[Study on isoniazid-resistant Mycobacterium tuberculosis isolates by multiple-polymerase chain reaction-single strand
Xiao-dong Cheng1, Wen-bin Yu, Liang-feng Bie
1Department of Clinical Laboratories, Xijing Hospital, Fouth Military Medical University, Xi'an 710032, China.
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.
