Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-13
2003-04-01
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06541201
ABSTRACT:
FIELD OF THE INVENTION
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
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.
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. 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.
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° C. is required to maximize the culture of organisms.
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.
Oligonucleotides for the amplification of nucleic acid for detection of Neisseria have been described. Biken-meyer 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 gonnorhoeae
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
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.
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.
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:
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 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: GAACGGCCGC CGATATTGGC
SEQ ID NO 32: GCCAAUAUCG GCGGCCGAUG
SEQ ID NO 33: CCGCUACCCG GUACGUUC
SEQ ID NO 34: CAUCGGCCGC CGAUAUUGGC
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:
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, and
SEQ ID NO: 28 CGCUGGUAUU AGCAACAGCC.
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:
SEQ ID NO: 1: GAACGTACCG GGTAGCGG
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
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:
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
Bee Gary
McDonough Sherrol
Yang Yeasing
Cappellari Charles B.
Fredman Jeffrey
Gen-Probe Incorporated
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