RNA恒温扩增实时检测技术对结核分枝杆菌利福平耐药性的研究

Evaluation of the RNA simultaneous amplification and testing for detection of rifampin resistance in Mycobacterium tuberculosis

目的建立RNA恒温扩增实时检测技术（SAT）检测MTB利福平耐药性的方法，初步评价其检测效果。方法以pre．16SrRNA为检测靶标设计RNA探针及带有T7启动子的逆转录扩增引物，42℃恒温扩增实时检测在药物作用下MTB中pre-16SrRNA的变化。通过SAT检测20株已知药物敏感结果的临床分离株在利福平分别作用1、2和3d后pre-16SrRNA的变化，确定SAT检测利福平耐药性的最佳药物作用时间。抽取50株已知药物敏感结果的临床分离株，加药培养至最佳药物作用时间，经SAT检测后，建立受试者工作特征曲线，确定利福平耐药性的判断界值（cutoff），并对128株临床分离株进行SAT检测，以BactecMGIT960为金标准，评价SAT检测MTB利福平耐药性方法的效果。结果SAT检测利福平耐药性的最佳药物作用时间为2d，cutoff值为2．95；以BactecMGIT960为判定标准时，该方法检测51株利福平耐药菌株的敏感度为100％（51／51），检测77株利福平敏感菌株的特异度为97．4％（75／77），阳性预测值及阴性预测值均分别为96．2％（51／53）和100％（75／75），符合率为98．4％（126／128），约登指数为0．974。结论SAT检测利福平耐药性是一种新型耐药性检测方法，具有较高的敏感度和特异度。

Objective To establish and evaluate a method for detection of rifampin resistance in Mycobacterium tuberculosis （ M. tuberculosis ） by the RNA simultaneous amplification and testing （SAT）. Methods RNA probe and primer of reverse transcription with T7 promoter targets pre-16S rRNA were designed, and the isothermal RNA amplification at 42 ℃ was performed for real-time detection of the levels of pre-16S rRNA in drug exposed MTB. Twenty clinical isolates were detected by the SAT after treatment with rifampicin 1,2 and 3 d in order to determine the best drug effect time. Fifty clinical isolates with known drug susceptibility results were used to determine the best cutoff value of rifampicin drug susceptibility by SAT. In total, 128 clinical isolates were detected by SAT to evaluate the accuracy compared with the Bactec MGIT 960. Results The best drug effect time and cutoff value for the detection of rifampin resistance by SAT were 2 d and 2. 95. With the result of Bactec MGIT 960 as the reference, the sensitivity and the specificity of the assay was 100% （ 51/51 ） and 97.4% （ 75/77 ）, respectively. Conclusions The isothermal RNA amplification assay is a highly sensitive and specific tool for the detection of rifampicin resistance in M. tuberculosis, and therefore, it may be a new method to detect rifampicin resistance in M. tuberculosis.