应用巢氏PCR-RFLP直接检测结核病临床标本中结核分支杆菌链霉素耐药基因型

DETECTION OF STREPTOMYCIN-RESISTANT GENOTYPE IN MYCOBACTERIUM TUBERCULOSIS FROM CLINICAL SAMPLES BY NESTED PCR-RFLP

目的：了解结核分支杆菌链霉素耐药基因突变情况，建立快速检测耐药突变株的方法。方法：通过ＰＣＲ－ＳＳＣＰ、ＰＣＲ－ＲＦＬＰ和巢氏ＰＣＲ－ＲＦＬＰ分析４０株结核分支杆菌临床分离株和１５例临床标本的ｒｐｓＬ基因突变的部位和性质。结果：１０株药物敏感株的ｒｐｓＬ双链泳动正常、并可被ＭｂｏⅡ消化；３０株耐ＳＭ分离株中，２５株双链泳动异常，２４株不被消化；１５例临床标本中，常规ＰＣＲ扩增７例ｒｐｓＬ阳性，巢氏ＰＣＲ１５例均阳性，４例ｒｐｓＬ双链泳动异常、并不被ＭｂｏⅡ消化。结论：通过常规ＰＣＲ－ＳＳＣＰ和ＰＣＲ－ＲＦＬＰ可快速检测结核分支杆菌耐ＳＭ分离株４３位密码子点突变，而通过巢氏ＰＣＲ－ＲＦＬＰ可直接检测大多数临床标本中结核分支杆菌ＳＭ耐药基因型。

Object:To observe the mutations of rpsL gene in streptomycin-resistant M.tuberculosis,and to develop a new method for detecting drug resistance.Method:The rpsL gene in 40 M.tuberculosis clinical isolates and 15 clinical samples were analysed with PCR-SSCP,PCR-RFLP and nested PCR-RFLP.Results:M.tuberculosis H 37 R v was used as a control.The rpsL genes from 10 drugsensitive isolates displayed normal SSCP profile,and could be digested by MboII.Of 30 streptomycin+resistant isolates,25 isolates displayed abnormal rpsL SSCP profiles,and 24 were not digested by MboII.Of 15 clinical samples,7 were rpsL genes positive by PCR and 15 were positive by nested PCR,4 had abnormal SSCP profiles and were not digested by MboII.Conclusions:The mutations of rpsL genes in streptomycin-resistant M.tuberculosis isolates could be detected rapidly by PCR-SSCP and PCR-RFLP.The streptomycin-resistant genotype in M.tuberculosis could be analysed directly from most clinical samples by nested PCR-RFLP.