Abstract：
We aimed to develop a novel diagnostic method called multiplex fluorescence of loop primer upon self-dequenching loop-mediated isothermal amplification (mFLOS-LAMP) for the rapid detection of Mycobacterium tuberculosis complex (MTBC). A set of specific primers was designed to target the detection of IS1081 and IS6110 genes, which are insertion sequences within the MTBC. The 110 sputum specimens collected were assessed using the established mFLOS-LAMP method, multiplex polymerase chain reaction, Xpert MTB/RIF, and smear microscopy. The optimal reaction temperature and duration for mFLOS-LAMP were determined to be 65°C and 30 min, respectively, by optimizing the entire system. The detection sensitivity of mFLOS-LAMP was 6.0 × 101 CFU/mL, by Bacillus Calmette-Guerin, and the mFLOS-LAMP sensitivity of M. tuberculosis H37Rv genomic DNA was 500 fg, and the specificity was 100%. The sensitivity of mFLOS-LAMP was 94.2% and the specificity was 96.6%, when Xpert MTB/RIF was used as the reference method. There was no statistically significant difference in their detection rate (χ2 = 0, P = 1.000), and the consistency was good (kappa = 0.909, P < 0.001). The receiver operating characteristic analysis yielded the maximum area under the curve of 0.954. The mFLOS-LAMP method demonstrated high sensitivity and specificity, allowing for swift and accurate detection of MTBC.
摘要：
我们旨在开发一种新的诊断方法，称为自我去猝灭环介导的等温扩增（mFLOS-LAMP）的多重环引物荧光，用于快速检测结核分枝杆菌复合体（MTBC）。设计了一套特异性引物来靶向检测IS1081和IS6110基因，它们是MTBC内的插入序列。收集的110份痰标本采用已建立的mFLOS-LAMP方法进行评估,多重聚合酶链反应，XpertMTB/RIF,和涂片显微镜。mFLOS-LAMP的最佳反应温度和持续时间确定为65°C和30分钟，分别,通过优化整个系统。mFLOS-LAMP的检测灵敏度为6.0×101CFU/mL,卡介苗，结核分枝杆菌H37Rv基因组DNA的mFLOS-LAMP敏感性为500fg，特异性为100%。mFLOS-LAMP的敏感性为94.2%,特异性为96.6%,当使用XpertMTB/RIF作为参考方法时。检出率差异无统计学意义(χ2=0,P=1.000),一致性良好(kappa=0.909,P<0.001)。接收器工作特性分析得出曲线下的最大面积为0.954。mFLOS-LAMP方法具有较高的灵敏度和特异性,允许MTBC的快速和准确的检测。
