结核分支杆菌利福平药物敏感性的直接快速检测  

Direct, rapid detection for rifampin susceptibility to M. tuberculosis

作　　者：鱼瑛[1,2,3] 康熙雄 金玲[1,2,3] 王淑华 马琳[1,2,3] 

机构地区：[1]长春职工医科大学 [2]白求恩医科大学第三临床医院 [3]白求恩医科大学微生物教研室

出　　处：《中华结核和呼吸杂志》1999年第5期299-301,共3页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：长春市科委攻关项目;长春市卫生局基金

摘　　要：目的探讨利用分子生物学方法直接快速检测临床标本结核分支杆菌利福平（ＲＦＰ）药物敏感性的可行性。方法自行设计针对ｒｐｏＢ基因特异扩增的引物（扩增产物为１８３ｂｐ片段），运用聚合酶链反应单链构象多态性（ＰＣＲＳＳＣＰ）银染色法，检测４５株结核分支杆菌临床分离株，对其中部分菌株进行ｒｐｏＢ基因ＤＮＡ序列分析；运用ＰＣＲＳＳＣＰ银染色法对７０份结核病患者痰标本进行直接快速检测，并与药敏试验结果做对照分析。结果４５株临床分离株中１６株ＲＦＰ敏感结核分支杆菌ＳＳＣＰ带谱与结核分支杆菌标准株（Ｈ３７Ｒｖ）相同，２９株ＲＦＰ耐药株中有２６株检测到突变图谱，阳性率为９０％。从具有ＳＳＣＰ特征性带谱的菌株中筛选８株临床分离株进行ｒｐｏＢ基因序列分析显示：１株ＲＦＰ敏感株无ｒｐｏＢ基因突变，７株耐药株均发生碱基突变（符合率１００％）。碱基以单点突变为主，以５３１位碱基突变最为多见［４株５３１位ＴＣＧ→ＴＴＧ，Ｓｅｒ→Ｌｅｕ（５７％）；１株５３１位ＴＣＧ→ＣＡＧ，Ｓｅｒ→Ｇｌｎ（１４％）；２株５２６位ＣＡＣ→ＴＡＣ，Ｈｉｓ→Ｔｙｒ（２９％）］。与ＢＡＣＴＥＣ对照临床痰标本直接快速检测阳性率为６４％。结论结核分支杆菌耐ＲＦＰ?Objective To evaluate the use of molecular biotechnology for direct, rapid detection of rifampicinresistance mutations in M.tuberculosis. Methods 45 M.tuberculosis clinical isolates and 70 sputum samples were tested by polymerase chain reactionsingle stranded conformation polymorphism (PCRSSCP) technique. M.tuberculosis strain H37Rv was used as control and compared with the result of susceptibility test.DNA sequencing was also performed in some of the strains. Results All tested susceptible isolates displayed identical SSCP patterns. Of 29 RFP resistance strains, 26(90%) had distinct mobility shifts that can be discriminated from susceptible isolates. 9 sputum samples which were successfully evaluated by PCRSSCP showed concordant result acquired from BACTEC 460 method.As the result of DNA sequencing,it was observed that seven RFPresistance phenotype of M. tuberculosis strains had missense mutation,in which 5 isolates displayed TCGTTG or CAG mutations at codon 531,2 had CAC TAC mutation at codon 526. On the other hand, one strain which was susceptible to rifampin exhibited identical nucleotide alignment to the sequence of rpoB gene. Conclusions PCRSSCP could be used as a method for simple,rapid,and reliable detection of rifampicinresistance mutations in clinical samples of M.tuberculosis.

关 键 词：结核分支杆菌 利福平 药物敏感性 聚合酶链反应 

分 类 号：R978.3[医药卫生—药品] R378.911[医药卫生—药学]