Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1995-05-26
2002-11-19
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S091200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320
Reexamination Certificate
active
06482589
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to mycoplasmas associated with the genito-urinary tract, believed to be the cause of nongonococcal urethritis, pelvic inflammatory diseases, septic abortion, and a wide array of diseases of other tissues. More specifically, embodiments of the present invention provide nucleic acid probes and compositions, methods for their use for the specific detection or identification of
Mycoplasma hominis, Ureaplasma urealyticum
, and
Mycoplasma genitalium
in clinical and other samples, and packaged components suitable for use as kits.
BACKGROUND OF THE INVENTION
Mycoplasmas are small wall-less bacteria, primarily isolated from animal sources including humans. There are over 70 members of the genus Mycoplasma, and several related genera which are also characterized by small wall-less bacteria; these are Spiroplasma, Acholeplasma, Ureaplasma, Anaeroplasma, and Asteroleplasma. Only a handful of the species within these genera have been found associated with humans-some presumed to be “normal flora”, others occasionally pathogenic, and still others always believed to be clinically significant. Among the mycoplasmas known to be pathogenic,
Mycoplasma pneumoniae
is historically the most well studied, it is the major infectious agent of primary atypical pneumonia. Nucleic acid compositions and methods for the detection of
Mycoplasma pneumoniae
and a further mycoplasma pathogen,
Mycoplasma fermentans
, are the subject of two concurrently filed applications U.S. Ser. No. 07/673,686 and U.S. Ser. No. 07/673,687, entitled “Nucleic Acid Probes For The Detection Of Mycoplasma Pneumoniae” and “Nucleic Acid Probes For The Detection Of Mycoplasma Fermentans Or The Aids-Associated Virus-Like Infectious Agent.” At least one inventor is common to all these applications and the present application.
Three other mycoplasma species, which can be isolated from the human genito-urinary tract,
Mycoplasma hominis, Mycoplasma genitalium
and
Ureaplasma urealyticum
, are somewhat more enigmatic in the clinical implications of the detection of these organisms in the human body. Ureaplasma urealyticum, for example, although it is implicated in significant and serious human morbidity and mortality, may be found in asymptomatic “normal” individuals as well. The three species,
Mycoplasma hominis, Mycoplasma genitalium
, and
Ureaplasma urealyticum
will be referred to herein as the genital mycoplasmas.
Mycoplasma genitalium
was initially isolated from the urethras of two males with nongonococcal urethritis (Tully, et. al., Int. J. Syst. Bacteriol. vol. 33, 1983). Subsequently,
M. genitalium
was isolated from the human respiratory tract, in co-culture with
Mycoplasma pneumoniae
(Baseman, et. al., J. Clinical Micro. vol.26, 1988). However, studies have shown (for example Hooton, et.al., Lancet, 1988) that
M. genitalium
plays a role in at least some of the cases of acute urethritis, particularly in homosexual males. It is an aspect of the present invention to describe methods for the design and manufacture of probes specific for the detection of
Mycoplasma genitalium
. Nucleic acid probes which recognize both
M. genitalium
and
Mycoplasma pneumoniae
, evolutionary close relatives, are described in an application filed concurrently.
Mycoplasma hominis
has been implicated as a causative agent of salpingitis, amnionitis, nonspecific vaginitis, and postpartum septic fever.
Mycoplasma hominis
can be isolated from a significant number of asymptomatic women. It is an aspect of the present invention to describe methods for the design and manufacture of probes specific for the detection of
Mycoplasma hominis.
Ureaplasma urealyticum
has been implicated in nongonococcal urethritis, chorioamnionitis, premature delivery, and perinatal morbidity and mortality. Some clinical investigators estimate that the etiological agency of nongonococcal urethritis (NGU) by
Ureaplasma urealyticum
may approach the same levels as that of
Chlamydia trachomatis
. As much as 40% of acute NGU may be caused by ureaplasma (Bowie, Urological Clinics of North America, vol. 11, 1984). This translates to approximately 3-4 million United States cases per year. Like
Mycoplasma hominis, Ureaplasma urealyticum
can be isolated from a significant number of both male and female asymptomatic individuals. There are at least 15 serotypes of
Ureaplasma urealyticum
. The combination of serotype, numerical prevalence and other factors that contribute to ureaplasma pathogenesis is, at present, totally unknown. It is an aspect of the present invention to describe methods for the design and manufacture of probes specific for the detection of
Ureaplasma urealyticum.
The mycoplasmas, such as those described above, 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 require the addition of carbon dioxide. For these reasons, many clinical laboratories are unable to perform culture isolation of mycoplasmas, and consequently are left with no real ability to diagnose the presence of these important pathogenic bacteria. 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 the presence of one or more of these bacteria, particularly if the clinical presentation is predictive, and prescribe the appropriate antibiotic.
Ribosomes are of profound importance to all organisms. Ribosomes serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Bacterial ribosomes contain three distinct RNA molecules which, at least in
Escherichia coli
, are referred to as 5S, 16S and 23S rRNAs. In eukaryotic organisms, there are four distinct rRNA species, generally referred to as 5S, 18S, 28S, and 5.8S. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary substantially in size between organisms. Nonetheless, 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacterium, including the mycoplasmas, and this convention will be continued herein. The probes of the present invention target the 16S and 23S rRNA molecules of the genital mycoplasmas.
Hybridization is a process by which, under appropriate reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together In an antiparallel fashion (one oriented 5′ to 3′, the other 3′ to 5′) to form a double-stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acid bases may pair with one another.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under hybridization conditions, specifically and preferentially, to target nucleic acid sequences. The term “preferentially” is used In a relative sense, one hybridization reaction product is more stable than another under identical conditions. Under some conditions, a hybridization reaction product may be formed with respect to one target, but not to another potential binding partner. In addition to their hybridization properties, probes also may contain certain constituents that pertain to their proper or optimal functioning under particular assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (e.g. by end capping), to carry detection ligands (e.g. fluorescein, biotin, etc.), to facilitate detection (e.g. chemiluminescent, flu
Pelletier Dale A.
Weisburg William G.
Fredman Jeffrey
Galloway Norval B.
Vysis, Inc.
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