RPA-LFD技术快速检测结核分枝杆菌利福平耐药性的应用

Application of RPA-LFD for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis

目的基于重组酶聚合酶扩增(RPA)技术结合侧向流动试纸条(LFD),建立可快速检测结核分枝杆菌(MTB)利福平耐药的方法,并初步评价其临床应用价值。方法采用比例法对182株结核分枝杆菌临床分离株进行利福平耐药性检测。建立RPA-LFD技术,检测182株菌株中利福平耐药决定区(RRDR)常见突变位点rpoB 531、526和516的突变情况,同时使用PCR测序进行验证。用SPSS 24.0分析,以比例法为金标准,评价RPA-LFD对利福平耐药的检测效率。结果以比例法药敏结果为金标准,RPA-LFD检测利福平耐药的灵敏度为84.2%,特异度为100.0%,Kappa检验显示Kappa值为0.880,两种方法检测结果具有高度一致性。同样以比例法为金标准,以DNA测序结果判断利福平耐药性的灵敏度为91.2%,特异度为100.0%,Kappa值为0.935。结论RPA-LFD技术用于结核分枝杆菌利福平耐药性的检测具有好的灵敏度、很高的特异度,且检测时间短,具有一定的临床应用价值。

We established an RPA-LFD assay for the rapid detection of 531,526 and 516 mutations in the Mycobacterium tuberculosis(MTB)rpoB gene,and evaluated the assay’s clinical application.We analyzed rifampicin resistance in 182 M.tuberculosis isolates preserved in the laboratory using drug sensitivity tests.Further analysis was performed by sequencing of the rifampin resistance-determining region(RRDR)in these strains and screening their mutation sites.The mutation sites in codons 516,526 and 531 of rpoB were chosen and used to design primers for RPA-LFD.Finally,statistical analysis was conducted on the test results for the three methods to evaluate the detection efficiency of RPA-LFD.Relative to those of traditional DST,the sensitivity and specificity of RPA-LFD in the detection of rifampicin resistance were 84.2%and 100.0%,respectively.The Kappa value was 0.880,thus indicating high coincidence between the two methods.With respect to those of traditional DST,the sensitivity and specificity of DNA sequencing in the detection of rifampin resistance were 91.2%and 100.0%,respectively.RPA-LFD for the detection of resistance of M.tuberculosis to ri fampicin showed good sensitivity and specificity,and was not time consuming;thus,it should have value in the rapid diagnosis of rifampicin-resistant tuberculosis.