Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-08
2004-01-27
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024320, C536S024330
Reexamination Certificate
active
06682889
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for determining the presence or absence of organisms of the Chlamydiaceae family in endocervical, throat and urethral swabs, urine, sputa, bronchoalveolar lavage fluids, eye secretions or other patient and animal specimens, cultures, food, and environmental samples. The method involves using nucleic acid primers to amplify specifically a target sequence within the ribonuclease P RNA gene (rnpB), preferably using one of the techniques of Strand Displacement Amplification (SDA), thermophilic Strand Displacement Amplification (tSDA) or fluorescent real time tSDA.
BACKGROUND OF THE INVENTION
Three species in the family Chlamydiaceae:
Chlamydophila pneumoniae
(formerly
Chlamydia pneumoniae
),
Chlamydia trachomatis
and
Chlamydophila psittaci
(formerly
Chlamydia psittaci
) (Everett, et al., 1999
, Int. J. Syst. Bacteriol
. 49:415-440), cause diseases in humans, which include trachoma; respiratory infection (pneumonia); and sexually transmitted infections of the reproductive organs, such as urethritis, cervicitis, pelvic inflammatory disease, and epididymitis. Pneumonia can be caused by
C. pneumoniae
and
C. psittaci
(psittacosis) in adults, and by
C. trachomatis
in newborn infants (Guo, et al., 1995
, Clin. Microbiol. Rev
. 8:451-461 and Madico, et al., 2000
, J. Clin. Microbiol
. 38(3):1085-1093). Some species of the Chlamydiaceae family may also be involved in infections of the heart (Odeh, et al., 1992
, Eur. J. Clin. Microbiol. Infect. Dis
. 11:885-893). The two genera in the family Chlamydiaceae, Chlamydia and Chlamydophila, include several species that can cause diseases in animals (Everett, et al.,
Vet. Microbiol
. 75(2):109-126). Among these are,
C. psittaci
and
C. pecorum
which give rise to a wide variety of conditions in animals including abortion, pneumonia, enteritis, polyarthretis, encephalomyelitis, and conjunctivitis (Sheehy, et al., 1996
, J. Clin. Microbiol
. 34(2):3175-3179). There is therefore a clinical need for the detection of all pathogens or potential pathogens within the Chlamydiaceae in a variety of clinical samples.
Endoribonuclease P (RNase P) is a ribonucleoprotein complex that removes 5′ leader sequences from tRNA precursors during tRNA biosynthesis. RNase P is an essential riboenzyme found in all living cells and subcellular compartments that synthesize tRNA, although catalytic proficiency of the RNA alone has been demonstrated only for the bacterial RNase P (Brown, et al., 1992
, Nucl. Acids Res
. 20:1451-1456 and Haas, et al., 1998
, Nucl. Acids Res
. 26:4093-4099). Sequencing of the RNase P RNA genes provides a potential tool for the identification of bacteria and eukaryotic organisms in clinical diagnostics. The RNase P RNA gene (rnpB) has recently been used as a marker to differentiate chlamydial strains and species (Herrmann, et al., 1996
, J. Clin. Microbiol
. 34(8):1897-1902). Characterization of the rnpB gene in the order Chlamydiales (Herrmann, et al., 2000
, Int. J. Syst. Evol. Microbiol
. 50:149-158) revealed similarities of 76.6% between
C. trachomatis
and
C. pneumoniae
, 79.5% between
C. trachomatis
and
C. psittaci
, and 84.7% between
C. pneumoniae
and
C. psittaci
. It is therefore possible to use the rnpB gene as a genus or familial marker to identify organisms within the Chlamydiaceae.
Nucleic acid amplification is a powerful technology, which allows rapid detection of specific target sequences and it is therefore a promising technology for the rapid detection and identification of species in the Chlamydiaceae family. Various nucleic acid amplification methods have been described previously for the detection of some or all of the species within this group of organisms. Touchdown enzyme time release-PCR has been used to amplify specific DNA sequences in the variable regions of the 16S and 16-23S spacer rRNA genes of
C. trachomatis, C. pneumoniae
, and
C. psittaci
(Madico, et al., 2000
, J. Clin. Microbiol
. 38:1085-1093). Rapid detection of the Chlamydiaceae and other families in the order Chlamydiales has also been reported using three different PCR assays targeting the ompA gene and the rRNA intergenic spacer region (Everett, et al., 1999
, J. Clin. Microbiol
. 37:575-580). Identification of nine species of the Chlamydiaceae using PCR-Restriction Fragment Length Polymorphism analysis was also reported by Everett, et al. (1999
, Int. J. Syst. Bacteriol
. 49:803-813), and Jantos, et al. described detection of
C. pneumoniae
using a PCR-enzyme immunoassay based on the 16S rRNA gene (1998
, J. Clin. Microbiol
. 36:1890-1894). Kaltenböck, et al. established a nested PCR for genus-specific amplification of the Chlamydia ompl locus (1997
, J. Clin. Microbiol
. 35:1835-1841) and the application of a nested, multiplex PCR based on the 16S rRNA gene, in the investigation of psittacosis outbreaks was reported by Messmer, et al. (1997
, J. Clin. Microbiol
. 35:2043-2046). Differentiation of
C. psittaci
and
C. pecorum
by species-specific PCR targeting the outer membrane protein genes has been reported by Sheehy, et al. (1996
, J. Clin. Microbiol.
34:3175-3179). Use of the ligase chain reaction (LCR) has also been described for the amplification and detection of
C. trachomatis
(Dille, et al., 1993
, J. Clin. Microbiol
. 31:729-731 and Schachter, et al., 1994
, J. Clin. Microbiol
. 32:2540-2543). The oligonucleotide primers of the present invention are applicable to nucleic acid amplification and detection of organisms belonging to the Chlamydiaceae family.
The following terms are defined herein as follows:
An amplification primer is a primer for amplification of a target sequence by extension of the primer after hybridization to the target sequence. Amplification primers are typically about 10-75 nucleotides in length, preferably about 15-50 nucleotides in length. The total length of an amplification primer for SDA is typically about 25-50 nucleotides. The 3′ end of an SDA amplification primer (the target binding sequence) hybridizes at the 3′ end of the target sequence. The target binding sequence is about 10-25 nucleotides in length and confers hybridization specificity on the amplification primer. The SDA amplification primer further comprises a recognition site for a restriction endonuclease 5′ to the target binding sequence. The recognition site is for a restriction endonuclease, which will nick one strand of a DNA duplex, when the recognition site is hemimodified, as described by G. Walker, et al. (1992
. Proc. Natl. Acad. Sci. USA
89:392-396 and 1992
Nucl. Acids Res
. 20:1691-1696). The nucleotides 5′ to the restriction endonuclease recognition site (the “tail”) function as a polymerase repriming site when the remainder of the amplification primer is nicked and displaced during SDA. The repriming function of the tail nucleotides sustains the SDA reaction and allows synthesis of multiple amplicons from a single target molecule. The tail is typically about 10-25 nucleotides in length. Its length and sequence are generally not critical and can be routinely selected and modified. As the target binding sequence is the portion of a primer which determines its target-specificity, for amplification methods which do not require specialized sequences at the ends of the target the amplification primer generally consists essentially of only the target binding sequence. For example, amplification of a target sequence according to the invention using the Polymerase Chain Reaction (PCR) will employ amplification primers consisting of the target binding sequences of the amplification primers described herein. For amplification methods that require specialized sequences appended to the target other than the nickable restriction endonuclease recognition site and the tail of SDA (e.g., an RNA polymerase promoter for Self-Sustained Sequence Replication (3SR), Nucleic Acid Sequence-Based Amplification (NASBA) or the Transcription-Based Amplification System (TAS)), the required specialized sequence may be linked to the target bindin
Wang Sha-Sha
Wolfe David
Becton Dickinson and Company
Horlick Kenneth R.
Kiang Allan M.
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