Development and Application of Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation of Mycobacterium Species from Isolates and Sputum Specimens
| Autor: | Seong Yong Lim, Tae Sun Shim, Bum Joon Kim, Won-Jung Koh, Kijeong Kim, Wonyong Kim, Yoon Hoh Kook, Hyungki Lee, Seoung Ae Lee, Sang In Chung, Mi Kyung Lee, Bazarragchaa Munkhtsetseg, Jae Joon Yim |
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| Rok vydání: | 2010 |
| Předmět: |
Male
Microbiology (medical) Tuberculosis Polymerase Chain Reaction Sensitivity and Specificity Mycobacterium law.invention Microbiology Mycobacterium tuberculosis Bacterial Proteins Tuberculosis diagnosis law medicine Humans Polymerase chain reaction DNA Primers Bacteriological Techniques biology Sputum Mycobacteriology and Aerobic Actinomycetes Chaperonin 60 DNA-Directed RNA Polymerases Middle Aged bacterial infections and mycoses biology.organism_classification rpoB medicine.disease Real-time polymerase chain reaction Female medicine.symptom Oligonucleotide Probes |
| Zdroj: | Journal of Clinical Microbiology. 48:3073-3080 |
| ISSN: | 1098-660X 0095-1137 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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