Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-11-05
2001-03-20
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C536S022100, C536S024300, C536S024320
Reexamination Certificate
active
06204026
ABSTRACT:
FEDERAL FUNDING LEGEND
This invention was produced in part using funds obtained through a grant from the National Institutes of Health. Consequently, the federal government has certain rights in this invention.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of clinical microbiology. Specifically, the present invention relates to the detection of viable organisms of the
Mycobacterium tuberculosis
complex using a reverse transcriptase polymerase chain reaction assay.
2. Description of the Related Art
The resurgence of tuberculosis in the United States over the past decade and its continued worldwide dominance as a cause of morbidity and mortality (Raviglione et al, 1995) have focused attention on the need for more rapid and reliable means of diagnosis. Traditionally, diagnosis is dependent upon acid-fast staining and culture of the causative agent,
Mycobacterium tuberculosis
(
M. tuberculosis
), in broth or on solid media. However, this process may require up to 6 weeks owing to the slow growth rate of the organism. In contrast, nucleic acid amplification assays have the potential to reduce the time for definitive diagnosis to as little as one day. Several assays have been described for the detection of nucleic acid sequences that are specific for the
M. tuberculosis
complex which comprises
M. tuberculosis, M. bovis, M. bovis bacille
Calmette-Guérin (BCG),
M. africanum
and
M. microti
(Eisenach et al, 1991; Iovannisci et al, 1993; Jonas et al, 1993; Shah et al, 1995; van der Vliet et al, 1993; Walker et al, 1992). Although beneficial to the initial diagnosis of infection, such assays have so far proven unsuitable for monitoring the response of patients to therapy.
Typically, successful treatment of a patient with tuberculosis results in conversion of smears and cultures to negative within 3-4 months. However, recently it has been demonstrated that DNA-based amplification assays such as the Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR) and Strand Displacement Amplification (SDA) are an inappropriate substitute for conventional microbiological methods of patient follow-up since
M. tuberculosis
DNA may persist for long periods after smears and cultures have become negative (Hellyer et al, 1996). Similarly, a poor correlation has been observed between smear and culture results and those obtained with the Gen-Probe Amplified
Mycobacterium Tuberculosis
Direct Test for
M. tuberculosis
16S ribosomal RNA (Moore et al, 1996).
In prokaryotic cells, messenger RNA (mRNA) is degraded rapidly with a typical half-life of 3 min (Belasco et al, 1986; von Gabain et al, 1983). Consequently an mRNA-based amplification assay is likely to detect only living organisms and thus be a good indicator of therapeutic efficacy. Thus, the prior art is deficient in methods for diagnosis of and determination of efficacy of treatment for
M. tuberculosis.
The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention provides a reverse transcriptase-mediated polymerase chain reaction assay (RT-PCR) for
M. tuberculosis
&agr;-antigen mRNA (also termed the US-Japan antigen 6 or the 30 kd, 85B or MPB59 protein). This target was selected because the &agr;-antigen is one of the most abundant proteins produced by
M. tuberculosis
in broth cultures as well as in human mononuclear phagocytes (Lee et al, 1995; Harth et al, 1996). &agr;-antigen may comprise up to 41% of protein in culture supernatants (Wiker et al., 1992) and it is reasonable to expect viable cells to possess a corresponding abundance of the encoding mRNA.
The present invention provides specifically a reverse transcriptase-mediated polymerase chain reaction (RT-PCR) assay for
M. tuberculosis
&agr;-antigen using primers selected from sequences disclosed in Table 1. A preferred embodiment comprises a method for the detection of viable organisms of the
M. tuberculosis
complex by RT-PCR in clinical specimens or in vitro cultures, comprising the steps of: adding mRNA isolated from the specimens or cultures to an appropriate buffer containing a primer, nucleotides and a reverse transcriptase enzyme to form a reaction mixture; incubating the reaction mixture at a suitable temperature to permit synthesis of EDNA by the reverse transcriptase; transfering an aliquot of said cDNA to a suitable buffer containing one or more additional primers, nucleotides and a DNA polymerase to form a PCR reaction mixture; incubating the PCR mixture over successive cycles of heating and cooling to facilitate generation of specific products by the DNA polymerase; and detecting the products by gel electrophoresis, autoradiography or emission of fluorescence, wherein a presence of said products indicates the presence of viable
M. tuberculosis
complex organisms and an absence of said products indicates an absence of said viable
M. tuberculosis
complex organisms in said sample.
A preferred embodiment is to perform the method wherein the sequence of the primer for reverse transcription is SEQ ID No. 13, and wherein the sequences of the primers used for PCR amplification of the cDNA are SEQ ID No. 12 and SEQ ID No. 13, and wherein the products are detected by incorporation of radiolabeled SEQ ID No. 12 and SEQ ID No. 13 in said reaction mixture, followed by gel electrophoresis of the products of the reaction and autoradiography. A particularly preferred embodiment of the objective is to detect the products using a fluorescently-labeled probe wherein the sequence of said probe is SEQ ID No. 14 and wherein the PCR amplification is performed using a 5′ fluorogenic exonuclease assay (Holland et al, 1991; Livak et al, 1995).
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.
REFERENCES:
Matsuo et al. Cloning and expression of theMycobacterium bovisBCG gene for extracellular alpha antigen J. Bacteriology, vol. 170(9). pp. 3847-3854, 1988.*
Jou et al. Single-tube, nested, reverse transcriptase PCR for detection of viableMycobacterium tuberculosis, vol. 35(5), pp. 1161-1165, J. Clinical Microbiology, 1997.*
Douglas F. Moore, et al. Amplification of rRNA for Assessment of Treatment Response of Pulmonary Tuberculosis Patients during AntimicrobialTherapy. Journal of Clinical Microbiology, vol. 34, No. 7, pp. 1745-1749 (Jul. 1996).
A. K. Bej, et al. Detection of viableLegionella pneumophilain Water by Polymerase Chain Reaction and Gene Probe Methods. Applied and Environmental Microbiology, vol. 57, No. 2, pp. 597-600 (Feb. 1991).
L. E. DesJardin, et al. Alkaline Decontamination of Sputum Specimens Adversely Affects Stability of Mycobacterial mRNA. vol. 34, No. 10, pp. 2435-2439 (Oct. 1996).
Gabrielle M. E. Van Der Vliet, et al. Assessment of Mycobacterial Viability by RNA Amplification. Antimicrobial Agents and Chemotherapy, vol. 38, No. 9, pp. 1959-1965 (Sep. 1994).
N. Martin-Casabona, et al. Rapid Method for Testing Susceptibility ofMycobacterium tuberculosisby Using DNA Probes. Journal of Clinical Microbiology, vol. 35, No. 10, pp. 2521-2525 (Oct. 1997).
Nainn-Tsyr Jou, et al. Single-Tube, Nested Reverse Transcriptase PCR for Detection of ViableMycobacterium tuberculosis. Journal of Clinical Microbiology, vol. 35, No. 5, pp. 1161-1165 (May 1997).
Gerard A. Cangelosi, et al. Detection of Rifampin- and Ciprofoxacin-ResistantMycobacterium tuberculosisby Using Species-Specific Assays for Precursor rRNA. Antimicrobial Agents and Chemotherapy, vol. 40, No. 8, pp. 1790-1795 (Aug. 1996).
Diane E. Kawa, et al. Development of a Rapid Method for Determining the Susceptibility ofMycobacterium tuberculosisto Isoniazid Using the Gen-Probe DNA Hybridization System. Anitmicrobial Agents and Chemotherapy, vol. 40, No. 8, pp. 1790-1795 (Aug. 1996).
Tobin J. Hellyer, et al. Strand Displacement Amplification and the Polymerase Chain Reaction for Monitoring Respo
Cave Mac Donald
DesJardin Lucy Ellen
Eisenach Kathleen Davis
Adler Benjamin Aaron
Horlick Kenneth R.
The Board of Trustees of the University of Arkansas
Tung Joyce
LandOfFree
Detection of M. tuberculosis complex via reverse... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Detection of M. tuberculosis complex via reverse..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Detection of M. tuberculosis complex via reverse... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2516573