title: Detection of Mycobacterium tuberculosis by PCR amplification with pan-Mycobacterium primers and hybridization to an M. tuberculosis-specific probe.
abstract: Nucleic acid amplification techniques such as the PCR are very useful in the rapid diagnosis of infections by Mycobacterium tuberculosis. However, recent studies have shown that the accuracy of results can vary widely when tests are performed with nonstandardized reagents. We have developed a PCR assay for the detection of M. tuberculosis that is both rapid and accurate. The assay reagents are standardized and quality controlled. False-positive results due to carryover contamination are prevented by the incorporation of dUTP coupled with uracil-N-glycosylase restriction. This assay also employs pan-Mycobacterium amplification primers, allowing for flexibility in the mycobacterial species that can be identified from a single amplification reaction. The amplification is very sensitive; amplification products generated from as few as three bacteria can be detected by agarose gel electrophoresis. DNAs isolated from 33 of 34 mycobacterial species tested were amplified efficiently. Only DNA from Mycobacterium simiae did not amplify. The amplification is also very specific. Amplification products were generated only from the DNAs of bacteria in closely related genera such as Corynebacterium. The nonmycobacterial amplicons do not pose a problem, as they do not hybridize to mycobacterium-specific probes. Hybridization of amplicons to an M. tuberculosis-specific probe allows for the unambiguous identification of M. tuberculosis complex organisms. The clinical performance of this PCR assay was evaluated against that of culture in 662 respiratory specimens. Sensitivities of 100 and 73.1% were obtained from smear-positive and -negative respiratory specimens, respectively. The corresponding specificities were 100 and 99.8%. The high sensitivity and specificity, coupled with the potential for detecting a wide range of mycobacteria, make this assay a useful tool in the clinical management of mycobacterial infections.
authors.affiliation: Roche Molecular Systems, Inc., Branchburg, New Jersey, USA.
authors.forename: V J
authors.initials: VJ
authors.lastname: Tevere
authors.forename: P L
authors.initials: PL
authors.lastname: Hewitt
authors.forename: A
authors.initials: A
authors.lastname: Dare
authors.forename: P
authors.initials: P
authors.lastname: Hocknell
authors.forename: A
authors.initials: A
authors.lastname: Keen
authors.forename: J P
authors.initials: JP
authors.lastname: Spadoro
authors.forename: K K
authors.initials: KK
authors.lastname: Young
mesh_terms: D001431:Bacteriological Techniques / Q000706:statistics & numerical data; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D003951:Diagnostic Errors; D005069:Evaluation Studies as Topic; D006801:Humans; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis / Q000382:microbiology
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial