title: Development and application of multiprobe real-time PCR method targeting the hsp65 gene for differentiation of Mycobacterium species from isolates and sputum specimens.
abstract: We developed a multiprobe real-time PCR assay targeting hsp65 (HMPRT-PCR) to detect and identify mycobacterial isolates and isolates directly from sputum specimens. Primers and probes for HMPRT-PCR were designed on the basis of the hsp65 gene sequence, enabling the recognition of seven pathogenic mycobacteria, including Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansasii, M. abscessus, M. massiliense, and M. fortuitum. This technique was applied to 24 reference and 133 clinical isolates and differentiated between all strains with 100% sensitivity and specificity. Furthermore, this method was applied to sputum specimens from 117 consecutive smear-positive patients with smear results of from a trace to 3+. These results were then compared to those obtained using the rpoB PCR-restriction analysis method with samples from cultures of the same sputum specimens. The HMPRT-PCR method correctly identified the mycobacteria in 89 samples (76.0%, 89/117), and moreover, the sensitivity level was increased to 94.3% (50/53) for sputa with an acid-fast bacillus score equal to or greater than 2+. Our data suggest that this novel HMPRT-PCR method could be a promising approach for detecting pathogenic mycobacterial species from sputum samples and culture isolates routinely in a clinical setting.
authors.affiliation: Department of Internal Medicine and Lung Institute, Seoul National University College of Medicine, Seoul 110-799, Republic of Korea.
authors.forename: Kijeong
authors.initials: K
authors.lastname: Kim
authors.forename: Hyungki
authors.initials: H
authors.lastname: Lee
authors.forename: Mi-Kyung
authors.initials: MK
authors.lastname: Lee
authors.forename: Seoung-Ae
authors.initials: SA
authors.lastname: Lee
authors.forename: Tae-Sun
authors.initials: TS
authors.lastname: Shim
authors.forename: Seong Yong
authors.initials: SY
authors.lastname: Lim
authors.forename: Won-Jung
authors.initials: WJ
authors.lastname: Koh
authors.forename: Jae-Joon
authors.initials: JJ
authors.lastname: Yim
authors.forename: Bazarragchaa
authors.initials: B
authors.lastname: Munkhtsetseg
authors.forename: Wonyong
authors.initials: W
authors.lastname: Kim
authors.forename: Sang-In
authors.initials: SI
authors.lastname: Chung
authors.forename: Yoon-Hoh
authors.initials: YH
authors.lastname: Kook
authors.forename: Bum-Joon
authors.initials: BJ
authors.lastname: Kim
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D018834:Chaperonin 60 / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D017931:DNA Primers; D015345:Oligonucleotide Probes; C074119:heat-shock protein 65, Mycobacterium; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit