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Journal of Clinical Microbiology, November 2004, p. 5277-5285, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5277-5285.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Dual-Probe Assay for Rapid Detection of Drug-Resistant Mycobacterium tuberculosis by Real-Time PCR

Takayuki Wada,1 Shinji Maeda,2* Aki Tamaru,3 Shigeyoshi Imai,4 Atsushi Hase,1 and Kazuo Kobayashi2

Department of Microbiology, Osaka City Institute of Public Health and Environmental Sciences,1 Department of Host Defense, Osaka City University Graduate School of Medicine,2 Department of Microbiology, Osaka Prefectural Institute of Public Health,3 Central Clinical Laboratory, Osaka City University Hospital, Osaka, Japan4

Received 24 March 2004/ Returned for modification 9 May 2004/ Accepted 11 July 2004

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.

* Corresponding author. Mailing address: Department of Host Defense, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. Phone: 81-6-6645-3746. Fax: 81-6-6645-3747. E-mail: smaeda{at}med.osaka-cu.ac.jp.

Journal of Clinical Microbiology, November 2004, p. 5277-5285, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5277-5285.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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