U.S. patent application number 10/365034 was filed with the patent office on 2003-10-23 for hybridization assay probes and methods for detecting the presence of neisseria meningitidis subtypes a,c and l in a sample.
Invention is credited to Bee, Gary, McDonough, Sherrol, Yang, Yeasing.
Application Number | 20030198984 10/365034 |
Document ID | / |
Family ID | 23924852 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030198984 |
Kind Code |
A1 |
Yang, Yeasing ; et
al. |
October 23, 2003 |
Hybridization assay probes and methods for detecting the presence
of Neisseria meningitidis subtypes A,C and L in a sample
Abstract
The present invention discloses hybridization assay probes,
amplification primers, nucleic acid compositions and methods useful
for detecting Neisseria nucleic acids. Hybridization assay probes
and amplification primers that selectively detect Neisseria
meningitidis and distinguish those Neisseria meningitidis from
Neisseria gonorrohoeae are disclosed. Other hybridization probes
selectively detect Neisseria gonorrohoeae and not Neisseria
meningitidis are also described.
Inventors: |
Yang, Yeasing; (San Diego,
CA) ; Bee, Gary; (Vista, CA) ; McDonough,
Sherrol; (San Diego, CA) |
Correspondence
Address: |
GEN PROBE INCORPORATED
10210 GENETIC CENTER DRIVE
SAN DIEGO
CA
92121
|
Family ID: |
23924852 |
Appl. No.: |
10/365034 |
Filed: |
February 12, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10365034 |
Feb 12, 2003 |
|
|
|
09394175 |
Sep 13, 1999 |
|
|
|
6541201 |
|
|
|
|
09394175 |
Sep 13, 1999 |
|
|
|
08962369 |
Oct 31, 1997 |
|
|
|
6100027 |
|
|
|
|
08962369 |
Oct 31, 1997 |
|
|
|
08484607 |
Jun 7, 1995 |
|
|
|
5747252 |
|
|
|
|
Current U.S.
Class: |
435/6.15 ;
536/24.3 |
Current CPC
Class: |
C12Q 1/689 20130101 |
Class at
Publication: |
435/6 ;
536/24.3 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
1. A hybridization assay probe for use in detecting the presence of
Neisseria meningitidis in a sample, said probe comprising a base
sequence region up to 100 bases in length which is able to form a
detectable hybrid with a first target nucleic acid region present
in nucleic acid from or derived from Neisseria meningitidis
subtypes A, C and L and which is unable to form a detectable hybrid
with nucleic acid from Neisseria gonorrhoeae under stringent
hybridization assay conditions, wherein said first target region
has a sequence selected from the group consisting of SEQ ID NO. 11,
SEQ ID NO. 15, SEQ ID NO. 25 and SEQ ID NO. 27.
2. The probe of claim 1, wherein the base sequence of said base
sequence region is at least 80% complementary to the base sequence
of said first target region.
3. The probe of claim 1, wherein the base sequence of said base
sequence region is fully complementary to the base sequence of said
first target region.
4. The probe of claim 1, wherein the base sequence of said probe is
at least 80% complementary to the base sequence of said first
target region.
5. The probe of claim 1 further comprising a detectable label.
6. A composition comprising a nucleic acid hybrid formed between
said probe and said first target region of claim 1.
7. A composition comprising a nucleic acid hybrid formed between
said probe and said first target region of claim 2.
8. A composition comprising a nucleic acid hybrid formed between
said probe and said first target region of claim 3.
9. A composition comprising a nucleic acid hybrid formed between
said probe and said first target region of claim 4.
10. A probe mix comprising the probe of claim 1 and at least one
helper probe having from 12 to 100 bases which is able to hybridize
to a second target nucleic acid region present in the nucleic acid
containing said first target region under said conditions.
11. The probe mix of claim 10, wherein said second target region
has a sequence selected from the group consisting of SEQ ID NO. 13,
SEQ ID NO. 14, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 35, SEQ ID
NO. 36, SEQ ID NO. 37 and SEQ ID NO. 38.
12. The probe mix of claim 11, wherein the base sequence of said
helper probe is at least 80% complementary to the base sequence of
said second target region.
13. The probe mix of claim 12, wherein the base sequence of said
base sequence region is at least 80% complementary to the base
sequence of said first target region.
14. The probe mix of claim 12, wherein the base sequence of said
base sequence region is fully complementary to the base sequence of
said first target region
15. The probe mix of claim 12, wherein the base sequence of said
probe is at least 80% complementary to the base sequence of said
first target region.
16. The probe mix of claim 12, wherein said at least one helper
probe includes first and second helper probes, wherein the base
sequence of said second target region for said first helper probe
is selected from the group consisting of SEQ ID NO. 13, SEQ ID NO.
17, SEQ ID NO. 35 and SEQ ID NO. 37, and wherein the base sequence
of said second target region for said second helper probe is
selected from the group consisting of SEQ ID NO. 14, SEQ ID NO. 18,
SEQ ID NO. 36 and SEQ ID NO. 38.
17. The probe mix of claim 16, wherein the base sequence of said
base sequence region is at least 80% complementary to the base
sequence of said first target region.
18. The probe mix of claim 16, wherein the base sequence of said
base sequence region is fully complementary to the base sequence of
said first target region
19. The probe mix of claim 16, wherein the base sequence of said
probe is at least 80% complementary to the base sequence of said
first target region.
20. A kit for use in detecting the presence of Neisseria
meningitidis in a sample, said kit comprising: a hybridization
assay probe comprising a base sequence region which is able to form
a detectable hybrid with nucleic acid from Neisseria meningitidis
subtypes A, C and L and which is unable to form a detectable hybrid
with nucleic acid from Neisseria gonorrhoeae under stringent
hybridization assay conditions, wherein the base sequence of said
region is at least 80% homologous to the base sequence of a first
sequence selected from the group consisting of SEQ ID NO. 11, SEQ
ID NO. 15, SEQ ID NO. 25 and SEQ ID NO. 27, and wherein said probe
does not comprise any other base sequence region which is able to
form a detectable hybrid with nucleic acid from Neisseria
meningitidis subtype A, C or L or Neisseria gonorrhoeae under said
conditions; and at least one helper probe which is able to
hybridize to nucleic acid from Neisseria meningitidis under
stringent hybridization assay conditions, wherein the base sequence
of said helper probe is at least 80% homologous to a the base
sequence of a second sequence selected from the group consisting of
SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID
NO. 35, SEQ ID NO. 36, SEQ ID NO. 37 and SEQ ID NO. 38.
21. The kit of claim 20, wherein the base sequence of said region
is fully homologous to the base sequence of said first
sequence.
22. The kit of claim 20, wherein the base sequence of said probe is
at least 80% homologous to the base sequence of said first
sequence.
23. The kit of claim 20, wherein said at least one helper probe
includes first and second helper probes, wherein the base sequence
of said second sequence for said first helper probe is selected
from the group consisting of SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID
NO. 35 and SEQ ID NO. 37, and wherein the base sequence of said
second sequence for said second helper probe is selected from the
group consisting of SEQ ID NO. 14, SEQ ID NO. 18, SEQ ID NO. 36 and
SEQ ID NO. 38.
24. The kit of claim 23, wherein the base sequence of said region
is fully homologous to the base sequence of said first
sequence.
25. The kit of claim 23, wherein the base sequence of said probe is
at least 80% homologous to the base sequence of said first
sequence.
26. A kit for use in detecting the presence of Neisseria
meningitidis in a sample, said kit comprising: a hybridization
assay probe comprising a base sequence region which is able to form
a detectable hybrid with nucleic acid from Neisseria meningitidis
subtypes A, C and L and which is unable to form a detectable hybrid
with nucleic acid from Neisseria gonorrhoeae under stringent
hybridization assay conditions, wherein the base sequence of said
region is at least 80% homologous to the base sequence of a first
sequence selected from the group consisting of SEQ ID NO. 11, SEQ
ID NO. 15, SEQ ID NO. 25 and SEQ ID NO. 27, and wherein said probe
does not comprise any other base sequence region which is able to
form a detectable hybrid with nucleic acid from Neisseria
meningitidis subtype A, C or L or Neisseria gonorrhoeae under said
conditions; and at least one amplification oligonucleotide which is
able to bind to nucleic acid from Neisseria meningitidis under
amplification conditions, said amplification oligonucleotide
comprising a first base sequence which is at least 80% homologous
to the base sequence of a second sequence selected from the group
consisting of SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 43 and SEQ ID
NO. 45, wherein the base sequence of said amplification
oligonucleotide consists of said first base sequence and,
optionally, a 5' sequence which is recognized by an RNA polymerase
or which enhances initiation or elongation by an RNA
polymerase.
27. The kit of claim 26, wherein the base sequence of said region
is fully homologous to the base sequence of said first
sequence.
28. The kit of claim 27, wherein the base sequence of said probe is
at least 80% homologous to the base sequence of said first
sequence.
29. The kit of claim 26, wherein said at least one amplification
oligonucleotide includes first and second amplification
oligonucleotides, wherein the base sequence of said second sequence
for said first amplification oligonucleotide is SEQ ID NO. 7 or SEQ
ID NO. 43, and wherein the base sequence of said second sequence
for said second amplification oligonucleotide is SEQ ID NO. 9 or
SEQ ID NO. 45.
30. The kit of claim 29, wherein the base sequence of said region
is fully homologous to the base sequence of said first
sequence.
31. The kit of claim 32, wherein the base sequence of said probe is
at least 80% homologous to the base sequence of said first
sequence.
32. A method for detecting the presence of Neisseria meningitidis
subtypes A, C and L in a sample, said method comprising the steps
of: (a) contacting said sample with said probe of claim 1 under
stringent hybridization assay conditions; and (b) detecting the
presence of said probe as an indication of the presence of at least
one of Neisseria meningtidis subtypes A, C and L in said
sample.
33. The method of claim 32, wherein the base sequence of said base
sequence region is at least 80% complementary to the base sequence
of said first target region.
34. The method of claim 32, wherein the base sequence of said base
sequence region is fully complementary to the base sequence of said
first target region.
35. The method of claim 32, wherein the base sequence of said probe
is at least 80% complementary to the base sequence of said first
target region.
36. The method of claim 32 further comprising the step of providing
to said sample at least one amplification oligonucleotide able to
bind to nucleic acid from Neisseria meningitidis under
amplification conditions prior to said detecting step.
37. The method of claim 36, wherein said amplification
oligonucleotide comprises a first base sequence which is at least
80% homologous to the base sequence of a second sequence selected
from the group consisting of SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO.
43 and SEQ ID NO. 45, wherein the base sequence of said
amplification oligonucleotide consists of said first base sequence
and, optionally, a 5' sequence which is recognized by an RNA
polymerase or which enhances initiation or elongation by an RNA
polymerase.
38. The method of claim 37, wherein the base sequence of said base
sequence region is at least 80% complementary to the base sequence
of said first target region.
39. The method of claim 37, wherein the base sequence of said base
sequence region is fully complementary to the base sequence of said
first target region.
40. The method of claim 37, wherein the base sequence of said probe
is at least 80% complementary to the base sequence of said first
target region.
41. The method of claim 40, wherein said at least one amplification
oligonucleotide includes first and second amplification
oligonucleotides, wherein the base sequence of said second sequence
for said first amplification oligonucleotide is SEQ ID NO. 7 or SEQ
ID NO. 43, and wherein the base sequence of said second sequence
for said second amplification oligonucleotide is SEQ ID NO. 9 or
SEQ ID NO. 45.
Description
[0001] This application is a continuation of application Ser. No.
09/394,175, filed Sep. 13, 1999, now pending, the contents of which
are hereby incorporated by reference in their entirety, which is a
continuation of application Ser. No. 08/962,369, filed Oct. 31,
1997, now U.S. Pat. No. 6,100,027, which is a continuation of
application Ser. No. 08/484,607, filed Jun. 7, 1995, now U.S. Pat.
No. 5,747,252.
FIELD OF THE INVENTION
[0002] The inventions described and claimed herein relate to the
design and use of amplification oligonucleotides and nucleic acid
probes to Neisseria gonorrhoeae and Neisseria meningitidis which
allow detection of these organisms in test samples.
BACKGROUND OF THE INVENTION
[0003] The genus Neisseria includes two gram-negative species of
pyogenic cocci that are pathogenic for man, and that have no other
known reservoir: the meningococcus (Neisseria meningitidis) and the
gonococcus (Neisseria gonorrhoeae). A number of non-pathogenic
species also inhabit the upper respiratory tract of humans and may
be easily confused with meningococci. Meningococcal meningitis was
recognized as a contagious disease early in the 19th century and is
especially prevalent among military personnel. The causative agent
of meningococcal meningitis is Neisseria meningitidis.
[0004] Neisseria gonorrhoeae is one of the main causes of epidemic
sexually transmitted disease and is prevalent in the United States.
Infection with Neisseria gonorrhoeae causes many common symptoms
including urethritis, cervicitis, and proctitis. In addition,
chronic infection with Neisseria gonorrhoeae can cause pelvic
inflammatory disease.
[0005] Meningococci have polysaccharide-containing capsules.
Gonococcis may also possess capsules, but the exact chemical
composition of such a capsule is unknown. In addition, both
gonococci and meningococci may have pili which play a role in
virulence.
[0006] Meningococci and gonococci are difficult to cultivate and
require special techniques to grow the organisms from body fluids.
In addition, selective culture medium, (for example, Thayer-Martin
medium) and growth in 3-10% carbon dioxide at approximately
35.degree. C. is required to maximize the culture of organisms.
[0007] In addition to the difficult cultivation, the gonococcus and
meningococcus detection by immunoassay suffers a lack of
sensitivity and specificity. This appears to be due to the cross
reaction between various other pathogens and non-pathogenic
microorganisms often found in the same clinical specimens.
[0008] Oligonucleotides for the amplification of nucleic acid for
detection of Neisseria have been described. Bikenmeyer and
Armstrong, J. Clin. Microbiol. 30:3089-3094 (1992), describe probe
sets for use in the ligase chain reaction directed to the Opa and
pilin genes of Neisseria gonorrhoeae. Kristiansen et al. Lancet
340:1432-1434 (1992) describe primers directed to an insertion
element referred to as IS1106 for amplification and detection of
Neisseria meningitidis. McLaughlin et al., Mol. and Cell Probes
7:7-17 (1993) describe primers for use in the polymerase chain
reaction directed to the 16S-23S rRNA internal transcribed spacer
and a set of primers directed to a subregion of the 16S rRNA of
Neisseria meningitidis. Probes for the detection of rRNA or rDNA
sequences of Neisseria gonorrhoeae and/or Neisseria meningitidis
have been described by Granato and Franz J. Clin. Microbiol.
28:944-948, (1990), Wolff, U.S. Pat. No. 5,173,401 (Dec. 22, 1992),
Rossau and Van Heuverswijn, European Patent Application Publication
No. 0 337 896, Hogan et al. PCT/US87/03009, and Barns et al., U.S.
Pat. No. 5,217,862 (Jun. 8, 1993).
SUMMARY OF INVENTION
[0009] The featured invention discloses and claims novel and useful
amplification oligonucleotides, helper oligonucleotides, and
oligonucleotide hybridization assay probes which are designed to be
complementary to specific regions of the rRNA (ribosomal RNA) or
rDNA (ribosomal DNA) nucleotide sequences of Neisseria, or
oligonucleotides having a nucleic acid sequence substantially
corresponding to a specific portion of Neisseria rRNA or rDNA
nucleotide sequence or its complement. Because these amplification
oligonucleotides, helper oligonucleotides and hybridization assay
probes are derived from the rRNA of pathogenic Neisseria, a
superior detection assay is obtained due to the higher level of RNA
expressed from these rRNA genes and the lack of lateral transfer of
the rRNA sequences between organisms.
[0010] The amplification oligonucleotides and oligonucleotide
hybridization assay probes function by hybridizing to target
Neisseria 16S and 23S rRNA and/or rDNA gene sequences under
stringent hybridization assay conditions. In preferred embodiments,
the probes and amplification oligonucleotides described herein,
when used together, can distinguish Neisseria meningitidis from
other microorganisms found in clinical samples such as blood or
tissues and from Neisseria gonorrhoeae species. Accordingly, the
amplification oligonucleotides and hybridization assay probes may
be used in an assay to specifically detect and/or amplify Neisseria
meningitidis-derived nucleic acids. In preferred embodiments, the
hybridization assay probes described herein are able to selectively
hybridize to nucleic acids from Neisseria meningitidis over those
from Neisseria gonorrhoeae under stringent hybridization
conditions. In some embodiments of the present invention, the
hybridization assay probe comprises an oligonucleotide that
contains a reporter group such as an acridinium ester or a
radioisotope to help identify hybridization of the probe to its
target sequence. In some embodiments of the present invention, the
amplification oligonucleotide optionally has a nucleic acid
sequence recognized by an RNA polymerase or which enhances
transcription initiation by an RNA polymerase.
[0011] The present invention features hybridization assay probes
useful for detecting the presence of nucleic acids from Neisseria.
Preferably, the hybridization assay probes are selected from the
following nucleotide sequences:
1 GGCTGTTGCT AATACCAGCG: SEQ ID NO 12 CGCTGATATT AGCAACAGCC: SEQ ID
NO 15 CGCTGGTATT AGCAACAGCC: SEQ ID NO 16 GGCUGUUGCU AAUAUCAGCG:
SEQ ID NO 25 GGCUGUUGCU AAUACCAGCG: SEQ ID NO 26 CGCUGAUAUU
AGCAACAGCC: SEQ ID NO 27 CGCUGGUAUU AGCAACAGCC: SEQ ID NO 28
GAACGTACCG GGTAGCGG: SEQ ID NO 1 GCCAATATCG GCGGCCGATG: SEQ ID NO 3
CCGCTACCCG GTACGTTC: SEQ ID NO 29 CATCGGCCGC CGATATTGGC: SEQ ID NO
30 GAACGUACCG GGUAGCGG: SEQ ID NO 31 GCCAAUAUCG GCGGCCGAUG: SEQ ID
NO 32 CCGCUACCCG GUACGUUC: SEQ ID NO 33 CAUCGGCCGC CGAUAUUGGC: SEQ
ID NO 34
[0012] The present invention features hybridization assay probes
useful for detecting nucleic acids from Neisseria meningitidis.
These hybridization assay probes are preferably selected from the
following nucleotide sequences:
2 GGCTGTTGCT AATATCAGCG SEQ ID NO:11 GGCTGTTGCT AATACCAGCG SEQ ID
NO:12 CGCTGATATT AGCAACAGCC SEQ ID NO:15 CGCTGGTATT AGCAACAGCC SEQ
ID NO:16 GGCUGUUGCU AAUAUCAGCG SEQ ID NO:25 GGCUGUUGCU AAUACCAGCG
SEQ ID NO:26 CGCUGAUAUU AGCAACAGCC, and SEQ ID NO:27 CGCUGGUAUU
AGCAACAGCC. SEQ ID NO:28
[0013] The present invention also features hybridization assay
probes useful for detecting Neisseria gonorrhoeae nucleic acids.
Preferably, these hybridization assay probes have a nucleotide
sequence selected from one of the following nucleotide
sequences:
3 GAACGTACCG GGTAGCGG SEQ ID NO:1 GCCAATATCG GCGGCCGATG SEQ ID NO:3
CCGCTACCCG GTACGTTC SEQ ID NO:29 CATCGGCCGC CGATATTGGC SEQ ID NO:30
GAACGUACCG GGUAGCGG SEQ ID NO:31 GCCAAUAUCG GCGGCCGAUG SEQ ID NO:32
CCGCUACCCG GUACGUUC, and SEQ ID NO:33 CAUCGGCCGC CGAUAUUGGC. SEQ ID
NO:34
[0014] Another aspect of the present invention is a probe mix
comprising a hybridization assay probe of the present invention
together with a helper oligonucleotide (probe). Preferably, helper
oligonucleotides are used to facilitate the specific hybridization
of the assay probe to its target nucleic acid; helper
oligonucleotides are described by Hogan and Milliman U.S. Pat. No.
5,030,557 which is hereby incorporated by reference and enjoys
common ownership with the present invention. Oligonucleotides used
as helper probes in this invention include the following
sequences:
4 SEQ ID NO:2 GGGATAACTG ATCGAAAGAT CAGCTAATAC CGCATACG SEQ ID NO:4
ACGGTACCTG AAGAATAAGC ACCGGCTAAC TACGTG SEQ ID NO:39 GGGAUAACUG
AUCGAAAGAU CAGCUAAUAC CGCAUACG SEQ ID NO:40 ACGGUACCUG AAGAAUAAGC
ACCGGCUAAC UACGUG SEQ ID NO:13 GCCTTCGGGT TGTAAAGGAC TTTTGTCAGG
GAAGAAAA SEQ ID NO:14 GCTGATGACG GTACCTGAAG AATAAGCACC GGC SEQ ID
NO:35 GCCUUCGGGU UGUAAAGGAC UUUUGUCAGG GAAGAAAA SEQ ID NO:36
GCUGAUGACG GUACCUGAAG AAUAAGCACC GGC SEQ ID NO:17 TTTTCTTCCC
TGACAAAAGT CCTTTACAAC CCGAAGGC SEQ ID NO:18 GCCGGTGCTT ATTCTTCAGG
TACCGTCATC AGC SEQ ID NO:37 UUUUCUUCCC UGACAAAAGU CCUUUACAAC
CCGAAGGC, and SEQ ID NO:38 GCCGGUGCUU AUUCUUCAGG UACCGUCAUC AGC
[0015] Another aspect of the present invention includes
compositions for detecting Neisseria meningitidis and Neisseria
gonorrhoeae that are nucleic acid hybrids formed between an
oligonucleotide of the present invention and a specific region of a
nucleotide polymer from a Neisseria meningitidis or Neisseria
gonorrhoeae. Generally, the nucleotide polymer contains a nucleic
acid sequence that substantially corresponds to an oligonucleotide
sequence of the present invention or its complement and is derived
from the rRNA or the rDNA encoding the ribosomal RNA of the
Neisseria meningitidis or Neisseria gonorrhoeae. The
oligonucleotide present in these compositions may be an
amplification oligonucleotide, a helper oligonucleotide, a
hybridization assay probe, or a combination thereof. Thus,
compositions of the present invention may contain one or more
amplification oligonucleotides, one or more helper
oligonucleotides, and one or more hybridization assay probes.
[0016] The compositions of the present invention containing a probe
hybridized to its target sequence are useful for detecting the
presence of a nucleic acid sequence. Compositions of the present
invention containing a helper oligonucleotide hybridized to its
target nucleic acid sequence are useful for making a particular
portion of the target nucleic acid available for hybridization.
Compositions of the present invention containing an oligonucleotide
primer hybridized to its target sequence are useful for creating an
initiation site for a polymerase at the 3' end of the primer,
and/or providing a template for extension of the 3' end of the
target sequence.
[0017] The present invention also contemplates methods for
detecting the presence of Neisseria in which a test sample is
contacted with a nucleic acid hybridization assay probe under
stringent hybridization assay conditions wherein the nucleic acid
hybridization assay probe is capable of hybridizing to Neisseria
meningitidis target nucleic acid sequences and not to the nucleic
acid sequences from Neisseria gonorrhoeae. The present invention
also contemplates oligonucleotides and the equivalents thereof used
in these methods that optionally contain a reporter molecule that
aids in the identification of the hybridization of the probe to its
target sequence. This invention is useful for detecting the
presence of Neisseria nucleic acids in test samples from humans
such as blood, blood derived samples, tissues, tissue derived
samples, other body fluids and body samples.
[0018] The present invention also contemplates methods for
detecting the presence of Neisseria meningitidis in which the
nucleic acid is amplified using at least one amplification
oligonucleotide of the present invention. In preferred embodiments,
that amplification is then followed by a detection step in which
the amplified nucleic acid is detected using an oligonucleotide
hybridization assay probe of the present invention. The methods of
the present invention also contemplate the use of amplification
oligonucleotides which include the nucleotide sequence for an RNA
promoter.
[0019] In another aspect, the invention features amplification
oligonucleotides useful for detection of organisms of the genus
Neisseria in an amplification assay. Such oligomers preferably
substantially correspond to one of the following nucleotide
sequences:
5 SEQ ID NO:5 GTCCCCTGCT TTCCCTCTCA AGAC SEQ ID NO:6 GGCGAGTGGC
GAACGGGTGA GTAACATA SEQ ID NO:7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT
CTTCAG SEQ ID NO:8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG SEQ ID
NO:9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT SEQ ID NO:10
GAAGGCCTTC GGGTTGTAAA GGAC SEQ ID NO:41 GUCCCCUGCU UUCCCUCUCA AGAC
SEQ ID NO:42 GGCGAGUGGC GAACGGGUGA GUAACAUA SEQ ID NO:43 GCUGCUGCAC
GUAGUUAGCC GGUGCUUAUU CUUCAG SEQ ID NO:44 GUUAGCCGGU GCUUAUUCUU
CAGGUACCGU CAUCG SEQ ID NO:45 CGGGUUGUAA AGGACUUUUG UCAGGGAAGA
AAAGGCCGUU, and SEQ ID NO:46 GAAGGCCUUC GGGUUGUAAA GGAC
[0020] where the oligomer may be unmodified or contain a
modification such as addition of a specific nucleic acid sequence
to 5' terminus that is recognized by an RNA polymerase, (including
but not limited to the promoter sequence for T7, T3, or SP6 RNA
polymerase), and/or sequences which enhance initiation of RNA
transcription by an RNA polymerase. One example of a promoter
sequence includes the sequence SEQ ID NO. 53
5'-AATTTAATACGACTCACTATAGGGAGA-3'. Other examples of useful
promoter sequences are contained in various commercially available
vectors including, for example, pBluescript.RTM. vectors from
Stratagene Cloning Systems (San Diego, Calif.) or the pGEM.TM.
vectors from Promega Corp. (Madison, Wis.)
[0021] In another aspect of the present invention the amplification
oligonucleotides bind to or cause elongation through sequences
substantially corresponding to the following sequences:
6 SEQ ID NO:23 GTCTTGAGAG GGAAAGCAGG GGAC SEQ ID NO:24 TATGTTACTC
ACCCGTTCGC CACTCGCC SEQ ID NO:19 CTGAAGAATA AGCACCGGCT AACTACGTGC
AGCAGC SEQ ID NO:21 CGATGACGGT ACCTGAAGAA TAAGCACCGG CTAAC SEQ ID
NO:20 AACGGCCTTT TCTTCCCTGA CAAAAGTCCT TTACAACCCG SEQ ID NO:22
GTCCTTTACA ACCCGAAGGC CTTC SEQ ID NO:47 GUCUUGAGAG GGAAAGCAGG GGAC
SEQ ID NO:48 UAUGUUACUC ACCCGUUCGC CACUCGCC SEQ ID NO:49 CUGAAGAAUA
AGCACCGGCU AACUACGUGC AGCAGC SEQ ID NO:50 CGAUGACGGU ACCUGAAGAA
UAAGCACCGG CUAAC SEQ ID NO:51 AACGGCCUUU UCUUCCCUGA CAAAAGUCCU
UUACAACCCG SEQ ID NO:52 GUCCUUUACA ACCCGAAGGC CUUC
[0022] Another aspect of the present invention includes kits that
contain one or more of the oligonucleotides of the present
invention including amplification oligonucleotides, helper
oligonucleotides and hybridization assay probes. In preferred
embodiments, a kit of the present invention includes at least one
amplification oligonucleotide and one hybridization assay probe
capable of distinguishing Neisseria, Neisseria meningitidis or
Neisseria gonorrhoeae from other microorganisms.
[0023] Background descriptions of the use of nucleic acid
hybridization to detect particular nucleic acid sequences are given
in Kohne, U.S. Pat. No. 4,851,330 issued Jul. 25, 1989, and by
Hogan et al., International Patent Application No. PCT/US87/03009,
entitled "Nucleic Acid Probes for Detection and/or Quantitation of
Non-Viral Organisms", both references hereby incorporated by
reference herein. Hogan et al., supra, describe methods for
determining the presence of a non-viral organism or a group of
non-viral organisms in a sample (e.g., sputum, urine, blood and
tissue sections, food, soil and water).
[0024] In the most preferred embodiments, the compositions, probe
mixes, probes, amplification primers, helper oligonucleotides and
the like have a nucleotide sequence that consists of the specified
nucleic acid sequence rather than substantially corresponding to
the nucleic acid sequence. These most preferred embodiments use the
sequence listed in the sequence listing which forms part of the
present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0025] A. Definitions
[0026] The following terms have the indicated meanings in the
specification unless expressly indicated to have a different
meaning.
[0027] By "target nucleic acid" is meant a nucleic acid having a
target nucleotide sequence.
[0028] By "oligonucleotide" is meant a single-stranded nucleotide
polymer made of more than 2 nucleotide subunits covalently joined
together. Preferably between 10 and 100 nucleotide units are
present, most preferably between 12 and 50 nucleotides units are
joined together. The sugar groups of the nucleotide subunits may be
ribose, deoxyribose or modified derivatives thereof such as
2'-O-methyl ribose. The nucleotide subunits of an oligonucleotide
may be joined by phosphodiester linkages, phosphorothioate
linkages, methyl phosphonate linkages or by other rare or
non-naturally-occurring linkages that do not prevent hybridization
of the oligonucleotide. Furthermore, an oligonucleotide may have
uncommon nucleotides or non-nucleotide moieties. An oligonucleotide
as defined herein is a nucleic acid, preferably DNA, but may be RNA
or have a combination of ribo- and deoxyribonucleotides covalently
linked. Oligonucleotide probes and amplification oligonucleotides
of a defined sequence may be produced by techniques known to those
of ordinary skill in the art, such as by chemical or biochemical
synthesis, and by in vitro or in vivo expression from recombinant
nucleic acid molecules, e.g., bacterial or retroviral vectors. As
intended by this disclosure, an oligonucleotide does not consist of
wild-type chromosomal DNA or the in vivo transcription products
thereof. One use of a probe is as a hybridization assay probe;
probes may also be used as in vivo or in vitro therapeutic
amplification oligomers or antisense agents to block or inhibit
gene transcription, or translation in diseased, infected, or
pathogenic cells.
[0029] By "target nucleic acid sequence", "target nucleotide
sequence" or "target sequence" is meant a specific
deoxyribonucleotide or ribonucleotide sequence comprising all or a
part of the nucleotide sequence of a single-stranded nucleic acid
molecule, and the deoxyribonucleotide or ribonucleotide sequence
complementary thereto.
[0030] Nucleic acid hybridization is the process by which two
nucleic acid strands having completely or partially complementary
nucleotide sequences come together under predetermined reaction
conditions to form a stable, double-stranded hybrid with specific
hydrogen bonds. Either nucleic acid strand may be a
deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or an analog
of one of these nucleic acids; thus hybridization can involve
RNA:RNA hybrids, DNA:DNA hybrids, or RNA:DNA hybrids.
[0031] The term "hybridization" as used in this application, refers
to the ability of two completely or partly complementary single
nucleic acid strands to come together in an antiparallel
orientation to form a stable structure having a double-stranded
region. The two constituent strands of this double-stranded
structure, sometimes called a hybrid, are held together with
hydrogen bonds. Although these hydrogen bonds most commonly form
between nucleotides containing the bases adenine and thymine or
uracil (A and T or U) or cytosine and guanine (C and G) on single
nucleic acid strands, base pairing can form between bases who are
not members of these "canonical" pairs. Non-canonical base pairing
is well-known in the art. See e.g., The Biochemistry of the Nucleic
Acids (Adams et al., eds., 1992). "Stringent" hybridization assay
conditions refer to conditions wherein a specific hybridization
assay probe is able to hybridize with target nucleic acids
(preferably rRNA or rDNA of a Neisseria, Neisseria meningitidis or
Neisseria gonorrhoeae) over other nucleic acids present in the test
sample derived either from other microorganisms or from humans. It
will be appreciated that these conditions may vary depending upon
factors including the GC content and length of the probe, the
hybridization temperature, the composition of the hybridization
reagent or solution, and the degree of hybridization specificity
sought. Specific stringent hybridization conditions are provided in
the disclosure below.
[0032] As an example of specific stringent hybridization conditions
useful in detecting Neisseria, Neisseria meningitidis or Neisseria
gonorrhoeae, for the hybridization assay probes of this invention,
a set of preferred stringent hybridization assay conditions was
used. One preferred set comprised hybridizing the target nucleic
acid and hybridization probe together in 100 .mu.l of 0.05 M
lithium succinate (pH 5.0), 6 M LiCl, 1% (w/v) lithium lauryl
sulfate, (L.L.S.) 10 mM ethylene diamine tetraacetic acid (EDTA),
10 mM ethylene glycol bis (.beta.-amino ethyl ether) N,N,N',N'
tetraacetic acid (EGTA) at 60.degree. C. for 15 minutes, then
adding 300 .mu.l of 0.15 M sodium tetraborate (pH 8.5), 1% (v/v)
TRITON.RTM. X-100 at 60.degree. C. for 5-7 minutes. Additional sets
of stringent hybridization conditions can be determined after
reading the present disclosure by those or ordinary skill in the
art.
[0033] By "probe" is meant a single-stranded oligonucleotide having
a sequence partly or completely complementary to a nucleic acid
sequence sought to be detected, so as to hybridize thereto under
stringent hybridization conditions. The term "probe" is meant to
exclude nucleic acids normally existing in nature. Purified
oligonucleotide probes may be produced by techniques known in the
art such as chemical synthesis and by in vitro or in vivo
expression from recombinant nucleic acid molecules, e.g.,
retroviral vectors. Preferably probes are 10 to 100 nucleotides in
length. Probes may or may not have regions which are not
complementary to a target sequence, so long as such sequences do
not substantially affect hybridization under stringent
hybridization conditions. If such regions exist they may contain a
5' promoter sequence and/or a binding site for RNA transcription, a
restriction endonuclease recognition site, or may contain sequences
which will confer a desired secondary or tertiary structure, such
as a catalytic active site or a hairpin structure on the probe, on
the target nucleic acid, or both. A probe may be labeled with a
reporter group moiety such as a radioisotope, a fluorescent or
chemiluminescent moiety, with an enzyme or ligand, which can be
used for detection or confirmation that the probe has hybridized to
the target sequence.
[0034] As used in this disclosure, the phrase "a probe (or
oligonucleotide) having a nucleic acid sequence consisting
essentially of a sequence selected from" a group of specific
sequences means that the probe, as a basic and novel
characteristic, is capable of stably hybridizing to a nucleic acid
having the exact complement of one of the listed nucleic acid
sequences of the group under stringent hybridization conditions. An
exact complement includes the corresponding DNA or RNA
sequence.
[0035] The phrase "substantially corresponding to a nucleic acid
sequence" means that the referred-to nucleic acid is sufficiently
similar to the nucleic acid sequence such that the referred-to
nucleic acid has similar hybridization properties to a nucleic acid
sequence in that it would hybridize with the same target nucleic
acid sequences under stringent hybridization conditions.
[0036] One skilled in the art will understand that substantially
corresponding probes and primers of the invention can vary from the
referred-to sequence and still hybridize to the same target nucleic
acid sequence. This variation from the nucleic acid may be stated
in terms of a percentage of identical bases within the sequence or
the percentage of perfectly complementary bases between the probe
or primer and its target sequence. One skilled in the art will also
understand that this variation could be expressed as the number of
bases in a probe or primer or the number of mismatched bases of a
probe that do not hybridize to a corresponding base of a target
nucleic acid sequence. Probes or primers of the present invention
substantially correspond to a nucleic acid sequence if these
percentages are from 100% to 80% or from 0 base mismatches in a 10
nucleotide target sequence to 2 bases mismatched in a 10 nucleotide
target sequence.
[0037] In preferred embodiments, the percentage is from 100% to
85%. In more preferred embodiments, this percentage is from 90% to
100%; in other preferred embodiments, this percentage is from 95%
to 100%. One skilled in the art will understand the various
modifications to the hybridization conditions that might be
required at various percentages of complementarity to allow
hybridization to a specific target sequence without causing an
unacceptable level of non-specific hybridization.
[0038] By "nucleic acid hybrid" or "hybrid" is meant a nucleic acid
structure containing a double-stranded, hydrogen-bonded region,
preferably of between 10 and 100 nucleotides in length, most
preferably of between about 12 and 50 nucleotides in length,
wherein each strand is complementary to the other and wherein the
region is sufficiently stable under stringent hybridization
conditions to be detected by means including but not limited to
chemiluminescent or fluorescent light detection, autoradiography,
or gel electrophoresis. Such hybrids may comprise RNA:RNA, RNA:DNA,
or DNA:DNA duplex molecules or duplex molecules containing analogs
of these nucleic acids.
[0039] By "complementary" is meant that the nucleotide sequences of
similar regions of two single-stranded nucleic acids, or to
different regions of the same single-stranded nucleic acid have a
nucleotide base composition that allow the single strands to
hybridize together in a stable double-stranded hydrogen-bonded
region under stringent hybridization conditions. When a contiguous
sequence of nucleotides of one single-stranded region is able to
form a series of "canonical" hydrogen-bonded base pairs with an
analogous sequence of nucleotides of the other single-stranded
region, such that A is paired with U or T and C is paired with G.
the nucleotides sequences are "perfectly" complementary.
[0040] By "conservatively modified variants" is meant nucleic acids
or oligonucleotides having a nucleotide sequence that is
complementary to a nucleic acid region of another nucleic acid,
such region in turn being perfectly complementary to a reference
nucleic acid. Such conservatively modified variants are able to
stably hybridize to a target nucleic acid region having a
Neisseria, Neisseria meningitidis or Neisseria gonorrhoeae
nucleotide sequence under stringent hybridization conditions.
[0041] By "amplification oligonucleotide" is meant an
oligonucleotide capable of hybridizing to a target nucleic acid
sequence and acting as a primer for nucleic acid synthesis or a
promoter template (e.g., for synthesis of a complementary strand,
thereby forming a functional promoter sequence), or both, for the
initiation of nucleic acid synthesis. If the amplification
oligonucleotide is designed to initiate RNA synthesis, the
oligonucleotide may contain nucleotide sequences which are
non-complementary to the target sequence, but are recognized by an
RNA polymerase (such as T7, T3 and SP6 RNA polymerase). An
amplification oligonucleotide may or may not have a 3' terminus
which is modified to prevent or lessen the amount of primer
extension. An amplification oligonucleotide as defined herein will
preferably be between 10 and 100 nucleotides in length; most
preferably between about 12 and 50 nucleotides in length. While the
amplification oligonucleotides of the present invention may be
chemically synthesized or derived from a vector, such
oligonucleotides are not naturally-occurring nucleic acids.
[0042] By "nucleic acid amplification" or "target amplification" is
meant increasing the number of nucleic acid molecules having at
least one target nucleic acid sequence.
[0043] By "antisense" or "negative sense" is meant having a nucleic
sequence complementary to that of a reference nucleic acid
sequence.
[0044] By "sense", "same-sense" or "positive sense" is meant having
a nucleic acid sequence analogous to that of a reference nucleic
acid sequence.
[0045] By "helper oligonucleotide" is meant a nucleic acid probe
designed to hybridize with the target nucleic acid at a different
locus than that of a labeled probe, thereby either increasing the
rate of hybridization of the labeled probe, increasing the melting
temperature (Tm) of the target:labeled probe hybrid, or both.
[0046] "Phylogenetically closely related" means that the organisms
are closely related to each other in an evolutionary sense and
therefore would have a higher total nucleic acid sequence homology
than organisms that are more distantly related. Organisms occupying
adjacent and next to adjacent positions on the phylogenetic tree
are closely related. Organisms occupying positions further away
than adjacent or next to adjacent positions on the phylogenetic
tree will still be closely related if they have significant total
nucleic acid sequence homology.
[0047] B. Hybridization Conditions and Probe/Primer Design
[0048] Hybridization reaction conditions, most importantly the
temperature of hybridization and the concentration of salt in the
hybridization solution, can be selected to allow the amplification
oligonucleotides or hybridization probes of the present invention
to preferentially hybridize to nucleic acids having a target
Neisseria nucleotide sequence, and not to other non-target nucleic
acids suspected of being present in the test sample. At decreased
salt concentrations and/or increased temperatures (called increased
stringency) the extent of nucleic acid hybridization decreases as
hydrogen bonding between paired nucleotide bases in the
double-stranded hybrid molecule is disrupted; this process is
called "melting".
[0049] Generally speaking, the most stable hybrids are those having
the largest number of contiguous perfectly matched (i.e.,
hydrogen-bonded) nucleotide base pairs. Thus, such hybrids would
usually be expected to be the last to melt as the stringency of the
hybridization conditions increases. However, a double-stranded
nucleic acid region containing one or more mismatched,
"non-canonical", or imperfect base pairs (resulting in weaker or
non-existent base pairing at that position in the nucleotide
sequence of a nucleic acid) may still be sufficiently stable under
conditions of relatively high stringency to allow the nucleic acid
hybrid to be detected in a hybridization assay without cross
reacting with other, non-selected nucleic acids present in the test
sample.
[0050] Hence, depending on the degree of similarity between the
nucleotide sequences of the target nucleic acid and those of
non-target nucleic acids belonging to phylogenetically distinct,
but closely-related organisms on one hand, and the degree of
complementarity between the nucleotide sequences of a particular
amplification oligonucleotide or hybridization probe and those of
the target and non-target nucleic acids on the other, one or more
mismatches will not necessarily defeat the ability of the
oligonucleotide to hybridize to that nucleic acid and not to
non-target nucleic acids.
[0051] The hybridization assay probes of the present invention were
chosen, selected, and/or designed to maximize the difference
between the melting temperatures of the probe:target hybrid (Tm,
defined as the temperature at which half of the potentially
double-stranded molecules in a given reaction mixture are in a
single-stranded, denatured state) and the Tm of a mismatched hybrid
formed between the probe and the rRNA or rDNA of the
phylogenetically most closely-related organisms expected to be
present in the test sample, but not sought to be detected. While
the unlabeled amplification oligonucleotides and helper
oligonucleotides need not have such an extremely high degree of
specificity as the labeled hybridization assay probe to be useful
in the present invention, they are designed in a similar manner to
preferentially hybridize to one or more target nucleic acids over
other nucleic acids.
[0052] Probes specific for Neisseria meningitidis were designed
using sequences determined in prospective target areas using
primers complementary to the 16S rRNAs of strains of Neisseria
including Neisseria gonorrhoeae (ATCC No. 19424), Neisseria
meningitidis serogroup A (ATCC No. 13077), serogroup C (ATCC No.
23248) and serogroup L (ATCC No. 43828), clinical isolates of
Neisseria meningitidis, Neisseria lactamica (ATCC NO. 29193),
Neisseria cinerea (ATCC NO. 14685), Neisseria mucosa (ATCC NO.
19696), Neisseria sicca (ATCC NO. 29193) and Kingella kingae (ATCC
No. 23330). The nucleic acid sequence from phylogenetically near
neighbors, including the published sequence of Neisseria
gonorrhoeae NCTC 8375, Rossau et al. Nuc. Acids Res. 16:6227 were
also used as comparisons with the nucleic sequences from Neisseria
meningitidis to determine variable regions.
[0053] To facilitate the identification of nucleic acid sequences
to be used as probes and amplification oligonucleotides, the
nucleotide sequences from different species of organisms were first
aligned to maximize homology. Within the rRNA molecule there is a
close relationship between the overall structure and function. This
imposes restrictions on evolutionary changes in the primary
sequence so that the secondary structure is maintained. For
example, if a base is changed on one side of a helix, a
compensating change may be evolutionarily made to the other side to
preserve the complementarity (this is referred to as co-variance).
This allows two very different sequences to be aligned using the
conserved primary sequence and also the conserved secondary
structure elements as points of reference. Potential target
sequences for the hybridization probes were identified by noting
variations in the homology of the aligned sequences in certain
discrete regions (variable regions) of the rRNA and rDNA
sequences.
[0054] The sequence evolution at each of the variable regions is
mostly divergent. Because of the divergence, more distant
phylogenetic relatives of Neisseria meningitidis or Neisseria
gonorrhoeae tend to show greater variability in a given variable
region than phylogenetically closer relatives. The observed
sufficient variation between Neisseria meningitidis and Neisseria
gonorrhoeae species was used to identify preferred target sites and
design useful probes.
[0055] We have identified sequences which vary between Neisseria
meningitidis and Neisseria gonorrhoeae, between these and other
Neisseria species, and between members of the genus Neisseria and
other organisms by comparative analysis of rRNA sequences published
in the literature or determined in the laboratory. Computers and
computer programs which may be used or adapted for the purposes
herein disclosed are commercially available. We have seen
sufficient variation between the target organisms and the closest
phylogenetic relative likely to be found in the same sample to
design the present probes. The Neisseria meningitidis strains have
been classified into three sequence groups in the probe region
represented by serogroups A, C and L.
[0056] Merely identifying putatively unique potential target
nucleotide sequences does not guarantee that a functionally
species-specific hybridization assay probe may be made to hybridize
to Neisseria rRNA or rDNA comprising that sequence. Various other
factors will determine the suitability of a nucleic acid locus as a
target site for species-specific probes. 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 oligonucleotide, whether perfectly
complementary to its target or not. The importance and effect of
various assay conditions are known to those skilled in the art as
described in Hogan et al., PCT/US87/03009, and Hogan and Hammond,
U.S. Pat. No. 5,216,143, and Kohne, U.S. Pat. No. 4,851,330 which
share the same assignee as the present application and are hereby
incorporated by reference herein.
[0057] The desired temperature of hybridization and the
hybridization solution composition (such as salt concentration,
detergents and other solutes) can also greatly affect the stability
of double-stranded hybrids. Conditions such as ionic strength and
the temperature at which a probe will be allowed to hybridize to
target must be taken into account in constructing a group- or
species-specific probe. The thermal stability of hybrid nucleic
acids generally increases with the ionic strength of the reaction
mixture. On the other hand, chemical reagents which disrupt
hydrogen bonds, such as formamide, urea, dimethyl sulfoxide and
alcohols, can greatly reduce the thermal stability of the
hybrids.
[0058] To maximize the specificity of a probe for its target, the
subject probes of the present invention were designed to hybridize
with their targets under conditions of high stringency. Under such
conditions only single nucleic acid strands having a high degree of
complementarity will hybridize to each other; single nucleic acid
strands without such a high degree of complementarity will not form
hybrids. Accordingly, the stringency of the assay conditions
determines the amount of complementarity which should exist between
two nucleic acid strands in order to form a hybrid. Stringency is
chosen to maximize the difference in stability between the hybrid
formed between the probe and the target nucleic acid and potential
hybrids between the probe and any non-target nucleic acids
present.
[0059] Proper specificity may be achieved by minimizing the length
of the probe having perfect complementarity to sequences of
non-target organisms, by avoiding G and C rich regions of homology
to non-target sequences, and by constructing the probe to contain
as many destabilizing mismatches to nontarget sequences as
possible. Whether a probe sequence is useful to detect only a
specific type of organism depends largely on the thermal stability
difference between probe:target hybrids versus potential
probe:nontarget hybrids. In designing probes, the differences in
the Tm values between these hybrids should be made as large as
possible (preferably about 5.degree. C. or more). Manipulation of
the Tm can be accomplished by changes to probe length and probe
composition (GC content vs. AT content).
[0060] In general, the optimal hybridization temperature for
oligonucleotide probes of about 10-50 nucleotides in length is
approximately 5.degree. C. below the melting temperature for a
given duplex. Incubation at temperatures below the optimum
temperature may allow mismatched base sequences to hybridize and
can therefore decrease specificity. The longer the probe, the more
hydrogen bonding between base pairs and, in general, the higher the
Tm. Increasing the percentage of G and C also increases the Tm
because G-C base pairs exhibit additional hydrogen bonding and
therefore greater thermal stability than A-T base pairs.
[0061] A preferred method to determine Tm measures hybridization
using a Hybridization Protection Assay (HPA) according to Arnold et
al., U.S. Pat. No. 5,283,174 which enjoys exclusive ownership with
the present application and is incorporated by reference herein. Tm
can be measured using HPA in the following manner. A probe:target
hybrid is formed in lithium succinate buffered solution (0.1 M
lithium succinate buffer, pH 5.0, 2 mM ethylenediamine tetraacetic
acid (EDTA), 2 mM ethylene glycol-bis(.beta.-amino-ethyl ether)
N,N,N',N'-tetraacetic acid (EGTA), 10% (w/v) lithium lauryl
sulfate) using an excess amount of target. Aliquots of the hybrid
are then diluted in the lithium succinate buffered solution and
incubated for five minutes at various temperatures starting below
that of the anticipated Tm for example, 55.degree. C. and
increasing in 2-5.degree. C. increments. This solution is then
diluted with a mild alkaline borate buffer (0.15 M sodium
tetraborate, pH 7.6, 5% (v/v) TRITON.RTM. X-100) and incubated at a
lower temperature (for example 50.degree. C.) for ten minutes.
Under these conditions the acridinium ester attached to a
single-stranded probe is hydrolyzed while the acridinium ester
attached to hybridized probe is relatively protected from
hydrolysis. Thus, the amount of acridinium ester remaining is
proportional to the amount of hybrid and can be measured by the
chemiluminescence produced from the acridinium ester upon the
addition of hydrogen peroxide followed by alkali. Chemiluminescence
can be measured in a luminometer (e.g., Gen-Probe LEADER.RTM. I or
LEADER.RTM. 50). The resulting data are plotted as percent of
maximum signal (usually from the lowest temperature), versus
temperature. The Tm is defined as the temperature at which 50% of
the maximum signal remains. In addition to the method above, Tm may
be determined by isotopic methods well known to those skilled in
the art (e.g., Hogan et al., supra).
[0062] It should be noted that the Tm for a given hybrid varies
depending on the hybridization solution used. Factors such as the
salt concentration, detergents, and other solutes can effect hybrid
stability during thermal denaturation (J. Sambrook, E. F. Fritsch
and T. Maniatis, 2 Molecular Cloning, ch. 11 (2d ed. 1989)).
Conditions such as ionic strength and incubation temperature under
which a probe will be used to hybridize to target should be taken
into account in constructing a probe. On the other hand, chemical
reagents which disrupt hydrogen bonds such as formamide, urea,
dimethylsulfoxide and alcohols, can greatly reduce the thermal
stability of the hybrids.
[0063] To ensure the probe is specific for its target, it is
desirable to have probes which hybridize only under conditions of
high stringency. Under conditions of high stringency 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 to form a
hybrid. Stringency is chosen to maximize the difference in
stability between the hybrid formed with the target and other
nucleic acid sequences.
[0064] The length of the target nucleic acid sequence and,
accordingly, the length of the probe sequence can also be
important. In some cases, there may be several sequences from a
particular region, for example, a variable region varying in
location and length, which yield probes with the desired
hybridization characteristics. In other cases, one probe may be
significantly better than another probe with a nucleotide sequence
differing by a single base. While it is possible for nucleic acids
that are not perfectly complementary to hybridize, the longest
stretch of perfectly homologous base sequence will generally
determine hybrid stability, with the composition of the base pairs
also playing a role.
[0065] Regions of rRNA which form strong internal structures
inhibitory to hybridization are less preferred target regions at
least in assays in which helper probes are not used. Likewise,
probe designs which result in extensive self complementarity should
be avoided. If one of the two strands is wholly or partially
involved in an intramolecular or intermolecular hybrid it will be
less able to participate in the formation of a new intermolecular
probe:target hybrid. Ribosomal RNA molecules are known to form very
stable intramolecular helices and secondary structures by hydrogen
bonding. By designing a hybridization assay so that a substantial
portion of the targeted sequence remains in a single-stranded state
until hybridization with the probe, the rate and extent of
hybridization between probe and target may be greatly increased.
One way this may be accomplished is by choosing as a target
nucleotide sequence a sequence that is relatively uninvolved in
intramolecular hydrogen-bonding. Alternatively or additionally, the
hybridization assay probe may be used in a probe mix with helper
oligonucleotides which can make the target site more accessible for
hybridization with the hybridization assay probe.
[0066] A DNA target occurs naturally in a double-stranded form as
does the product of the polymerase chain reaction (PCR). These
double-stranded targets are naturally inhibitory to hybridization
with a probe and require denaturation prior to hybridization.
Appropriate denaturation and hybridization conditions are known in
the art (e.g., E. M. Southern, J. Mol. Bio. 98:503 (1975)).
[0067] A number of formulae are available which will provide an
estimate of the melting temperature for perfectly matched
oligonucleotides to their target nucleic acids. One such
formula,
T.sub.m=81.5+16.6(log.sub.10[Na.sup.+])+0.41(fraction
G+C)-(600/N)
[0068] (where N=the length of the oligonucleotide in number of
nucleotides) provides a good estimate for the T.sub.m for
oligonucleotides between 14 and 60 or 70 nucleotides in length.
From such calculations, subsequent empirical verification or "fine
tuning" of the Tm may be made using screening techniques well known
in the art. For further information on hybridization and
oligonucleotide probes see, e.g., Sambrook et al., 2 Molecular
Cloning: A Laboratory Manual (Cold Springs Harbor Laboratory Press
1989) hereby incorporated by reference herein (at Chapter 11). This
reference, among others well known in the art, also provides
estimates of the effect of mismatches on the Tm of a hybrid. Thus,
from the known nucleotide sequence of a given region of the
ribosomal RNA (or rDNA) of two or more organisms, oligonucleotides
may be designed which will distinguish these organisms from one
another.
[0069] C. Nucleic Acid Amplification
[0070] Preferably, the amplification oligonucleotides of the
present invention are oligodeoxynucleotides and are sufficiently
long to be used as a substrate for the synthesis of extension
products by a nucleic acid polymerase. Optimal primer length should
take into account several factors, including the temperature of
reaction, the structure and base composition of the primer, and how
the primer is to be used. For example, for optimal specificity the
oligonucleotide primer generally should contain at least about 12
nucleotides depending on the complexity of the target nucleic acid
sequence. If such specificity is not essential, shorter primers may
be used; in such a case, it may be desirable to carry out reaction
at cooler temperatures in order to form stable hybrid complexes
with the template nucleic acid.
[0071] Useful guidelines for designing amplification
oligonucleotides and probes with desired characteristics are
described herein. Our best mode target regions contain at least two
and preferably three conserved regions of Neisseria meningitidis or
Neisseria gonorrhoeae nucleic acid. These regions are about 15-350
in length; preferably 15-150 nucleotides in length.
[0072] The degree of amplification observed with a set of primers
or promoter primers depends on several factors, including the
ability of the oligonucleotides to hybridize to their complementary
sequences and their ability to be extended or copied enzymatically.
While oligonucleotides of different lengths and base composition
may be used, oligonucleotides preferred in this invention have
target binding regions of 18-40 bases with a predicted Tm to target
of about 65.degree. C.
[0073] Parameters which affect hybridization of a probe such as
T.sub.m, complementarity and secondary structure of the target
sequence also affect primer hybridization and therefore
performance. The degree of non-specific extension (primer-dimer or
non-target copying) can also affect amplification efficiency,
therefore primers are selected to have low self- or
cross-complementarity, particularly at the 3' ends of the sequence.
Long homopolymer tracts and high GC content are avoided to reduce
spurious primer extension. Computer programs are available to aid
in this aspect of the design.
[0074] A nucleic acid polymerase used in conjunction with the
amplification oligonucleotides of the present invention refers to a
chemical, physical or biological agent which incorporates either
ribo- or deoxyribonucleotides, or both, into a nucleic acid
polymer, or strand, in a template-dependent manner. Examples of
nucleic acid polymerases include DNA-directed DNA polymerases,
RNA-directed DNA polymerases, and RNA-directed RNA polymerases. DNA
polymerases bring about nucleic acid synthesis in a
template-dependent manner and in a 5' to 3' direction. Because of
the antiparallel orientation of the two strands in a
double-stranded nucleic acid, this direction is from a 3' region on
the template to a 5' region on the template. Examples of
DNA-directed DNA polymerases include E. coli DNA polymerase I, the
thermostable DNA polymerase from Thermus aquaticus (Taq), and the
large fragment of DNA polymerase I from Bacillus stearothermophilus
(Bst). Examples of RNA directed DNA polymerases include various
retroviral reverse transcriptases, such as Moloney murine leukemia
virus (MMLV) reverse transcriptase or avian myeloblastosis virus
(AMV) reverse transcriptase.
[0075] During most nucleic acid amplification reactions, a nucleic
acid polymerase adds nucleotide residues to the 3' end of the
primer using the target nucleic acid as a template, thus
synthesizing a second nucleic acid strand having a nucleotide
sequence partially or completely complementary to a region of the
target nucleic acid. In many nucleic acid amplification reactions,
the two strands comprising the resulting double-stranded structure
must be separated by chemical or physical means in order to allow
the amplification reaction to proceed. Alternatively, the
newly-synthesized template strand may be made available for
hybridization with a second primer or promoter-primer by other
means--e.g. through strand displacement or the use of a nucleolytic
enzyme which digests part or all of the original target strand. In
this way the process may be repeated through a number of cycles,
resulting in a large increase in the number of nucleic acid
molecules having the target nucleotide sequence.
[0076] Either the first or second amplification oligonucleotide, or
both, may be a promoter-primer. Such a promoter-primer usually
contains nucleotide sequences that are not complementary to those
of the target nucleic acid molecule, or primer extension
product(s). For example, Kacian and Fultz, U.S. Pat. No. 5,399,491
which is hereby incorporated by reference, describes various such
oligonucleotides. These non-complementary sequences may be located
5' to the complementary sequences on the amplification
oligonucleotide, and may provide a locus for initiation of RNA
synthesis when made double-stranded through the action of a nucleic
acid polymerase. The promoter thus provided may allow for the in
vitro transcription of multiple RNA copies of the target nucleic
acid sequence. It will be appreciated that when reference is made
to a primer in this specification, such reference is intended to
include the primer aspect of a promoter-primer as well unless the
context of the reference clearly indicates otherwise.
[0077] In some amplification systems, for example the amplification
method of Dattagupta et al., supra, the amplification
oligonucleotides may contain 5' non-complementary nucleotides which
assist in strand displacement. Furthermore, when used in
conjunction with a nucleic acid polymerase having 5' exonuclease
activity, the amplification oligonucleotides may have modifications
at their 5' end to prevent enzymatic digestion. Alternatively, the
nucleic acid polymerase may be modified to remove the 5'
exonuclease activity, such as by treatment with a protease that
generates an active polymerase fragment with no such nuclease
activity. In such a case the oligonucleotides need not be modified
at their 5' end.
[0078] 1. Preparation of Oligonucleotides
[0079] All of the amplification oligonucleotides of the present
invention can be readily prepared by methods known in the art.
Preferably, the primers are synthesized using solid phase methods.
For example, Caruthers, et al., describe using standard
phosphoramidite solid phase chemistry to join nucleotides by
phosphodiester linkages. Automated solid-phase chemical synthesis
using cyanoethyl phosphoramidite precursors has been described by
Barone, et al., Nucleic Acids Research, 12:405 (1984). (Methods in
Enzymology, Volume 143, pg. 287 (1987)). Likewise, Bhatt describes
a procedure for synthesizing oligonucleotides containing
phosphorothioate linkages. (WO92/04358, entitled "Method and
Reagent for Sulphurization of Organophosphorous Compounds", which
enjoys common ownership with the present invention.) Also, Klem et
al., entitled "Improved Process for the Synthesis of Oligomers",
PCT WO 92/07864, describe the synthesis of oligonucleotides having
different linkages including methylphosphonate linkages. The latter
three references are hereby incorporated by reference herein. In
addition, methods for the organic synthesis of oligonucleotides are
known to those of skill in the art, and are described in Sambrook,
et al., supra, previously incorporated by reference herein.
[0080] Following synthesis and purification of a particular
oligonucleotide, several different procedures may be utilized to
purify and control the quality of the oligonucleotide. Suitable
procedures include polyacrylamide gel electrophoresis or high
pressure liquid chromatography. Both of these procedures are well
known to those skilled in the art.
[0081] All of the oligonucleotides of the present invention,
whether hybridization assay probes, amplification oligonucleotides,
or helper oligonucleotides, may be modified with chemical groups to
enhance their performance or to facilitate the characterization of
amplification products. For example, backbone-modified
oligonucleotides such as those having phosphorothioate or
methylphosphonate groups which render the oligonucleotides
resistant to the nucleolytic activity of certain polymerases or to
nuclease enzymes may allow the use of such enzymes in an
amplification or other reaction. Another example of modification
involves using non-nucleotide linkers (e.g., Arnold, et al.,
"Non-Nucleotide Linking Reagents for Nucleotide Probes", EP 0 313
219 hereby incorporated by reference herein) incorporated between
nucleotides in the nucleic acid chain which do not interfere with
hybridization or the elongation of the primer. Amplification
oligonucleotides may also contain mixtures of the desired modified
and natural nucleotides.
[0082] The 3' end of an amplification oligonucleotide may be
blocked to prevent initiation of DNA synthesis as described by
McDonough, et al., entitled "Nucleic Acid Sequence Amplification",
WO94/03472 which enjoys common ownership with the present invention
and is hereby incorporated by reference herein. A mixture of
different 3' blocked amplification oligonucleotides, or of 3'
blocked and unblocked oligonucleotides may increase the efficiency
of nucleic acid amplification, as described therein.
[0083] As disclosed above, the 5' end of the oligonucleotides may
be modified to be resistant to the 5'-exonuclease activity present
in some nucleic acid polymerases. Such modifications can be carried
out by adding a non-nucleotide group to the terminal 5' nucleotide
of the primer using techniques such as those described by Arnold,
et al., supra, entitled "Non-Nucleotide Linking Reagents for
Nucleotide Probes", previously incorporated by reference
herein.
[0084] Once synthesized, selected oligonucleotide probes may be
labeled by any of several well known methods (e.g., J. Sambrook,
supra). Useful labels include radioisotopes as well as
non-radioactive reporting groups. Isotopic labels include .sup.3H,
.sup.35S, .sup.32P, .sup.125I, .sup.57Co and .sup.14C. Isotopic
labels can be introduced into the oligonucleotide by techniques
known in the art such as nick translation, end labeling, second
strand synthesis, the use of reverse transcription, and by chemical
methods. When using radiolabeled probes hybridization can be
detected by autoradiography, scintillation counting, or gamma
counting. The detection method selected will depend upon the
particular radioisotope used for labeling.
[0085] Non-isotopic materials can also be used for labeling and may
be introduced internally into the nucleic acid sequence or at the
end of the nucleic acid sequence. Modified nucleotides may be
incorporated enzymatically or chemically. Chemical modifications of
the probe may be performed during or after synthesis of the probe,
for example, through the use of non-nucleotide linker groups as
described by Arnold, et al., supra "Non-Nucleotide Linking Reagents
for Nucleotide Probes", previously incorporated by reference
herein. Non-isotopic labels include fluorescent molecules,
chemiluminescent molecules, enzymes, cofactors, enzyme substrates,
haptens or other ligands.
[0086] Preferably, the probes are labeled with an acridinium ester.
Acridinium ester labeling may be performed as described by Arnold
et al., U.S. Pat. No. 5,185,439, entitled "Acridinium Ester
Labeling and Purification of Nucleotide Probes" issued Feb. 9, 1993
and hereby incorporated by reference herein.
[0087] 2. Amplification of Neisseria rRNA and rDNA
[0088] The amplification oligonucleotides of the present invention
are directed to particular Neisseria 16S rRNA nucleotide sequences,
or their rDNA counterparts. These amplification oligonucleotides
may flank, overlap or be contained within at least one of the
target nucleotide sequences used as a hybridization assay probe to
detect the presence of Neisseria in a nucleic acid amplification
assay. The amplification oligonucleotides described and claimed
herein comprise two sets of amplification oligonucleotides. Members
of the set of amplification oligonucleotides are able to hybridize
with a nucleic acid having or substantially corresponding to one of
the following nucleotide sequences:
7 SEQ ID NO: 23 GTCTTGAGAG GGAAAGCAGG GGAC SEQ ID NO: 24 TATGTTACTC
ACCCGTTCGC CACTCGCC SEQ ID NO: 19 CTGAAGAATA AGCACCGGCT AACTACGTGC
AGCAGC SEQ ID NO: 21 CGATGACGGT ACCTGAAGAA TAAGCACCGG CTAAC SEQ ID
NO: 20 AACGGCCTTT TCTTCCCTGA CAAAAGTCCT TTACAACCCG SEQ ID NO: 22
GTCCTTTACA ACCCGAAGGC CTTC SEQ ID NO: 47 GUCUUGAGAG GGAAAGCAGG GGAC
SEQ ID NO: 48 UAUGUUACUC ACCCGUUCGC CACUCGCC SEQ ID NO: 49
CUGAAGAAUA AGCACCGGCU AACUACGUGC AGCAGC SEQ ID NO: 50 CGAUGACGGU
ACCUGAAGAA UAAGCACCGG CUAAC SEQ ID NO: 51 AACGGCCUUU UCUUCCCUGA
CAAAAGUCCU UUACAACCCG and SEQ ID NO: 52 GUCCUUUACA ACCCGAAGGC
CUUC
[0089] In preferred embodiments, these amplification
oligonucleotides have or substantially correspond to the following
sequences:
8 SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC SEQ ID NO: 6 GGCGAGTGGC
GAACGGGTGA GTAACATA SEQ ID NO: 7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT
CTTCAG SEQ ID NO: 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG SEQ ID
NO: 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT SEQ ID NO: 10
GAAGGCCTTC GGGTTGTAAA GGAC SEQ ID NO: 41 GUCCCCUGCU UUCCCUCUCA AGAC
SEQ ID NO: 42 GGCGAGUGGC GAACGGGUGA GUAACAUA SEQ ID NO: 43
GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU CUUCAG SEQ ID NO: 44 GUUAGCCGGU
GCUUAUUCUU CAGGUACCGU CAUCG SEQ ID NO: 45 CGGGUUGUAA AGGACUUUUG
UCAGGGAAGA AAAGGCCGUU SEQ ID NO: 46 GAAGGCCUUC GGGUUGUAAA GGAC
[0090] These oligonucleotides may also have additional,
non-complementary bases at their 5' end comprising a promoter
sequence able to bind an RNA polymerase and direct RNA
transcription using the target nucleic acid as a template. For
example the promoter, SEQ ID NO: 53 AATTTAATAC GACTCACTAT AGGGAGA
may be used.
[0091] All of the amplification oligonucleotides of the present
invention may have sequences which do not contain modifications or
additions to these sequences. The amplification oligonucleotides
may also or alternatively have modifications, such as blocked 3'
and/or 5' termini or additions including but not limited to the
addition of a specific nucleotide sequence that is recognized by an
RNA polymerase, (e.g., the promoter sequence for T7, T3, or SP6 RNA
polymerase), addition of sequences which enhance initiation or
elongation of RNA transcription by an RNA polymerase, or sequences
which may provide for intramolecular base pairing and encourage the
formation of secondary or tertiary nucleic acid structures.
[0092] Amplification oligonucleotides are used in a nucleic acid
amplification procedure, such as the polymerase chain reaction or
an amplification reaction using RNA polymerase, DNA polymerase and
RNAse H or its equivalent, as described by Kacian and Fultz supra,
Dattagupta et al., supra, and by Sninsky et al., U.S. Pat. No.
5,079,351; all hereby incorporated by reference herein, the first
two of which enjoy common ownership with the present invention.
[0093] A wide variety of methods are available to detect an
amplified target sequence. For example, the nucleotide substrates
or the primers can include a detectable label which is incorporated
into newly synthesized DNA. The resulting labeled amplification
product is then separated from the unused labeled nucleotides or
primers and the label is detected in the separated product
fraction.
[0094] Substances which can serve as useful detectable labels are
well known in the art and include radioactive isotopes, fluorescent
compounds, chemiluminescent compounds, chromophores, as well as
ligands such as biotin and haptens which, while not directly
detectable, can be readily detected by a reaction with labeled
forms of their specific binding partners, e.g., avidin and
antibodies, respectively.
[0095] Another approach is to detect the amplification product by
hybridization with a detectably labeled nucleic acid probe and
measuring the resulting hybrids in any conventional manner. In
particular, the product can be assayed by hybridizing a
chemiluminescent acridinium ester-labeled nucleic acid probe to the
target sequence, selectively hydrolyzing the acridinium ester
present on unhybridized probe, and measuring the chemiluminescence
produced from the remaining acridinium ester in a luminometer.
(see, e.g., Arnold, et al., supra, U.S. Pat. No. 5,283,174, and
Nelson, et al., "Non-Isotopic DNA Probe Technologies", Academic
Press, San Diego (Kricka, ed. 1992) both references hereby
incorporated by reference herein.)
[0096] D. Oligonucleotide Hybridization Assay Probes to Neisseria
meningitidis or Neisseria gonorrhoeae rRNA and rDNA
[0097] The oligonucleotide hybridization assay probes disclosed and
claimed herein are able to preferentially hybridize to target
nucleic acids of Neisseria meningitidis rRNA or rDNA nucleotide
sequences over nucleic acids of phylogenetically closely related
bacterial species. These hybridization assay probes were designed,
selected and/or chosen based upon a comparison of the nucleotide
sequences of corresponding regions of the ribosomal RNA of
Neisseria meningitidis and said phylogenetically closely-related
species. In preferred embodiments these probes selectively
hybridize to the nucleic acids of Neisseria meningitidis over the
nucleic acids of Neisseria gonorrhoeae.
[0098] The present invention contemplates oligonucleotide
hybridization probes that selectively hybridize to the nucleic
acids of Neisseria meningitidis and not to the nucleic acids of
Neisseria gonorrhoeae and include Neisseria meningitidis nucleic
acid sequences having or substantially corresponding to the
following nucleic acid sequences:
9 GGCTGTTGCT AATATCAGCG SEQ ID NO: 11 GGCTGTTGCT AATACCAGCG SEQ ID
NO: 12 CGCTGATATT AGCAACAGCC SEQ ID NO: 15 CGCTGGTATT AGCAACAGCC
SEQ ID NO: 16 GGCUGUUGCU AAUAUCAGCG SEQ ID NO: 25 GGCUGUUGCU
AAUACCAGCG SEQ ID NO: 26 CGCUGAUAUU AGCAACAGCC SEQ ID NO: 27
CGCUGGUAUU AGCAACAGCC SEQ ID NO: 28
[0099] A number of oligonucleotide hybridization assay probes of
the present invention preferably hybridize to target nucleic acids
containing Neisseria gonorrhoeae rRNA or rDNA nucleotide sequences
over nucleic acids of other phylogenetically closely related
bacterial species. In preferred embodiments, these hybridization
assay probes can distinguish Neisseria gonorrhoeae nucleic acids
from Neisseria meningitidis.
[0100] The hybridization probes of the present invention that
selectively hybridize to nucleic acids derived from Neisseria
gonorrhoeae and not to the nucleic acids of Neisseria meningitidis
have or substantially correspond to the following nucleotide
sequences:
10 GAACGTACCG GGTAGCGG SEQ ID NO: 1 GCCAATATCG GCGGCCGATG SEQ ID
NO: 3 CCGCTACCCG GTACGTTC SEQ ID NO: 29 CATCGGCCGC CGATATTGGC SEQ
ID NO: 30 GAACGUACCG GGUAGCGG SEQ ID NO: 31 GCCAAUAUCG GCGGCCGAUG
SEQ ID NO: 32 CCGCUACCCG GUACGUUC SEQ ID NO: 33 CAUCGGCCGC
CGAUAUUGGC SEQ ID NO: 34
[0101] The oligonucleotide hybridization assay probes of the
present invention are preferably labeled with a reporter group
moiety such as a radioisotope, a fluorescent or chemiluminescent
moiety, with an enzyme or other ligand, which can be used for
detection or confirmation that the probe has hybridized to the
target sequence. The Applicant most prefers the use of
chemiluminescent acridinium esters as labels. See e.g. Arnold et
al., U.S. Pat. No. 5,185,439, previously incorporated by reference
herein. The assay probe is mixed with a sample suspected of
containing a nucleic acid having the target sequence under
hybridization conditions suitable for allowing annealing of the two
strands by hydrogen bonding in the region of complementarity.
[0102] The probe may also be combined with one or more unlabeled
helper oligonucleotides to facilitate binding to the nucleic acid
having the target Neisseria meningitidis or Neisseria gonorrhoeae
nucleotide sequence. The probe then hybridizes to the target
nucleic acid present in the sample; the resulting hybrid duplex may
be separated and detected by various techniques well known in the
art, such as hydroxyapatite adsorption and radioactive monitoring.
Also included among these techniques are those that involve
selectively degrading the label present on unhybridized probe and
then measuring the amount of label associated with the remaining
hybridized probe, as disclosed in Arnold et al., U.S. Pat. No.
5,283,174, which enjoys common ownership with the present
application and is incorporated by reference herein. This latter
technique is presently preferred by the Applicants.
[0103] E. Helper Oligonucleotides Used in the Detection of
Neisseria
[0104] Specific helper oligonucleotides were used to facilitate the
hybridization of the hybridization assay probes to the target
nucleic acid. Helper oligonucleotides are described in Hogan and
Milliman, U.S. Pat. No. 5,030,557 entitled Means and Method for
Enhancing Nucleic Acid Hybridization, which enjoys common ownership
with the present application and is hereby incorporated by
reference herein.
[0105] Helper probes are selected to hybridize to nucleic acid
sequences located near the region targeted by the hybridization
assay probe. Hybridization of the helper probe alters the secondary
and tertiary structure of the target nucleic acid, facilitating the
hybridization of the probe to the target nucleic acid.
[0106] Specific helper oligonucleotides for facilitating the
specific detection of Neisseria meningitidis nucleic acids have or
substantially correspond to one of these nucleotide sequences:
11 SEQ ID NO: 13 GCCTTCGGGT TGTAAAGGAC TTTTGTCAGG GAAGAAAA SEQ ID
NO: 14 GCTGATGACG GTACCTGAAG AATAAGCACC GGC SEQ ID NO: 17
TTTTCTTCCC TGACAAAAGT CCTTTACAAC CCGAAGGC SEQ ID NO: 18 GCCGGTGCTT
ATTCTTCAGG TACCGTCATC AGC SEQ ID NO: 35 GCCUUCGGGU UGUAAAGGAC
UUUUGUCAGG GAAGAAAA SEQ ID NO: 36 GCUGAUGACG GUACCUGAAG AAUAAGCACC
GGC SEQ ID NO: 37 UUUUCUUCCC UGACAAAAGU CCUUUACAAC CCGAAGGC SEQ ID
NO: 38 GCCGGUGCUU AUUCUUCAGG UACCGUCAUC AGC
[0107] In preferred embodiments, hybridization probes directed to
Neisseria meningitidis nucleic acids substantially correspond to
SEQ ID NOS: 11, 12, 25 or 26
[0108] used in a probe mixture together with a helper
oligonucleotide having or substantially corresponding to the
nucleotide sequence of:
[0109] SEQ ID NOS: 13, 14, 35 and 36
[0110] In other embodiments, a hybridization assay probe directed
to Neisseria meningitidis nucleic acids substantially corresponding
to
[0111] SEQ ID NOS: 15, 16, 27 or 28
[0112] is used in a probe mixture together with a helper
oligonucleotide having or substantially corresponding to a
nucleotide sequence of:
[0113] SEQ ID NOS: 17, 18, 37 and 38
[0114] In a preferred embodiment, a hybridization probe directed to
Neisseria gonorrhoeae ribosomal nucleic acid substantially
corresponding to
[0115] SEQ ID NOS: 1 or 31
[0116] is used in a mixture together with a helper oligonucleotide
having or substantially corresponding to the nucleotide sequence
of:
[0117] SEQ ID NOS: 2 or 39
[0118] In other preferred embodiments, a hybridization probe
directed to Neisseria gonorrhoeae nucleic acids substantially
corresponding to
[0119] SEQ ID NOS: 3 or 32
[0120] is used in a probe mixture together with a helper
oligonucleotide having or substantially corresponding to a
nucleotide sequence of:
[0121] SEQ ID NOS: 4 or 40
[0122] Helper oligonucleotides generally may be used under
stringent hybridization conditions, but are not necessarily species
specific.
[0123] F. Nucleic Acid Compositions
[0124] In another related aspect, the invention features
compositions comprising a nucleic acid hybrid between a
hybridization assay probe and a nucleic acid sequence substantially
complementary thereto (probe:target). One use of the hybrid formed
between probe and target is to detect the presence of a target
sequence. For example, acridinium ester ("AE") present in hybrids
is resistant to hydrolysis in alkali solution whereas AE present in
single-stranded nucleic acid is hydrolyzed in alkali solution
(Arnold et al., entitled "Homogenous Protection Assay," EPO
application number 88308767.8, publication number 309230, and by
U.S. Pat. No. 5,238,174 hereby incorporated by reference). Thus,
presence of target nucleic acids can be detected, after hydrolysis
of the unbound AE-labeled probe, by measuring chemiluminescence of
acridinium ester remaining associated with the nucleic acid
hybrid.
[0125] The present invention also contemplates compositions
comprising a nucleic acid hybrid between an amplification
oligonucleotide and a nucleic acid sequence substantially
complementary thereto (primer:target). One use the nucleic acid
hybrid formed between primer and target is to provide an initiation
site for a nucleic acid polymerase at the 3' end of the
amplification oligonucleotide. For example, hybrids may form an
initiation site for reverse transcriptase, DNA polymerases such as
Taq polymerase or T4 DNA polymerase and RNA polymerases such as, T7
polymerase, SP6 polymerase, T3 polymerases and the like.
[0126] The present invention also features compositions comprising
nucleic acid hybrids between a helper oligonucleotide and a nucleic
acid sequence substantially complementary thereto (helper
oligonucleotide:target). One use of the hybrid between the helper
oligonucleotide and target is to make available a particular
nucleic acid sequence for hybridization. For example, a hybrid
between a helper oligonucleotide and its target may make a nucleic
acid sequence capable of hybridizing to the target sequence
available for hybridization with a hybridization probe. A full
description of the use of helper oligonucleotides is provided in
Hogan and Milliman, U.S. Pat. No. 5,030,557.
[0127] Compositions of the present invention include compositions
for detecting Neisseria meningitidis nucleic acid comprising a
nucleic acid hybrid formed between a nucleic acid derived from
Neisseria meningitidis and an oligonucleotide having a nucleic acid
sequence substantially corresponding to at least one of the nucleic
acid sequences that follows:
12 SEQ ID NO: 11 GGCTGTTGCT AATATCAGCG SEQ ID NO: 12 GGCTGTTGCT
AATACCAGCG SEQ ID NO: 15 CGCTGATATT AGCAACAGCC SEQ ID NO: 16
CGCTGGTATT AGCAACAGCC SEQ ID NO: 25 GGCUGUUGCU AAUAUCAGCG SEQ ID
NO: 26 GGCUGUUGCU AAUACCAGCG SEQ ID NO: 27 CGCUGAUAUU AGCAACAGCC
SEQ ID NO: 28 CGCUGGUAUU AGCAACAGCC SEQ ID NO: 13 GCCTTCGGGT
TGTAAAGGAC TTTTGTCAGG GAAGAAAA SEQ ID NO: 14 GCTGATGACG GTACCTGAAG
AATAAGCACC GGC SEQ ID NO: 17 TTTTCTTCCC TGACAAAAGT CCTTTACAAC
CCGAAGGC SEQ ID NO: 18 GCCGGTGCTT ATTCTTCAGG TACCGTCATC AGC SEQ ID
NO: 35 GCCUUCGGGU UGUAAAGGAC UUUUGUCAGG GAAGAAAA SEQ ID NO: 36
GCUGAUGACG GUACCUGAAG AAUAAGCACC GGC SEQ ID NO: 37 UUUUCUUCCC
UGACAAAAGU CCUUUACAAC CCGAAGGC SEQ ID NO: 38 GCCGGUGCUU AUUCUUCAGG
UACCGUCAUC AGC SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC SEQ ID NO: 6
GGCGAGTGGC GAACGGGTGA GTAACATA SEQ ID NO: 7 GCTGCTGCAC GTAGTTAGCC
GGTGCTTATT CTTCAG SEQ ID NO: 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT
CATCG SEQ ID NO: 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT SEQ
ID NO: 10 GAAGGCCTTC GGGTTGTAAA GGAC SEQ ID NO: 41 GUCCCCUGCU
UUCCCUCUCA AGAC SEQ ID NO: 42 GGCGAGUGGC GAACGGGUGA GUAACAUA SEQ ID
NO: 43 GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU CUUCAG SEQ ID NO: 44
GUUAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG SEQ ID NO: 45 CGGGUUGUAA
AGGACUUUUG UCAGGGAAGA AAAGGCCGUU SEQ ID NO: 46 GAAGGCCUUC
GGGUUGUAAA GGAC
[0128] Preferred compositions of the present invention include
compositions for detecting Neisseria meningitidis comprising a
nucleic acid hybrid formed between a nucleic acid derived from
Neisseria meningitidis and an oligonucleotide having a nucleic acid
sequence substantially corresponding to at least one of the nucleic
acid sequences that follows:
13 GGCTGTTGCT AATATCAGCG SEQ ID NO: 11 GGCTGTTGCT AATACCAGCG SEQ ID
NO: 12 CGCTGATATT AGCAACAGCC SEQ ID NO: 15 CGCTGGTATT AGCAACAGCC
SEQ ID NO: 16 GGCUGUUGCU AAUAUCAGCG SEQ ID NO: 25 GGCUGUUGCU
AAUACCAGCG SEQ ID NO: 26 CGCUGAUAUU AGCAACAGCC SEQ ID NO: 27
CGCUGGUAUU AGCAACAGCC SEQ ID NO: 28
[0129] The present invention also contemplates compositions for
detecting Neisseria meningitidis having a nucleic acid hybrid
formed between a Neisseria meningitidis-derived nucleic acid and a
hybridization assay probe having a nucleic acid sequence
substantially corresponding to:
[0130] SEQ ID NO: 11 or SEQ ID NO: 25;
[0131] and which may also optionally contain a helper
oligonucleotide hybridized to said nucleic acid which has a nucleic
acid sequence which substantially corresponds to one of the
following nucleic acid sequences:
[0132] SEQ ID NOS: 13, 14, 35 or 36.
[0133] The present invention also contemplates compositions for
detecting Neisseria meningitidis having a nucleic acid hybrid
formed between a Neisseria meningitidis-derived nucleic acid and a
hybridization assay probe having a nucleic acid sequence
substantially corresponding to:
[0134] SEQ ID NO: 12 or SEQ ID NO: 26;
[0135] and which may also optionally contain a helper
oligonucleotide hybridized to said nucleic acid which has a nucleic
acid sequence which substantially corresponds to one of the
following nucleic acid sequences:
[0136] SEQ ID NOS: 13, 14, 35 or 36.
[0137] The present invention also contemplates compositions for
detecting Neisseria meningitidis having a nucleic acid hybrid
formed between a Neisseria meningitidis-derived nucleic acid and a
hybridization assay probe having a nucleic acid sequence
substantially corresponding to:
[0138] SEQ ID NO: 15 or SEQ ID NO: 27;
[0139] and which may also optionally contain a helper
oligonucleotide hybridized to said nucleic acid which has a nucleic
acid sequence which substantially corresponds to one of the
following nucleic acid sequences:
[0140] SEQ ID NOS: 17, 18, 37 or 38.
[0141] The present invention also contemplates compositions for
detecting Neisseria meningitidis having a nucleic acid hybrid
formed between a Neisseria meningitidis-derived nucleic acid and a
hybridization assay probe having a nucleic acid sequence
substantially corresponding to:
[0142] SEQ ID NO: 16 or SEQ ID NO: 28;
[0143] and which may also optionally contain a helper
oligonucleotide hybridized to said nucleic acid which has a nucleic
acid sequence which substantially corresponds to one of the
following nucleic acid sequences:
[0144] SEQ ID NOS: 17, 18, 37 or 38.
[0145] The present invention also contemplates compositions for
detecting Neisseria gonorrhoeae having a nucleic acid hybrid formed
between a Neisseria gonorrhoeae-derived nucleic acid and a
hybridization assay probe having a nucleic acid sequence
substantially corresponding to:
[0146] SEQ ID NO: 1 or SEQ ID NO: 31;
[0147] and which may also optionally contain a helper
oligonucleotide hybridized to said nucleic acid which has a nucleic
acid sequence which substantially corresponds to one of the
following nucleic acid sequences:
[0148] SEQ ID NOS: 2 or 39.
[0149] The present invention also contemplates compositions for
detecting Neisseria gonorrhoeae having a nucleic acid hybrid formed
between a Neisseria gonorrhoeae-derived nucleic acid and a
hybridization assay probe having a nucleic acid sequence
substantially corresponding to:
[0150] SEQ ID NO: 3 or SEQ ID NO: 32;
[0151] and which may also optionally contain a helper
oligonucleotide hybridized to said nucleic acid which has a nucleic
acid sequence which substantially corresponds to one of the
following nucleic acid sequences:
[0152] SEQ ID NOS: 4 or 40.
[0153] The present invention also contemplates compositions for
detecting Neisseria meningitidis having a nucleic acid derived from
Neisseria meningitidis and an oligonucleotide having a nucleic acid
sequence substantially corresponding to
[0154] SEQ ID NOS: 7 or 43
[0155] and/or an oligonucleotide having nucleic acid sequence
substantially corresponding to at least one nucleic acid sequence
that follows:
[0156] SEQ ID NOS: 9 or 45
[0157] and optionally has a hybridization assay probe capable of
hybridizing to a Neisseria meningitidis nucleic acid and which has
a nucleic acid sequence substantially corresponding to one of the
following nucleic acid sequences:
[0158] SEQ ID NOS: 11, 15, 25 or 27
[0159] and which may also optionally contain a helper
oligonucleotide which has a nucleic acid sequence which
substantially corresponds to one of the following nucleic acid
sequences:
[0160] SEQ ID NOS: 13, 14, 35 or 36.
[0161] The present invention also contemplates compositions for
detecting Neisseria meningitidis having a nucleic acid derived from
Neisseria meningitidis and an oligonucleotide with a nucleic acid
sequence substantially corresponding to
[0162] SEQ ID NOS: 7 or 43
[0163] and/or which also has an oligonucleotide having nucleic acid
sequence substantially corresponding to at least one nucleic acid
sequence that follows:
[0164] SEQ ID NOS: 9 or 45
[0165] and optionally has a hybridization assay probe capable of
hybridizing to Neisseria meningitidis nucleic acids and which has a
nucleic acid sequence substantially corresponding to one of the
following nucleic acid sequences:
[0166] SEQ ID NOS: 12, 16, 26 or 28
[0167] and which may also optionally contain a helper
oligonucleotide which has a nucleic acid sequence which
substantially corresponds to one of the following nucleic acid
sequences:
[0168] SEQ ID NOS: 13, 14, 35 or 36.
[0169] The present invention also contemplates compositions for
detecting Neisseria meningitidis having a nucleic acid derived from
Neisseria meningitidis and an oligonucleotide with a nucleic acid
sequence substantially corresponding to
[0170] SEQ ID NOS: 7 or 43
[0171] and/or which also has an oligonucleotide having nucleic acid
sequence substantially corresponding to at least one nucleic acid
sequence that follows:
[0172] SEQ ID NOS: 9 or 45
[0173] and optionally has a hybridization assay probe capable of
hybridizing to a Neisseria meningitidis nucleic acid which has a
nucleic acid sequence substantially corresponding to one of the
following nucleic acid sequences:
[0174] SEQ ID NOS: 15, 11, 27 or 25
[0175] and which may also optionally contain a helper
oligonucleotide which has a nucleic acid sequence which
substantially corresponds to one of the following nucleic acid
sequences:
[0176] SEQ ID NOS: 17, 18, 37 or 38.
[0177] The present invention also contemplates compositions for
detecting Neisseria meningitidis having a nucleic acid derived from
Neisseria meningitidis and an oligonucleotide with a nucleic acid
sequence substantially corresponding to
[0178] SEQ ID NOS: 7 or 43
[0179] and/or which also has an oligonucleotide having nucleic acid
sequence substantially corresponding to at least one nucleic acid
sequence that follows:
[0180] SEQ ID NOS: 9 or 45
[0181] and optionally has a hybridization assay probe capable of
hybridizing to a Neisseria meningitidis nucleic acid which has a
nucleic acid sequence substantially corresponding to one of the
following nucleic acid sequences:
[0182] SEQ ID NOS: 16, 12, 28 or 20
[0183] and which may also optionally contain a helper
oligonucleotide which has a nucleic acid sequence which
substantially corresponds to one of the following nucleic acid
sequences:
[0184] SEQ ID NOS: 17, 18, 37 or 38.
[0185] The present invention also contemplates compositions for
detecting Neisseria meningitidis having a nucleic acid derived from
Neisseria meningitidis and an oligonucleotide with a nucleic acid
sequence substantially corresponding to
[0186] SEQ ID NOS: 8 or 44
[0187] and/or which also has an oligonucleotide having nucleic acid
sequence substantially corresponding to at least one nucleic acid
sequence that follows:
[0188] SEQ ID NOS: 10 or 46
[0189] and optionally has a hybridization assay probe capable of
hybridizing to a Neisseria meningitidis nucleic acid which has a
nucleic acid sequence substantially corresponding to one of the
following nucleic acid sequences:
[0190] SEQ ID NOS: 15, 11, 27 or 25
[0191] and which may also optionally contain a helper
oligonucleotide which has a nucleic acid sequence which
substantially corresponds to one of the following nucleic acid
sequences:
[0192] SEQ ID NOS: 17, 18, 37 or 38.
[0193] The present invention also contemplates compositions for
detecting Neisseria meningitidis having a nucleic acid derived from
Neisseria meningitidis and an oligonucleotide with a nucleic acid
sequence substantially corresponding to
[0194] SEQ ID NOS: 8 or 44
[0195] and/or which also has an oligonucleotide having nucleic acid
sequence substantially corresponding to at least one nucleic acid
sequence that follows:
[0196] SEQ ID NOS: 10 or 46
[0197] and optionally has a hybridization assay probe capable of
hybridizing to a Neisseria meningitidis nucleic acid which has a
nucleic acid sequence substantially corresponding to one of the
following nucleic acid sequences:
[0198] SEQ ID NOS: 16, 12, 28 or 26
[0199] and which may also optionally contain a helper
oligonucleotide which has a nucleic acid sequence which
substantially corresponds to one of the following nucleic acid
sequences:
[0200] SEQ ID NOS: 17, 18, 37 or 38.
[0201] Preferred compositions of the present invention include
compositions for detecting Neisseria gonorrhoeae comprising a
nucleic acid hybrid formed between a nucleic acid derived from
Neisseria gonorrhoeae and an oligonucleotide having a nucleic acid
sequence substantially corresponding to at least one of the nucleic
acid sequences that follow:
14 SEQ ID NO: 1 GAACGTACCG GGTAGCGG SEQ ID NO: 3 GCCAATATCG
GCGGCCGATG SEQ ID NO: 29 CCGCTACCCG GTACGTTC SEQ ID NO: 30
CATCGGCCGC CGATATTGGC SEQ ID NO: 31 GAACGUACCG GGUAGCGG SEQ ID NO:
32 GCCAAUAUCG GCGGCCGAUG SEQ ID NO: 33 CCGCUACCCG GUACGUUC SEQ ID
NO: 34 CAUCGGCCGC CGAUAUUGGC SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA
AGAC SEQ ID NO: 6 GGCGAGTGGC GAACGGGTGA GTAACATA SEQ ID NO: 7
GCTGCTGCAC GTAGTTAGCC GGTGCTTATT CTTCAG SEQ ID NO: 8 GTTAGCCGGT
GCTTATTCTT CAGGTACCGT CATCG SEQ ID NO: 9 CGGGTTGTAA AGGACTTTTG
TCAGGGAAGA AAAGGCCGTT SEQ ID NO: 10 GAAGGCCTTC GGGTTGTAAA GGAC SEQ
ID NO: 41 GUCCCCUGCU UUCCCUCUCA AGAC SEQ ID NO: 42 GGCGAGUGGC
GAACGGGUGA GUAACAUA SEQ ID NO: 43 GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU
CUUCAG SEQ ID NO: 44 GUUAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG SEQ ID
NO: 45 CGGGUUGUAA AGGACUUUUG UCAGGGAAGA AAAGGCCGUU SEQ ID NO: 46
GAAGGCCUUC GGGUUGUAAA GGAC SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT
CAGCTAATAC CGCATACG SEQ ID NO: 4 ACGGTACCTG AAGAATAAGC ACCGGCTAAC
TACGTG SEQ ID NO: 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG SEQ
ID NO: 40 ACGGUACCUG AAGAAUAAGC ACCGGCUAAC UACGUG
[0202] More preferred compositions of the present invention include
compositions for detecting Neisseria gonorrhoeae comprising a
nucleic acid hybrid formed between a nucleic acid derived from
Neisseria gonorrhoeae and an oligonucleotide having a nucleic acid
sequence substantially corresponding to at least one of the nucleic
acid sequences that follow:
15 SEQ ID NO: 1 GAACGTACCG GGTAGCGG SEQ ID NO: 3 GCCAATATCG
GCGGCCGATG SEQ ID NO: 31 GAACGUACCG GGUAGCGG SEQ ID NO: 32
GCCAAUAUCG GCGGCCGAUG SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC
CGCATACG SEQ ID NO: 4 ACGGTACCTG AAGAATAAGC ACCGGCTAAC TACGTG SEQ
ID NO: 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG SEQ ID NO: 40
ACGGUACCUG AAGAAUAAGC ACCGGCUAAC UACGUG
[0203] The present invention also contemplates compositions for
detecting Neisseria gonorrhoeae having a nucleic acid derived from
Neisseria gonorrhoeae and an oligonucleotide with a nucleic acid
sequence substantially corresponding to
[0204] SEQ ID NOS: 5 or 41
[0205] and/or which also optionally has an oligonucleotide having
nucleic acid sequence substantially corresponding to at least one
nucleic acid sequence that follows:
[0206] SEQ ID NOS: 6 or 42
[0207] and optionally has a hybridization assay probe capable of
hybridizing to a Neisseria gonorrhoeae nucleic acid and which has a
nucleic acid sequence substantially corresponding to one of the
following nucleic acid sequences:
[0208] SEQ ID NOS: 1, 29, 31 or 33
[0209] and which may also optionally contain a helper
oligonucleotide which as a nucleic acid sequence which
substantially corresponds to one of the following nucleic acid
sequences:
[0210] SEQ ID NOS: 2 or 39
[0211] The present invention also contemplates compositions for
detecting Neisseria gonorrhoeae having a nucleic acid derived from
Neisseria gonorrhoeae and an oligonucleotide with a nucleic acid
sequence substantially corresponding to
[0212] SEQ ID NOS: 7 or 42
[0213] and/or which optionally has an oligonucleotide nucleic acid
sequence substantially corresponding to at least one nucleic acid
sequence that follows:
[0214] SEQ ID NOS: 9 or 45
[0215] and which optionally has a hybridization assay probe capable
of hybridizing to a Neisseria gonorrhoeae nucleic acid and which
has a nucleic acid sequence substantially corresponding to one of
the following nucleic acid sequences:
[0216] SEQ ID NOS: 3, 30, 32 or 34
[0217] and which may also optionally contain a helper
oligonucleotide which has a nucleic acid sequence which
substantially corresponds to one of the following nucleic acid
sequences:
[0218] SEQ ID NOS: 4 or 40
[0219] and which also optionally has an oligonucleotide having a
nucleic acid sequence substantially corresponding to at least one
nucleic acid sequence that follows:
[0220] SEQ ID NOS: 8 or 44
[0221] The present invention also contemplates compositions for
detecting Neisseria gonorrhoeae having a nucleic acid derived from
Neisseria gonorrhoeae and an oligonucleotide with a nucleic acid
sequence substantially corresponding to
[0222] SEQ ID NOS: 10 or 46
[0223] and optionally has a hybridization assay probe capable of
hybridizing to Neisseria gonorrhoeae nucleic acids and which has a
nucleic acid sequence substantially corresponding to one of the
following nucleic acid sequences:
[0224] SEQ ID NOS: 3, 30, 32 or 34
[0225] and/or which may also optionally contain a helper
oligonucleotide which has a nucleic acid sequence which
substantially corresponds to one of the following nucleic acid
sequences:
[0226] SEQ ID NOS: 4 or 40
[0227] The present invention also contemplates nucleic acid hybrids
comprising probes of the present invention and also at least one
helper oligonucleotide that has a nucleic acid sequence
substantially corresponding to at least one of the nucleic acid
sequences that follows:
16 SEQ ID NO: 13 GCCTTCGGGT TGTAAAGGAC TTTTGTCAGG GAAGAAAA SEQ ID
NO: 14 GCTGATGACG GTACCTGAAG AATAAGCACC GGC SEQ ID NO: 17
TTTTCTTCCC TGACAAAAGT CCTTTACAAC CCGAAGGC SEQ ID NO: 18 GCCGGTGCTT
ATTCTTCAGG TACCGTCATC AGC SEQ ID NO: 35 GCCUUCGGGU UGUAAAGGAC
UUUUGUCAGG GAAGAAAA SEQ ID NO: 36 GCUGAUGACG GUACCUGAAG AAUAAGCACC
GGC SEQ ID NO: 37 UUUUCUUCCC UGACAAAAGU CCUUUACAAC CCGAAGGC SEQ ID
NO: 38 GCCGGUGCUU AUUCUUCAGG UACCGUCAUC AGC SEQ ID NO: 2 GGGATAACTG
ATCGAAAGAT CAGCTAATAC CGCATACG SEQ ID NO: 4 ACGGTACCTG AAGAATAAGC
ACCGGCTAAC TACGTG SEQ ID NO: 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC
CGCAUACG SEQ ID NO: 40 ACGGUACCUG AAGAAUAAGC ACCGGCUAAC UACGUG
[0228] The present invention also contemplates compositions for
amplifying Neisseria nucleic acids comprising a nucleic acid hybrid
formed between a Neisseria nucleic acid and an oligonucleotide
having a nucleic acid sequence substantially corresponding to a
nucleic acid sequence selected from the group consisting of:
17 SEQ ID NO:5 GTCCCCTGCT TTCCCTCTCA AGAC SEQ ID NO:6 GGCGAGTGGC
GAACGGGTGA GTAACATA SEQ ID NO:41 GUCCCCUGCU UUCCCUCUCA AGAC SEQ ID
NO:7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT CTTCAG SEQ ID NO:8 GTTAGCCGGT
GCTTATTCTT CAGGTACCGT CATCG SEQ ID NO:43 GCUGCUGCAC GUAGUUAGCC
GGUGCUUAUU CUUCAG SEQ ID NO:44 GUUAGCCGGU GCUUAUUCUU CAGGUACCGU
CAUCG SEQ ID NO:9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT SEQ
ID NO:10 GAAGGCCTTC GGGTTGTAAA GGAC SEQ ID NO:45 CGGGUUGUAA
AGGACUUUUG UCAGGGAAGA AAAGGCCGUU SEQ ID NO:46 GAAGGCCUUC GGGUUGUAAA
GGAC
[0229] G. Assay Methods
[0230] The present invention contemplates various methods for
assaying for the presence of Neisseria meningitidis or Neisseria
gonorrhoeae nucleic acid within a sample. One skilled in the art
will understand that the exact assay conditions, probes or primers
used will vary depending on the particular assay format used and
the source of the sample.
[0231] Generally, the present invention contemplates methods for
detecting the presence of Neisseria meningitidis by contacting a
test sample under stringent hybridization conditions with a nucleic
acid hybridization assay probe capable of preferentially
hybridizing under stringent hybridization assay conditions to a
Neisseria meningitidis target nucleic acid over nucleic acids from
Neisseria gonorrhoeae, said target nucleic acid having a nucleic
acid sequence substantially corresponding to a sequence selected
from the group consisting of:
18 GGCTGTTGCT AATATCAGCG SEQ ID NO:11 GGCTGTTGCT AATACCAGCG SEQ ID
NO:12 CGCTGATATT AGCAACAGCC SEQ ID NO:15 CGCTGGTATT AGCAACAGCC SEQ
ID NO:16 GGCUGUUGCU AAUAUCAGCG SEQ ID NO:25 GGCUGUUGCU AAUACCAGCG
SEQ ID NO:26 CGCUGAUAUU AGCAACAGCC SEQ ID NO:27 CGCUGGUAUU
AGCAACAGCC SEQ ID NO:28
[0232] Preferred methods for detecting the presence of Neisseria
meningitidis include the step of contacting a test sample under
stringent hybridization conditions with a nucleic acid
hybridization assay probe capable of preferentially hybridizing
under stringent hybridization assay conditions to a Neisseria
meningitidis target nucleic acid sequence over nucleic acid
sequences of Neisseria gonorrhoeae, said target nucleic acid
sequence substantially corresponding to a sequence selected from
the group consisting of:
19 GGCTGTTGCT AATATCAGCG SEQ ID NO:11 GGCTGTTGCT AATACCAGCG SEQ ID
NO:12 CGCTGATATT AGCAACAGCC SEQ ID NO:15 CGCTGGTATT AGCAACAGCC SEQ
ID NO:16
[0233] Preferred methods for detecting the presence of Neisseria
gonorrhoeae include the step of contacting a test sample under
stringent hybridization conditions with a nucleic acid
hybridization assay probe capable of preferentially hybridizing
under stringent hybridization assay conditions to a Neisseria
gonorrhoeae target nucleic acid sequence over a nucleic acid
sequence of Neisseria meningitidis, said target nucleic acid
sequence substantially corresponding to a sequence selected from
the group consisting of:
20 GAACGTACCG GGTAGCGG SEQ ID NO:1 GCCAATATCG GCGGCCGATG SEQ ID
NO:3 CCGCTACCCG GTACGTTC SEQ ID NO:29 CATCGGCCGC CGATATTGGC SEQ ID
NO:30 GAACGUACCG GGUAGCGG SEQ ID NO:31 GCCAAUAUCG GCGGCCGAUG SEQ ID
NO:32 CCGCUACCCG GUACGUUC SEQ ID NO:33 CAUCGGCCGC CGAUAUUGGC SEQ ID
NO:34
[0234] In other embodiments, the present invention also
contemplates methods for detecting the presence of Neisseria
gonorrhoeae microorganisms by contacting a test sample under
stringent hybridization conditions with a nucleic acid
hybridization assay probe capable of preferentially hybridizing
under stringent hybridization assay conditions to a Neisseria
gonorrhoeae nucleic acid sequence over nucleic acid sequences from
Neisseria meningitidis, said target nucleic acid sequences
substantially corresponding to a sequence selected form the group
consisting of:
21 GAACGTACCG GGTAGCGG SEQ ID NO:1 GCCAATATCG GCGGCCGATG SEQ ID
NO:3 GAACGUACCG GGUAGCGG SEQ ID NO:31 GCCAAUAUCG GCGGCCGAUG SEQ ID
NO:32
[0235] The present invention also contemplates methods of detecting
Neisseria by first amplifying a portion of the Neisseria nucleic
acid and then optionally using a hybridization assay probe of the
present invention to assay for a specific Neisseria-derived nucleic
acid amplified by the primers of the present invention. The
amplified nucleic acid can be detected by a number of methods
including gel electrophoresis.
[0236] In preferred embodiments, the present invention contemplates
methods of detecting Neisseria-derived nucleic acid by first
amplifying said nucleic acid with at least one amplification
oligonucleotide that will bind to or cause elongation through one
or more of the following sequences:
22 SEQ ID NO:47 GUCUUGAGAG GGAAAGCAGG GGAC SEQ ID NO:48 UAUGUUACUC
ACCCGUUCGC CACUCGCC SEQ ID NO:49 CUGAAGAAUA AGCACCGGCU AACUACGUGC
AGCAGC SEQ ID NO:50 CGAUGACGGU ACCUGAAGAA UAAGCACCGG CUAAC SEQ ID
NO:51 AACGGCCUUU UCUUCCCUGA CAAAAGUCCU UUACAACCCG SEQ ID NO:52
GUCCUUUACA ACCCGAAGGC CUUC
[0237] wherein said amplification oligonucleotide optionally has a
nucleic acid sequence recognized by an RNA polymerase or which
enhances initiation of elongation by an RNA polymerase.
[0238] This first method step is then optionally followed by
detecting the amplified nucleic acid produced in the amplification
step with an oligonucleotide hybridization assay probe able to
specifically hybridize to nucleic acids derived from Neisseria
species, Neisseria cinerea, Neisseria meningitidis or Neisseria
gonorrhoeae under stringent hybridization conditions.
[0239] The amplification oligonucleotide used in the methods of the
present invention may optionally have a nucleic acid sequence for
example, a promoter sequence, recognized by an RNA polymerase or
which enhances initiation by an RNA polymerase.
[0240] In other preferred embodiments, the present invention
contemplates a method for amplifying Neisseria nucleic acids in a
test sample by amplifying the nucleic acid with one or more
amplification oligonucleotide that will bind to or cause elongation
through a nucleic acid sequence substantially corresponding to the
following nucleotide sequences:
[0241] SEQ ID NOs: 19 or 49,
[0242] SEQ ID NOs: 21 or 50, or
[0243] with a second amplification oligonucleotide that will bind
to or cause elongation through a nucleic acid sequence
substantially corresponding to one of the following sequences:
[0244] SEQ ID NOs: 20 or 51,
[0245] SEQ ID NOs: 22 or 52 or both, said amplification
oligonucleotides, wherein at least one of said amplification
oligonucleotides optionally has a nucleic acid sequence recognized
by an RNA polymerase or which enhances initiation or elongation by
an RNA polymerase.
[0246] In other more preferred embodiments, the present invention
contemplates a method for amplifying Neisseria-derived nucleic
acids in a test sample comprising amplifying said nucleic acid with
one or more amplification oligonucleotide that will bind to or
cause elongation through a nucleic acid sequence substantially
corresponding to one of the following nucleotide sequences:
[0247] SEQ ID NOS: 19 or 49, or
[0248] with a second amplification oligonucleotide that will bind
to or cause elongation through a nucleic acid sequence
substantially corresponding to the following sequences:
[0249] SEQ ID NOS: 20 or 51, or both said amplification
oligonucleotides, wherein at least one of said amplification
oligonucleotides optionally has a nucleic acid sequence recognized
by an RNA polymerase or which enhances initiation or elongation by
an RNA polymerase.
[0250] In other preferred embodiments, the present invention
contemplates a method for increasing the number of
Neisseria-derived nucleic acid sequences in a test sample
comprising amplifying said nucleic acid with one or more
amplification oligonucleotides that will bind to or cause
elongation through a nucleic acid sequence substantially
corresponding to the following nucleotide sequences:
[0251] SEQ ID NOS: 21 or 50,
[0252] or with a second amplification oligonucleotide that will
bind to or cause elongation through a nucleic acid sequence
substantially corresponding to the following sequences:
[0253] SEQ ID NOS: 22 or 52, or both said amplification
oligonucleotides, wherein at least one of said amplification
oligonucleotides optionally has a nucleic acid sequence recognized
by an RNA polymerase or which enhances initiation or elongation by
an RNA polymerase.
[0254] The above methods may also include the further step of
detecting the amplified nucleic acid with an oligonucleotide
hybridization assay probe able to specifically hybridize to
Neisseria meningitidis nucleic acids under stringent hybridization
conditions.
[0255] Specifically, the methods may detect Neisseria meningitidis
using oligonucleotide hybridization assay probes which will
hybridize under stringent hybridization conditions to a nucleic
acid sequence substantially corresponding to a sequence selected
from the group consisting of:
23 GGCTGTTGCT AATATCAGCG SEQ ID NO:11 CGCUGAUAUU AGCAACAGCC SEQ ID
NO:27 GGCTGTTGCT AATACCAGCG SEQ ID NO:12 CGCUGGUAUU AGCAACAGCC SEQ
ID NO:28 CGCTGATATT AGCAACAGCC SEQ ID NO:15 GGCUGUUGCU AAUAUCAGCG
SEQ ID NO:25 CGCTGGTATT AGCAACAGCC SEQ ID NO:16 GGCUGUUGCU
AAUACCAGCG SEQ ID NO:26
[0256] The present invention also contemplates methods for
increasing the number of Neisseria gonorrhoeae-derived nucleic
acids in a test sample by amplifying said nucleic acid with one or
more amplification oligonucleotides that will bind to or cause
elongation through a nucleic acid sequence substantially
corresponding to one or more of the following nucleotide
sequences:
[0257] SEQ ID NOs 23 or 47,
[0258] SEQ ID NOs 24 or 48,
[0259] and where the amplification oligonucleotide optionally has a
nucleic acid sequence recognized by an RNA polymerase or which
enhances initiation or elongation by an RNA polymerase.
[0260] Additional methods are contemplated for amplifying Neisseria
gonorrhoeae-derived nucleic acids in a test sample with a first
amplification oligonucleotide that will bind to or cause elongation
through a nucleic acid sequence substantially corresponding to one
of the following nucleotide sequences:
[0261] SEQ ID NOs: 23 or 47, or
[0262] with a second amplification oligonucleotide that will bind
to or cause elongation through a nucleic acid sequence
substantially corresponding to one of the following sequences:
[0263] SEQ ID NOs: 24 or 48, or
[0264] with both said first and second amplification
oligonucleotides wherein one of the amplification oligonucleotides
optionally has a nucleic acid sequence recognized by an RNA
polymerase or which enhances initiation or elongation by an RNA
polymerase.
[0265] These methods of amplifying a Neisseria gonorrhoeae-derived
nucleic acid may be followed by the step of detecting the amplified
nucleic acid with an oligonucleotide hybridization assay probe able
to specifically hybridize to Neisseria gonorrhoeae nucleic acids
under stringent hybridization conditions.
[0266] Preferably the oligonucleotide hybridization assay probe has
a nucleic acid sequence substantially corresponding to a sequence
selected from the group consisting of:
[0267] SEQ ID NOS: 1, 29, 31, and 33.
[0268] The detecting of Neisseria gonorrhoeae nucleic acid may
include the use of a helper oligonucleotide having a nucleic acid
sequence substantially corresponding to a nucleic acid sequence
selected from the group consisting of:
[0269] SEQ ID NO: 2, and
[0270] SEQ ID NO: 39.
[0271] Other methods of detecting Neisseria gonorrhoeae nucleic
acid are contemplated by increasing the number of Neisseria
gonorrhoeae-derived nucleic acids in a test sample by amplifying
said nucleic acid with one or more amplification oligonucleotide
that will bind to or cause elongation through a nucleic acid
sequence substantially corresponding to one or more of the
following nucleotide sequences: SEQ ID NOS: 19 or 49, SEQ ID NOS:
20 or 51,
[0272] SEQ ID NOS: 21 or 50, SEQ ID NOS: 22 or 52, and where the
amplification oligonucleotide optionally has a nucleic acid
sequence recognized by an RNA polymerase or which enhances
initiation or elongation by an RNA polymerase.
[0273] Preferred methods for amplifying Neisseria nucleic acids in
a test sample include amplifying the nucleic acid with one or more
amplification oligonucleotide that will bind to or cause elongation
through a nucleic acid sequence substantially corresponding to one
of the following nucleotide sequences:
[0274] SEQ ID NOS: 19 or 49, or
[0275] with a second amplification oligonucleotide that will bind
to or cause elongation through a nucleic acid sequence
substantially corresponding to one of the following sequences:
[0276] SEQ ID NOS: 20 or 51, or
[0277] with both said first and second amplification
oligonucleotides.
[0278] Alternatively the present invention contemplates amplifying
Neisseria nucleic acids in a test sample comprising amplifying the
nucleic acid with one or more amplification oligonucleotide that
will bind to or cause elongation through a nucleic acid sequence
substantially corresponding to one of the following nucleotide
sequences:
[0279] SEQ ID NOS: 21 or 50, or
[0280] with a second amplification oligonucleotides that will bind
to or cause elongation through a nucleic acid sequence
substantially corresponding to one of the following sequences:
[0281] SEQ ID NOS: 22 or 52, or
[0282] with both said first and second amplification
oligonucleotides.
[0283] The amplification of the Neisseria nucleic acid is
preferably followed by detecting the amplified nucleic acid with an
oligonucleotide hybridization assay probe able to specifically
hybridize to Neisseria gonorrhoeae nucleic acids under stringent
hybridization conditions. The oligonucleotide hybridization assay
probe used preferably has a nucleic acid sequence substantially
corresponding to a sequence selected from the group consisting of:
SEQ ID NOS: 3, 32, 30, 34
[0284] H. Diagnostic Systems
[0285] The present invention also contemplates diagnostic systems
in kit form. A diagnostic system of the present invention may
include a kit which contains, in an amount sufficient for at least
one assay, amplification primers and/or hybridization assay probes
of the present invention in a packaging material. Typically, the
kits would also include instructions for use of the packaged
primers and/or probes.
[0286] The various components of the diagnostic system may be
provided in various forms. For example, the required enzymes, the
nucleotide triphosphates, the primers and probes may be provided as
a lyophilized reagent. These lyophilized reagents may be premixed
before lyophilization so that when reconstituted form a complete
mixture with the proper ratio of each of the components ready for
use in the assay. In addition, the diagnostic systems of the
present invention may contain a reconstitution reagent for
reconstituting the lyophilized reagents of the kit. In preferred
kits, the enzymes, nucleotides, triphosphates and required
cofactors for the enzymes are provided as a single lyophilized
reagent that when reconstituted forms a proper reagent for use in
the present methods. In these preferred kits, a lyophilized primer
agent may also be provided. In other preferred kits, lyophilized
probe reagents are provided.
[0287] Typical packaging materials would include solid matrices
such as glass, plastic, paper, foil, micro particles and the like,
capable of holding within fixed limits hybridization assay probe or
amplification primer of the present invention. Thus, for example, a
package made from packaging materials can be a glass vial used to
contain sub-milligram (i.e. picogram, nanogram etc.) quantities of
a contemplated primer or hybridization assay probe or it could be a
microtiter plate well to which the probes and/or primers of the
present invention have been operatively affixed, i.e., linked so as
to be capable of participating in a detection method of the present
invention.
[0288] Instructions for use typically include a tangible expression
describing the various reagents and/or concentrations of reagents
and at least one assay method parameter which, for example, would
be the relative amounts of reagents to use per amount of sample. In
addition, such specifics as maintenance, time periods, temperature
and buffer conditions may also be included.
[0289] The present invention contemplates diagnostic systems or
kits containing the oligonucleotides of a composition of the
present invention. The present invention also contemplates
diagnostic systems or kits containing the oligonucleotides required
to perform a method of the present invention.
[0290] This method preferably also uses a helper oligonucleotide
having a nucleic acid sequence substantially corresponding to a
nucleic acid sequence selected from the group consisting of:
[0291] SEQ ID NO. 4: and
[0292] SEQ ID NO. 40:
[0293] The present invention contemplates diagnostic systems or a
kit containing at least one oligonucleotide having a nucleic acid
sequence substantially corresponding to a nucleic acid sequence
selected from the group consisting of:
[0294] SEQ ID NOS: 1, 3, 11, 12, 15, 16, 29, 30, 33, 34, 27, 28,
25, 26.
[0295] The present invention contemplates diagnostic systems or a
kit having an oligonucleotide hybridization assay probe having at
least one helper probe having a nucleic acid sequence substantially
corresponding to the sequence selected from the group consisting
of:
[0296] SEQ ID NOS: 2 or 39,
[0297] when said oligonucleotide substantially corresponds to SEQ
ID NOS: 1 or 31;
[0298] or
[0299] SEQ ID NOS: 4 or 40,
[0300] when said oligonucleotide substantially corresponds to SEQ
ID NOS: 3 or 32;
[0301] or
[0302] SEQ ID NOS: 13 or 35, or
[0303] SEQ ID NOS: 14 or 36,
[0304] when said oligonucleotide substantially corresponds to
[0305] SEQ ID NOS: 11 or 25, or
[0306] SEQ ID NOS: 12 or 26;
[0307] or
[0308] SEQ ID NOS: 17 or 37,
[0309] SEQ ID NOS: 18 or 38,
[0310] when said oligonucleotide substantially corresponds to
[0311] SEQ ID NOS: 15 or 27, or
[0312] SEQ ID NOS: 16 or 28.
[0313] The present invention contemplates diagnostic systems or a
kit containing two oligonucleotides having a nucleic acid sequence
substantially corresponding to a nucleic acid sequence selected
from the group consisting of:
24 SEQ ID NO:7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT CTTCAG SEQ ID NO:8
GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG SEQ ID NO:9 CGGGTTGTAA
AGGACTTTTG TCAGGGAAGA AAAGGCCGTT SEQ ID NO:10 GAAGGCCTTC GGGTTGTAAA
GGAC SEQ ID NO:43 GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU CUUCAG SEQ ID
NO:44 GUUAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG SEQ ID NO:45
CGGGUUGUAA AGGACUUUUG UCAGGGAAGA AAAGGCCGUU SEQ ID NO:46 GAAGGCCUUC
GGGUUGUAAA GGAC
[0314] optionally having a 5' sequence recognized by an RNA
polymerase or which enhances initiation or elongation by an RNA
polymerase.
[0315] The present invention contemplates diagnostic systems or a
kit containing oligonucleotides having a nucleic acid sequence
substantially corresponding to the following sequences:
[0316] SEQ ID NOS: 7 or 43,
[0317] SEQ ID NOS: 9 or 45,
[0318] SEQ ID NOS: 11 or 25,
[0319] SEQ ID NOS:. 13 or 35,
[0320] SEQ ID NOS: 14 or 36.
[0321] The present invention contemplates diagnostic systems or a
kit containing oligonucleotides having a nucleic acid sequence
substantially corresponding to the following sequences:
[0322] SEQ ID NOS: 15 or 27,
[0323] SEQ ID NOS: 16 or 26,
[0324] SEQ ID NOS: 17 or 37,
[0325] SEQ ID NOS: 18 or 38.
[0326] The present invention contemplates diagnostic systems or a
kit containing oligonucleotides having a nucleic acid sequence
substantially corresponding to the following sequences:
[0327] SEQ ID NOS: 7 or 43,
[0328] SEQ ID NOS: 9 or 45,
[0329] SEQ ID NOS: 15 or 27,
[0330] SEQ ID NOS: 16 or 28,
[0331] SEQ ID NOS: 17 or 37,
[0332] SEQ ID NOS: 18 or 38.
[0333] The present invention contemplates diagnostic systems or a
kit containing oligonucleotides having a nucleic acid sequence
substantially corresponding to the following sequences:
[0334] SEQ ID NOS: 5 or 41,
[0335] SEQ ID NOS: 6 or 42,
[0336] SEQ ID NOS: 2 or 39,
[0337] SEQ ID NOS: 1 or 31.
[0338] The present invention contemplates diagnostic systems or a
kit containing oligonucleotides having a nucleic acid sequence
substantially corresponding to the following sequences:
[0339] SEQ ID NOS: 5 or 41,
[0340] SEQ ID NOS: 2 or 39,
[0341] SEQ ID NOS: 1 or 31.
EXAMPLES
[0342] Examples are provided below to illustrate different aspects
and embodiments of the present invention. These examples are not
intended in any way to limit the disclosed invention, which is
limited only by the claims.
[0343] Probes specific for Neisseria meningitidis were designed
using sequences determined in prospective target areas using
primers complementary to the 16S rRNAs of Neisseria gonorrhoeae
(ATCC NO. 19424), Neisseria meningitidis sero group A (ATCC NOs
13077), serogroup C (ATCC No. 23248) and serogroup L (ATCC No.
43828), and clinical isolates, Neisseria lactamica (ATCC NO.
23970), Neisseria cinerea (ATCC NO. 14685), Neisseria mucosa (ATCC
NO. 19696), Neisseria sicca (ATCC NO. 29193) and Kingella kingae
(ATCC NO. 23330). The nucleic acid sequence from phylogenetically
near neighbors, including the published sequence of Neisseria
gonorrhoeae NCTC 8375 Rossau et al., Nuc. Acids Res. 16:6227 were
also used as comparisons with the nucleic sequence from Neisseria
meningitidis to determine variable regions.
[0344] An example of such an alignment follows: A specific sequence
in which Neisseria meningitidis varied from E. coli and Neisseria
gonorrohoeae was chosen for probe design. Two different probes were
designed to Neisseria meningitidis (SEQ ID NO: 11) and (SEQ ID NO:
12). The rRNA sequences are shown below:
25 E. coli GAGUAAAG(UUAAUAC)CUUUG SEQ ID NO:54 . . .. .. . .
GGCTGTTG(CTAATAC)CAGCG SEQ ID NO:12 ........ ...... .....
GGCTGTTG(CTAATAT)CAGCG SEQ ID NO:11 N. meningitidis.c ... . . . ..
. ..... GGCUGUUG(CUAAUAU)CAGCG SEQ ID NO:55 N. gonorrhoeae.P
........ . ..... . . . GGCUGUUG(CCAAUAU)CGGGG SEQ ID NO:56
[0345] The following hybridization assay probe sequences are
featured in the examples described below:
26 GAACGTACCG GGTAGCGG, SEQ ID NO:1 GCCAATATCG GCGGCCGATG, SEQ ID
NO:3 GGCTGTTGCTAATATCAGCG, SEQ ID NO:11 GGCTGTTGCTAATACCAGCG, SEQ
ID NO:12 CGCTGATATTAGCAACAGCC, and SEQ ID NO:15
CGCTGGTATTAGCAACAGCC SEQ ID NO:16
Example 1
[0346] In this experiment, purified N. gonorrhoeae rRNA (ATCC NO.
19424) was amplified with oligonucleotides containing sequences
complementary to N. gonorrhoeae rRNA using the techniques described
in Kacian et al. U.S. Pat. No. 5,399,491. Two promoter primers were
synthesized, each containing a T7 RNA polymerase promoter sequence
5'-AATTTAATACGACTCACTATA- GGGAGA-3' SEQ ID NO. 53 at the 5' end,
covalently attached to a target complementary sequence
5'-GTCCCCTGCTTTCCCTCTCAAGAC-3' (SEQ ID NO. 5) at the 3' end. One
promoter primer was synthesized with a free 3' OH group, and was
used at two pmol per reaction. The second promoter primer was
synthesized with an alkane diol group at the 3' end and was used at
13 pmol per reaction. The target nucleic acid and primers were
heated to 95.degree. C. for 15 minutes and cooled to 42.degree. C.
Moloney Murine Leukemia Virus reverse transcriptase (MMLV RT), 900
units, and 400 units of T7 RNA polymerase were added. The final
amplification mixture contained 50 mM Tris HCl (pH 8.5), 35 mM
potassium chloride, 4 mM GTP, 4 mM ATP, 4 mM UTP, 4 mM CTP, 1 mM
DATP, 1 mM dTTP, 1 mM dCTP, 1 mM dGTP, 20 mM MgCl2, 20 mM
N-Acetyl-L-Cysteine, and 5% (w/v) glycerol. After a two hour
incubation at 42.degree. C., the entire one hundred .mu.l
amplification reaction was assayed by hybridization with an
acridinium ester labeled probe of sequence 5'-GAACGTACCGGGTAGCGG-3'
(SEQ. ID. NO. 1) and an unlabeled helper probe of sequence
5'-GGGATAACTGATCGAAAGATCAGCTAAT- ACCGCATACG-3' (SEQ. ID. NO. 2)
Hybridization was performed in 200 .mu.l of a solution containing
0.05 M lithium succinate (pH 5), 0.6 M LiCl, 1% (w/v) lithium
lauryl sulfate, 10 mM EDTA, 10 nM EGTA, at 60.degree. C. for 10
minutes, followed by addition of 300 .mu.l of 0.15 M sodium
tetraborate pH 8.5, 1% TRITON.RTM. X-100. This mixture was
incubated at 60.degree. C. for 10 minutes, and cooled to room
temperature. The remaining chemiluminescence in each tube was
assayed in a Gen-Probe LEADER.RTM. I luminometer equipped with
automatic injection of 1 mM nitric acid and 0.1% (v/v) hydrogen
peroxide followed by injection of a solution containing 1 N sodium
hydroxide. Results were given in Relative Light Units (RLU), a
measure of the photons detected by the luminometer.
27TABLE 1 Amplification of Neisseria gonorrhoeae nucleic acid with
amplification oligonucleotides comprising SEQ ID NO. 5 followed by
detection with a probe comprising SEQ ID NO. 1. Amount of target
RLU 0.1 pg* 103,596 99,931 123,512 0.025 pg 25,636 39,454 29,594 0
pg 1,084 1,012 640 *pg = picogram
Example 2
[0347] This experiment demonstrates amplification of N. gonorrhoeae
rRNA with two primers of opposite sense. The promoter-primer
described in Example 1 containing a T7 RNA polymerase promoter
sequence and a 3' target hybridizing region of sequence
5'-GTCCCCTGCTTTCCCTCTCAAGAC-3' (SEQ. ID. NO. 5) was used at 15 pmol
per reaction and a primer containing a sequence of the same sense
as N. gonorrhoeae rRNA, 5'-GGCGAGTGGCGAACGGGTGAGTAACATA-3' (SEQ.
ID. NO. 6) was used at 15 pmol per reaction. Reactions were
performed in triplicate. The amplification conditions were as
described in Example 1, and samples were heated to 95.degree. C.
for 5 minutes, then cooled to 42.degree. C. Enzymes were added, and
after a two hour incubation at 42.degree. C., 20 .mu.l of the
amplification reaction was assayed by hybridization with an
acridinium ester labeled probe synthesized with sequence SEQ. ID.
NO. 1 and an unlabeled helper probe synthesized with sequence SEQ.
ID. NO. 2. The primers amplified N. gonorrhoeae RNA and allowed
detection of less than 100 copies of target.
28TABLE 2 Amplification of N. gonorrhoeae rRNA with primers
comprising SEQ. ID. NOs. 5 and 6 and detection with a probe
comprising SEQ ID NO. 1. Amount of rRNA target added RLU 500 copies
(0.0012 pg) 334,838 343,107 1,320,194 80 copies (0.0002 pg) 255,898
1,512,486 377,938 0 pg 2,354 2,454 2,440
Example 3
[0348] In this experiment, two promoter primers of identical
sequence were again used. Each promoter primer was synthesized with
a 5' T7 RNA polymerase promoter sequence
5'-AATTTAATACGACTCACTATAGGGAGA-3' (SEQ ID NO: 53) at the 5' end and
a target hybridizing region
5'-GCTGCTGCACGTAGTTAGCCGGTGCTTATTCTTCAG-3' (SEQ ID NO. 7) at the 3'
end. The promoter primers were synthesized either with a
3'-hydroxyl group and used at 2 pmol per reaction, or with a
3'-alkane diol and used at 13 pmol per reaction. Samples were
heated to 95.degree. C. for 5 minutes and cooled to 42.degree. C.
prior to enzyme addition. The amplification conditions were as
described in Example 1. After a two hour incubation at 42.degree.
C., 100 .mu.l of the amplification reaction was assayed by
hybridization with an acridinium ester labeled probe synthesized
with sequence 5'GCCAATATCGGCGGCCGATG-3' (SEQ. ID. NO. 3) and an
unlabeled helper probe with the sequence
5'-ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3' (SEQ. ID. NO. 4) using
the conditions described in Example 1.
29TABLE 3 Amplification of N. gonorrhoeae rRNA using primers
comprising SEQ ID NO. 7. Amount of rRNA target added RLU with probe
SEQ ID NO. 3 0.025 pg 95,905 49,717 59,774 0.0125 pg 10,520 12,576
12,322 0.005 pg 19,498 40,962 21,722 0 pg 2,888 2,792 2,777
Example 4
[0349] In this experiment, N. gonorrhoeae rRNA was amplified with a
mixture of two oligonucleotides, one a promoter primer
complementary to N. gonorrhoeae rRNA and one primer of the same
sense as N. gonorrhoeae rRNA. The promoter primer contained a T7
RNA polymerase promoter sequence at the 5' end and a target
hybridizing region 5'-GCTGCTGCACGTAGTTAGCCGGTG- CTTATTCTTCAG-3'
(SEQ ID NO. 7) at the 3' end and was used with a primer of sequence
5'-CGGGTTGTAAAGGACTTTTGTCAGGGAAGAAAAGGCCGTT-3' (SEQ. ID. NO. 9) at
30 pmol per reaction. Alternatively, a promoter primer containing a
target hybridizing region of sequence
5'-GTTAGCCGGTGCTTATTCTTCAGGTACCGTCA- TCG-3' (SEQ. ID. NO. 8) was
used at 15 pmol per reaction with the promoter primer with a
sequence 5'-GAAGGCCTTCGGGTTGTAAAGGAC-3' (SEQ. ID. NO. 10), at 15
pmol per reaction. Amplification conditions were as described for
Example 1. Twenty 1 .mu.l of the product was assayed by
hybridization with an acridinium ester labeled probe synthesized
with sequence 5'-GCCAATATCGGCGGCCGATG-3' (SEQ. ID. NO. 3) and an
unlabeled helper probe synthesized with the sequence
5'-ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3' (SEQ. ID. NO. 4) as
described in Example 1.
30TABLE 4 Amplification of N. gonorrhoeae rRNA using primers
comprising SEQ ID NOs. 7 and 9 or 8 and 10. RLU with probe SEQ ID
NO. 3 Amount of rRNA target added SEQ ID NOS. 7 SEQ ID NOS. 8
Primer sequences: and 9 and 10 0.5 pg 817,344 680,836 802,901
603,811 785,206 619,969 0.05 pg 188,661 132,359 192,656 157,509
204,878 87,161 0.005 pg 25,276 51,398 26,451 40,032 24,915 55,968 0
pg 3,600 2,189 3,366 2,205 888
Example 5
[0350] This example demonstrates the reactivity of the
amplification and detection assay. Fresh cultures of thirteen
strains of N. gonorrhoeae were suspended in 0.9% sodium chloride at
a density of approximately 10.sup.10 cells/ml and lysed in a
solution containing 3% (w/v) lithium lauryl sulfate, 30 mm sodium
phosphate pH 6.8, 1 mM EDTA and 1 mM EGTA. Release of nucleic acid
was confirmed by hybridization with a probe directed to a conserved
region of ribosomal RNA of all bacteria. The cell lysates were
further diluted in water and added to amplification reactions
containing 30 pmol of a promoter-primer containing a 5' T7 RNA
promoter sequence SEQ ID NO: 53 5'-AATTTAATACGACTCACTATAGGGAGA-3'
and a 3' target binding sequence comprising SEQ. ID. NO. 7, and 30
pmol of primer comprising sequence SEQ. ID. NO. 9. Duplicate
reactions containing lysate from at least 10.sup.5 cells were
performed using an amplification mixture containing 50 mM Tris HCl
(pH 8.5), 35 mM potassium chloride, 4 mM GTP, 4 mM ATP, 4 mM UTP, 4
mM CTP, 1 mM DATP, 1 mM dTTP, 1 mM dCTP, 1 mM dGTP, 20 mM MgCl2, 20
mM N-acetyl-L-cysteine, 5% (v/v) glycerol and the oligonucleotide
primers described above. The mixture was heated to 95.degree. C.
for 5 minutes, cooled to 42.degree. C. and 900 units of MMLV
reverse transcriptase and 400 units of T7 RNA polymerase were
added. After a one hour incubation at 42.degree. C., 20 .mu.l of
amplification reaction was assayed by hybridization with an
acridinium ester labeled probe synthesized with sequence
5'GCCAATATCGGCGGCCGATG-3' (SEQ. ID. NO. 3) and an unlabeled helper
probe containing sequence
5'-ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3' (SEQ. ID. NO. 4).
31TABLE 5 Amplification of different strains of N. gonorrhoeae
using primers comprising SEQ ID NOs. 7 and 9. N. gonorrhoeae ATCC
No. RLU with probe SEQ ID NO. 3 9793 1,150,477 1,162,284 9826
1,173,586 1,149,251 9827 1,093,440 1,080,405 9828 1,143,960
1,149,465 9830 1,165,108 1,143,063 10150 1,105,754 1,131,598 10874
1,139,487 1,103,912 11688 1,024,195 1,112,160 11689 1,141,404
1,116,069 19424 1,104,256 1,116,832 27628 1,133,696 1,117,624 27630
1,132,496 1,146,161 27631 1,089,105 1,070,058
Example 6
[0351] Sequence analysis of other Neisseria species indicated that
the amplification oligonucleotides of this invention could amplify
nucleic acids of other species. This example demonstrates the
utility of the amplification oligonucleotides of this invention to
amplify nucleic acid from another Neisseria species, N.
meningitidis. In the course of development of a specific probe for
N. meningitidis, it became clear that the members of the species N.
meningitidis were not homogeneous in the probe region of choice.
The sequences of 16S rRNAs of representative N. meningitidis
species which showed low reactivity to the initial probe were
determined and a second probe was designed. These data demonstrate
the differential reactivity of three N. meningitidis species to the
two probes. In this example, purified RNA from Neisseria
gonorrhoeae (ATCC No. 19424), or lysates from Neisseria
meningitidis serogroup A (ATCC No. 13077), serogroup C (ATCC No.
13102) and serogroup L, (ATCC No. 43828) representing approximately
1,000 cells were amplified with a promoter-primer and primer
described in Example 5 under the conditions described in Example 5.
Ten .mu.l samples of the 100 .mu.l amplification reactions were
assayed by hybridization with an acridinium ester labeled probe
synthesized with sequence 5'-GCCAATATCGGCGGCCGATG-3' (SEQ ID NO. 3)
and an unlabeled helper probe synthesized with the sequence
5'-ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3, (SEQ ID NO. 4), or an
acridinium ester labeled probe synthesized with the sequence
5'-GGCTGTTGCTAATATCAGCG-3' (SEQ ID NO. 11) and two unlabeled helper
probes, one synthesized with sequence
5'-GCCTTCGGGTTGTAAAGGACTTTTGTCAGGGA- AGAAAA-3' (SEQ ID NO. 13) and
one synthesized with the sequence
5'-GCTGATGACGGTACCTGAAGAATAAGCACCGGC-3' (SEQ ID NO. 14), or an
acridinium ester labeled probe synthesized with sequence
5'-GGCTGTTGCTAATACCAGCG-3' (SEQ ID NO. 12) with unlabeled helper
probes SEQ ID NO: 13 and 14 or with a combination of labeled probes
SEQ ID NO: 11 and 12 used with unlabeled helper probes SEQ ID NO:
13 and 14. Sequence analysis indicated that other strains of
Neisseria will also amplify with these primers.
32TABLE 6 Amplification of Neisseria gonorrhoeae and Neisseria
meningitidis strains using primers comprising SEQ ID NOs. 7 and 9.
RLU Probe SEQ ID NOs: 3 11 12 11+ 12 Helper probe SEQ ID NOs:
Organism 4 13 + 14 13 + 14 13 + 14 N. gonorrhoeae 1,017,626 1,660
820 1,603 994,788 1,448 809 1,559 1,030,242 1,743 805 1,792 N.
meningitidis 2,059 1,208,967 3,534 829,251 Serogroup A 1,861
1,115,956 3,700 760,360 2,183 1,138,675 3,546 775,675 N.
meningitidis 1,931 1,164,254 2,819 749,502 Serogroup C 2,130
1,068,489 2,477 687,517 1,963 1,110,933 3,103 803,732 N.
meningitidis 1,833 85,321 1,206,045 1,537,314 Serogroup L 1,972
79,555 1,199,815 1,474,016 1,814 77,797 1,211,022 1,645,742
[0352] The data show that strains of N. meningitidis and N.
gonorrhoeae can be amplified using primers comprising SEQ ID NOs. 7
and 9 and detected with probes of SEQ ID NOs. 3, 11, and 12.
Example 7
[0353] The sensitivity of the amplification and detection assay for
N. meningitidis were demonstrated in this experiment. In this
example, Neisseria meningitidis serogroup C cells were cultured and
suspended in 0.9% sodium chloride to a density of approximately
10.sup.9 cells per ml. Cells were lysed following addition of an
equal volume of a solution containing 3% (w/v) lithium lauryl
sulfate, 30 mM sodium phosphate (pH 6.8), 1 mM EDTA, 1 mM EGTA and
diluted with water prior to addition to the amplification
reactions. Amplifications were performed as described for Example 5
using the promoter primer and primer described in Example 5 (SEQ ID
NOs. 7 and 9, respectively). Twenty .mu.l of the reaction was
analyzed by hybridization in the HPA format using an acridinium
ester labeled probe synthesized with the sequence
5'-GGCTGTTGCTAATATCAGCG-3' (SEQ ID NO. 11) and two unlabeled helper
probes, one synthesized with the sequence
5'-GCCTTCGGGTTGTAAAGGACTTTTGTCAGGGAAGAAAA-3' (SEQ ID NO. 13) and
one synthesized with the sequence
5'-GCTGATGACGGTACCTGAAGAATAAGCACCGGC-3' (SEQ ID NO. 14).
33TABLE 7 Amplification of N. meningitidis serogroup A with
amplification oligomers comprising SEQ ID NOs. 7 and 9, followed by
detection with probe SEQ ID NO. 11. Amount of target RLU with probe
added SEQ ID NO. 11 40 cells 723,645 648,069 686,492 4 cells
195,370 189,451 162,128 0.4 cells 28,585 23,253 824,742 64,945 0
cells 1,432 1,202 1,258
Example 8
[0354] To demonstrate the reactivity and specificity of the probes
directed to N. meningitidis 16S rRNA, a mixture of probes
containing acridinium ester labeled oligonucleotides synthesized
with the sequence 5'-CGCTGATATTAGCAACAGCC-3', (SEQ ID NO. 15) or
sequence 5'-CGCTGGTATTAGCAACAGCC-3', (SEQ ID NO. 16), and unlabeled
helper probes synthesized with the sequence
5'-TTTTCTTCCCTGACAAAAGTCCTTTACAACCCGAAGGC-3- ' (SEQ ID NO. 17 and
5'-GCCGGTGCTTATTCTTCAGGTACCGTCATCAG-3' (SEQ ID NO. 18), were
hybridized to nucleic acid in lysates prepared from fresh cultures
of the Neisseria species listed below. Each lysate was tested with
a probe directed to a conserved region of 23S rRNA to confirm the
lysis of the organism and integrity of the rRNA.
34TABLE 8 Reactivity and specificity of probes directed to N.
meningitidis 16S rRNA. RLU with RLU with conserved Organism ATCC
No. probe mix* probe N. cinerea 14685 736,927 59,831 N.
denitrificans 14686 581 50,391 N. elongata 25295 1,511 52,017 N.
elongata subspecies 29315 618 53,312 glycolytica N. flavescens
13120 1,316 53,397 N. gonorrhoeae 9793 1,826 62,658 N. gonorrhoeae
9827 753 60,252 N. gonorrhoeae 9830 4,832 58,346 N. gonorrhoeae
10150 1,139 61,573 N. gonorrhoeae 10874 759 58,291 N. gonorrhoeae
11689 4,824 60,039 N. gonorrhoeae 19088 910 53,594 N. gonorrhoeae
19424 851 60,372 N. gonorrhoeae 21824 746 62,153 N. gonorrhoeae
27630 1,829 53,241 N. gonorrhoeae 33084 784 62,696 N. gonorrhoeae
35541 431 59,229 N. lactamica 23970 3,497 54,255 N. meningitidis
13077 844,739 54,292 serogroup A N. meningitidis 23255 722,108
61,439 serogroup B N. meningitidis 13090 704,890 57,321 serogroup B
N. meningitidis 23251 761,475 58,545 serogroup B N. meningitidis
13103 770,221 63,704 serogroup C N. meningitidis 13106 761,099
60,928 serogroup C N. meningitidis 13102 752,743 62,351 serogroup C
N. meningitidis 13111 711,196 59,635 serogroup C N. meningitidis
13109 768,874 63,295 serogroup C N. meningitidis 13110 676,060
58,150 serogroup C N. meningitidis 13112 543,492 54,921 serogroup C
N. meningitidis 23248 321,600 59,308 serogroup C N. meningitidis
13113 770,893 56,429 serogroup D N. meningitidis group E 35558
797,072 58,882 N. meningitidis 43828 559,406 61,534 serogroup L N.
meningitidis 43744 705,798 62,152 serogroup W-135 N. meningitidis
35561 778,600 54,938 serogroup Y N. meningitidis 35562 749,756
61,793 serogroup Z N. meningitidis 13095 726,612 52,614 N.
meningitidis 13101 775,912 59,839 N. meningitidis 13804 785,737
61,790 N. meningitidis 43743 734,400 61,357 N. mucosa 19696 1,560
53,427 N. mucosa subspecies 25999 1,761 59,306 heidelbergensis N.
sicca 29193 1,205 58,260 N. sicca 9913 2,203 57,764 N. subflava
14799 2,046 50,832 Negative sample 5,251 124 467 132 1,691 138
*probe mix contained acridinium ester labeled probes synthesized
with sequences of SEQ ID NO. 15 and SEQ ID NO. 16 and unlabeled
helper probes synthesized with sequences of SEQ ID NO. 17 and SEQ
ID NO. 18.
[0355] The data show that the mixture of probes allowed detection
of all of the N. meningitidis strains tested. The probe mix did
show a cross reaction with N. cinerea, an organism unlikely to be
found in the same clinical samples as N. meningitidis. Treatment of
patients with N. cinerea infections would be the same as for
patients infected with N. meningitidis.
Example 9
[0356] This example demonstrates the specificity of the
amplification and detection assay. Thirty pmol of the
promoter-primer comprising SEQ. ID. NO. 7 and 30 pmol of the primer
comprising SEQ. ID. NO. 9 were used in the assay with eleven
different Neisseria species. Cell lysates were prepared as
described in Example 5 and amplified and analyzed by hybridization
using the conditions described in Example 1. Twenty microliters of
the amplification reactions were hybridized to an acridinium ester
labeled probe synthesized with sequence 5'-GCCAATATCGGCGGCCGATG-3'
(SEQ ID NO. 3) and an unlabeled helper probe synthesized with the
sequence 5'-ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3' (SEQ ID NO. 4),
or an acridinium ester labeled probe synthesized with the sequence
5'-GGCTGTTGCTAATATCAGCG-3' (SEQ ID NO. 11) in the presence of
unlabeled helper probes synthesized with sequences comprising SEQ
ID NOs. 13 and 14, or an acridinium ester labeled probe synthesized
with the sequence 5'-GGCTGTTGCTAATACCAGCG-3' (SEQ ID NO. 12) in the
presence of unlabeled helper probes of SEQ ID NOs. 13 and 14.
35TABLE 9 Specificity of an assay using amplification with
oligonucleotides comprising SEQ ID NOs. 7 and 9 followed by
detection with probes comprising SEQ ID NOs. 3, 11 or 12. RLU Probe
to conserved regions Probe of SEQ ID NOs.: Helpers bacterial 11 12
3 Organism ATCC No. rRNA 13 + 14 13 + 14 4 Neisseria 14685
2,468,721 540,699 1,804 1,633 cinerea 609,648 2,484 1,536 575,050
1,943 1,494 Neisseria 14686 2,339,034 740 644 1,563 denitrificans
659 578 1,539 Neisseria 25295 2,486,745 772 428 1,521 elongata 738
3,297 1,528 Neisseria 29315 2,397,697 697 431 1,443 elongata 954
813 1,528 subspecies glycolytica Neisseria 13120 2,622,452 780 493
1,547 flavescens 874 481 1,610 969 429 1,589 Neisseria 23970
2,299,619 736 410 1,621 lactamica 839 425 1,544 1,583 428 1,559
Neisseria 19696 2,565,699 1,021 981 1,596 mucosa 1,408 559 6,781
851 5,260 1,574 Neisseria 25999 2,927,147 653 367 1,430 mucosa 664
390 1,971 heidelber gensis Neisseria 9913 2,427,561 699 777 1,609
sicca 847 477 1,552 834 437 1,642 Neisseria 29193 2,804,642 954 423
1,588 sicca 615 388 1,505 Neisseria 19424 N.T. 3,826 419 586,358
gonorrhoeae* 1,092 411 564,987 2,390 388 554,134 Neisseria 13077
N.T. 557,656 1,287 1,492 menirigitidis* 621,180 1,009 1,509
Serogroup A 539,592 954 1,617 *purified RNA used at 500 pg per
reaction. N.T. = Not tested.
[0357] The data shown in the examples described above confirm that
the novel amplification oligonucleotides herein described and
claimed are capable of amplifying Neisseria nucleic acid and can be
used in an assay to distinguish Neisseria meningitidis or Neisseria
gonorrhoeae from each other, the closest known phylogenetic
neighbours. None of the examples described herein are intended to
limit the present invention to the embodiments of this disclosure,
said invention being limited exclusively by the claims which
follow.
Sequence CWU 1
1
* * * * *