应用聚合酶链反应-寡核苷酸探针快速检测结核菌耐药基因突变

Rapid detection of mutations of drug-resistant genes in Mycobacterium tuberculosis by PCR-oligonucleotide probes

目的 应用聚合酶链反应 (PCR) 寡核苷酸探针快速检测结核菌耐药基因突变,建立一种新的分子药敏试验方法。方法 以传统药敏试验、PCR 单链构象多态性和PCR 直接测序方法为对照,对利福平、链霉素(SM)和乙胺丁醇耐药的结核菌基因rpoB、rpsL、rrs和embB的寡核苷酸探针,通过反向斑点杂交检测 78株结核菌临床分离株PCR产物。结果 18株结核菌药物敏感株的 4种耐药基因均为野生型。60株结核菌耐药分离株中, 59株为耐RFP株,rpoB基因突变率为 93. 2%,最常见的突变位点为 531位和 526位密码子, 33株为耐SM株,rpsL基因突变率为 75. 8%,均为 43位密码子AAG→AGG突变,rrs基因突变率为 9 .1%,均为 513位A→C突变,rpsL和rrs基因总突变率为 84 .8%; 43株为耐EMB株,embB基因突变率为 58 .1%,均为 306位密码子突变。结论 应用PCR 寡核苷酸探针反向斑点杂交技术可简便、快速、准确地分析大多数结核菌耐药基因突变,检测其耐药性。

Objective To detect the mutations of drug resistant genes in Mycobacterium tuberculosis isolates by PCR oligonucleotide probes, and develop a new, rapid method for drug resistance Method Using traditional drug susceptibility testing, PCR SSCP and PCR DNA sequencing as control, rpoB, rpsL, rrs, and embB genes from 78 M tuberculosis clinical isolates were analyzed by a reverse hybridization with PCR oligonucleotide probes Results The rpoB, rpsL, rrs and embB genes from 18 drug sensitive M tuberculosis isolates were all wild types Of 60 drug resistant M tuberculosis isolates, 93 2% (55/59) rifampicin resistant isolates had rpoB gene mutations Codon 531 and 526 of the rpoB were the most common sites of nucleotide substitutions. 75 8% of 33 streptomycin resistant isolates had an identical mutation (AAG→AGG) at codon 43 of the rpsL gene Only 9 1% of these streptomycin resistant isolates had A to C transversions at position 513 of the rrs gene 58 1% of 43 ethambutol resistant isolates had nucleotide substitutions (ATG→GTG or ATA or CTG) at codon 306 Conclusion The reverse hybridization with PCR oligonucleotide probes might become a rapid and simple method to detect the mutations of drug resistant genes in the most drug resistant Mycobacterium tuberculosis isolates