title: Rapid and simultaneous detection of Mycobacterium tuberculosis complex and Beijing/W genotype in sputum by an optimized DNA extraction protocol and a novel multiplex real-time PCR.
abstract: Rapid diagnosis and genotyping of Mycobacterium tuberculosis by 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. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains 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. tuberculosis culture-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. tuberculosis bacilli, 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.
authors: Department of Microbiology1 and Department of Medicine and Therapeutics,2 The Chinese University of Hong Kong, Hong Kong, China. ericleung@cuhk.edu.hk
authors: Eric T Y
authors: ET
authors: Leung
authors: L
authors: L
authors: Zheng
authors: Rity Y K
authors: RY
authors: Wong
authors: Edward W C
authors: EW
authors: Chan
authors: T K
authors: TK
authors: Au
authors: Raphael C Y
authors: RC
authors: Chan
authors: Grace
authors: G
authors: Lui
authors: Nelson
authors: N
authors: Lee
authors: Margaret
authors: M
authors: Ip
references: J Interferon Cytokine Res. 2005 Nov;25(11):694-701
references: 16318583
references: Microbiology (Reading). 2009 Apr;155(Pt 4):1272-1281
references: 19332828
references: J Clin Microbiol. 2006 Dec;44(12):4384-8
references: 17035487
references: J Clin Microbiol. 1995 Dec;33(12):3234-8
references: 8586708
references: J Bacteriol. 2007 Apr;189(7):2583-9
references: 17237171
references: Clin Microbiol Infect. 2011 Sep;17(9):1391-6
references: 21091972
references: J Clin Microbiol. 2001 Oct;39(10):3768-71
references: 11574614
references: J Appl Microbiol. 2009 Nov;107(5):1433-9
references: 19426272
references: Trends Microbiol. 2002 Jan;10(1):45-52
references: 11755085
references: Emerg Infect Dis. 2009 Jul;15(7):1061-7
references: 19624921
references: Nucleic Acids Res. 1989 Oct 11;17(19):7843-53
references: 2798131
references: BMC Infect Dis. 2006 Jul 05;6:105
references: 16820066
references: Nature. 2004 Sep 2;431(7004):84-7
references: 15343336
references: J Clin Microbiol. 1996 Jun;34(6):1407-11
references: 8735089
references: Tuberculosis (Edinb). 2007 Sep;87(5):446-9
references: 17632035
references: J Clin Microbiol. 2005 May;43(5):2471-3
references: 15872286
references: J Clin Microbiol. 1990 Mar;28(3):495-503
references: 1691208
references: Emerg Infect Dis. 2003 Jul;9(7):838-45
references: 12890325
references: APMIS. 2010 Jan;118(1):60-5
references: 20041872
references: Microbes Infect. 2007 Feb;9(2):241-6
references: 17224291
references: Emerg Infect Dis. 2006 May;12(5):736-43
references: 16704829