 54, 55, 59, 61, 62, 64, 67, 68, 71 and 74 as
well as all genital high-risk types including HPV types 16, 18, 31,
33, 35, 39, 45, 51, 52, 56-58, 66 and 69 (zur Hausen, 1996). It
should be clear to one skilled in the art that other probes than
those listed in table 7 or 12 may be chosen within said region D,
provided that they specifically hybridize to only one HPV-type. It
should also be clear that in some cases probes may be chosen that
overlap with the primers used in the amplification step. In this
case, however, the region of overlap between primer and probe
should not be as long as to allow by itself duplex formation under
the experimental conditions used. It should furthermore be clear
that, if presently unknown types are detected that differ in the D
region from all presently known types, the methods of this
invention will also enable detection and/or identification of said
presently unknown HPV types. The present invention furthermore
discloses novel sequences in said 22 bp region, as shown in example
5 and in FIG. 1 (SEQ ID NO 135-153). Probes or primers that are
designed to specifically hybridize to these sequences, may be used
in a method to detect and/or to identify HPV polynucleic acids
comprising any of these sequences, when these polynucleic acids are
present in a biological sample.
[0047] According to another preferred embodiment of the present
invention, probes are used that specifically hybridize to the D
region, or more particularly to the E region of more than one HPV
type. Examples of such probes are given in table 9 and in table 10.
The probes in table 9 have been designed for hybridization in a
microtiter plate, e.g. according to the DEIA technique (see below),
whereas the probes in table 10 are more suitable for the LiPA
technique (see below). These probes hybridize to the E region of
more than one HPV type, and hence may be used to detect the
presence in a biological sample of any of the types to which they
hybridize. It should be clear to one skilled in the art that,
according to this embodiment, other probes than those listed in
table 9 and table 10 may be chosen within the D region, provided
that they hybridize to one or more than one HPV type.
[0048] According to another preferred embodiment of the present
invention, the aforementioned methods of detection and/or
identification of HPV are characterized further in that the
hybridization step involves a reverse hybridization format. This
format implies that the probes are immobilized to certain locations
on a solid support and that the amplified HPV polynucleic acids are
labelled in order to enable the detection of the hybrids formed.
According to this embodiment, at least one probe, or a set of a
least 2, preferably at least 3, more preferably at least 4 and most
preferably at least 5 probes is used. When at least 2 probes are
used, said probes are meticulously designed in such a-way that they
specifically hybridize to their target sequences under the same
hybridization and wash conditions.
[0049] According to an even more preferred embodiment of the
present invention, the aforementioned hybridization step is
performed according to the LiPA technique. Said technique involves
a reverse hybridization assay, characterized in that the
oligonucleotide probes are immobilized on a solid support as
parallel lines (Stuyver et al., 1993; international application WO
94/12670). The reverse hybridization format and particularly the
LiPA format have many practical advantages as compared to other DNA
techniques or hybridization formats, especially when the use of a
combination of probes is preferable or unavoidable to obtain the
relevant information sought.
[0050] Alternatively, detection of HPV polynucleic acids in a
biological sample may be performed by use of the DNA Enzyme Immuno
Assay (DEIA). This method is used for rapid and specific detection
of PCR products. PCR products are generated by a primer set, of
which either the forward or the reverse primer contain biotin at
the 5' end. This allows binding of the biotinylated amplimers to
streptavidin-coated microtiter wells. PCR products are denatured by
sodium hydroxide, which allows removal of the non-biotinylated
strand. Specific labelled oligonucleotide probes (e.g. with
digoxigenin) are hybridized to the single-stranded immobilized PCR
product and hybrids are detected by enzyme-labelled conjugate and
calorimetric methods.
[0051] The present invention also relates to sets of
oligonucleotides, said sets comprising at least one primer and/or
at least one probe that may be used to perform the methods for
detection and/or identification of HPV as described above.
Preferred primers according to the present invention can for
instance be chosen from table 1, table 4 and table 11. Preferred
probes are shown in tables 7, 9, 10 and 12. These probes can be
optimized to be used together in a given format, e.g. a LiPA
format, under the same hybridization and washing conditions.
Evidently, when other hybridization conditions would be preferred,
all probes should be adapted accordingly by adding or deleting one
or more nucleotides at their extremities. It should be understood
that these concomitant adaptations should give rise to the same
result, namely that the probes still hybridize specifically to
their respective type-specific target sequences. Such adaptations
may also be necessary if the amplified material is RNA and not DNA
as is the case in the NASBA system.
[0052] The present invention also relates to diagnostic kits for
detection and/or identification of HPV, possibly present in a
biological sample, comprising the following components:
[0053] (i) at least one suitable primer or at least one suitable
primer pair;
[0054] (ii) at least one suitable probe, preferably at least 2,
more preferably at least 3, even more preferably at least 4 and
most preferably at least 5 suitable probes, possibly fixed to a
solid support;
[0055] (iii) a hybridization buffer, or components necessary for
the production of said buffer, or instructions to prepare said
buffer;
[0056] (iv) a wash solution, or components necessary for the
production of said solution, or instructions to prepare said
solution;
[0057] (v) optionally a means for detection of the hybrids
formed;
[0058] (vi) optionally a means for attaching the probe(s) to a
known location on a solid support.
[0059] The following definitions and explanations will permit a
better understanding of the present invention.
[0060] HPV isolates that display a sequence difference of more than
10%:to any previously known type in the combined nucleotide
sequences of E6, E7 and L1 genes (Chan et al., 1995, de Villiers,
1994) are classified as different HPV "genotypes". HPV isolates
that differ between 2 and 10% are classified as different
"subtypes". If the sequence variation is below 2%, the isolates are
classified within the same subtype as different "variants". The
term "type" when applied to HPV refers to any of the three
categories defined above.
[0061] The target material in the samples to be analyzed may either
be DNA or RNA, e.g. genomic DNA, messenger RNA, viral RNA or
amplified versions thereof. These molecules are in this application
also termed "polynucleic acids".
[0062] Well-known extraction and purification procedures are
available for the isolation of RNA or DNA from a sample (e.g. in
Sambrook et al., 1989).
[0063] The term "probe" according to the present invention refers
to a single-stranded oligonucleotide which is designed to
specifically hybridize to HPV polynucleic acids. The term "primer"
refers to a single stranded oligonucleotide sequence capable of
acting as a point of initiation for synthesis of a primer extension
product which is complementary to the nucleic acid strand to be
copied. The length and the sequence of the primer must be such that
they allow to prime the synthesis of the extension products.
Preferably the primer is about 5-50 nucleotides long. Specific
length and sequence will depend on the complexity of the required
DNA or RNA targets, as well as on the conditions at which the
primer is used, such as temperature and ionic strength.
[0064] The expression "suitable primer pair" in this invention
refers to a pair of primers allowing the amplification of part or
all of the HPV polynucleic acid fragment for which probes are
immobilized.
[0065] The term "target sequence" of a probe or a primer according
to the present invention is a sequence within the HPV polynucleic
acids to which the probe or the primer is completely complementary
or partially complementary (i.e. with some degree of mismatch). It
is to be understood that the complement of said target sequence is
also a suitable target sequence in some cases. Probes of the
present invention should be complementary to at least the central
part of their target sequence. In most cases the probes are
completely complementary to their target sequence. The term
"type-specific target sequence" refers to a target sequence within
the polynucleic acids of a given HPV type that contains at least
one nucleotide difference as compared to any other HPV-type.
[0066] "Specific hybridization" of a probe to a region of the HPV
polynucleic acids means that, after the amplification step, said
probe forms a duplex with part of this region or with the entire
region under the experimental conditions used, and that under those
conditions said probe does not form a duplex with other regions of
the polynucleic acids present in the sample to be analysed. It
should be understood that probes that are designed for specific
hybridization to a region of HPV polynucleic acids, may fall within
said region or may to a large extent overlap with said region (i.e.
form a duplex with nucleotides outside as well as within said
region). For instance, some of the probes that are shown in table 7
and that are designed for specific hybridization to the 22 bp
region between the B and the C regions (FIG. 1), extend up to 5
nucleotides beyond the 3'-end of said 22 bp region and other probes
of table 7 extend up to 3 nucleotides beyond the 5'-end of said 22
bp region.
[0067] "Specific hybridization" of a primer to a region of the HPV
polynucleic acids means that, during the amplification step, said
primer forms a duplex with part of this region or with the entire
region under the experimental conditions used, and that under those
conditions said primer does not form a duplex with other regions of
the polynucleic acids present in the sample to be analysed. It
should be understood that primers that are designed for specific
hybridization to a region of HPV polynucleic acids, may fall within
said region or may to a large extent overlap with said region (i.e.
form a duplex with nucleotides outside as well as within said
region).
[0068] Since the current application requires the detection of
single base pair mismatches, stringent conditions for hybridization
of probes are required, allowing only hybridization of exactly
complementary sequences. However, it should be noted that, since
the central part of the probe is essential for its hybridization
characteristics, possible deviations of the probe sequence versus
the target sequence may be allowable towards the extremities of the
probe when longer probe sequences are used. Variations are possible
in the length of the probes. Said deviations and variations, which
may be conceived from the common knowledge in the art, should
however always be evaluated experimentally, in order to check if
they result in equivalent hybridization characteristics as the
exactly complementary probes.
[0069] Preferably, the probes of the invention are about 5 to 50
nucleotides long, more preferably from about 10 to 25 nucleotides.
Particularly preferred lengths of probes include 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. The
nucleotides as used in the present invention may be
ribonucleotides, deoxyribonucleotides and modified nucleotides such
as inosine or nucleotides containing modified groups which do not
essentially alter their hybridization characteristics.
[0070] Probe sequences are represented throughout the specification
as single stranded DNA oligonucleotides from the 5' to the 3' end.
It is obvious to the man skilled in the art that any of the
below-specified probes can be used as such, or in their
complementary form, or in their RNA form (wherein T is replaced by
U).
[0071] The probes according to the invention can be prepared by
cloning of recombinant plasmids containing inserts including the
corresponding nucleotide sequences, if need be by excision of the
latter from the cloned plasmids by use of the adequate nucleases
and recovering them, e.g. by fractionation according to molecular
weight. The probes according to the present invention can also be
synthesized chemically, for instance by the conventional
phospho-triester method.
[0072] The fact that amplification primers do not have to match
exactly with the corresponding target sequence in the template to
warrant proper amplification is amply documented in the literature
(Kwok et al., 1990). However, when the primers are not completely
complementary to their target sequence, it should be taken into
account that the amplified fragments will have the sequence of the
primers and not of the target sequence. Primers may be labelled
with a label of choice (e.g. biotine). The amplification method
used can be either polymerase chain reaction (PCR; Saiki et al.,
1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu &
Wallace, 1989; Barany, 1991), nucleic acid sequence-based
amplification (NASBA; Guatelli et al., 1990; Compton, 1991),
transcription-based amplification system (TAS; Kwoh et al., 1989),
strand displacement amplification (SDA; Duck, 1990; Walker et al.,
1992) or amplification by means of Q.beta. replicase (Lizardi et
al., 1988; Lomeli et al., 1989) or any other suitable method to
amplify nucleic acid molecules known in the art.
[0073] The oligonucleotides used as primers or probes may also
comprise nucleotide analogues such as phosphorothiates (Matsukura
et al., 1987), alkylphosphorothiates (Miller et al., 1979) or
peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993)
or may contain intercalating agents (Asseline et al., 1984). As
most other variations or modifications introduced into the original
DNA sequences of the invention these variations will necessitate
adaptions with respect to the conditions under which the
oligonucleotide should be used to obtain the required specificity
and sensitivity. However the eventual results of hybridization will
be essentially the same as those obtained with the unmodified
oligonucleotides. The introduction of these modifications may be
advantageous in order to positively influence characteristics such
as hybridization kinetics, reversibility of the hybrid-formation,
biological stability of the oligonucleotide molecules, etc.
[0074] The term "solid support" can refer to any substrate to which
an oligonucleotide probe can be coupled, provided that it retains
its hybridization characteristics and provided that the background
level of hybridization remains low. Usually the solid substrate
will be a microtiter plate (e.g. in the DEIA technique), a membrane
(e.g. nylon or nitrocellulose) or a microsphere (bead) or a chip.
Prior to application to the membrane or fixation it may be
convenient to modify the nucleic acid probe in order to facilitate
fixation or improve the hybridization efficiency. Such
modifications may encompass homopolymer tailing, coupling with
different reactive groups such as aliphatic groups, NH.sub.2
groups, SH groups, carboxylic groups, or coupling with biotin,
haptens or proteins.
[0075] The term "labelled" refers to the use of labelled nucleic
acids. Labelling may be carried out by the use of labelled
nucleotides incorporated during the polymerase step of the
amplification such as illustrated by Saiki et al. (1988) or Bej et
al. (1990) or labelled primers, or by any other method known to the
person skilled in the art. The nature of the label may be isotopic
(.sup.32P, .sup.35S, etc.) or non-isotopic (biotin, digoxigenin,
etc.).
[0076] The "sample" may be any biological material taken either
directly from the infected human being (or animal), or after
culturing (enrichment). Biological material may be e.g. scrapes or
biopsies from the urogenital tract or any part of the human or
animal body.
[0077] The sets of probes of the present invention will include at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 93, 24, 25, 26, 27, 28, 29, 30 or more probes.
Said probes may be applied in two or more (possibly as many as
there are probes) distinct and known positions on a solid
substrate. Often it is preferable to apply two or more probes
together in one and the same position of said solid support.
[0078] For designing probes with desired characteristics, the
following useful guidelines known to the person skilled in the art
can be applied.
[0079] Because the extent and specificity of hybridization
reactions such as those described herein are affected by a number
of factors, manipulation of one or more of those factors will
determine the exact sensitivity and specificity of a particular
probe, whether perfectly complementary to its target or not. The
importance and effect of various assay conditions are explained
further herein.
[0080] **The stability of the [probe:target] nucleic acid hybrid
should be chosen to be compatible with the assay conditions. This
may be accomplished by avoiding long AT-rich sequences, by
terminating the hybrids with G:C base pairs, and by designing the
probe with an appropriate Tm. The beginning and end points of the
probe should be chosen so that the length and % GC result in a Tm
about 2-10.degree. C. higher than the temperature at which the
final assay will be performed. The base composition of the probe is
significant because G-C base pairs exhibit greater thermal
stability as compared to A-T base pairs due to additional hydrogen
bonding. Thus, hybridization involving complementary nucleic acids
of higher G-C content will be more stable at higher
temperatures.
[0081] **Conditions such as ionic strength and incubation
temperature under which a probe will be used should also be taken
into account when designing a probe. It is known that the degree of
hybridization will increase as the ionic strength of the reaction
mixture increases, and that the thermal stability of the hybrids
will increase with increasing ionic strength. On the other hand,
chemical reagents, such as formamide, urea, DMSO and alcohols,
which disrupt hydrogen bonds, will increase the stringency of
hybridization. Destabilization of the hydrogen bonds by such
reagents can greatly reduce the Tm. In general, optimal
hybridization for synthetic oligonucleotide probes of about 10-50
bases in length occurs approximately 5.degree. C. below the melting
temperature for a given duplex. Incubation at is temperatures below
the optimum may allow mismatched base sequences to hybridize and
can therefore result in reduced specificity.
[0082] **It is desirable to have probes which hybridize only under
conditions of high stringency. Under high stringency conditions
only highly complementary nucleic acid hybrids will form; hybrids
without a sufficient degree of complementarity will not form.
Accordingly, the stringency of the assay conditions determines the
amount of complementarity needed between two nucleic acid strands
forming a hybrid. The degree of stringency is chosen such as to
maximize the difference in stability between the hybrid formed with
the target and the nontarget nucleic acid. In the present case,
single base pair changes need to be detected, which requires
conditions of very high stringency.
[0083] **The length of the probe sequence can also be important. In
some cases, there may be several sequences from a particular
region, varying in location and length, which will yield probes
with the desired hybridization characteristics. In other cases, one
sequence may be significantly better than another which differs
merely by a single base. While it is possible for nucleic acids
that are not perfectly complementary to hybridize, the longest
stretch of perfectly complementary base sequence will normally
primarily determine hybrid stability. While oligonucleotide probes
of different lengths and base composition may be used, preferred
oligonucleotide probes of this invention are between about 5 to 50
(more particularly 10-25) bases in length and have a sufficient
stretch in the sequence which is perfectly complementary to the
target nucleic acid sequence.
[0084] **Regions in the target DNA or RNA which are known to form
strong internal structures inhibitory to hybridization are less
preferred. Likewise, probes with extensive self-complementarity
should be avoided. As explained above, hybridization is the
association of two single strands of complementary nucleic acids to
form a hydrogen bonded double strand. It is implicit that if one of
the two strands is wholly or partially involved in a hybrid that it
will be less able to participate in formation of a new hybrid.
There can be intramolecular and intermolecular hybrids formed
within the molecules of one type of probe if there is sufficient
self complementarity. Such structures can be avoided through
careful probe design. By designing a probe so that a substantial
portion of the sequence of interest is single stranded, the rate
and extent of hybridization may be greatly increased. Computer
programs are available to search for this type of interaction.
However, in certain instances, it may not be possible to avoid this
type of interaction.
[0085] **Standard hybridization and wash conditions are disclosed
in the Materials & Methods section of the Examples. Other
conditions are for instance 3.times.SSC (Sodium Saline Citrate),
20% deionized FA (Formamide) at 50.degree. C. Other solutions (SSPE
(Sodium saline phosphate EDTA), TMAC (Tetramethyl ammonium
Chloride), etc.) and temperatures can also be used provided that
the specificity and sensitivity of the probes is maintained. When
needed, slight modifications of the probes in length or in sequence
have to be carried out to maintain the specificity and sensitivity
required under the given circumstances.
[0086] In order to identify different HPV types with the selected
set of oligonucleotide probes, any hybridization method known in
the art can be used (conventional dot-blot, Southern blot,
sandwich, etc.). However, in order to obtain fast and easy results
if a multitude of probes are involved, a reverse hybridization
format may be most convenient. In a preferred embodiment the
selected probes are immobilized to a solid support in known
distinct locations (dots, lines or other figures). In another
preferred embodiment the selected set of probes are immobilized to
a membrane strip in a line fashion. Said probes may be immobilized
individually or as mixtures to delineated locations on the solid
support. A specific and very user-friendly embodiment of the
above-mentioned preferential method is the LiPA method, where the
above-mentioned set of probes is immobilized in parallel lines on a
membrane, as further described in Example 4. The HPV polynucleic
acids can be labelled with biotine, and the hybrid can then, via a
biotine-streptavidine coupling, be detected with a non-radioactive
colour developing system.
[0087] The term "hybridization buffer" means a buffer allowing a
hybridization reaction between the probes and the polynucleic acids
present in the sample, or the amplified products, under the
appropriate stringency conditions.
[0088] The term "wash solution" means a solution enabling washing
of the hybrids formed under the appropriate stringency
conditions.
[0089] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
stated integers or steps but not to the exclusion of any other
integer or step or group of integers or steps.
FIGURE AND TABLE LEGENDS
[0090] FIG. 1. Alignment of HPV sequences
[0091] Alignment of sequences of genital HPV types and previously
unknown sequences within the region from position 6553 to position
6646 (numbered according to HPV 16, Genbank locus name PPH16,
accession number K02718), denoted region D. Hyphens indicate the
presence of identical nucleotides as in HPV 16. The primer target
regions A, B and C are boxed. The sequences identified as 95M or
97M followed by a number are novel sequences disclosed by the
present invention. The SEQ ID NO of the novel squences is shown
between brackets.
[0092] FIG. 2. Outline of the HPV DNA genome
[0093] Schematic outline of the HPV genome. The Early (E) and Late
(L) antigens are boxed. The length of the amplimers that can be
synthesized by the different general primer sets in the L1 region
is shown by a horizontal bar (bp stands for base pairs).
[0094] FIG. 3. Phylogenetic tree of HPV sequences in the MY11/MY09
region
[0095] Phylogenetic analyses were performed with the Phylip 3.5c
software (Felsenstein, 1995). The numbers correspond to the
different HPV types; the HPV groups are also indicated.
[0096] FIG. 4. Phylogenetic tree of HPV sequences between regions B
and C.
[0097] Phylogenetic analyses of the region between B and C
(corresponding to position 6602 to 6623 of HPV 16) were performed
with the Phylip 3.5c software (Felsenstein, 1995). The numbers
correspond to the HPV types.
[0098] FIG. 5. Phylogenetic tree of HPV sequences between regions A
and C.
[0099] Phylogenetic analyses of the region between A and C
(corresponding to position 6573 to 6623 of HPV 16) were performed
with the Phylip 3.5c software (Felsenstein, 1995). The numbers
correspond to the HPV types.
[0100] FIG. 6. Outline of a HPV LiPA
[0101] The bottom panel shows a possible configuration of a LiPA
strip enabling detection and identification of HPV types 16, 18,
31, 33, 45, 6 and 11 (ook 52, 56, 58, 40?). The lines correspond to
the positions of type-specific probes. "Control" indicates the
position of biotinlyated DNA that is used as a control for the
conjugate and substrate reaction. "General HPV" indicates the
position of probes that enable detection of almost all HPV types.
For the amplification step, primers SGP1 and SGP2 can be used; the
position of these primers is indicated in the top panel.
[0102] FIG. 7. LiPA experiment
[0103] Plasmids containing complete genomic sequences from the HPV
types 6, 11, 16, 18, 31, 33 and 45 were subjected to PCR with
primer set SGP1-bio/SGP2-bio. Subsequently, the amplimers were
analysed in a LiPA assay containing type-specific probes for
recognition of the HPV types 6, 11, 16, 18, 31, 33 and 45. The
strips A and B contained 5 probes for each of these types, as
indicated. Of each probe, two amounts (0.2 and 1 pmol) were present
on the strip. The probes for recognition of types 6, 11, 16 and 18
were applied to strip A and those for types 31, 33 and 45 were
applied to strip B.
[0104] FIG. 8. LiPA experiment
[0105] Amplimers synthesized by use of primer set SGP1-bio/MY09-bio
from HPV types 6, 16, 31, 33 and 45 were analysed by means of a
LiPA experiment. The strip contained 5 probes for each of the
types; of each probe two amounts were present. Strip A contains the
probes for recognition of types 6, 11, 16 and 18, whereas strip B
contains the probes for types 31, 33 and 45.
[0106] FIG. 9. Nucleotide sequence alignments of 39 HPV
genotypes
[0107] Alignment of HPV sequences within the region from position
6582 to position 6646 (numbered according to HPV 16, GenBank locus
name PPH16, accession number K02718). Hyphens indicate the presence
of identical nucleotides as in HPV 16. The primer target regions B
and C are boxed.
[0108] FIG. 10. Outline HPV-LiPA for identification of 25 types
[0109] The LiPA strip shows a possible configuration enabling
detection and identification of HPV types 6, 11, 16, 18, 31, 33,
34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68,
70 and 74. The lines correspond to the positions of type-specific
probes. "Control" indicates the position of biotinylated DNA that
is used as a control for the conjugate and substrate reaction.
[0110] FIG. 11. Typical HPV-LiPA patterns
[0111] Plasmids containing genomic sequences of HPV genotypes 6,
11, 16, 18, 33, 35, 45, 51, 52, 53, 56, 59, 66, 68, and 70 were
subjected to PCR using primers directed to the B and C region in
FIG. 9. Subsequently, the amplimers were analysed in a LiPA
experiment containing type-specific probes for identification of 25
HPV genotypes. The colored bands indicate hybridization of the
amplimer to the type-specific probe.
[0112] Table 1. HPV L1 primers for the A, B and C regions
[0113] Selection of preferred primers specifically hybridizing to
the A, B or C regions. HPV16, MY16s and SGP16 as represent the
corresponding sequence of HPV type 16. MY11 was described by Manos
et al. (1989). A + sign indicates that the primer is a sense
(forward) primer; a - sign refers to an antisense (reverse)
primer.
[0114] Table 2. HPV DNA detection by the novel general primers
SGP1/SGP2.
[0115] Plasmids containing HPV polynucleic acids were subjected to
PCR with primer sets SGP1/SGP2 and SGP3/GP6. + indicates that an
amplimer was obtained. - indicates that no amplimer was obtained.
n.d. indicates that this HPV plasmid was not subjected to PCR with
the SGP3/GP6 primer set. An amplimer was obtained for all HPV
plasmids with the SGP1/SGP2 primer set, although the amount of
PCR-product was different. Sequence analysis revealed that the
PCR-product was obtained from the corresponding HPV plasmid and
matched the published sequence. Primer set SGP3/GP6 was used to
confirm proper isolation of the HPV plasmids.
[0116] Table 3. HPV DNA detection by type-specific primers and
general primer sets
[0117] To evaluate the efficacy of the novel primers SGP1 and SGP2
in biological samples, 92 formalin-fixed, paraffin-embedded
cervical cancer biopsies were tested. A total of 61 out of the 92
biopsies were positive by type-specific PCR. Of these 61 biopsies,
54 contained HPV type 16 and 7 contained HPV type 18. The remaining
31 biopsies were assayed by HPV 31 and HPV 33 type-specific primers
and remained negative. These 31 samples, negative by type-specific
PCR, were also analyzed by two general primer sets described
previously. By using the MY11/MY09 and GP5/GP6 primer sets only
1/31 and 3/31 biopsies were found positive, respectively. All 92
biopsies were found positive by the newly developed SGP1/SGP2
primerset.
[0118] Table 4. HPV L1 primers for the A, B and C regions
[0119] Selection of preferred primers specifically hybridizing to
the A, B or C regions.
[0120] Table 5. PCR amplification with primers in the B and in the
C region
[0121] The specificity of the primers listed in table 4 for the
regions B and C was tested on plasmids containig polynucleic acids
of HPV types 6, 11, 16, 18, 31, 33, 45, 3D, 39, 58, 57 and 59. PCR
was performed by all 20 possible primer combinations for the
regions B and C. The results are indicated as follows: .+-.=poor
amplification; +=good; ++=very good; blank=no amplification.
[0122] Table 6. HPV genotyping of 77 isolates by type-specific PCR
and sequence analysis of the SGP1/SGP2 amplimer
[0123] 77 HPV isolates positive with specific primers for type 16
or 18, were studied for sequence variability in the SGP1/SGP2
amplimer. Samples identified as type 16 by type-specific PCR were
all identically typed by sequence analysis of the SGP1/SGP2
amplimer. There was no intratypic sequence variation in the small
SGP1/SGP2 amplimer. Identical results were obtained for HPV 18.
[0124] Table 7. Type-specific HPV probes
[0125] Selection of preferred probes specifically hybridizing to
the 22 bp region between regions B and C. "+" indicates that the
probe is a sense probe; "-" refers to an antisense probe. The
underlined G or C residues represent non-specific nucleotides that
were added to facilitate tailing of the probes.
[0126] Table 8. HPV primers for the synthesis of biotinviated PCR
products
[0127] SGP1-bio and SGP2-bio are the biotinylated versions of SGP1
and SGP2, shown in table 1. MY09-bio is the biotinylated version of
MY09, the sequence of which was disclosed in Manos et
al.(1989).
[0128] Table 9. Probes for general HPV detection
[0129] Selection of preferred probes that enable detection of more
than one HPV type. The types detected by each probe are listed next
to the probe.
[0130] Table 10. Probes for general HPV detection
[0131] Selection of preferred probes that enable detection of more
than one HPV type.
[0132] Table 11. PCR primers
[0133] Selection of preferred primers specifically hybridizing to
the B or C regions.
[0134] Table 12. HPV type-specific probes
[0135] Selection of preffered probes specifically hybridizing to
the region between position 6582-6646 (numbers according to HPV 16,
GenBank locus name PPH16, accession number K02718). "+" indicates
that the probe is a sense probe; "-" refers to an antisense probe.
The underlined residues represent non HPV type-specific
nucleotides.
[0136] Table
1TABLE 1 HPV L1 primers in the A, B and C regions SEQ ID NO/ Name
polarity 5'-sequence-3' reference A region HPV16 +
TATTCAATAAACCTTATTGC 1 SGP3 + -D--T-----R--W------ 2 SGP3A +
----T--------A------ 3 SGP3B + ----T-----G--A------ 4 B region
MY16s + GCACAGGGCCACAATAATGG 5 MY11 + --M-----W--T--Y----- Manos et
al., 1989 SGP1 + --M-----H--T--Y----- 6 C region SGP16as -
GTATCAACAACAGTAACAAA 7 SGP2A - -----T--C----------- 8 SGP2 -
-----H--H----------- 9 D = G, A or T; R = A or G; W = A or T; M = A
or C; Y = C or T; H = A, C or T.
[0137]
2TABLE 2 HPV DNA detection by the novel general primers SGP1/SGP2
HPV plasmid SGP1/SGP2 SGP3/GP6.sup.a reference.sup.b 3 + + Ostrow 4
+ - de Villiers 5 + + Ostrow 5/48 + - de Villiers 6 + n.d. de
Villiers 7/4 + + de Villiers 7/5 + + de Villiers 8 + - de Villiers
11 + + de Villiers 13 + + de Villiers 16 + + de Villiers 18 + + de
Villiers 26 + + Ostrow 27 + + Ostrow 30 + + Orth 31 + + Lorinz 33 +
n.d. Orth 35s + + Lorincz 35l + + Lorincz 37 + + de Villiers 39 +
n.d. Orth 43s + + Lorincz 43l + + Lorincz 45 + + de Villiers 51 + +
de Villiers 52 + n.d. Orth 53 + + de Villiers 56 + + Lorincz 57 + +
de Villiers 58 + + Matsukura 59 + + Matsukura 62.2 + + Matsukura 64
+ + Matsukura 65 + + de Villiers 67 + + Matsukura .sup.aGeneral
primer GP6 (van den Brule et al., 1990) .sup.bThe HPV plasmids were
kindly provided by the Dr's: Lorincz, de Villiers, Matsukura,
Ostrow, ter Schegget and Orth.
[0138]
3TABLE 3 HPV DNA detection by type-specific primers and general
primer sets HPV primer set number HPV pos. HPV neg. amplimer 16 92
54 38 96 bp 18 92 7 85 115 bp 31 31.sup.a 0 31 110 bp 33 31.sup.a 0
31 114 bp MY11/MY09 31.sup.b 1 30 .+-.450 bp GP5/GP6 31.sup.b 3 28
.+-.142 bp SGP1/SGP2 92 92 0 62 bp .sup.aSamples negative by
type-specific primers for HPV 16 and 18 .sup.bSamples negative by
type-specific primers for HPV 16, 18, 31 and 33
[0139]
4TABLE 4 HPV L1 primers for the A, B and C regions Name
5'-sequence-3' SEQ ID NO Forward primers region A SGP3A
TATTTAATAAACCATATTGG 3 SGP3B TATTTAATAAGCCATATTGG 4 SGP3C
TATTTAATAAGCCTTATTGG 10 SGP3D TATTCAATAAACCTTATTGG 11 SGP3E
TATTTAATAAACCTTACTGG 12 SGP3F TATTTAATAAICCITATTGG 13 SGP3G
TATTTAATAAICCITACTGG 14 Forward primers region B SGP1A
GCICAGGGTCACAATAATGG 15 SGP1B GCICAGGGICATAACAATGG 16 SGP1C
GCICAGGGICATAATAATGG 17 SGP1D GCICAAGGICATAATAATGG 18 Reverse
primers region C SGP2B-bio bio-GTIGTATCIACAACAGTAACAAA 19 SOP2C-bio
bio-GTIGTATCTACCACAGTAACAAA 20 SGP2D-bio
bio-GTIGTATCIACTACAGTAACAAA 21 SCP2-bio bio-GTIGTATCIACGACAGTIACAAA
22 SGP2F-bio bio-GTIGTATCIACAACAGTIAIAAA 23 I stands for inosine
"bio-" indicates that the primer is biotinylated
[0140]
5TABLE 5 PCR amplification with primers in the B and in the C
region SGP1C SGP1C SGP1C SGP1C SGP1D SGP1D SGP1D SGP1D SGP1D
SGP1tot MY11Q SGP1AB Primerset SGP2C- SGP2D- SGP2E- SGP2F- SGP2B-
SGP2C- SGP2D- SGP2E- SGP2F- SGP2tot- SGP2- SGP2BD- HPV blo blo blo
blo blo blo blo blo blo blo blo blo 6 ++ ++ .+-. + ++ .+-. ++ ++ ++
11 ++ ++ ++ + ++ .+-. ++ + ++ .+-. ++ 16 ++ ++ .+-. .+-. + ++ ++ ++
18 ++ ++ ++ + ++ + ++ ++ ++ ++ ++ 31 ++ ++ ++ + ++ + ++ + ++ ++ ++
33 ++ ++ ++ + ++ ++ ++ ++ + ++ ++ ++ 45 .+-. + .+-. + .+-. ++ ++ ++
35 .+-. ++ ++ ++ ++ ++ ++ + ++ .+-. ++ 39 ++ + ++ ++ ++ ++ .+-. ++
58 .+-. + + .+-. ++ ++ ++ + ++ ++ ++ 57 + ++ + + + ++ ++ + ++ 59 +
++ .+-. + + .+-. ++ ++ ++ SGP1A SGP1A SGP1A SGP1A SGP1A SGP1B SGP1B
SGP1B SGP1B SGP1B SGP1B Primerset SGP2B- SGP2C- SGP2D- SGP2E-
SGP2F- SGP2B- SGP2C- SGP2D- SGP2E- SGP2F- SGP2B- HPV blo blo blo
blo blo blo blo blo blo blo blo 6 + + + ++ ++ ++ ++ + ++ 11 ++ ++ +
++ ++ ++ ++ + ++ 16 ++ ++ ++ + ++ + + ++ 18 ++ + ++ ++ .+-. ++ ++
++ ++ + ++ 31 ++ ++ ++ ++ + ++ + ++ ++ ++ 33 ++ + ++ ++ ++ + ++ ++
++ 45 ++ ++ + 35 + ++ .+-. .+-. ++ + ++ 39 + + .+-. + + + 58 .+-.
++ + ++ + + 57 + ++ + ++ + 59 .+-. + .+-. + + ++ ++ .+-. +
[0141]
6TABLE 6 HPV genotyping of 77 isolates by type-specific PCR and
sequence analysis of the SGP1/SGP2 amplimer. HPV-type type-specific
PCR SGP1/SGP2 16 70 70 18 7 7
[0142]
7TABLE 7 Type-specific HPV probes po- lar- SEQ Name 5'-sequence-3'
ity ID NO HPV6 Pr1 TTGGGGTAATCAACTGTGG + 24 HPV6 Pr2
GTTGGGGTAATCAACTGTGG + 25 HPV6 Pr3 TTGGGGTAATCAACTGTTG + 26 HPV6
Pr4 GTTGGGGTAATCAACTGTTG + 27 HPV6 Pr5 TTGGGGTAATCAACTGTTT + 28
HPV11 Pr1 TGCTGGGGAAACCACTG + 29 HPV11 Pr2 TGCTGGGGAAACCACTTAGG +
30 HPV11 Pr3 TTGTTGGGGAAACCACTG + 31 HPV11 Pr4
TTGCTGGGGAAACCACTTAGG + 32 HPV11 Pr5 TGCTGGGGAAACCACTTGGG + 33
HPV16 Pr1 TTGGGGTAACCAACTATGG + 34 HPV16 Pr2 GTTGGGGTAACCAACTATGG +
35 HPV16 Pr3 TTGGGGTAACCAACTATTG + 36 HPV16 Pr4
GTTGGGGTAACCAACTATTG + 37 HPV16 Pr5 TTGGGGTAACCAACTATTT + 38 HPV18
Pr1 GTGTTTGCTGOCATAAT + 39 HPV18 Pr2 GGTGTTTGCTGGCATAAG + 40 HPV18
Pr3 GTGTTTGCTGGCATAATC + 41 HPV18 Pr4 TGGTGTTTGCTGGCATAAG + 42
HPV18 Pr5 GGTGTTTGCTGGCATAAT + 43 HPV31 Pr1 TTGGGGCAATCAGTTATGG +
44 HPV31 Pr2 GTTGGGGCAATCAGTTATGG + 45 HPV31 Pr3
TTGGGGCAATCAGTTATTG + 46 HPV31 Pr4 GTTGGGGCAATCAGTTATTG + 47 HPV31
Pr5 GTTGGGGCAATCAGTTATTT + 48 HPV31 Pr21 GGGCAATCAGTTATTG + 49
HPV31 Pr22 AATAACTGATTGCCC - 50 HPV31 Pr23 GGCAATCAGTTATTTCC + 51
HPV31 Pr24 AAATAACTGATTGCC - 52 HPV31 Pr25 GCAATCAGTTATTTGG + 53
HPV31 Pr26 CAAATAACTGATTGC - 54 HPV31 Pr31 GGCAATCAGTTATTTGG + 55
HPV31 Pr32 GCAATCAGTTATTTGTG + 56 HPV33 Pr1 TTGGGGCAATCAGGTATGG +
57 HPV33 Pr2 GTTGGGGCAATCAGGTATGG + 58 HPV33 Pr3
TTGGGGCAATCAGGTATTG + 59 HPV33 Pr4 GTTGGGGCAATCAGGTATTG + 60 HPV33
Pr5 GTTGGGGCAATCAGGTATTT + 61 HPV33 Pr21 GGGCAATCAGGTATTG + 62
HPV33 Pr22 AATACCTGATTGCCC - 63 HPV33 Pr23 GGCAATCAGGTATTTCC + 64
HPV33 Pr24 AAATACCTGATTGCC - 65 HPV33 Pr25 GCAATCAGGTATTTGG + 66
HPV33 Pr26 CAAATACCTGATTGC - 67 HPV40 Pr1 CATATGTTTTGGCAATC + 68
HPV45 Pr1 = SGPP68 GTATTTGTTGGCATAAT + 69 H2V45 Pr2
GGTATTTGTTGGCATAAG + 70 HPV45 Pr3 GTATTTGTTGGCATAATC + 71 HPV45 Pr4
TGGTATTTGTTGGCATAAG + 72 HPV45 Pr5 GGTATTTGTTGGCATAAT + 73 HPV45
Pr11 TGGCATAATCAGTTGGG + 74 HPV45 Pr12 GGCATAATCAGTTGTG + 75 HPV45
Pr13 GCATAATCAGTTGTTT + 76 HPV52 Pr1 GCAATCAGTTGTTTGC + 77 HPV52
Pr2 CAATCAGTTGTTTGTC + 78 HPV52 Pr3 ATGGCATATGTTGGG + 79 HPV52 Pr4
TGGCATATGTTGGGGG + 80 HPV52 Pr5 GGCATATGTTGGGGC + 81 HPVS2 Pr6
GCATATGTTGGGGCA + 82 HPV56 Pr1 GGGGTAATCAATTATC + 83 HPVS6 Pr2
GGGGTAATCAATTATTC + 84 HPV5G Pr3 GGGGTAATCAATTATTT + 85 HPV56 Pr11
TGGGGTAATCAATTATTT + 86 HPV56 Pr12 GGGGTAATCAATTATTTGG + 87 HPV58
Pr1 CATTTGCTGGGGCAAG + 88 HPV58 Pr2 ATTTGCTGGGGCAAT + 89 HPV58 Pr3
TTTGCTGGGGCAATC + 90 HPV58 Pr4 TTGCTGGGGCAATCA + 91 SGPP35
GTTGGAGTAACCAATTG + 92 SGPP39 GTATATGTTGGCATAAT + 93 SGPP51 = HPV51
Pr1 GCATTTGCTGGAACAAT + 94 SGPP54 GGGGCAATCAGGTGTTT + 95 SGPP59
GGTATATGTTGGCACAA + 96 SGPP66 GCATATGCTGGGGTA + 97 SGPP68 = HPV45
Pr1 GTATTTGTTGGCATAAT + 69 SGPP70 = HPV70 Pr11 CATTTGTTGGCATAACC +
99 SGPP13 TGGGGCAATCACTTG + 100 SGPP34 GCATTTGCTGGCATA + 101 SGPP42
TGGGGAAATCAGCTATT + 102 SGPP43 GGCATTTGTTTTGGGAA + 103 SGPP44
TTGGGGAAATCAGTTATT + 104 SGPP53 GCATCTGTTGGAACAA + 105 SGPP55
GTTGGGGGAATCAGT + 106 SGPP69 GTTGGGGCAACCAATTG + 107 SGPP61
TGGTTTAATGAATTGTTT + 108 SGPP62 GGTTTAATGAACTGTTT + 109 SGPP64
AATGGAATTTGTTGGCA + 110 SGPP67 GTATATGCTGGGGTAAT + 111 SGPP74 =
HPV74 Pr13 ATTTGTTGGGGTAATCA + 112 MM4 = HPVM4 Pr11
TGCTGGAATAATCAGCT + 113 MM7 TGGTTTAATGAGTTATTT + 114 MM8
ATATGCTGGTTTAATCA + 115
[0143]
8TABLE 8 HPV primers for synthesis of biotinylated PCR products.
SEQ ID NO/ Name polarity 5'-sequence-3+ reference SGP1-bio +
bio-GCMCAGGGHCATAAYAATGG 6 SGP2-bio - bio-GTATCHACHACAGTAACAAA 9
MY09-bio - bio-CGTCCMARRGGAWACTGATC Manos et al., 1989 M = A or C;
H = A, C or T; Y = C or T; R = A or G; W = A or T
[0144]
9TABLE 9 Probes for general HPV detection HPV types Name
5'-sequence-3'.sup.1 position.sup.2 recognized SEQ ID NO HPVuni1
AATAATGGCATITGTTGG 6594-6611 16, 30, 52,53, 116 70, MM7, 72, 43
HPVuni2 AATAATGGTATITGTTGG 6594-6611 31, 33, 26, 35, 117 13, 42,
44, 55, 62, 73 HPVuni3 AACAATGGTATITGTTGG 6594-6611 45, 6, 59, 68,
118 54, 61, 39 HPVuni4 AACAATGGTATITGCTGG 6594-6611 11, 67, MM8 119
HPVuni5 AACAATGGTGTTTGCTGG 6594-6611 18 120 HPVuni6
AATAATGGCATTTGCTGG 6594-6611 51, 56, 66, MM4 121 HPVuni7
AACAATGGCATITGCTGG 6594-6611 34, 57, 58 122 HPVuni1A
CAIAATAATGGCATITGTTGGC 6591-6612 16, 30, 52, 53, 220 70, MM7, 72,
43 HPVuni1B CAIAACAATGGCATTTGTTGGC 6591-6612 16, 30, 40, 52, 5 221
3, 69, 70, MM7 72, 43 HPVuni1c CACAATAATGGCATTTGTTGGGG 6591-6613
16, 30, 52, 53, 222 70, MM7, 72, 43 HPVuni2A CAIAATAATGGTATITGTTGGG
6591-6612 31, 33, 26, 35, 223 13, 42, 44, 55, 62, 73 HPVuni3A
CAIAACAATGGTATITGTTGGC 6591-6612 45, 6, 59, 68, 224 54, 61, 39
.sup.1I = inosine .sup.2Sequence positions according to HPV
genotype 16 sequence PPH16, Genbank accession number K02718 HPV
type 64 is theoretically not recognised.
[0145]
10TABLE 10 Probes for general HPV detection po- lar- SEQ Name
5'-sequence-3' ity ID NO HPVuni2L2 CAIAATAATGGTATITGTTGG + 123
HPVuni2L3 AIAATAATGGTATITGTTGG + 124 HPVuni2L4
CAIAATAATGGTATTTGTTGG + 125 HPVuni2L5 AIAATAATGGTATTTGTTGG + 126
HPVuni2LG CACAATAATGGTATTTGTTGG + 127 HPVuni2L7
ACAATAATGGTATTTGTTGG + 128 HPVuni4L1 CAIAACAATGGTATITGTTGG + 129
HPVuni4L2 AIAACAATGGTATITCTTGG + 130 HPVuni4L3
CAIAACAATGCTATTTGTTGG + 131 HPVuni4L4 AIAACAATGGTATTTGTTGG + 132
HPVuni4L5 CATAACAATGGTATTTGTTGG + 133 HPVuni4LG
ATAACAATGGTATTTGTTGG + 134 HPV G1 AATGGCATTTGTTGGGGTAACCA- ACTATTT
+ 225 HPV G1A1 TTGTTGGGGTAACCAACTATG + 226 HPV G1A2
ATTTGTTGGGGTAACCAACTATTG + 227 HPV G1A3 GCATTTGTTGGGGTAACCAACTA +
228 HPV G1A4 TGGCATTTGTTGGGGTAACCAACTA + 229 HPV G2
AATGGTATTTGTTGGGGCAATCAGTTATTT + 230 HPV G3
AATGGTATTTGTTGGCATAATCAGTTGTTT + 231 HPV G4
AATGGTATTTGTTGGTTTAATGAATTGTTT + 232 HPV G5
AATGGCATTTGCTGGAACAATCAGCTTTTT + 233 HPV G6
AATGGTATATGTTGGGGCAATCACTTGTTT + 234 HPV R1 AATGGCATTTGTTGGGGC +
235 HPV R10 AATGGCATATGCTGGAATAATC + 236 HPV R11
AATGGTATATGTTGGGGCAATC + 237 HPV R2 AATGGTATTTGTTGGGGC + 238 HPV R3
AATGGAATTTCTTGGCATAATC + 239 HPV R4 GGTATCTGCTGGCATAAT + 240 HPV R5
AATGGCATTTGTTGGTTTAATC + 241 HPV R6 AATGGTATTTGTTGGTTTAATG + 242
HPV R7 AATGGCATCTGTTGGTTTAATG + 243 HPV R8 TGTTGGTTTAATGAGCTCTG +
244 HPV R9 TGCTGGTTTAATCAATTGTTG + 245 Underlined sequences are not
complementary to HPV.
[0146]
11TABLE 11 PCR primers SEQ Primer ID designation
5'-sequence-3'.sup.1 position.sup.2 NO SGP1A GCICAGGGICACAATAATGG
6582-6601 15 SGP1B GCICAGGGICATAACAATGG 6582-6601 16 SGP1C
GCICAGGGICATAATAATGG 6582-6601 17 SGP1D CCICAAGGICATAATAATGG
6582-6601 18 SGP2B-bio GTIGTATCIACAACAGTAACAAA 6624-6646 19
SGP2D-bio GTIGTATCIACTACAGTAACAAA 6624-6646 21 SGP2H-bio
GTIGTATCIACAACTGTAACAAA 6624-6646 98 SGP2I-bio
GTIGTATCCACAACAGTTACAAA 6624-6646 154 SGP2J-bio
GTGGTATCCACAACIGTGACAAA 6624-6646 155 SGP2K-bio
GTAGTTTCCACAACAGTAAGAAA 6624-6646 156 SGP2L-bio
GTAGTATCAACCACACTTAAAAA 6624-6646 157 SGP2M-bio
CTIGTATCTACAACIGTTAAAAA 6624-6646 158 SGP2N-bio
GTAGTATCTACACAAGTAACAAA 6624-6646 159 SGP2P-bio
GTAGTATCAACACAGGTAATAAA 6624-6646 160 .sup.1I = inosine
.sup.2Sequence positions according to HPV genotype 16 sequence
PPH16, Genbank accession number K02718.
[0147]
12TABLE 12 HFV type-specific probes +HC,28SEQ HPV PROBE
5'-sequence-3' polarity ID NO HPV18b Pr1 GGTATCTGCTGGCATAAG + 161
HPV18b Pr2 TGGTATCTGCTGGCATA + 162 HPV31 Vs40-1 TATTTGTTGGGGCAATC +
163 HPV31 Vs40-2 ATTTGTTGGGGCAATC + 164 HPV31 Vs40-3
TATTTGTTGGGGCAAT + 165 HPV34 Pr1 GGCATTTGCTGGCATA + 166 HPV35 Pr1
GTTGGAGTAACCAATTGGG + 167 HPV35 Pr2 TGTTGGAGTAACCAATTCC + 168 HPV35
Pr3 TTGTTGGAGTAACCAATG + 169 HPV39 Pr1 GGTATATGTTGGCATAAT + 170
HPV42 Pr1 GGGGAAATCAGCTATTG + 171 HPV42 Pr2 GGGAAATCAGCTATTT + 172
HPV43 Pr1 GGCATTTGTTTTGGGAAG + 173 HPV43 Pr2 GCATTTGTTTTGGGAAT +
174 HPV43 Pr3 CATTTGTTTTGGGAATC + 175 HPV44 Pr1 GGGGAAATCAGTTATTG +
176 HPV44 Pr2 GGGGAAATCACTTATTT + 177 HPV44 Pr3 GGGAAATCAGTTATTT +
178 HPV44 Pr4 TGGGGAAATCAGTTATG + 179 HPV45 Pr5 GGTATTTGTTGGCATAAT
+ 73 HPV51 Pr1= GCATTTGCTGGAACAAT + 94 SGPP51 HPV51 Pr2
CATTTGCTGGAACAATC + 180 HPV53 Pr1 GGCATCTGTTGGAACAA + 181 HPV54 Pr1
GGCAATCAGGTGTTTC + 182 HPV54 Pr11 CGGCAATCAGGTGTTTC + 183 HPV54
Pr11as AAACACCTGATTGCCC - 184 HPV54 Pr12 GGCAATCAGGTGTTTTG + 185
HPV55 Pr1 GGGGGAATCAGTTATTG + 186 HPV55 Pr11 GGGGGAATCAGTTATG + 187
HPV55 Pr12 TGGGGGAATCAGTTATG + 188 HPV55 Pr13 TGGGGGAATCAGTTAG +
189 HPV56 Vs74-1 CATTTGCTGGGGTAAT + 190 HPV59 Pr1
TGGTATATGTTGGCACAA + 191 HPV59 Pr11 GGTATATGTTGGCACAAT + 192 HPV59
Pr12 GTATATGTTGGCACAATC + 193 HPV59 Pr13 TATATGTTGGCACAATC + 194
HPV66 Pr1 GGCATATGCTGGGGTA + 195 HPV67 Pr1 GGTATATGCTGGGGTAAT + 196
HPV67 Pr11 GGTATATGCTGGGGTA + 197 HPV67 Pr12 TGGTATATGCTGGGGT + 198
HPV67 Pr13 ATGGTATATGCTGGGGG + 199 HPV67 Pr21 GGTATATGCTGGGGT + 200
HPV67 Pr22 TGGTATATGCTGGGGG + 201 HPV67 Pr23 AATGGTATATGCTGGG + 202
HPV68 Pr1 TGGTATTTGTTGGCATA + 203 HPV68 Pr2 ATGGTATTTGTTGGCATA +
204 HPV68 Pr3 ATGGTATTTGTTGGCAT + 205 HPV68 Vs45-1
TTGGCATAATCAATTATTT + 206 HPV68 Vs45-2 TTGGCATAATCAATTATTTCG + 207
HPV70 Pr1 GCATTTGTTGGCATAACC + 208 HPV70 Pr11 = CATTTGTTGGCATAACC +
99 SGPP70 HPV70 Pr12 GCATTTGTTGGCATAAC + 209 HPV70 Pr13
CATTTGTTGGCATAAC + 210 HPV74 Pr1 TATTTGTTGGGGTAAT + 211 HPV74 Pr11
ATTTGTTGGGGTAATC + 212 HPV74 Pr12 TTTGTTGGGGTAATCA + 213 HPV74 Pr13
= ATTTGTTGGGGTAATCA + 112 SGPP74 HPV74 Pr2 GTATTTGTTGGGGTAAT + 214
HPV74 Pr3 TATTTGTTGGGGTAATC + 215 HPVM4 Pr1 TTGCTGGAATAATCAGCT +
216 HPVM4 Pr11 = TGCTGGAATAATCAGCT + 113 MM4 HPVM4 Pr12
TGCTGGAATAATCAGC + 217 HPVM4 Pr21 TGCTGGAATAATCAGCTG + 218 HPVM4
Pr22 TGCTGGAATAATCAGCG + 219 Underlined sequences are not
complementary to HPV
EXAMPLES
[0148] The following examples only serve to illustrate the present
invention. These examples are in no way intended to limit the scope
of the present invention.
Example 1
Development of Novel General HPV PCR Primers
[0149] Introduction
[0150] The aim of the present example was to deduce PCR primers
that allow general PCR amplification of sequences from multiple HPV
types.
[0151] Materials and Methods
[0152] Design of Primers
[0153] HPV sequences were obtained from the GenBank database.
[0154] Alignment of all available L1 sequences revealed that there
are several regions that show a high degree of conservation among
the different HPV genotypes. These regions are indicated in FIG. 1
and are designated A, B and C, respectively.
[0155] In order to obtain universal amplification of all HPV
sequences, several primers were selected in these three regions.
The locations and sequences of the different primers are
represented in FIG. 2 and table 1, respectively. Primer
combinations from the A (SGP3) and C (SGP2) region and those from
the B (SGP1) and C (SGP2) region will yield an expected amplimer of
91 basepairs (bp) or 62 bp, respectively. Type-specific primers for
HPV types 16, 18, 31 and 33 were described in Baay et al. (1996).
The MY11-MY09 primer set was described in Manos et al. (1989). The
GP5/GP6 primer set was described in van den Brule et al.
(1990).
[0156] DNA Isolation
[0157] DNA was isolated from the 92 formalin fixed and
paraffin-embedded cervical cancer biopsies by a modified version of
the method described by Claas et al (1989). A 10 .mu.m section was
collected in a 1.5 ml tube and deparaffinized by 500 .mu.l Xylol.
After gently shaking for 2 minutes and centrifugation for 5 minutes
the pellet was again treated with 500 .mu.l Xylol. The pellet was
washed twice with 500 .mu.l alcohol 96% and once with 500 .mu.l
acetone. Subsequently, the pellet was air-dried and treated with a
200 .mu.l proteinase K solution (1 mg/ml) overnight at 37.degree.
C.
[0158] PCR
[0159] The PCR was performed essentially as described by Saiki
(1988). Briefly, the final volume of 100 .mu.l contained 10 .mu.l
of the isolated DNA, 10 mM Tris-HCl pH 9.0, 50 mM KCl, 2.5 mM
MgCl.sub.2, 0.1% Triton X-100, 0.01% gelatin, 200 .mu.M of each
deoxynucleoside triphosphate, 50 pmol of forward and reverse
primer, and 0.25U of SuperTaq (Sphaero Q, Cambridge, United
Kingdom). For the MY11/MY09 primerset (Manos et al., 1989) 0.5U
SuperTaq was used. PCR conditions were a preheating step for 1 min
94.degree. C. followed by 40 cycles of 1 min 94.degree. C., 1 min
52.degree. C. and 1 min 72.degree. C. For the primerset SGP3/SGP2
the 40 cycles of amplification consisted of 1 min 94.degree. C., 1
min 40.degree. C. and 1 min 72.degree. C. For the primerset GP5/GP6
(van den Brule et al., 1990) the 40 cycles of amplification
consisted of and 1 min 94.degree. C., 2 min 40.degree. C. and 1.5
min 72.degree. C. As a control for succesful DNA isolation a PCR
was performed using .beta.-globin primers described by Saiki
(1986).
[0160] Southern Blot Analysis
[0161] The Southern blot hybridization experiments were performed
according to standard procedures (Sambrook et al., 1989). Briefly,
20 .mu.l of the PCR-product was electrophoresed on a 2% agarose
gel. Amplimers produced by the primer sets SGP1/SGP2 and SGP3/SGP2
were applied on a 3% agarose gel. Subsequently, amplimers were
transferred to a nylon membrane (Hybond N+, Amersham, Little
Chalfont, United Kingdom) by vacuum blotting in the presence of
0.4N NaOH. The Southern blots were hybridized with a .sup.32P
5'-end labeled probe(s) for 16 hours at 42.degree. C. in a solution
containing 5.times.SSC (1.times.SSC: 15 mM Na-citrate and 150 mM
NaCl, pH 7.0), 5.times. Denhardt's (1.times. Denhardt: 0.02% bovine
serum albumin, 0.02% polyvinyl pyrolidone and 0.02% ficoll), 0.5%
SDS, 75 MM EDTA and 0.1 mg/ml herring sperm DNA. Subsequently, the
blots were washed twice in 2.times.SSC/0.1% SDS at 42.degree. C.
for 15 minutes. Autoradiography was performed for 3.5 hours using
the Kodak X-Omat AR film.
[0162] Samples that were negative by type-specific primers were
also analyzed by the L1 directed general primer sets MY11/MY09 and
GP5/GP6.
[0163] Sequence Analysis
[0164] PCR products were analyzed by direct sequencing, using a
cycle-sequencing kit (Perkin Elmer). Sequences were analyzed by the
PC-Gene software (Intelligenetics, USA)
[0165] Results
[0166] In order to develop a general set of PCR primers that would
allow universal amplification of HPV sequences, we aimed at the L1
region. Primers SGP1 and SGP2 were tested on a number of plasmids,
containing partial or complete genomic sequences from various HPV
types. The results are summarized in table 2. An amplimer was
obtained for all HPV plasmids by the SGP1/SGP2 primer set, although
the amount of PCR-product was different. Sequence analysis revealed
that the PCR-product was obtained from the corresponding HPV
plasmid and matched the published sequence. Primer set SGP3/GP6 was
used to confirm proper isolation of the HPV plasmids.
[0167] To evaluate the efficacy of the novel primers SGP1 and SGP2
in biological samples, 92 formalin-fixed, paraffin-embedded
cervical cancer biopsies were tested. DNA isolated from these
biopsies was subjected to different PCR assays: .beta.-globin
primers PCO3 and PCO4 (Saiki et al., 1988), SGP1/SGP2, and
type-specific PCR for HPV types 16, 18, 31, and 33. The results are
summarized in table 3.
[0168] 1. All biopsies contained amplifiable DNA as determined with
PCR directed to the .beta.-globin gene.
[0169] 2. A total of 61 (66%) of the 92 biopsies were positive by
type-specific PCR. Of these 61 biopsies, 54 contained HPV type 16
and 7 contained HPV type 18. Subsequently, the remaining 31
biopsies were assayed by HPV 31 and HPV 33 type-specific primers
and remained negative.
[0170] 3. The 31 samples, negative by type-specific PCR were also
analyzed by two general primer sets described previously (Manos et
al., 1989; van den Brule et al., 1990). By using the MY11/MY09 and
GP5/GP6 primersets only 1/31 and 3/31 biopsies were found positive,
respectively.
[0171] 4. All 92 biopsies were found positive by the newly
developed SGP1/SGP2 primerset.
[0172] Discussion
[0173] In general, amplification of a small genomic fragment is
likely to increase the sensitivity of the PCR. This is of
particular importance when using biological samples that contain a
very low copy number of HPV. Furthermore, cervical biopsies that
have been formalin-fixed and paraffin-embedded are a poor source of
amplifiable DNA.
[0174] In this high-risk group for HPV, the novel primer
combination SGP1/SGP2 was more sensitive than the type-specific PCR
and the general PCRs that were also directed to the L1 region of
HPV.
[0175] In conclusion the newly developed primer sets are highly
sensitive for detection of HPV DNA.
Example 2
Optimization of PCR Primers from the A, B and C region
[0176] Introduction
[0177] Example 1 describes the selection of semi-conserved regions
in the L1 gene of the HPV genome, that permitted the development of
a general PCR system. Degenerated primers were used for universal
amplification of HPV sequences from different genotypes.
[0178] The present example describes the optimization of the
primers aimed at these regions. Instead of degenerated primers,
this study aimed at the development of several distinct and defined
forward and reverse primers.
[0179] Materials and Methods
[0180] Alignments of L1 sequences were used to deduce PCR primers
from the three regions A, B and C (FIG. 1). Primers were tested by
PCR in different combinations on plasmids, containing partial or
complete genomic inserts from the genital HPV types 6, 11, 16, 18,
31, 33, 35, 39, 43, 45, 51, 52, 53, 56, 57, 58, 59, 62, 64 and 67
as listed in table 2.
[0181] HPV DNA amplification was performed according to the
following protocol. The final PCR volume of 100 .mu.l contained 10
.mu.l of HPV plasmid DNA, 75 mM Tris-HCl pH 9.0, mM
(NH.sub.4).sub.2SO.sub.4, 2.5 mM MgCL.sub.2, 0.1% (w/v) Tween 20,
200 .mu.M of each deoxynucleoside triphosphate, 100 pmol of forward
and reverse primer, and 3U of Taq-DNA polymerase (Pharmacia,
Uppsala, Sweden). After a preheating step for 1 min 94.degree. C.
amplification was performed by 1 min 94.degree. C., 1 min
52.degree. C. and 1 min 72.degree. C. for 40 cycles. Subsequently,
PCR-products were analyzed on a 3% agarose gel.
[0182] Results
[0183] Based on the alignments of the L1 sequences as shown in FIG.
1, primers were selected, as shown in table 4. The specificity of
the primers in the regions B and C was tested on the plasmids HPV
6, 11, 16, 18, 31, 33, 45, 35, 39, 58, 57 and 59. PCR was performed
by all 20 possible primer combinations for the regions B and C and
results are summarized in table 5. Poor results were only obtained
when using the primer SGP2F-bio that contains an inosine residu at
four positions. Although some primer sets had mismatches with the
target HPV sequences, amplimers were synthesized for all tested HPV
plasmids. From the tested nine primers in the regions B and C, four
of them (SGP1A, SGP1B, SGP2B-bio and SGP2D-bio) could be used for
efficient HPV amplification. PCR performance of the primer set
containing the four primers SGP1A, SGP1B, SGP2B and SGP2D revealed
amplification from all tested HPV plasmids: 6, 11, 16, 18, 31, 33,
45, 35, 39, 58, 57 and 59.
[0184] Sequence analysis of the amplimers revealed the expected
sequence for each plasmid. This result indicates that the four
primers are able to detect the various HPV types. The mismatches
with especially type 57 and 59 apparently did not hamper
amplification.
[0185] Discussion
[0186] Despite the presence of mismatches between primer and target
sequence, successful amplification by PCR may occur if there are no
mismatches at the 3' end of the primer. The PCR and sequence data
obtained in this study indicate that the primers SGP1A, SGP1B,
SGP2B-bio and SGP2D-bio are able to detect efficiently the various
HPV genotypes. Therefore, these four primers can be used for
universal amplification of HPV.
Example 3
Identification of Different HPV Types by Analysis of a Small PCR
Fragment Derived from the L1 region
[0187] Introduction
[0188] Identification of the different HPV genotypes may have great
clinical and epidemiological importance. Current classification
methods are for instance based on either type-specific PCR or
sequence analysis of larger DNA fragments. Therefore, there is a
clear need for a simple, rapid and reliable genotyping assay for
the different HPV genotypes.
[0189] This assay should preferably be combined with the detection
of HPV DNA, aiming at the same genomic region. Therefore, we aimed
at the development of a screening assay to detect the presence of
HPV DNA in clinical samples, and (in case of a positive screening
result) the subsequent use of the same amplimer in a genotyping
assay.
[0190] The theoretical requirements for such an assay would be as
follows:
[0191] 1. The amplimer should be small, to allow highly sensitive
detection and to permit amplification from formalin-fixed,
paraffin-embedded materials. The development of such a PCR assay
has been described in examples 1 and 2.
[0192] 2. The amplified fragment should contain sufficient sequence
variation to permit specific detection of the different
genotypes.
[0193] The present study describes: (i) the relationship between
sequences from the various HPV types by phylogenetic analyses of
the regions MY11/MY09, the sequence between region A and C (51 bp)
and between B and C (22 bp); (ii) the analysis of the small
amplimer of 62 bp generated by primers from the region B and C;
(iii) The development of HPV type-specific probes from this
region.
[0194] Materials and Methods
[0195] 1. Sequences from the different HPV genotypes were obtained
from the GenBank database.
[0196] 2. Phylogenetic analyses were performed with the Phylip 3.5c
software (Felsenstein 1995).
[0197] 3. Type-specific HPV PCR and general HPV amplification by
SGP1/SGP2 were performed according to the protocol as described in
examples 1 and 2.
[0198] 4. Sequence analysis of the PCR-products was performed by
manual sequencing, using the cycle-sequencing kit (Perkin Elmer).
Sequences were analyzed by the PC-Gene software (Intelligenetics,
USA)
[0199] Results
[0200] Phylogenetic Analyses
[0201] In order to study the relationships between the HPV-derived
sequences, several phylogenetic trees were constructed.
[0202] 1. Sequences between primers MY11 and MY09 were selected
from all available HPV sequences. The phylogenetic tree is shown in
FIG. 3. Sequence variation in this .+-.410 bp region permits
discrimination between most, if not all HPV genotypes. The
different groups of HPV (indicated with an A followed by a number)
are indicated in the figure (Chan et al., 1995).
[0203] 2. Sequences between the regions A and C and those between B
and C were also subjected to phylogenetic analysis, and both trees
are shown in the FIG. 4 and FIG. 5, respectively. Sequence
variation enclosed by the primers in regions B and C (22 bp) allows
discrimination between the genital HPV types. HPV68 (a genital
type) and HPV73 (an oral 15-type) show an identical sequence in
this region. However these two types can be recognized in the
region flanked by primers in the regions A and C, for instance by
use of probes HPV 68 (CAGGGACACAACAATG) and HPV 73
(CAGGGTCATAACAATGG).
[0204] Intratypic Variation
[0205] Since the aim of this study is to determine whether the
intratypic sequence variation in the small PCR product is
sufficient to identify the different HPV genotypes, the intratypic
variation should also be investigated.
[0206] Therefore, 77 HPV isolates positive with specific primers
for type 16 or 18, were studied for sequence variability in the
SGP1/SGP2 amplimer. Samples identified as type 16 by type-specific
PCR were all identically typed by sequence analysis of the
SGP1/SGP2 amplimer (table 6). There was no intratypic sequence
variation in the small SGP1/SGP2 amplimer. Identical results were
obtained for HPV 18. Sequence analysis of the SGP1/SGP2 amplimers
in the group of 31 samples negative by HPV type-specific PCR, as
described in example 1, revealed different HPV sequences. The
obtained sequences were identical to HPV types 16 (n=9), 18 (n=4),
31 (n=2), 35 (n=1), 45 (n=5), 52 (n=2), 56 (n=3) and 58 (n=2). This
indicates that PCR with SGP1/SGP2 is more sensitive than HPV
type-specific PCRs. Aberrant sequences, not matching any known HPV
type, were found in three cases. It was not possible to amplify
these isolates by other previously described general primer sets
(MY11/MY09, GP5/GP6 and CPI/CPIIg). For these samples the HPV
specificity was confirmed by performing a semi-nested PCR with the
primer sets SGP3/SGP2 and SGP1/SGP2.
[0207] Discussion
[0208] Phylogenetic analyses of the various HPV types revealed
heterogeneity in the region between primers SGP1 and SGP2. Sequence
variation was found to be sufficient for consistent discrimination
between all genital HPV types. In order to investigate the
reproducibility of this region for HPV genotyping, 77 samples were
typed by type-specific PCR and sequence analysis of the SGP1/SGP2
amplimer. No intratypic variation was observed in the SGP1/SGP2
amplimers.
[0209] From these results and that of already reported sequences,
in particular HPV type 16 variants, it might be suggested that
intratypic variability in the 22 bp between the SGP1 and SGP2
primers is very limited. This observation supports the use of
sequence variation in the SGP1/SGP2 amplimer for HPV
genotyping.
Example 4
Development of the HPV INNO-LiPA Genotyping Assay
[0210] Introduction
[0211] An aim of the invention was to develop a simple and reliable
system for detection as well as identification of HPV genotypes. A
possible format of such a system could comprise a single PCR using
universal primers, that amplify a small genomic fragment with very
high sensitivity. Subsequently, the same PCR product can be used to
discriminate between the HPV genotypes.
[0212] For analysis of the PCR products, sequence analysis is a
very accurate method, but it is not very convenient. Therefore we
aimed at the development of type-specific probes, that would permit
positive recognition of the different HPV genotypes.
[0213] Materials and Methods
[0214] Selection of Probes:
[0215] Based on the 22 bp sequences located between the regions B
and C (FIG. 1), a number of type-specific probes were proposed.
These probes are listed in table 7.
[0216] HPV Plasmids and Clinical Isolates
[0217] The selected probes were analysed for analytical and
clinical specificity. First, plasmids, containing complete genomic
sequences of different HPV types, were used as target for PCR
amplification with primers SGP1-bio and SGP2-bio, and with primers
SGP1-bio and MY09-bio.
[0218] PCR Reactions
[0219] PCR was performed using the primer sets SGP1-bio/SGP2-bio
and SGP1-bio/MY09-bio. All primers contained a biotin moiety at the
5' end (table 8). The PCR conditions were similar to those
described in example 1. The final volume of 100 .mu.l contained 10
.mu.l of plasmid DNA, 75 mM Tris-HCl pH 9.0, 20 mM
(NH.sub.4).sub.2SO.sub.4 2.5 mM MgCl.sub.2 0.1% (w/v) Tween 20, 200
.mu.M of each deoxynucleoside triphosphate, 100 pmol of forward and
reverse primer, and 3U of Taq-DNA polymerase (Pharmacia, Uppsala,
Sweden). After a preheating step for 1 min 94.degree. C.
amplification was performed by 1 min 94.degree. C., 1 min
52.degree. C. and 1 min 72.degree. C. for 40 cycles.
[0220] Development of a Reverse Hybridization Format
[0221] In order to permit analysis of multiple probes in a single
hybridization step, a reverse hybridization assay was developed.
This requires the selection of type-specific probes that have very
similar hybridization characteristics. For this experiment probes
were chosen for HPV types 6, 11, 16, 18, 31, 33 and 45.
[0222] Oligonucleotide probes were provided with a poly-(dT) tail
at the 3' end. Twenty pmol primer was incubated in 25 .mu.l buffer
containing 3.2 mM dTTP, 25 mM Tris-HCl (pH 7.5), 0.1 M sodium
cacodylate, 1 mM CoCl.sub.2, 0.1 mM dithiothreitol and 60 U
terminal desoxynucleotidyl transferase for 1 h at 37.degree. C. The
reaction was stopped by adding 2.5 .mu.l 0.5 M EDTA (pH 8.0) and
diluted with 20.times.SSC (Sambrook et al., 1989), until a final
concentration of 6.times.SSC and 2.5 pmol oligonucleotide/.mu.l was
reached. The tailed probes were immobilized on a nitrocellulose
strip as parallel lines. As a control for the conjugate,
biotinylated DNA was also applied. A possible outline of the strip
is shown in FIG. 6.
[0223] Ten .mu.l of the PCR amplification product, containing
biotin at the 5' end of each primer, was mixed with 10 .mu.l of
denaturation solution (400 mM NaOH, 10 mM EDTA) and incubated at
room temperature for 10 minutes. After denaturation of the DNA, 1
ml of preheated hybridization buffer, 3.times.SSC, 0.1% SDS,
(1.times.SSC: 15 mM Na-citrate and 150 mM NaCl) was added. The
hybridization was performed at 50.degree. C. in a shaking waterbath
for 1 h. The strips were washed once with hybridization buffer at
50.degree. C. for 30 minutes. The strips were then washed by rinse
solution (phosphate buffer containing NaCl, Triton and 0.5%
NaN.sub.3). Alkaline phosphatase labelled streptavidin was added in
conjugate diluent (phosphate buffer containing NaCl, Triton,
protein stabilizers and 0.1% NaN.sub.3) and incubated at 37.degree.
C. for 1 h. Strips were washed again three times with rinse
solution and once with substrate buffer (Tris buffer containing
NaCl and MgCl.sub.2). Colour development was achieved by addition
of BCIP and NBT in substrate buffer and incubation for 30 minutes
at room temperature. Colour development was stopped by incubation
in water and drying of the strips. Reverse hybridization results
were interpreted visually.
[0224] Results and Discussion
[0225] In order to develop a novel HPV typing assay, we selected
probes from a small part of the L1 region. This approach would
first require detection of HPV sequences in general by PCR using
universal primers, such as SGP1/SGP2, generating a fragment of 62
bp or MY11/MY09, generating a fragment of approximately 450 bp.
Subsequently, the same PCR product can be analysed using
type-specific probes from this L1 region. PCR fragments of 62 bp
and 450 bp were generated by primer sets SGP1-bio/SGP2-bio and
SGP1-bio/MY09-bio, respectively from different target DNA.
[0226] First, plasmids containing complete genomic sequences from
the HPV types 6, 11, 16, 18, 31, 33 and 45 were subjected to PCR
with primerset SGP1-bio/SGP2-bio. Subsequently, the amplimers were
analysed in the reverse hybridization assay containing
type-specific probes for recognition of the HPV types 6, 11, 16,
18, 31, 33 and 45. Representative results of reverse hybridization
are shown in FIG. 7. Secondly, amplimers synthesized by the
primerset SGP1-bio/MY09-bio from HPV types 6, 16, 31, 33 and 45
were analysed in the reverse hybridization assay (FIG. 8).
[0227] The results show that the method has a high sensitivity and
allows detection of HPV sequences at very low concentrations or
from difficult clinical materials, such as formalin-fixed,
paraffin-embedded biopsies. The reverse hybridization method
permits positive identification of the main HPV genotypes 6, 11,
16, 18, 31, 33 and 45. This assay can easily be extended by adding
probes on the strip for recognition of all other genital HPV
genotypes.
Example 5
Sequencing of HPV Isolates
[0228] Introduction
[0229] In this study, the sequence of HPV isolates in the region
between primers SGP1 and SGP2 was analyzed.
[0230] Materials and Methods
[0231] DNA was isolated from formalin-fixed and paraffin-embedded
cervical cancer biopsies and cytologically abnormal scrapes
according to standard protocols. PCR was is performed as described
in example 1 by the use of primers SGP1 and SGP2. The obtained
amplimers were analyzed by direct sequencing.
[0232] Results
[0233] Sequencing of HPV-positive samples revealed that, within the
region between primers SGP1 and SGP2, 19 sequences from different
patients were aberrant from previously described full-length HPV
types. These previously unknown sequences are listed in FIG. 1.
Sequences having an identification number starting with 95, were
found in cervical cancer biopsies, whereas those starting with 97
were found in cytologically abnormal scrapes.
[0234] Discussion
[0235] Any of the 19 sequences disclosed in this study may be
representative for a new HPV type. Further investigation will be
carried out to determine whether indeed any of these sequences is
characteristic of a new HPV type that is possibly clinically
important. Probes that specifically hybridize to these sequences
can be used to detect and/or to identify the corresponding HPV
types according to the methods of the present invention.
Example 6
Broad-Spectrum Detection of HPV by Amplification of a Short PCR
Fragment Using a Mixture of 10 HPV Primers
[0236] Introduction
[0237] The examples 1 and 2 describe the selection and optimization
of a novel HPV PCR primerset. The selected primers from example 2,
SGP1A, SGP1B, SGP2B-bio and SGP2D-bio, could be used for efficient
HPV amplification. Additional broad spectrum primers were developed
for a more sensitive HPV DNA PCR assay. The current example
describes the use of a mixture of 10 primers for highly sensitive
detection of human papillomaviruses.
[0238] Materials and Methods
[0239] From alignments of HPV L1 sequences as shown in FIG. 1,
forward and reverse primers were selected for sensitive
amplification of HPVs, see table 11. The primers were tested on
plasmids containing HPV genotypes 6, 13, 16, 18, 26, 34, 35, 39,
40, 42, 43, 51, 52, 53, 54, 55, 68, 69, 70, 74. These HPV plasmids
were provided by Dr. E-M. de Villiers, Heidelberg, Germany (HPV
genotypes 6, 13, 16, 18, 40, 51 and 53), Dr. R. Ostrow,
Minneapolis, Minn. (HPV genotype 26), Dr. A. Lorincz, Silver
Springs, MD (HPV genotypes 35 and 43), Dr. T. Matsukura, Tokyo,
Japan (HPV genotype 69), and Dr. G. Orth, Paris, France (HPV
genotypes 34, 39, 42, 52, 54, 55, 68, 70 and 74).
[0240] HPV DNA amplification was performed in a final reaction
volume of 50 .mu.l, containing 10 .mu.l of small amounts of plasmid
DNA, 10 mM Tris-HCl pH 9.0, 50 MM KCl, 2.5 mM MgCl.sub.2, 0.1%
Triton X-100, 0.01% gelatin, 200 mM of each deoxynucleoside
triphosphate, 15 pmol of each forward (SGP1A-1D) and 15 pmol of
different reverse primers, and 1.5 U of AmpliTaq gold (Perkin
Elmer, Branchburg, N.J., USA). The PCR conditions were as follows:
preheating for 9 min 94.degree. C. was followed by 40 cycles of 30
seconds 94.degree. C., 45 seconds, at 50.degree. C. or 52.degree.
C. or 55.degree. C. and 45 seconds at 72.degree. C., and a final
extension of 5 min at 72.degree. C. PCR-products were analyzed on a
3% TBE agarose gel.
[0241] Results
[0242] Developed were 14 broad spectrum primers, 4 sense (SGP1A,
SGP1B, SGP1C, SGP1D) and 10 antisense (SGP2B-bio, SGP2D-bio,
SGP2H-bio, SGP2I-bio, SGP2J-bio, SGP2K-bio, SGP2L-bio, SGP2M-bio,
SGP2N-bio, SGP2P-bio), respectively. See table 11 for sequences and
positions. For selection of sensitive PCR primers, plasmid DNA from
HPV genotypes 6, 13, 16, 18, 26, 34, 35, 39, 40, 42, 43, 51, 52,
53, 54, 55, 68, 69, 70 and 74 were used as target. PCR experiments
were performed with the 4 sense primers (SGP1A, SGP1B, SGP1C,
SGP1D) in combination with one or more reverse primers at different
annealing temperatures, using low amounts of HPV plasmid DNA. The
reverse primers SGP2H-bio, SGP2I-bio, SGP2L-bio and SGP2N-bio
appeared to have no added value compared to a mixture of the
remaining 6 reverse primers (SGP2B-bio, SGP2D-bio, SGP2J-bio,
SGP2K-bio, SGP2M-bio and SGP2P-bio) as listed in table 11. Although
the sequences of the 10 primers, 4 sense (SGP1A-1D) and 6 antisense
(SGP2B-bio, SGP2D-bio, SGP2J-bio, SGP2K-bio, SGP2M-bio and
SGP2P-bio) showed minor mismatches compared to known HPV genotypes
(FIG. 1), still low amounts of HPV DNA could efficiently be
amplified.
[0243] Discussion
[0244] A mixture of 10 primers was developed for broad-spectrum
detection of HPV. Despite minor mismatches between primer and
target sequences of known HPVs, the 10 selected primers were
succesfull to detect various HPV genotypes at low levels.
Therefore, this mixture of 10 primers can be used for sensitive
broad-spectrum detection of HPV.
Example 7
A Line Probe Assay for Rapid Detection and Simultaneous
Identification of 25 Different HPV Genotypes
[0245] Introduction
[0246] Example 4 describes the development of the HPV INNO-LiPA
genotyping assay for simple detection and identification of HPV
genotypes. This example describes an HPV INNO-LiPA genotyping assay
for simultaneous detection and identification of 25 types. After
universal HPV amplification, synthesized amplimers can be detected
and identified by hybridization to type-specific probes that are
applied on a LiPA strip.
[0247] Materials and Methods
[0248] Based on the inner primer sequence of 22 bp which is located
between the regions B and C (FIG. 9), several type-specific probes
were proposed and tested for specificity reasons. The selected
probes are listed in tables 7 and 12. Plasmids containing HPV
sequences of different genotypes were used as target for
broad-spectrum amplification (see examples 4 and 6). LiPA
experiments were performed as described in example 4 using the
Auto-LiPA system.
[0249] Results
[0250] Amplimers obtained from well defined plasmids containing HPV
sequences of various genotypes were used in LiPA experiments in
order to determine the specificity of the selected probes (tables 7
and 12). Subsequently, 25 HPV type-specific probes and another 3
probes were selected for simultaneous identification of 25
different HPV genotypes. The outline of the HPV-LiPA is shown in
FIG. 10 and typical LiPA patterns are shown in FIG. 11.
[0251] In most cases the probe name is directly linked to the HPV
type (e.g. a purple color on probe lane 16 means hybridization of
an amplimer derived from HPV type 16). The probes c31, c56 and c68
are secundairy probes. These probes are of interest when there is a
positive hybridization with the probe line just above (31/40/58 or
56/74 or 68/45). These `c` probes were developed for exclusion of
type 40, 58, 74, and 45. Those types are also identified by
positive hybridization. The `c` probes c31, c56 and c68 will also
react with other types. Amplimers from type 33 and 54 will give a
positive reaction with probe c31. Similarly, the amplimer from type
58 hybridizes with c56. Therefore, amplimers of type 58 will give
three bands on a LiPA strip (positive on: 31/40/58 and c56 and 58).
Probe c68 is also reactive with amplimers from type 18 and 39. HPV
type 6 is identified by hybridization to the probes 6. HPV type 74
is identified by the probes 56/74 and 74. A sample contains type 54
when probe c31 is positive while probes 31/40/58, 33, 40, and 58
are negative.
[0252] Discussion
[0253] The described HPV LiPA genotyping assay detects and
identifies simultaneously the HPV genotypes 6, 11, 16, 18, 31, 33,
34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68,
70, and 74. These genotypes can be recognized after universal PCR
using the novel developed primerset as described in this patent and
the MY11/09 primerset which is discussed in example 4. This typing
assay can still be extended with type-specific probes for
recognition of other HPV genotypes.
[0254] In summary, the novel PCR system for highly sensitive
detection of HPV DNA in diverse clinical materials followed by a
HPV LiPA typing experiment could be a usefull tool to improve the
molecular diagnosis and epidemiology of HPV infections.
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and HLA-DQ-alfa DNA with allele-specific oligonucleotide probes.
Nature 324:163-166.
[0270] Sambrook et al. 1989 Molecular Cloning: A Laboratory Manual,
2nd Edition, Cold Spring Harbour Laboratory Press
[0271] Sommer, R., and D. Tautz. 1989. Minimal homology
requirements for PCR primers. Nucleic Acids Research 17:6749.
[0272] Stuyver, L., R. Rossau, A. Wyseur, M. Duhamel, B.
Vanderborght, H. Van Heuverswyn, and G. Maertens. 1993. Typing of
hepatitis C virus isolates and characterization of new subtypes
using a line probe assay. J. Gen. Virol. 74:1093-1102.
[0273] Tieben L. M., J. ter Schegget, R. P. Minnaar, J. N. Bouwes
Bavinck, R. J. M. Berkhout, B. J. Vermeer, M. f. Jebbink, and H. L.
Smits. 1993. Detection of cutaneous and genital HPV types in
clinical samples by PCR using consensus primers. J. Virol. Methods
42:265-280.
[0274] Van den Brule, A. J. C., P. J. F. Snijders, R. L. J.
Gordijn, O. P. Bleker, C. J. L. M. Meijer, and J. M. M. Walboomers.
1990. General primer-mediated polymerase chain reaction permits the
detection of sequenced and still unsequenced human papillomavirus
genotypes in cervical scrapes and carcinomas. Int. J. Cancer
45:644-649.
[0275] Young, L. S., I. S. Bevan, M. A. Johnson, P. I. Blomfield,
T. Bromidge, N. J. Maitland, and G. B. J. Woodman. 1989. The
polymerase chain reaction: A new epidemiological tool for
investigatiing cervical human papillomavirus infection. Brit Med.
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[0276] Woodworth, C. D., Waggoner, S., Barnes, W., Stoler, M. H.,
Di Paolo, J. A. 1990. Human cervical and foreskin eptithelial cells
immortalized by human papillomavirus DNAs exhibit displastic
differentiation in vivo. Cancer Res. 50: 3709-3715.
Sequence CWU 1
1
497 1 20 DNA Artificial Sequence Synthetic Primer derived from the
Human Papillomavirus (HPV) 1 tattcaataa accttattgg 20 2 20 DNA
Artificial Sequence Synthetic Primer derived from the Human
Papillomavirus (HPV) 2 tdtttaataa rccwtattgg 20 3 20 DNA Artificial
Sequence Synthetic Primer derived from the Human Papillomavirus
(HPV) 3 tatttaataa accatattgg 20 4 20 DNA Artificial Sequence
Synthetic Primer derived from the Human Papillomavirus (HPV) 4
tatttaataa gccatattgg 20 5 20 DNA Artificial Sequence Synthetic
Primer derived from the Human Papillomavirus (HPV) 5 gcacagggcc
acaataatgg 20 6 20 DNA Artificial Sequence Synthetic Primer derived
from the Human Papillomavirus (HPV) 6 gcmcaggghc ataayaatgg 20 7 20
DNA Artificial Sequence Synthetic Primer derived from the Human
Papillomavirus (HPV) 7 gtatcaacaa cagtaacaaa 20 8 20 DNA Artificial
Sequence Synthetic Primer derived from the Human Papillomavirus
(HPV) 8 gtatctacca cagtaacaaa 20 9 20 DNA Artificial Sequence
Synthetic Primer derived from the Human Papillomavirus (HPV) 9
gtatchacha cagtaacaaa 20 10 20 DNA Artificial Sequence Synthetic
Primer for the Human Papillomavirus (HPV) 10 tatttaataa gccttattgg
20 11 20 DNA Artificial Sequence Synthetic Primer for the Human
Papillomavirus (HPV) 11 tattcaataa accttattgg 20 12 20 DNA
Artificial Sequence Synthetic Primer for the Human Papillomavirus
(HPV) 12 tatttaataa accttactgg 20 13 20 DNA Artificial Sequence
Synthetic Primer for the Human Papillomavirus (HPV) 13 tatttaataa
nccntattgg 20 14 20 DNA Artificial Sequence Synthetic Primer for
the Human Papillomavirus (HPV) 14 tatttaataa nccntactgg 20 15 20
DNA Artificial Sequence Synthetic Primer for the Human
Papillomavirus (HPV) 15 gcncagggnc acaataatgg 20 16 20 DNA
Artificial Sequence Synthetic Primer for the Human Papillomavirus
(HPV) 16 gcncagggnc ataacaatgg 20 17 20 DNA Artificial Sequence
Synthetic Primer for the Human Papillomavirus (HPV) 17 gcncagggnc
ataataatgg 20 18 20 DNA Artificial Sequence Synthetic Primer for
the Human Papillomavirus (HPV) 18 gcncaaggnc ataataatgg 20 19 23
DNA Artificial Sequence Synthetic Primer for the Human
Papillomavirus (HPV) 19 gtngtatcna caacagtaac aaa 23 20 23 DNA
Artificial Sequence Synthetic Primer for the Human Papillomavirus
(HPV) 20 gtngtatcta ccacagtaac aaa 23 21 23 DNA Artificial Sequence
Synthetic Primer for the Human Papillomavirus (HPV) 21 gtngtatcna
ctacagtaac aaa 23 22 23 DNA Artificial Sequence Synthetic Primer
for the Human Papillomavirus (HPV) 22 gtngtatcna cgacagtnac aaa 23
23 23 DNA Artificial Sequence Synthetic Primer for the Human
Papillomavirus (HPV) 23 gtngtatcna caacagtnan aaa 23 24 19 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 24 ttggggtaat caactgtgg 19 25 20 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 25 gttggggtaa tcaactgtgg 20 26 19 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 26 ttggggtaat caactgttg 19 27 20 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 27 gttggggtaa tcaactgttg 20 28 19 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 28 ttggggtaat caactgttt 19 29 17 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 29 tgctggggaa accactg 17 30 20 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 30 tgctggggaa accacttagg 20 31 18 DNA Artificial Sequence
Type specific probe derived from the Human Papillomavirus (HPV) 31
ttgttgggga aaccactg 18 32 21 DNA Artificial Sequence Type specific
probe derived from the Human Papillomavirus (HPV) 32 ttgctgggga
aaccacttag g 21 33 20 DNA Artificial Sequence Type specific probe
derived from the Human Papillomavirus (HPV) 33 tgctggggaa
accacttggg 20 34 19 DNA Artificial Sequence Type specific probe
derived from the Human Papillomavirus (HPV) 34 ttggggtaac caactatgg
19 35 20 DNA Artificial Sequence Type specific probe derived from
the Human Papillomavirus (HPV) 35 gttggggtaa ccaactatgg 20 36 19
DNA Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 36 ttggggtaac caactattg 19 37 20 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 37 gttggggtaa ccaactattg 20 38 19 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 38 ttggggtaac caactattt 19 39 17 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 39 gtgtttgctg gcataat 17 40 18 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 40 ggtgtttgct ggcataag 18 41 18 DNA Artificial Sequence Type
specific probe derived from the Human Papillomavirus (HPV) 41
gtgtttgctg gcataatc 18 42 19 DNA Artificial Sequence Type specific
probe derived from the Human Papillomavirus (HPV) 42 tggtgtttgc
tggcataag 19 43 18 DNA Artificial Sequence Type specific probe
derived from the Human Papillomavirus (HPV) 43 ggtgtttgct ggcataat
18 44 19 DNA Artificial Sequence Type specific probe derived from
the Human Papillomavirus (HPV) 44 ttggggcaat cagttatgg 19 45 20 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 45 gttggggcaa tcagttatgg 20 46 19 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 46 ttggggcaat cagttattg 19 47 20 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 47 gttggggcaa tcagttattg 20 48 20 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 48 gttggggcaa tcagttattt 20 49 16 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 49 gggcaatcag ttattg 16 50 15 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 50 aataactgat tgccc 15 51 17 DNA Artificial Sequence Type
specific probe derived from the Human Papillomavirus (HPV) 51
ggcaatcagt tatttcc 17 52 15 DNA Artificial Sequence Type specific
probe derived from the Human Papillomavirus (HPV) 52 aaataactga
ttgcc 15 53 16 DNA Artificial Sequence Type specific probe derived
from the Human Papillomavirus (HPV) 53 gcaatcagtt atttgg 16 54 15
DNA Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 54 caaataactg attgc 15 55 17 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 55 ggcaatcagt tatttgg 17 56 17 DNA Artificial Sequence Type
specific probe derived from the Human Papillomavirus (HPV) 56
gcaatcagtt atttgtg 17 57 19 DNA Artificial Sequence Type specific
probe derived from the Human Papillomavirus (HPV) 57 ttggggcaat
caggtatgg 19 58 20 DNA Artificial Sequence Type specific probe
derived from the Human Papillomavirus (HPV) 58 gttggggcaa
tcaggtatgg 20 59 19 DNA Artificial Sequence Type specific probe
derived from the Human Papillomavirus (HPV) 59 ttggggcaat caggtattg
19 60 20 DNA Artificial Sequence Type specific probe derived from
the Human Papillomavirus (HPV) 60 gttggggcaa tcaggtattg 20 61 20
DNA Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 61 gttggggcaa tcaggtattt 20 62 16 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 62 gggcaatcag gtattg 16 63 15 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 63 aatacctgat tgccc 15 64 17 DNA Artificial Sequence Type
specific probe derived from the Human Papillomavirus (HPV) 64
ggcaatcagg tatttcc 17 65 15 DNA Artificial Sequence Type specific
probe derived from the Human Papillomavirus (HPV) 65 aaatacctga
ttgcc 15 66 16 DNA Artificial Sequence Type specific probe derived
from the Human Papillomavirus (HPV) 66 gcaatcaggt atttgg 16 67 15
DNA Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 67 caaatacctg attgc 15 68 17 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 68 catatgtttt ggcaatc 17 69 17 DNA Artificial Sequence Type
specific probe derived from the Human Papillomavirus (HPV) 69
gtatttgttg gcataat 17 70 18 DNA Artificial Sequence Type specific
probe derived from the Human Papillomavirus (HPV) 70 ggtatttgtt
ggcataag 18 71 18 DNA Artificial Sequence Type specific probe
derived from the Human Papillomavirus (HPV) 71 gtatttgttg gcataatc
18 72 19 DNA Artificial Sequence Type specific probe derived from
the Human Papillomavirus (HPV) 72 tggtatttgt tggcataag 19 73 18 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 73 ggtatttgtt ggcataat 18 74 17 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 74 tggcataatc agttggg 17 75 16 DNA Artificial Sequence Type
specific probe derived from the Human Papillomavirus (HPV) 75
ggcataatca gttgtg 16 76 16 DNA Artificial Sequence Type specific
probe derived from the Human Papillomavirus (HPV) 76 gcataatcag
ttgttt 16 77 16 DNA Artificial Sequence Type specific probe derived
from the Human Papillomavirus (HPV) 77 gcaatcagtt gtttgc 16 78 16
DNA Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 78 caatcagttg tttgtc 16 79 15 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 79 atggcatatg ttggg 15 80 16 DNA Artificial Sequence Type
specific probe derived from the Human Papillomavirus (HPV) 80
tggcatatgt tggggg 16 81 15 DNA Artificial Sequence Type specific
probe derived from the Human Papillomavirus (HPV) 81 ggcatatgtt
ggggc 15 82 15 DNA Artificial Sequence Type specific probe derived
from the Human Papillomavirus (HPV) 82 gcatatgttg gggca 15 83 16
DNA Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 83 ggggtaatca attatc 16 84 17 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 84 ggggtaatca attattc 17 85 17 DNA Artificial Sequence Type
specific probe derived from the Human Papillomavirus (HPV) 85
ggggtaatca attattt 17 86 18 DNA Artificial Sequence Type specific
probe derived from the Human Papillomavirus (HPV) 86 tggggtaatc
aattattt 18 87 19 DNA Artificial Sequence Type specific probe
derived from the Human Papillomavirus (HPV) 87 ggggtaatca attatttgg
19 88 16 DNA Artificial Sequence Type specific probe derived from
the Human Papillomavirus (HPV) 88 catttgctgg ggcaag 16 89 15 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 89 atttgctggg gcaat 15 90 15 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 90 tttgctgggg caatc 15 91 15 DNA Artificial Sequence Type
specific probe derived from the Human Papillomavirus (HPV) 91
ttgctggggc aatca 15 92 17 DNA Artificial Sequence Type specific
probe derived from the Human Papillomavirus (HPV) 92 gttggagtaa
ccaattg 17 93 17 DNA Artificial Sequence Type specific probe
derived from the Human Papillomavirus (HPV) 93 gtatatgttg gcataat
17 94 17 DNA Artificial Sequence Type specific probe derived from
the Human Papillomavirus (HPV) 94 gcatttgctg gaacaat 17 95 17 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 95 ggggcaatca ggtgttt 17 96 17 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 96 ggtatatgtt ggcacaa 17 97 15 DNA Artificial Sequence Type
specific probe derived from the Human Papillomavirus (HPV) 97
gcatatgctg gggta 15 98 23 DNA Artificial Sequence Type specific
probe derived from the Human Papillomavirus (HPV) 98 gtngtatcna
caactgtaac aaa 23 99 17 DNA Artificial Sequence Type specific probe
derived from the Human Papillomavirus (HPV) 99 catttgttgg cataacc
17 100 15 DNA Artificial Sequence Type specific probe derived from
the Human Papillomavirus (HPV) 100 tggggcaatc acttg 15 101 15 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 101 gcatttgctg gcata 15 102 17 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 102 tggggaaatc agctatt 17 103 17 DNA Artificial Sequence Type
specific probe derived from the Human Papillomavirus (HPV) 103
ggcatttgtt ttgggaa 17 104 18 DNA Artificial Sequence Type specific
probe derived from the Human
Papillomavirus (HPV) 104 ttggggaaat cagttatt 18 105 16 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 105 gcatctgttg gaacaa 16 106 15 DNA Artificial
Sequence Type specific probe derived from the Human Papillomavirus
(HPV) 106 gttgggggaa tcagt 15 107 17 DNA Artificial Sequence Type
specific probe derived from the Human Papillomavirus (HPV) 107
gttggggcaa ccaattg 17 108 18 DNA Artificial Sequence Type specific
probe derived from the Human Papillomavirus (HPV) 108 tggtttaatg
aattgttt 18 109 17 DNA Artificial Sequence Type specific probe
derived from the Human Papillomavirus (HPV) 109 ggtttaatga actgttt
17 110 17 DNA Artificial Sequence Type specific probe derived from
the Human Papillomavirus (HPV) 110 aatggaattt gttggca 17 111 17 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 111 gtatatgctg gggtaat 17 112 17 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 112 atttgttggg gtaatca 17 113 17 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 113 tgctggaata atcagct 17 114 18 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 114 tggtttaatg agttattt 18 115 17 DNA
Artificial Sequence Type specific probe derived from the Human
Papillomavirus (HPV) 115 atatgctggt ttaatca 17 116 18 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 116 aataatggca tntgttgg 18 117 18 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 117 aataatggta tntgttgg 18 118 18 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 118 aacaatggta tntgttgg 18 119 18 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 119 aacaatggta tntgctgg 18 120 18 DNA
Artificial Sequence General Probe for Human Papillomavirus (HPV)
detectio 120 aacaatggtg tttgctgg 18 121 18 DNA Artificial Sequence
General probe derived from the Human Papillomavirus (HPV) 121
aataatggca tntgctgg 18 122 18 DNA Artificial Sequence General probe
derived from the Human Papillomavirus (HPV) 122 aacaatggca tntgctgg
18 123 21 DNA Artificial Sequence General probe derived from the
Human Papillomavirus (HPV) 123 canaataatg gtatntgttg g 21 124 20
DNA Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 124 anaataatgg tatntgttgg 20 125 21 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 125 canaataatg gtatttgttg g 21 126 20 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 126 anaataatgg tatttgttgg 20 127 21 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 127 cacaataatg gtatttgttg g 21 128 20 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 128 acaataatgg tatttgttgg 20 129 21 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 129 canaacaatg gtatntgttg g 21 130 20 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 130 anaacaatgg tatntgttgg 20 131 21 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 131 canaacaatg gtatttgttg g 21 132 20 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 132 anaacaatgg tatttgttgg 20 133 21 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 133 cataacaatg gtatttgttg g 21 134 20 DNA
Artificial Sequence General probe derived from the Human
Papillomavirus (HPV) 134 ataacaatgg tatttgttgg 20 135 20 DNA Human
Papillomavirus 135 catatgctgg aataatcaac 20 136 20 DNA Human
Papillomavirus 136 catatgctgg aataatcttc 20 137 22 DNA Human
Papillomavirus 137 tatctgctgg ggtaatcagc tt 22 138 21 DNA Human
Papillomavirus 138 atctgctggc ayaatcaatt a 21 139 20 DNA Human
Papillomavirus 139 tctgctggca taatcaatta 20 140 22 DNA Human
Papillomavirus 140 tatatgttgg cataatcaat ta 22 141 22 DNA Human
Papillomavirus 141 aatttgttgg cataatcaat tg 22 142 20 DNA Human
Papillomavirus 142 tttgttgggg taatcaattg 20 143 20 DNA Human
Papillomavirus 143 tttgctggtt taatcaattg 20 144 20 DNA Human
Papillomavirus 144 tatgttggtt taatgagctg 20 145 20 DNA Human
Papillomavirus 145 tttgttggtt taatgagttg 20 146 20 DNA Human
Papillomavirus 146 tttgttggtt taatgagtta 20 147 21 DNA Human
Papillomavirus 147 atttgttggt ttaatgagat g 21 148 20 DNA Human
Papillomavirus 148 tttgttggtt taatgaaatg 20 149 20 DNA Human
Papillomavirus 149 tatgttggtt taatgagctg 20 150 20 DNA Human
Papillomavirus 150 tytgttggtt taatgacctg 20 151 22 DNA Human
Papillomavirus 151 tatttgttgg tttaatgacc tg 22 152 20 DNA Human
Papillomavirus 152 tttgttggtt taatgaaatg 20 153 21 DNA Human
Papillomavirus 153 atctgttttg gyaaccaggt g 21 154 23 DNA Artificial
Sequence Synthetic Primer derived from the Human Papillomavirus
(HPV) 154 gtngtatcca caacagttac aaa 23 155 23 DNA Artificial
Sequence Synthetic Primer derived from the Human Papillomavirus
(HPV) 155 gtggtatcca caacngtgac aaa 23 156 23 DNA Artificial
Sequence Synthetic Primer derived from the Human Papillomavirus
(HPV) 156 gtagtntcca caacagtaag aaa 23 157 23 DNA Artificial
Sequence Synthetic Primer derived from the Human Papillomavirus
(HPV) 157 gtagtatcaa ccacagttaa aaa 23 158 23 DNA Artificial
Sequence Synthetic Primer derived from the Human Papillomavirus
(HPV) 158 gtngtatcta caacngttaa aaa 23 159 23 DNA Artificial
Sequence Synthetic Primer derived from the Human Papillomavirus
(HPV) 159 gtagtatcta cacaagtaac aaa 23 160 23 DNA Artificial
Sequence Synthetic Primer derived from the Human Papillomavirus
(HPV) 160 gtagtatcaa cacaggtaat aaa 23 161 18 DNA Artificial
Sequence Synthetic Probe derived from the Human Papillomavirus
(HPV) 161 ggtatctgct ggcataag 18 162 17 DNA Artificial Sequence
Synthetic Probe derived from the Human Papillomavirus (HPV) 162
tggtatctgc tggcata 17 163 17 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 163 tatttgttgg
ggcaatc 17 164 16 DNA Artificial Sequence Synthetic Probe derived
from the Human Papillomavirus (HPV) 164 atttgttggg gcaatc 16 165 16
DNA Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 165 tatttgttgg ggcaat 16 166 16 DNA Artificial
Sequence Synthetic Probe derived from the Human Papillomavirus
(HPV) 166 ggcatttgct ggcata 16 167 19 DNA Artificial Sequence
Synthetic Probe derived from the Human Papillomavirus (HPV) 167
gttggagtaa ccaattggg 19 168 19 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 168 tgttggagta
accaattcc 19 169 18 DNA Artificial Sequence Synthetic Probe derived
from the Human Papillomavirus (HPV) 169 ttgttggagt aaccaatg 18 170
18 DNA Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 170 ggtatatgtt ggcataat 18 171 17 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 171 ggggaaatca gctattg 17 172 16 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 172 gggaaatcag ctattt 16 173 18 DNA Artificial
Sequence Synthetic Probe derived from the Human Papillomavirus
(HPV) 173 ggcatttgtt ttgggaag 18 174 17 DNA Artificial Sequence
Synthetic Probe derived from the Human Papillomavirus (HPV) 174
gcatttgttt tgggaat 17 175 17 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 175 catttgtttt
gggaatc 17 176 17 DNA Artificial Sequence Synthetic Probe derived
from the Human Papillomavirus (HPV) 176 ggggaaatca gttattg 17 177
17 DNA Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 177 ggggaaatca gttattt 17 178 16 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 178 gggaaatcag ttattt 16 179 17 DNA Artificial
Sequence Synthetic Probe derived from the Human Papillomavirus
(HPV) 179 tggggaaatc agttatg 17 180 17 DNA Artificial Sequence
Synthetic Probe derived from the Human Papillomavirus (HPV) 180
catttgctgg aacaatc 17 181 17 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 181 ggcatctgtt
ggaacaa 17 182 16 DNA Artificial Sequence Synthetic Probe derived
from the Human Papillomavirus (HPV) 182 ggcaatcagg tgtttc 16 183 17
DNA Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 183 gggcaatcag gtgtttc 17 184 16 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 184 aaacacctga ttgccc 16 185 17 DNA Artificial
Sequence Synthetic Probe derived from the Human Papillomavirus
(HPV) 185 ggcaatcagg tgttttg 17 186 17 DNA Artificial Sequence
Synthetic Probe derived from the Human Papillomavirus (HPV) 186
gggggaatca gttattg 17 187 16 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 187 gggggaatca
gttatg 16 188 17 DNA Artificial Sequence Synthetic Probe derived
from the Human Papillomavirus (HPV) 188 tgggggaatc agttatg 17 189
16 DNA Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 189 tgggggaatc agttag 16 190 16 DNA Artificial
Sequence Synthetic Probe derived from the Human Papillomavirus
(HPV) 190 catttgctgg ggtaat 16 191 18 DNA Artificial Sequence
Synthetic Probe derived from the Human Papillomavirus (HPV) 191
tggtatatgt tggcacaa 18 192 18 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 192 ggtatatgtt
ggcacaat 18 193 18 DNA Artificial Sequence Synthetic Probe derived
from the Human Papillomavirus (HPV) 193 gtatatgttg gcacaatc 18 194
17 DNA Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 194 tatatgttgg cacaatc 17 195 16 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 195 ggcatatgct ggggta 16 196 18 DNA Artificial
Sequence Synthetic Probe derived from the Human Papillomavirus
(HPV) 196 ggtatatgct ggggtaat 18 197 16 DNA Artificial Sequence
Synthetic Probe derived from the Human Papillomavirus (HPV) 197
ggtatatgct ggggta 16 198 16 DNA Artificial Sequence Synthetic Probe
derived from the Human Papillomavirus (HPV) 198 tggtatatgc tggggt
16 199 17 DNA Artificial Sequence Synthetic Probe derived from the
Human Papillomavirus (HPV) 199 atggtatatg ctggggg 17 200 15 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 200 ggtatatgct ggggt 15 201 16 DNA Artificial
Sequence Synthetic Probe derived from the Human Papillomavirus
(HPV) 201 tggtatatgc tggggg 16 202 16 DNA Artificial Sequence
Synthetic Probe derived from the Human Papillomavirus (HPV) 202
aatggtatat gctggg 16 203 17 DNA Artificial Sequence Synthetic Probe
derived from the Human Papillomavirus (HPV) 203 tggtatttgt tggcata
17 204 18 DNA Artificial Sequence Synthetic Probe derived from the
Human Papillomavirus (HPV) 204 atggtatttg ttggcata 18 205 17 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 205 atggtatttg ttggcat 17 206 19 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 206 ttggcataat caattattt 19 207 21 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 207 ttggcataat caattatttc g 21 208 18 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 208 gcatttgttg gcataacc 18 209 17 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 209 gcatttgttg gcataac 17 210 16 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 210 catttgttgg cataac 16 211 16 DNA Artificial
Sequence Synthetic Probe derived from the Human Papillomavirus
(HPV) 211 tatttgttgg ggtaat 16 212 16 DNA Artificial Sequence
Synthetic Probe derived from the Human Papillomavirus (HPV) 212
atttgttggg gtaatc
16 213 16 DNA Artificial Sequence Synthetic Probe derived from the
Human Papillomavirus (HPV) 213 tttgttgggg taatca 16 214 17 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 214 gtatttgttg gggtaat 17 215 17 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 215 tatttgttgg ggtaatc 17 216 18 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 216 ttgctggaat aatcagct 18 217 16 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 217 tgctggaata atcagc 16 218 18 DNA Artificial
Sequence Synthetic Probe derived from the Human Papillomavirus
(HPV) 218 tgctggaata atcagctg 18 219 17 DNA Artificial Sequence
Synthetic Probe derived from the Human Papillomavirus (HPV) 219
tgctggaata atcagcg 17 220 22 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 220 canaataatg
gcatntgttg gc 22 221 22 DNA Artificial Sequence Synthetic Probe
derived from the Human Papillomavirus (HPV) 221 canaacaatg
gcatntgttg gc 22 222 23 DNA Artificial Sequence Synthetic Probe
derived from the Human Papillomavirus (HPV) 222 cacaataatg
gcatttgttg ggg 23 223 22 DNA Artificial Sequence Synthetic Probe
derived from the Human Papillomavirus (HPV) 223 canaataatg
gtatntgttg gg 22 224 22 DNA Artificial Sequence Synthetic Probe
derived from the Human Papillomavirus (HPV) 224 canaacaatg
gtatntgttg gc 22 225 30 DNA Artificial Sequence Synthetic Probe
derived from the Human Papillomavirus (HPV) 225 aatggcattt
gttggggtaa ccaactattt 30 226 21 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 226 ttgttggggt
aaccaactat g 21 227 24 DNA Artificial Sequence Synthetic Probe
derived from the Human Papillomavirus (HPV) 227 atttgttggg
gtaaccaact attg 24 228 23 DNA Artificial Sequence Synthetic Probe
derived from the Human Papillomavirus (HPV) 228 gcatttgttg
gggtaaccaa cta 23 229 25 DNA Artificial Sequence Synthetic Probe
derived from the Human Papillomavirus (HPV) 229 tggcatttgt
tggggtaacc aacta 25 230 30 DNA Artificial Sequence Synthetic Probe
derived from the Human Papillomavirus (HPV) 230 aatggtattt
gttggggcaa tcagttattt 30 231 30 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 231 aatggtattt
gttggcataa tcagttgttt 30 232 30 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 232 aatggtattt
gttggtttaa tgaattgttt 30 233 30 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 233 aatggcattt
gctggaacaa tcagcttttt 30 234 30 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 234 aatggtatat
gttggggcaa tcacttgttt 30 235 18 DNA Artificial Sequence Synthetic
Probe derived from the Human Papillomavirus (HPV) 235 aatggcattt
gttggggc 18 236 22 DNA Artificial Sequence Synthetic Probe derived
from the Human Papillomavirus (HPV) 236 aatggcatat gctggaataa tc 22
237 22 DNA Artificial Sequence Synthetic Probe derived from the
Human Papillomavirus (HPV) 237 aatggtatat gttggggcaa tc 22 238 18
DNA Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 238 aatggtattt gttggggc 18 239 22 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 239 aatggaattt gttggcataa tc 22 240 18 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 240 ggtatctgct ggcataat 18 241 22 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 241 aatggcattt gttggtttaa tg 22 242 22 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 242 aatggtattt gttggtttaa tg 22 243 22 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 243 aatggcatct gttggtttaa tg 22 244 20 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 244 tgttggttta atgagctgtg 20 245 21 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 245 tgctggttta atcaattgtt g 21 246 16 DNA
Artificial Sequence Synthetic Probe derived from the Human
Papillomavirus (HPV) 246 cagggacaca acaatg 16 247 17 DNA Artificial
Sequence Synthetic Probe derived from the Human Papillomavirus
(HPV) 247 cagggtcata acaatgg 17 248 65 DNA Human Papillomavirus 248
gcccagggac ataacaatgg tatttgttgg ggtaatcaac tgtttgttac tgtggtagat
60 accac 65 249 94 DNA Human Papillomavirus 249 tatttaataa
accatattgg cttcaaaagg ctcagggaca taacaatggt atttgctggg 60
gaaaccactt gtttgttact gtggtagata ccac 94 250 65 DNA Human
Papillomavirus 250 gctcagggac ataacaatgg tatttgctgg ggaaaccact
tgtttgttac tgtggtagat 60 accac 65 251 65 DNA Human Papillomavirus
251 gcccagggac acaataatgg tatatgttgg ggcaatcact tgtttgttac
tgtagttgat 60 actac 65 252 94 DNA Human Papillomavirus 252
tgtttaataa accatattgg ttacataagg cacagggtca taacaatggt gtttgctggc
60 ataatcaatt atttgttact gtggtagata ccac 94 253 65 DNA Human
Papillomavirus 253 gcacagggtc ataacaatgg tgtttgctgg cataatcaat
tatttgttac tgtggtagat 60 accac 65 254 65 DNA Human Papillomavirus
254 gcacagggtc ataataatgg tatctgttgg ggcaatcaat tgtttgttac
ctgtgttgat 60 accac 65 255 65 DNA Human Papillomavirus 255
gcacagggac acaataatgg catttgttgg ggcaaccagg tatttgttac tgttgtggac
60 accac 65 256 94 DNA Human Papillomavirus 256 tttttaataa
accatattgg atgcaacgtg ctcagggaca caataatggt atttgttggg 60
gcaatcagtt atttgttact gtggtagata ccac 94 257 65 DNA Human
Papillomavirus 257 gctcagggac acaataatgg tatttgttgg ggcaatcagt
tatttgttac tgtggtagat 60 accac 65 258 94 DNA Human Papillomavirus
258 tatttaataa gccatattgg ctacaacgtg cacaaggtca taataatggt
atttgttggg 60 gcaatcaggt atttgttact gtggtagata ccac 94 259 65 DNA
Human Papillomavirus 259 gcacaaggtc ataataatgg tatttgttgg
ggcaatcagg tatttgttac tgtggtagat 60 accac 65 260 94 DNA Human
Papillomavirus 260 tttttaataa gccttattgg ttgcaaaagg cccagggaca
aaacaatggc atttgctggc 60 ataatcaact gtttttaact gttgtagata ctac 94
261 65 DNA Human Papillomavirus 261 gcccagggac aaaacaatgg
catttgctgg cataatcaac tgtttttaac tgttgtagat 60 actac 65 262 94 DNA
Human Papillomavirus 262 tatttaataa accatattgg ttgcaacgtg
cacaaggcca taataatggt atttgttgga 60 gtaaccaatt gtttgttact
gtagttgata caac 94 263 65 DNA Human Papillomavirus 263 gcacaaggcc
ataataatgg tatttgttgg agtaaccaat tgtttgttac tgtagttgat 60 acaac 65
264 94 DNA Human Papillomavirus 264 tatttaataa gccttattgg
ctacataagg cccagggcca caacaatggt atatgttggc 60 ataatcaatt
atttcttact gttgtggaca ctac 94 265 65 DNA Human Papillomavirus 265
gcccagggcc acaacaatgg tatatgttgg cataatcaat tatttcttac tgttgtggac
60 accac 65 266 94 DNA Human Papillomavirus 266 tatttaacaa
gccattgtgg atacaaaagg cccagggcca taacaatggc atatgttttg 60
gcaatcagtt atttgttaca gttgtagaca ccac 94 267 65 DNA Human
Papillomavirus 267 gcccagggcc ataacaatgg catatgtttt ggcaatcagt
tatttgttac agttgtagac 60 accac 65 268 94 DNA Human Papillomavirus
268 tatttaataa accatattgg ttacaacaag cacaaggaca caataatggt
atatgttggg 60 gaaatcagct atttttaact gtggttgata ctac 94 269 65 DNA
Human Papillomavirus 269 gcacaaggac acaataatgg tatatgttgg
ggaaatcagc tatttttaac tgtggttgat 60 actac 65 270 59 DNA Human
Papillomavirus 270 ggacataata atggcatttg ttttgggaat cagttgtttg
ttacagtggt agataccac 59 271 59 DNA Human Papillomavirus 271
ggacataata atggcatttg ttttgggaat cagttgtttg ttacagtggt agataccac 59
272 65 DNA Human Papillomavirus 272 gcgcagggcc acaataatgg
tatttgttgg ggaaatcagt tatttgttac tgttgtagat 60 actac 65 273 65 DNA
Human Papillomavirus 273 gcgcagggcc acaataatgg tatttgttgg
ggaaatcagt tatttgttac tgttgtagat 60 actac 65 274 94 DNA Human
Papillomavirus 274 tatttaataa gccatattgg ttacataagg cccagggcca
taacaatggt atttgttggc 60 ataatcagtt gtttgttact gtagtggaca ctac 94
275 65 DNA Human Papillomavirus 275 gcccagggcc ataacaatgg
tatttgttgg cataatcagt tgtttgttac tgtagtggac 60 actac 65 276 94 DNA
Human Papillomavirus 276 tttttaataa gccttattgg ctccaccgtg
cgcagggtca caataatggc atttgctgga 60 acaatcagct ttttattacc
tgtgttgata ctac 94 277 65 DNA Human Papillomavirus 277 gcgcagggtc
acaataatgg catttgctgg aacaatcagc tttttattac ctgtgttgat 60 actac 65
278 94 DNA Human Papillomavirus 278 tatttaataa accgtactgg
ttacaacgtg cgcagggcca caataatggc atatgttggg 60 gcaatcagtt
gtttgtcaca gttgtggata ccac 94 279 65 DNA Human Papillomavirus 279
gcgcagggcc acaataatgg catatgttgg ggcaatcagt tgtttgtcac agttgtggat
60 accac 65 280 94 DNA Human Papillomavirus 280 tgtttaataa
gccatattgg ctgcaacgtg cccagggaca taataatggc atctgttgga 60
acaatcagtt atttgtaact gttgtggata ccac 94 281 65 DNA Human
Papillomavirus 281 gcccagggac ataataatgg catctgttgg aacaatcagt
tatttgtaac tgttgtggat 60 accac 65 282 94 DNA Human Papillomavirus
282 tatttaataa gccatactgg ttacaacggg cccagggtca aaacaatggt
atttgttggg 60 gcaatcaggt gtttttaaca gttgtagata ccac 94 283 65 DNA
Human Papillomavirus 283 gcccagggtc aaaacaatgg tatttgttgg
ggcaatcagg tgtttttaac agttgtagat 60 accac 65 284 65 DNA Human
Papillomavirus 284 gcgcagggcc acaataatgg tatttgttgg gggaatcagt
tatttgttac tgttgtagat 60 actac 65 285 65 DNA Human Papillomavirus
285 gcgcagggcc acaataatgg tatttgttgg gggaatcagt tatttgttac
tgttgtagat 60 actac 65 286 94 DNA Human Papillomavirus 286
tatttaataa accttattgg ttgcaacgtg cccaaggcca taataatggc atttgctggg
60 gtaatcaatt atttgttact gtagtagata ctac 94 287 65 DNA Human
Papillomavirus 287 gcccaaggcc ataataatgg catttgctgg ggtaatcaat
tatttgttac tgtagtagat 60 actac 65 288 94 DNA Human Papillomavirus
288 tatttaataa gccttattgg ctacagcgtg cacaaggtca taacaatggc
atttgctggg 60 gcaatcagtt atttgttacc gtggttgata ccac 94 289 65 DNA
Human Papillomavirus 289 gcacaaggtc ataacaatgg catttgctgg
ggcaatcagt tatttgttac cgtggttgat 60 accac 65 290 94 DNA Human
Papillomavirus 290 tatttaataa accatattgg ctgcacaagg ctcagggttt
aaacaatggt atatgttggc 60 acaatcaatt gtttttaaca gttgtagata ctac 94
291 65 DNA Human Papillomavirus 291 gctcagggtt taaacaatgg
tatatgttgg cacaatcaat tgtttttaac agttgtagat 60 actac 65 292 65 DNA
Human Papillomavirus 292 gcccagggcc acaacaatgg tatttgttgg
tttaatgaat tgtttgtaac cgttgtggat 60 accac 65 293 65 DNA Human
Papillomavirus 293 gcccagggcc acaacaatgg tatttgttgg tttaatgaat
tgtttgtaac cgttgtggat 60 accac 65 294 65 DNA Human Papillomavirus
294 gcacagggtc ataataatgg tatttgttgg tttaatgaac tgtttgttac
tgtggtggat 60 actac 65 295 65 DNA Human Papillomavirus 295
gcacagggtc ataataatgg tatttgttgg tttaatgaac tgtttgttac tgtggtggat
60 actac 65 296 65 DNA Human Papillomavirus 296 gcacagggac
ataacaatgg aatttgttgg cataatcaac tgtttctaac tgttgtatat 60 actac 65
297 65 DNA Human Papillomavirus 297 gcacagggac ataacaatgg
aatttgttgg cataatcaac tgtttctaac tgttgtatat 60 actac 65 298 65 DNA
Human Papillomavirus 298 gcacagggtc ataataatgg catatgctgg
ggtaatcagg tatttgttac tgttgtggat 60 actac 65 299 65 DNA Human
Papillomavirus 299 gcacagggtc ataataatgg catatgctgg ggtaatcagg
tatttgttac tgttgtggat 60 actac 65 300 65 DNA Human Papillomavirus
300 gcccagggac ataacaatgg tatatgctgg ggtaatcaaa tatttgttac
tgttgtagac 60 actac 65 301 65 DNA Human Papillomavirus 301
gcccagggac ataacaatgg tatatgctgg ggtaatcaaa tatttgttac tgttgtagac
60 actac 65 302 94 DNA Human Papillomavirus 302 tatttaacaa
gccctattgg ctgcacaagg cacagggaca caacaatggt atttgttggc 60
ataatcaatt atttcttact gttgtggata ccac 94 303 65 DNA Human
Papillomavirus 303 gcacagggac acaacaatgg tatttgttgg cataatcaat
tatttcttac tgttgtggat 60 accac 65 304 65 DNA Human Papillomavirus
304 gcacagggac ataacaatgg catttgttgg ggcaaccaat tgtttgttac
ttgtgtagat 60 actac 65 305 65 DNA Human Papillomavirus 305
gcacagggac ataacaatgg catttgttgg ggcaaccaat tgtttgttac ttgtgtagat
60 actac 65 306 94 DNA Human Papillomavirus 306 tgtttaataa
gccatattgg ctacaaaaag cccagggaca taacaatggt atttgttggg 60
gtaatcaact gtttgttact gtggtagata ccac 94 307 65 DNA Human
Papillomavirus 307 gcccagggaa ctaataatgg catttgttgg cataaccagt
tgtttattac tgtggtggac 60 actac 65 308 65 DNA Human Papillomavirus
308 gcccagggtc ataataatgg catctgttgg tttaatgagc tttttgtgac
agttgtagat 60 actac 65 309 65 DNA Human Papillomavirus 309
gcacagggtc ataataatgg tatttgttgg cataatcaat tatttttaac tgttgtagat
60 actac 65 310 94 DNA Human Papillomavirus 310 tgtttaataa
gccgttttgg ctgcaaaggg cgcaaggcca caataatggt atttgttggg 60
gtaatcaatt atttgttaca gttgtggata ccac 94 311 65 DNA Human
Papillomavirus 311 gcgcaaggcc acaataatgg tatttgttgg ggtaatcaat
tatttgttac agttgtggat 60 accac 65 312 94 DNA Human Papillomavirus
312 tattcaataa accttattgg ttacaacgag cacagggcca caataatggc
atttgttggg 60 gtaaccaact atttgttact gttgttgata ctac 94 313 65 DNA
Human Papillomavirus 313 gcacagggcc acaataatgg catttgttgg
ggtaaccaac tatttgttac tgttgttgat 60 actac
65 314 20 DNA Human Papillomavirus 314 gcmcagggwc ataayaatgg 20 315
65 DNA Human Papillomavirus 315 gcacagggac ataataatgg catttgctgg
aataatcagc tttttattac ttgtgttgac 60 actac 65 316 65 DNA Human
Papillomavirus 316 gcacagggac ataataatgg catttgctgg aataatcagc
tttttattac ttgtgttgac 60 actac 65 317 65 DNA Human Papillomavirus
317 gcccagggac ataataatgg catttgttgg tttaatgagt tatttgttac
agttgtagat 60 actac 65 318 65 DNA Human Papillomavirus 318
gcccagggac ataataatgg catttgttgg tttaatgagt tatttgttac agttgtagat
60 actac 65 319 65 DNA Human Papillomavirus 319 gcgcggggtc
ataacaatgg tatatgctgg tttaatcaat tgtttgtcac ggtggtggat 60 accac 65
320 65 DNA Human Papillomavirus 320 gcgcggggtc ataacaatgg
tatatgctgg tttaatcaat tgtttgtcac ggtggtggat 60 accac 65 321 20 DNA
Human Papillomavirus 321 tattcaataa accttattgg 20 322 20 DNA Human
Papillomavirus 322 tgtttaataa accatattgg 20 323 20 DNA Human
Papillomavirus 323 tttttaataa accatattgg 20 324 20 DNA Human
Papillomavirus 324 tatttaataa gccatattgg 20 325 20 DNA Human
Papillomavirus 325 tatttaataa accatattgg 20 326 20 DNA Human
Papillomavirus 326 tatttaataa gccttattgg 20 327 20 DNA Human
Papillomavirus 327 tatttaataa gccatattgg 20 328 20 DNA Human
Papillomavirus 328 tttttaataa gccttattgg 20 329 20 DNA Human
Papillomavirus 329 tatttaataa accgtactgg 20 330 20 DNA Human
Papillomavirus 330 tatttaataa accttattgg 20 331 20 DNA Human
Papillomavirus 331 tatttaataa gccttattgg 20 332 20 DNA Human
Papillomavirus 332 tgtttaataa gccatattgg 20 333 20 DNA Human
Papillomavirus 333 tatttaataa accatattgg 20 334 20 DNA Human
Papillomavirus 334 tttttaataa gccttattgg 20 335 20 DNA Human
Papillomavirus 335 tatttaacaa gccattgtgg 20 336 20 DNA Human
Papillomavirus 336 tatttaataa accatattgg 20 337 20 DNA Human
Papillomavirus 337 tgtttaataa gccatattgg 20 338 20 DNA Human
Papillomavirus 338 tatttaataa gccatactgg 20 339 20 DNA Human
Papillomavirus 339 tatttaataa accatattgg 20 340 20 DNA Human
Papillomavirus 340 tatttaacaa gccctattgg 20 341 20 DNA Human
Papillomavirus 341 tgtttaataa gccgttttgg 20 342 20 DNA Human
Papillomavirus 342 gcacagggcc acaataatgg 20 343 20 DNA Human
Papillomavirus 343 gcacagggtc ataacaatgg 20 344 20 DNA Human
Papillomavirus 344 gctcagggac acaataatgg 20 345 20 DNA Human
Papillomavirus 345 gcacaaggtc ataataatgg 20 346 20 DNA Human
Papillomavirus 346 gcacaaggcc ataataatgg 20 347 20 DNA Human
Papillomavirus 347 gcccagggcc acaacaatgg 20 348 20 DNA Human
Papillomavirus 348 gcccagggcc ataacaatgg 20 349 20 DNA Human
Papillomavirus 349 gcgcagggtc acaataatgg 20 350 20 DNA Human
Papillomavirus 350 gcgcagggcc acaataatgg 20 351 20 DNA Human
Papillomavirus 351 gcccaaggcc ataataatgg 20 352 20 DNA Human
Papillomavirus 352 gcacaaggtc ataacaatgg 20 353 20 DNA Human
Papillomavirus 353 gcacagggtc ataataatgg 20 354 20 DNA Human
Papillomavirus 354 gcacagggac ataacaatgg 20 355 20 DNA Human
Papillomavirus 355 gcccagggac ataacaatgg 20 356 20 DNA Human
Papillomavirus 356 gctcagggac ataacaatgg 20 357 20 DNA Human
Papillomavirus 357 gcccagggac aaaacaatgg 20 358 20 DNA Human
Papillomavirus 358 gcccagggcc ataacaatgg 20 359 20 DNA Human
Papillomavirus 359 gcacaaggac acaataatgg 20 360 14 DNA Human
Papillomavirus 360 ggacataata atgg 14 361 20 DNA Human
Papillomavirus 361 gcgcagggcc acaataatgg 20 362 20 DNA Human
Papillomavirus 362 gcccagggac ataataatgg 20 363 20 DNA Human
Papillomavirus 363 gcccagggtc aaaacaatgg 20 364 20 DNA Human
Papillomavirus 364 gcgcagggcc acaataatgg 20 365 20 DNA Human
Papillomavirus 365 gctcagggtt taaacaatgg 20 366 20 DNA Human
Papillomavirus 366 gcccagggcc acaacaatgg 20 367 20 DNA Human
Papillomavirus 367 gcacagggtc ataataatgg 20 368 20 DNA Human
Papillomavirus 368 gcacagggac ataacaatgg 20 369 20 DNA Human
Papillomavirus 369 gcccagggac ataacaatgg 20 370 20 DNA Human
Papillomavirus 370 gcacagggac acaacaatgg 20 371 20 DNA Human
Papillomavirus 371 gcgcaaggcc acaataatgg 20 372 20 DNA Human
Papillomavirus 372 gcacagggac ataataatgg 20 373 20 DNA Human
Papillomavirus 373 gcccagggac ataataatgg 20 374 20 DNA Human
Papillomavirus 374 gcgcggggtc ataacaatgg 20 375 23 DNA Human
Papillomavirus 375 tttgttactg ttgttgatac tac 23 376 23 DNA Human
Papillomavirus 376 tttgttactg tggtagatac cac 23 377 23 DNA Human
Papillomavirus 377 tttgttactg tggtagatac cac 23 378 23 DNA Human
Papillomavirus 378 tttgttactg tggtagatac cac 23 379 23 DNA Human
Papillomavirus 379 tttgttactg tagttgatac aac 23 380 23 DNA Human
Papillomavirus 380 tttcttactg ttgtggacac tac 23 381 23 DNA Human
Papillomavirus 381 tttgttactg tagtggacac tac 23 382 23 DNA Human
Papillomavirus 382 tttattacct gtgttgatac tac 23 383 23 DNA Human
Papillomavirus 383 tttgtcacag ttgtggatac cac 23 384 23 DNA Human
Papillomavirus 384 tttgttactg tagtagatac tac 23 385 23 DNA Human
Papillomavirus 385 tttgttaccg tggttgatac cac 23 386 23 DNA Human
Papillomavirus 386 tttgttactg ttgtggatac tac 23 387 23 DNA Human
Papillomavirus 387 tttgttactt gtgtagatac tac 23 388 23 DNA Human
Papillomavirus 388 tttgttactg tggtagatac cac 23 389 23 DNA Human
Papillomavirus 389 tttgttactg tggtagatac cac 23 390 23 DNA Human
Papillomavirus 390 tttttaactg ttgtagatac tac 23 391 23 DNA Human
Papillomavirus 391 tttgttacag ttgtagacac cac 23 392 23 DNA Human
Papillomavirus 392 tttttaactg tggttgatac tac 23 393 23 DNA Human
Papillomavirus 393 tttgttacag tggtagatac cac 23 394 23 DNA Human
Papillomavirus 394 tttgttactg ttgtagatac tac 23 395 23 DNA Human
Papillomavirus 395 tttgtaactg ttgtggatac cac 23 396 23 DNA Human
Papillomavirus 396 tttttaacag ttgtagatac cac 23 397 23 DNA Human
Papillomavirus 397 tttgttactg ttgtagatac tac 23 398 23 DNA Human
Papillomavirus 398 tttttaacag ttgtagatac tac 23 399 23 DNA Human
Papillomavirus 399 tttgtaaccg ttgtggatac cac 23 400 23 DNA Human
Papillomavirus 400 tttgttactg tggtggatac tac 23 401 23 DNA Human
Papillomavirus 401 tttctaactg ttgtatatac tac 23 402 23 DNA Human
Papillomavirus 402 tttgttactg ttgtagacac tac 23 403 23 DNA Human
Papillomavirus 403 tttcttactg ttgtggatac cac 23 404 23 DNA Human
Papillomavirus 404 tttgttacag ttgtggatac cac 23 405 23 DNA Human
Papillomavirus 405 tttattactt gtgttgacac tac 23 406 23 DNA Human
Papillomavirus 406 tttgttacag ttgtagatac tac 23 407 23 DNA Human
Papillomavirus 407 tttgtcacgg tggtggatac cac 23 408 22 DNA Human
Papillomavirus 408 catttgttgg ggtaaccaac ta 22 409 22 DNA Human
Papillomavirus 409 tgtttgctgg cataatcaat ta 22 410 22 DNA Human
Papillomavirus 410 tatttgttgg ggcaatcagt ta 22 411 22 DNA Human
Papillomavirus 411 tatttgttgg ggcaatcagg ta 22 412 22 DNA Human
Papillomavirus 412 tatttgttgg agtaaccaat tg 22 413 22 DNA Human
Papillomavirus 413 tatatgttgg cataatcaat ta 22 414 22 DNA Human
Papillomavirus 414 tatttgttgg cataatcagt tg 22 415 22 DNA Human
Papillomavirus 415 catttgctgg aacaatcagc tt 22 416 22 DNA Human
Papillomavirus 416 catatgttgg ggcaatcagt tg 22 417 22 DNA Human
Papillomavirus 417 catttgctgg ggtaatcaat ta 22 418 22 DNA Human
Papillomavirus 418 catttgctgg ggcaatcagt ta 22 419 22 DNA Human
Papillomavirus 419 catatgctgg ggtaatcagg ta 22 420 22 DNA Human
Papillomavirus 420 catttgttgg ggcaaccaat tg 22 421 22 DNA Human
Papillomavirus 421 tatttgttgg ggtaatcaac tg 22 422 22 DNA Human
Papillomavirus 422 tatttgctgg ggaaaccact tg 22 423 22 DNA Human
Papillomavirus 423 catttgctgg cataatcaac tg 22 424 22 DNA Human
Papillomavirus 424 catatgtttt ggcaatcagt ta 22 425 22 DNA Human
Papillomavirus 425 tatatgttgg ggaaatcagc ta 22 426 22 DNA Human
Papillomavirus 426 catttgtttt gggaatcagt tg 22 427 22 DNA Human
Papillomavirus 427 tatttgttgg ggaaatcagt ta 22 428 22 DNA Human
Papillomavirus 428 catctgttgg aacaatcagt ta 22 429 22 DNA Human
Papillomavirus 429 tatttgttgg ggcaatcagg tg 22 430 22 DNA Human
Papillomavirus 430 tatttgttgg gggaatcagt ta 22 431 22 DNA Human
Papillomavirus 431 tatatgttgg cacaatcaat tg 22 432 22 DNA Human
Papillomavirus 432 tatttgttgg tttaatgaat tg 22 433 22 DNA Human
Papillomavirus 433 tatttgttgg tttaatgaac tg 22 434 22 DNA Human
Papillomavirus 434 aatttgttgg cataatcaac tg 22 435 22 DNA Human
Papillomavirus 435 tatatgctgg ggtaatcaaa ta 22 436 22 DNA Human
Papillomavirus 436 tatttgttgg cataatcaat ta 22 437 22 DNA Human
Papillomavirus 437 tatttgttgg ggtaatcaat ta 22 438 22 DNA Human
Papillomavirus 438 catttgctgg aataatcagc tt 22 439 22 DNA Human
Papillomavirus 439 catttgttgg tttaatgagt ta 22 440 22 DNA Human
Papillomavirus 440 tatatgctgg tttaatcaat tg 22 441 65 DNA Human
Papillomavirus 441 gcacagggcc acaataatgg catttgttgg ggtaaccaac
tatttgttac tgttgttgat 60 actac 65 442 65 DNA Human Papillomavirus
442 gcacagggtc ataacaatgg tgtttgctgg cataatcaat tatttgttac
tgtggtagat 60 accac 65 443 65 DNA Human Papillomavirus 443
gctcagggac acaataatgg tatttgttgg ggcaatcagt tatttgttac tgtggtagat
60 accac 65 444 65 DNA Human Papillomavirus 444 gcacaaggtc
ataataatgg tatttgttgg ggcaatcagg tatttgttac tgtggtagat 60 accac 65
445 65 DNA Human Papillomavirus 445 gcacaaggcc ataataatgg
tatttgttgg agtaaccaat tgtttgttac tgtagttgat 60 acaac 65 446 65 DNA
Human Papillomavirus 446 gcccagggcc acaacaatgg tatatgttgg
cataatcaat tatttcttac tgttgtggac 60 actac 65 447 65 DNA Human
Papillomavirus 447 gcccagggcc ataacaatgg tatttgttgg cataatcagt
tgtttgttac tgtagtggac 60 actac 65 448 65 DNA Human Papillomavirus
448 gcgcagggtc acaataatgg catttgctgg aacaatcagc tttttattac
ctgtgttgat 60 actac 65 449 65 DNA Human Papillomavirus 449
gcgcagggcc acaataatgg catatgttgg ggcaatcagt tgtttgtcac agttgtggat
60 accac 65 450 65 DNA Human Papillomavirus 450 gcccaaggcc
ataataatgg catttgctgg ggtaatcaat tatttgttac tgtagtagat 60 actac 65
451 65 DNA Human Papillomavirus 451 gcacaaggtc ataacaatgg
catttgctgg ggcaatcagt tatttgttac cgtggttgat 60 accac 65 452 65 DNA
Human Papillomavirus 452 gcacagggtc ataataatgg catatgctgg
ggtaatcagg tatttgttac tgttgtggat 60 actac 65 453 65 DNA Human
Papillomavirus 453 gcacagggac ataacaatgg catttgttgg ggcaaccaat
tgtttgttac ttgtgtagat 60 actac 65 454 65 DNA Human Papillomavirus
454 gcccagggac ataacaatgg tatttgttgg ggtaatcaac tgtttgttac
tgtggtagat 60 accac 65 455 65 DNA Human Papillomavirus 455
gctcagggac ataacaatgg tatttgctgg ggaaaccact tgtttgttac tgtggtagat
60 accac 65 456 65 DNA Human Papillomavirus 456 gcccagggac
aaaacaatgg catttgctgg cataatcaac tgtttttaac tgttgtagat 60 actac 65
457 65 DNA Human Papillomavirus 457 gcccagggcc ataacaatgg
catatgtttt ggcaatcagt tatttgttac agttgtagac 60 accac 65 458 65 DNA
Human Papillomavirus 458 gcacaaggac acaataatgg tatatgttgg
ggaaatcagc tatttttaac tgtggttgat 60 actac 65 459 59 DNA Human
Papillomavirus 459 ggacataata atggcatttg ttttgggaat cagttgtttg
ttacagtggt agataccac 59 460 65 DNA Human Papillomavirus 460
gcgcagggcc acaataatgg tatttgttgg ggaaatcagt tatttgttac tgttgtagat
60 actac 65 461 65 DNA Human Papillomavirus 461 gcccagggac
ataataatgg catctgttgg aacaatcagt tatttgtaac tgttgtggat 60 accac 65
462 65 DNA Human Papillomavirus 462 gcccagggtc aaaacaatgg
tatttgttgg ggcaatcagg tgtttttaac agttgtagat 60 accac
65 463 65 DNA Human Papillomavirus 463 gcgcagggcc acaataatgg
tatttgttgg gggaatcagt tatttgttac tgttgtagat 60 actac 65 464 65 DNA
Human Papillomavirus 464 gctcagggtt taaacaatgg tatatgttgg
cacaatcaat tgtttttaac agttgtagat 60 actac 65 465 65 DNA Human
Papillomavirus 465 gcccagggcc acaacaatgg tatttgttgg tttaatgaat
tgtttgtaac cgttgtggat 60 accac 65 466 65 DNA Human Papillomavirus
466 gcacagggtc ataataatgg tatttgttgg tttaatgaac tgtttgttac
tgtggtggat 60 actac 65 467 65 DNA Human Papillomavirus 467
gcacagggac ataacaatgg aatttgttgg cataatcaac tgtttctaac tgttgtatat
60 actac 65 468 65 DNA Human Papillomavirus 468 gcccagggac
ataacaatgg tatatgctgg ggtaatcaaa tatttgttac tgttgtagac 60 actac 65
469 65 DNA Human Papillomavirus 469 gcacagggac acaacaatgg
tatttgttgg cataatcaat tatttcttac tgttgtggat 60 accac 65 470 65 DNA
Human Papillomavirus 470 gcgcaaggcc acaataatgg tatttgttgg
ggtaatcaat tatttgttac agttgtggat 60 accac 65 471 65 DNA Human
Papillomavirus 471 gcacagggac ataataatgg catttgctgg aataatcagc
tttttattac ttgtgttgac 60 actac 65 472 65 DNA Human Papillomavirus
472 gcccagggac ataataatgg catttgttgg tttaatgagt tatttgttac
agttgtagat 60 actac 65 473 65 DNA Human Papillomavirus 473
gcgcggggtc ataacaatgg tatatgctgg tttaatcaat tgtttgtcac ggtggtggat
60 accac 65 474 20 DNA Human Papillomavirus 474 gcccagggac
acaataatgg 20 475 20 DNA Human Papillomavirus 475 gcacagggtc
ataataatgg 20 476 20 DNA Human Papillomavirus 476 gcacagggac
acaataatgg 20 477 20 DNA Human Papillomavirus 477 gcccagggaa
ctaataatgg 20 478 20 DNA Human Papillomavirus 478 gcccagggtc
ataataatgg 20 479 20 DNA Human Papillomavirus 479 gcacagggtc
ataataatgg 20 480 23 DNA Human Papillomavirus 480 tttgttactg
tagttgatac tac 23 481 23 DNA Human Papillomavirus 481 tttgttacct
gtgttgatac cac 23 482 23 DNA Human Papillomavirus 482 tttgttactg
ttgtggacac cac 23 483 23 DNA Human Papillomavirus 483 tttattactg
tggtggacac tac 23 484 23 DNA Human Papillomavirus 484 tttgtgacag
ttgtagatac tac 23 485 23 DNA Human Papillomavirus 485 tttttaactg
ttgtagatac tac 23 486 22 DNA Human Papillomavirus 486 tatatgttgg
ggcaatcact tg 22 487 22 DNA Human Papillomavirus 487 tatctgttgg
ggcaatcaat tg 22 488 22 DNA Human Papillomavirus 488 catttgttgg
ggcaaccagg ta 22 489 22 DNA Human Papillomavirus 489 catttgttgg
cataaccagt tg 22 490 22 DNA Human Papillomavirus 490 catctgttgg
tttaatgagc tt 22 491 22 DNA Human Papillomavirus 491 tatttgttgg
cataatcaat ta 22 492 65 DNA Human Papillomavirus 492 gcccagggac
acaataatgg tatatgttgg ggcaatcact tgtttgttac tgtagttgat 60 actac 65
493 65 DNA Human Papillomavirus 493 gcacagggtc ataataatgg
tatctgttgg ggcaatcaat tgtttgttac ctgtgttgat 60 accac 65 494 65 DNA
Human Papillomavirus 494 gcacagggac acaataatgg catttgttgg
ggcaaccagg tatttgttac tgttgtggac 60 accac 65 495 65 DNA Human
Papillomavirus 495 gcccagggaa ctaataatgg catttgttgg cataaccagt
tgtttattac tgtggtggac 60 actac 65 496 65 DNA Human Papillomavirus
496 gcccagggtc ataataatgg catctgttgg tttaatgagc tttttgtgac
agttgtagat 60 actac 65 497 65 DNA Human Papillomavirus 497
gcacagggtc ataataatgg tatttgttgg cataatcaat tatttttaac tgttgtagat
60 actac 65
* * * * *