Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-08-27
2000-08-29
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 536 243, 536 2432, 536 2433, C12Q 168, C12P 1934, C07H 2104
Patent
active
06110681&
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention is directed to oligonucleotides that can be used as primers to amplify a region of the 16S rRNA of Mycoplasma pneumoniae. The amplified RNA can be detected with known probes for M. pneumoniae. However, with specific probes according to the present invention, not only detection of the amplified RNA but also further characterization with respect to the typing of M. pneumoniae strains is possible.
The primers, probes, methods and kits are especially useful as an aid in the diagnosis of M. pneunoniae.
BACKGROUND OF THE INVENTION
Mycoplasma pneumoniae is the causative agent of primary atypical pneumonia and is also responsible for other respiratory syndromes such as bronchitis, bronchiolitis, pharyngitis, croup and less severe upper respiratory tract infections with the highest incidence among school children.
Current methods for the diagnosis of M. pneumoniae infection include isolation of the organisms on complex media or demonstration of seroconversion during convalescent phases of infection (Leith et al., J. Exp. Med. 157:502-514 (1983)). The mycoplasmas, such as Mycoplasma pneumoniae, are fastidious organisms, requiring complex culture media containing peptone, yeast extract, expensive animal sera, and sterol. Growth is relatively slow and reaches low cell densities compared to most bacteria. In addition, atmospheric conditions for cell growth requires the addition of carbon dioxide. For these reasons, many clinical laboratories are unable to perform culture isolation of M. pneumoniae, and consequently are left with no real ability to diagnose the presence of this important pathogenic bacterium. Given that mycoplasmas lack cell walls, antibiotics that target the bacterial cell wall, such as penicillin, have no anti-mycoplasma activity.
Consequently, it is of importance for a physician to make a diagnosis of atypical pneumonia and prescribe the appropriate antibiotic. Initiation of appropriate therapy cannot be based on culture or serology.
Detection of genomic sequences have been proposed as rapid and specific alternatives. Different PCRs for the detection of M. pneumoniae have been described, using as targets the gene coding for the P1 adhesion protein (Jensen et al., Acta Pathol. Microbiol. Immunol. Scand. 97:1046-1048 (1989); Ursi et al., Acta Pathol. Microbiol.Immunol. Scand. 100:635-639 (1992)) or the 16S rRNA gene (van Kuppeveld et al., Appl. Environ. Microbiol. 58:2606-2615 (1992)) or a DNA sequence specific for M. pneumoniae selected from a genomic library (Bernet et al., J. Clin. Microbiol., 27:2492-2496 (1989)).
Although these methods have lesser drawbacks than culturing and serology, they are still too complex to be carried out in a routine diagnostic laboratory. False negative PCR results are rather common due to inhibitors of the PCR reaction in the clinical specimen, while false-positive results may occur due to contamination of the reagents with target DNA (Razin, Mot. and Cell. Probes, 8, 497-511 (1994)).
Based on sequence divergency of the major cytadhesin gene P1 (Su et al., Infect. Immun. 58:2669-2674 (1990)), restriction enzyme fingerprinting of genomic DNA (Su et al., J. Gen. Microbiol. 137:2727-2732 (1991); Su et al, J. Clin. Microbiol. 28:1538-1540 (1990)), two-dimensional gel electrophoresis of total proteins and PCR-mediated DNA fingerprinting (Ursi et al., J. Clin. Microbiol. 32:2873-2875 (1994)), only two types are presently recognized, indicating that M. pneumoniae as a species is genetically remarkably stable.
It was suggested by Ursi and coworkers that a switch in time from one type to another could be explained by the immune status of the population against one of these two types.
Typing M. pneumoniae is of major importance because unambiguous characterization is the basis for further identification of M. pneumoniae strains. Studies based on virulence differences between one strain and the other strain could be based on type-specificity. Furthermore, a relation may exist between type and sensitivity to antibiotics like macrolid
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Ovyn Caroline Louise Lucienne
Van Gemen Bob
van Strijp Dianne Arnoldina Margaretha Wilhelmina
Akzo Nobel N.V.
Horlick Kenneth R.
Sullivan Michael G.
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