| Autor: |
Leung, Eric T. Y., Zheng, L., Wong, Rity Y. K., Chan, Edward W. C., Au, T. K., Chan, Raphael C. Y., Lui, Grace, Lee, Nelson, Ip, Margaret |
| Zdroj: |
Journal of Clinical Microbiology; July 2011, Vol. 49 Issue: 7 p2509-2515, 7p |
| Abstrakt: |
ABSTRACTRapid diagnosis and genotyping of Mycobacterium tuberculosisby molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosiscomplex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosisIS6110and the Rv0927c-pstS3intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosisstrains from 17 different species of nontuberculosis Mycobacterium(NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosisculture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n= 31). The detection limit of the assay is 10 M. tuberculosisbacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
|
