关键词: 结核, 分枝杆菌,结核, 耐药性, 聚合酶链反应

Abstract: Objective To evaluate the value of detecting gene mutation by real-time PCR and its clinical application. Methods Targeting the most common mutation types at codon 526 and codon 531 of Rifampicin Resistance Determining Region (RRDR) of rpoB gene from Mycobacterium tuberculosis,labeled probes(526CAC, 526TAC,531TCG and 531TTG) were designed to detect 38 Rifampin-resistant clinical isolates with known RRDR sequence,24 Rifampin-sensitive clinical isolates with wild type sequence of RRDR and 5 nontuberculous mycobacteria isolates,then a method using real-time PCR was established.84 sputa from patients with pulmonary tuberculosis were analyzed by real-time PCR and drug susceptibility testing on Lowenstein-Jensen(L-J) medium.Some sputum samples were amplified by PCR with other primers and then sequenced. Results Using conventional drug susceptibility testing as control,the mutation type at codon 526 and codon 531 of rpoB gene were identified in 38 Rifampin-resistant clinical isolates,24 Rifampin-sensitive clinical isolates and 5 nontuberculous mycobacteria isolates by real-time PCR.The sensitivity and specificity were 100%,respectively.Of 84 sputum samples,62 were drug-resistant(including 48 Rifampicin-resistant samples) by conventional drug susceptibility testing,while 75 were positive by real-time PCR.Of 48 Rifampin-resistant samples,43 had TTG mutation of rpoB at codon 531,and 3 isolates had TAC mutation at codon 526 by real-time PCR.The result of sequencing showed that one mutant at codon 514 was inserted TTC,and another one had a CCG mutation at codon 511 with a GGC mutation at codon 516. Conclusion Real-time PCR is a rapid technique to detect mutation of target gene and could be an assistant tool to diagnose drug-resistant tuberculosis early.

Key words: Tuberculosis, Mycobacterium tuberculosis, Drug resistance, Polymerase chain reaction