结核分支杆菌临床分离株的基因检测研究

Study on determination for genes of mycobacterium tuberculosis clinical isolates strain

目的检测结核分支杆菌临床分离株的IS986和embB基因序列 ,了解结核分支杆菌及其药物敏感性的基因检测同Bactec 4 6 0自动检测系统的相关性。方法用Bactec 4 6 0检测系统对获自肺结核患者的 8株临床分离菌和 1株质控菌进行菌种及EMB药物敏感性的鉴定 ,采用PCR和PCR DS技术对菌株进行IS986和embB基因检测和序列分析。结果 8株临床分离菌经Bactec 4 6 0自动检测系统鉴定为结核分支杆菌 ,其中 7株对EMB敏感、1株耐药 ,质控株耐药。各分离菌株及质控株PCR检测出IS986和embB基因序列 ,符合率 10 0 %。 1株临床分离耐药株embB基因的第 30 6位密码子发生突变 ,1株敏感株embB基因的第 2 78位和 2 79位密码子有突变 ,其余 6株敏感菌及质控株未发现序列改变 ,符合率 77 8%。结论PCR扩增是一种快速、敏感、特异性的结核分支杆菌鉴定方法 ,鉴定结果同Bactec 4 6 0TB检测系统一致。结核分支杆菌对EMB耐药性的形成同embB基因突变有关。Bactec 4 6 0TB系统检测药物敏感性可发生漏诊或误诊 ,可能造成药敏结果与临床治疗效果不一致的情况。

Objective To detect IS986 and embB genies of M.tuberculosis clinical isolates strain and to understand the interrelation of the molecular identification methods with the Bactec-460TB system. Methods The properties and EMB susceptibility of a control strain of M.tuberculosis and 8 clinical isolates were determined by the Bactec-460TB system, the IS986 and embB genes from these bacteria were detected and analyzed by PCR and PCR-direct sequencing. Results 8 clinical isolates strain were identified as M.tuberculosis and seven of them were EMB sensitive isolates, one of the clinical isolates and control strain were EMB resistant stains. The IS986 and embB genes of the clinical isolates and the control strain were all amplified. The agreement rate of the two methods for M.tuberculosis determination was 100%. The mutations were showed in the drug-resistant clinical isolate at codon 306 and in the drug-sensitive isolates at both codon 278 and 279. Conclusion PCR is a rapid, sensitive and specific method to identification of M.tuberculosis and the result was agreed with the Bactec-460TB system.