Simultaneous detection for isoniazid and rifampin-resistanct Mycobacterium tuberculosis by Multiplex Allele-Specific PCR assay / 中华传染病杂志

ABSTRACT

Objective To develop a new Multiplex Allele-Specific polymerase chain reaction(MAS-PCR) assay to detect the main point mutations in the katG and rpoB genes, which has been reported to account for the majority of clinical Mycobaterium tuberculosis resistant to isoniazid and rifampin. Methods Based on the sequences of katG and rpoB genes, specific primers were designed to carry out the MAS-PCR to detect the most common mutations in codon315 of katG and codons 531,526,516 of rpoB gene. Results The purified DNA preparation of 96 clinical Mycobacterium tuberculosis strains were used to optimize PCR. No mutation was detected in 19 isoniazid-sensitive strains. The 315Ser point mutation was detected in 79.2%(61/77)of isoniazid-resistant strains, the type of mutation includes the most common S315T and the less common S315N, which could’t be detected by PCR-RFLP(polymerase chain reaction-restriction fragment length polymorphism). However, S315G couldn’t be detected by MAS-PCR and that will make a false negative. The mutations in codons 531,526,516 were detected by the MAS-PCR. Compared with the results of direct sequencing of rpoB gene, no mutation was detected in sensitive strains. For rifampin-resistant strains, the total sensitivity was 81.5%(66/81). Conclusions MAS-PCR is a new molecular method with high sensitivity and specificity, which can be used to detect the point mutation in katG and rpoB gene rapidly and economically. It can be used in clinical laboratories to detect drug-resistant tuberculosis strains. Simultaneous detection for katG and rpoB gene mutations in one MAS-PCR system will help to improve the efficiency of this method.