[Study on isoniazid-resistant Mycobacterium tuberculosis isolates by multiple-polymerase chain reaction-single strand conformation polymorphism].

Abstract

OBJECTIVE: To develop a new multiple-polymerase chain reaction-single strand conformation polymorphism (multi-PCR-SSCP) system for detecting the aphC promoter, inhA, and katG gene mutations in isoniazid-resistant Mycobacterium tuberculosis isolates in the single reaction, and for the quick diagnosis of isoniazid-resistant Mycobacterium tuberculosis isolates. METHODS: Three pairs of oligonucleotide primers were designed according to the aphC promoter, inhA, and katG genes of Mycobacterium tuberculosis to examine isoniazid-resistance by multi-PCR-SSCP. RESULTS: Isoniazid-sensitivity and resistance were analyzed with general PCR and multi-PCR at the same time, and H(37) Rv was used as a control. These two protocols amplified the anticipated fragments, the rate of consistency being 100%. By single gene PCR-SSCP, the mutation rates of aphC promoter, inhA, and katG gene were 17%, 20%, and 66%, respectively. The mutation rate detected by multi-PCR-SSCP was 83%. CONCLUSIONS: Multi-PCR-SSCP is a sensitive and specific method for rapid detection of aphC promoter, inhA, and katG gene mutations in isoniazid-resistant Mycobacterium tuberculosis isolates. Drug-resistant gene detection may be clinically useful in the therapy of tuberculosis.