Development and Application of Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation of Mycobacterium Species from Isolates and Sputum Specimens

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.This study was supported by grant A101205 from the Korean Healthcare Technology R&D project, Ministry for Health, Welfare & Family Affairs, Republic of Korea, and in part supported by grant 04-2008-0860 from the SNUH Research Fund.Halse TA, 2010, J CLIN MICROBIOL, V48, P1182, DOI 10.1128/JCM.02149-09Leung KL, 2009, J APPL MICROBIOL, V107, P1433, DOI 10.1111/j.1365-2672.2009.04324.xGopinath K, 2009, J APPL MICROBIOL, V107, P425, DOI 10.1111/j.1365-2672.2009.04218.xFoongladda S, 2009, J MOL DIAGN, V11, P42, DOI 10.2353/jmoldx.2009.080081Kim BJ, 2008, DIAGN MICR INFEC DIS, V62, P193, DOI 10.1016/j.diagmicrobio.2008.05.013Kim HY, 2008, J CLIN MICROBIOL, V46, P3384, DOI 10.1128/JCM.00319-08Pinsky BA, 2008, J CLIN MICROBIOL, V46, P2241, DOI 10.1128/JCM.00347-08Lim SY, 2008, LETT APPL MICROBIOL, V46, P101, DOI 10.1111/j.1472-765X.2007.02278.xNakanaga K, 2007, J CLIN MICROBIOL, V45, P3840, DOI 10.1128/JCM.01041-07Simmon KE, 2007, J CLIN MICROBIOL, V45, P1978, DOI 10.1128/JCM.00563-07Joh JS, 2007, J KOREAN MED SCI, V22, P26Mun HS, 2007, MICROBIOL IMMUNOL, V51, P105Kim HJ, 2006, J CLIN MICROBIOL, V44, P3855, DOI 10.1128/JCM.01214-06Espy MJ, 2006, CLIN MICROBIOL REV, V19, P165, DOI 10.1128/CMR.19.1.165-256.2006McNabb A, 2006, J CLIN MICROBIOL, V44, P60, DOI 10.1128/JCM.44.1.60-66.2006Koh WJ, 2005, J KOREAN MED SCI, V20, P913Kim H, 2005, INT J SYST EVOL MICR, V55, P1649, DOI 10.1099/ijs.0.63553-0TAYLOR Z, 2005, MMWR RECOMM REP, V54, P1Wagner D, 2004, INFECTION, V32, P257, DOI 10.1007/s15010-004-4001-4Adekambi T, 2003, J CLIN MICROBIOL, V41, P5699, DOI 10.1128/JCM.41.12.5699-5708.2003Coll P, 2003, INT J TUBERC LUNG D, V7, P886Miller N, 2002, J CLIN MICROBIOL, V40, P4143, DOI 10.1128/JCM.40.11.4143-4147.2002Cloud JL, 2002, J CLIN MICROBIOL, V40, P400, DOI 10.1128/JCM.40.2.400-406.2002Lee H, 2000, J CLIN MICROBIOL, V38, P2966Dye C, 1999, JAMA-J AM MED ASSOC, V282, P677Kim BJ, 1999, J CLIN MICROBIOL, V37, P1714ROSENZWEIG DY, 1996, SEMIN RESP INFECT, V11, P252HUMINER D, 1993, CLIN INFECT DIS, V17, P508BLOOM BR, 1992, SCIENCE, V257, P1055BLOOM BR, 1992, NATURE, V358, P538WOLINSKY E, 1992, CLIN INFECT DIS, V15, P1MASSENKEIL G, 1992, CLIN INFECT DIS, V14, P618BARNES PF, 1991, NEW ENGL J MED, V324, P1644KENT PT, 1985, PUBLIC HLTH MYCOBACT