结核分支杆菌利福平耐药基因突变的研究

Studies on mutation of rifampicin resistant genes in M. tuberculosis

目的了解我国结核分支杆菌耐利福平（ＲＦＰ）分离株ｒｐｏＢ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－单链构象多态性（ＰＣＲ－ＳＳＣＰ）和ＰＣＲ－直接测序法（ＰＣＲ－ＤＳ）分析５０株结核分支杆菌临床分离株的ｒｐｏＢ基因。结果３株结核分支杆菌药物敏感株和２株非ＲＦＰ耐药株中，仅１株ｒｐｏＢＳＳＣＰ图谱异常的药物敏感株出现５３１位密码子ＴＣＧ→ＴＴＧ突变。４５株ＲＦＰ耐药株中，１０株ＳＳＣＰ和ＤＳ分析均未见ｒｐｏＢ突变，３５株ＳＳＣＰ和ＤＳ分析异常，其中１４株为５３１位密码子ＴＣＧ→ＴＴＧ或ＴＧＧ或ＴＡＣ突变，１４株为５２６位ＣＡＣ→ＴＡＣ或ＧＡＣ或ＣＣＣ或ＣＴＣ或ＧＴＣ突变，２株为５１６位ＧＡＣ→ＧＴＣ或ＴＡＣ突变，２株为５１６位和５２６位及５１５位和５１６位密码子双点突变，３株ｒｐｏＢ序列与结核分支杆菌不同。结论大多数结核分支杆菌耐ＲＦＰ是由于其ｒｐｏＢ基因突变所致。

Objective To understand mutation of rifampicin resistant genes in M. tuberculosis clinical isolates,and to develop a new method for detecting drug resistance. Method Analyzing the rpoB genes in 50 M. tuberculosis clinical isolates with polymerase chain reaction single stranded conformation polymorphism (PCR SSCP) and PCR direct sequencing(PCR DS) techniques. M. tuberculosis strain H 37 Rv was used as control. Result Of 3 drug sensitive isolates and 2 non rifampicin resistant isolates selected,only 1 drug sensitive isolate with abnormal rpoB SSCP profile had TCG→TTG mutation at codon 531. Of 45 rifampicin resistant isolates,35 isolates with abnormal SSCP profiles had mutations in rpoB sequences,in which 14 isolates displayed TCG→TTG or TGG or TAC mutations at codon 531,14 had CAC→TAC or GAC or CCC or CTC or GTC mutations at codon 526,2 had GAC→GTC or TAC mutations at codon 516,2 had two point mutations,and 3 had different rpoB sequences from that of M. tuberculosis. Conclusion Resistance to rifampicin in most M. tuberculosis isolates is due to the mutations on the genes encoding the RNA polymerase subunit (rpoB). PCR SSCP and PCR DS techniques might become simple,rapid and reliable diagnostic tests for rifampicin resistance in clinical isolates.