结核病临床分离株及痰标本中embB基因型的快速测定

Rapid detection of embB genotype of Mycobacterium tuberculosis in clinical isolates and sputum

目的建立快速测定结核病耐乙胺丁醇(EMB)分离株和痰标本中结核分枝杆菌EMB耐药基因型的方法,以期为患者提供及时、有效地化验结果。方法应用16SrDNA聚合酶链反应(PCR)-单链构象多态性(SSCP)和PCR-限制性片段长度多态性(RFLP)对11株耐EMB分离株和46例结核病痰标本进行分子菌种鉴定和embB基因突变的部位与性质分析。结果以结核分枝杆菌H37Rv标准株为对照,11株耐EMB分离株和46例临床痰标本经16SrDNAPCR-SSCP分析电泳图谱均与结核分枝杆菌标准株相同;限制性内切酶Nla消化embB基因扩增产物显示,11株耐EMB分离株中4株(36.4%)不被Nla消化;46例结核病痰标本中10例(21.7%)不被Nla消化。结论部分结核分枝杆菌耐EMB是由于embB基因突变所致,采用PCR-SSCP和PCR-RFLP方法可直接快速测定结核病耐EMB分离株和痰标本中结核分枝杆菌耐EMB基因型。

Objective To develop a rapid method for detection of ethambutol resistance genotype ^(embB) in Mycobacterium tuberculosis clinical isolates and sputa. Methods In order to ~identify tuberculosis species and find mutation position and characterization of embB gene, 11 clinical isolates and 46 sputa were analyzed with 16S rDNA-Polymerase chain reaction (PCR), PCR-Single stranded conformation polymorphism (PCR-SSCP) and PCR-Restrictive fraction length polymerase (PCR-RFLP). Results Mycobacterium tuberculosis isolate H37Rv was used as control, 11 clinical isolates and 46 sputa specimens were analyzed with PCR-SSCP, they had the same 16S rDNA profiles as M.tuberculosis standard strain. Of 11 ethambutol-resistant clinical isolates, 4(36.4%) had abnormal RFLP profiles. of 46 sputa specimens, 10 (21.7%) had abnormal RFLP profiles. Conclusion Ethambutol resistances in some Mycobacterium tuberculosis isolates were due to mutations on embB genes. PCR-SSCP and PCR-RFLP methods might become rapid and effective diagnostic test for genotype of M.tuberculosis ethambutol-resistance clinical isolates and sputa.