Open AccessDevelopment and Application of Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation of Mycobacterium Species from Isolates and Sputum Specimens
Author(s) -
Ki-Jeong Kim,
Hyung-Ki Lee,
Eun Jig Lee,
Seoung Ae Lee,
Tae Sun Shim,
Seong Yong Lim,
Won Jung Koh,
JaeJoon Yim,
Munkhtsetseg Bazarragchaa,
Wonyong Kim,
Shin Chung,
Yoon Hoh Kook,
Bum Joon Kim
Publication year - 2010
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.00939-10
Subject(s) - rpob , sputum , biology , microbiology and biotechnology , nontuberculous mycobacteria , mycobacterium , mycobacterium tuberculosis , tuberculosis , mycobacterium kansasii , polymerase chain reaction , gene , bacteria , medicine , pathology , genetics
We developed a multiprobe real-time PCR assay targetinghsp65 (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 thehsp65 gene sequence, enabling the recognition of seven pathogenic mycobacteria, includingMycobacterium tuberculosis ,M. avium ,M. intracellulare ,M. kansasii ,M. abscessus ,M. massiliense , andM. 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 therpoB 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.