荧光定量PCR技术快速检测rpoB基因突变及其临床应用

Rapid detection of rpoB gene mutation from Mycobacterium tuberculosis by real-time PCR and its clinical application

目的探讨荧光定量PCR技术检测基因突变的价值及其临床应用。方法针对结核分枝杆菌rpoB基因利福平耐药决定区(Rifampicin Resistance Determining Region,RRDR)526密码子和531密码子常见的突变形式设计探针(526CAC,526TAC,531TCG和531TTG),应用已知rpoB基因RRDR区序列的38株利福平耐药临床分离株和24株利福平敏感临床分离株以及5株非结核分枝杆菌菌株建立荧光定量PCR检测基因突变的方法。继而,应用该技术检测84份肺结核病例痰标本,与痰罗氏培养以及药敏结果进行比较,部分标本进行DNA测序证实。结果在38株利福平耐药、24株利福平敏感的临床分离株和5株非结核分枝杆菌中,其检测526密码子和531密码子突变的敏感性和特异性达100%。在84份肺结核病例的痰标本中,罗氏培养阳性62株,其中利福平耐药株48份,荧光定量PCR检测痰标本结核分枝杆菌DNA阳性为75例。荧光定量PCR检测到531密码子TTG突变65例,526密码子TAC突变7例。在48株利福平耐药株中,荧光定量PCR检测43株为531TTG突变,3株为526TAC突变,另2株利福平耐药株,经测序证实,1株514密码子TTC插入突变,1株为511密码子CCG和516密码子GGC联合突变。结论荧光定量PCR技术能快速检测rpoB基因突变,并能作为临床肺结核病人早期快速耐药诊断的辅助手段。

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 Rifampiein 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 testing, while 75 were positive by real-time PCR at codon 531, and 3 isolates had TAC mutation Rifampicin-resistant samples） by conventional drug susceptibility . Of 48 Rifampin-resistant samples, 43 had TTG mutation of rpoB at codon 526 by real-time PCR. The result of sequencing showed that one mutant at codon 514 was inserted Trc, 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.