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应用PCR—直接测序法测定结核分支杆菌rpsL基因突变的研究
引用本文:吴雪琼,张俊仙.应用PCR—直接测序法测定结核分支杆菌rpsL基因突变的研究[J].中国抗生素杂志,1999,24(3):202-204.
作者姓名:吴雪琼  张俊仙
作者单位:解放军309医院
摘    要:目的:了解我国结核分支杆菌耐链霉素(Streptomycin,SM)分离株rpsL基因突变情况,建立快速检测结核分支杆菌耐药基因型的分子药敏试验方法。方法:通过聚合酶链反应(PolymeraseChainReaction,PCR)-单链构象多态性(Single-strandedConformationPolymorphism,SSCP)、PCR-限制性片段长度多态性(RestrictionFragmentLengthPolymorphism,RFLP)和PCR-直接测序法(DirectSequencing,DS)分析38株结核分支杆菌耐SM分离株的rpsL基因。结果38株耐SM分离株中,25株SSCP异常、不被MboⅡ消化、DS分析43位密码子AAG→AGG突变;1株SSCP异常、可被MboⅡ消化、DS分析33位密码子GTA→ATA突变;12株SSCP正常、可被MboⅡ消化、DS分析未见异常;未发现88位密码子突变。结论:大多数结核分支杆菌耐SM是由于其rpsL基因43位密码子突变所致,采用PCR-SSCP、PCR-RFLP和PCR-DS方法可快速测定部分结核分支杆菌SM耐药基因型。

关 键 词:聚合酶链反应  单链构象多态性  限制性片段长度多态性  直接测序法  药物耐受性  结核分支杆菌

Studies on the detection of rpsL gene mutations inM.tuberculosis by PCRdirect sequencing
Wu Xueqiong,Zhang Junxian,Zhuang Yuhui,Zhang JunziandJia Shulin.Studies on the detection of rpsL gene mutations inM.tuberculosis by PCRdirect sequencing[J].Chinese Journal of Antibiotics,1999,24(3):202-204.
Authors:Wu Xueqiong  Zhang Junxian  Zhuang Yuhui  Zhang JunziandJia Shulin
Abstract:Objective: To understand the mutations of streptomycinresistant genes in M.tuberculosis clinical isolates, and to develop a new molecular method for detecting drug resistance. Method: Analyzing the rpsL genes in 38 strains of M.tuberculosis streptomycinresistant isolates with PCRSSCP, PCRRFLP and PCRDS. Results M.tuberculosis strain H37Rv was used as a control. Of 38 streptomycinresistant isolates, 25 isolates displayed abnormal rpsL SSCP profiles, were not digested by MboII, and had AAGAGG mutations at codon 43; one displayed abnormal SSCP profile, was digested by MboII, and had GTAATA mutation at codon 33; 12 exhibited normal rpsL genes with PCRSSCP, PCRRFLP, PCRDS. There were no mutations found at codon 88. Conclusions: Resistance to streptomycin in most M.tuberculosis isolates is due to the mutations situated at codon 43 of genes encoding the ribosomal S12 protein (rpsL). PCRSSCP, PCRRFLP and PCRDS, ans it might become simple, rapid and reliable diagnostic tests for streptomycin resistance in some clinical isolates.
Keywords:Polymerase chain reaction  Singlestranded conformation polymorphism  Restriction    fragment length polymorphism  Direct sequencing  Drug resistance  M  tuberculosis
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