mFLOS-LAMP方法在快速检测结核分枝杆菌复合群中的应用价值

The value of the mFLOS-LAMP method in the rapid detection of Mycobacterium tuberculosis complexes

目的建立多重环介导等温扩增荧光环引物自淬灭探针(multiplex fluorescence of loop primer upon self dequenching-loop mediated isothermal amplification,mFLOS-LAMP)方法,并评价其快速检测结核分枝杆菌复合群(Mycobacterium tuberculosis complex,MTBC)的临床应用价值。方法选取2022年2月—2023年12月新疆医科大学第一附属医院及第八附属医院就诊的肺结核患者42例和患有其他呼吸道疾病的患者46例,收集晨痰标本,提取样本DNA。以MTBC插入序列IS1081和IS6110基因为检测靶标,分别设计一套特异性引物,利用FLOS探针的设计原理,在每套基因的环引物上分别标记相对应的荧光团,建立含有双靶标的mFLOS-LAMP方法,对其检测结果进行分析。结果利用已优化好的反应体系,用结核分枝杆菌标准菌株H37Rv基因组DNA系列稀释液检测mFLOS-LAMP检出下限为500 fg/μL,mFLOS-PCR检出下限为5 pg/μL,mFLOS-LAMP的检出下限比mFLOS-PCR的低10倍。mFLOS-LAMP方法对24种微生物菌株均无交叉反应,具有较强的特异性。临床标本检测显示,以临床诊断结果为参照标准,mFLOSLAMP方法的灵敏度和特异度分别为92.9%、100.0%,Kappa值为0.931,受试者工作特征曲线(receiver operating characteristic curve,ROC)曲线下面积为0.964。以培养法为金标准,mFLOS-LAMP方法的灵敏度为92.6%。mFLOS-LAMP方法与Xpert MTB/RIF检测的一致率为94.3%(83/88);Kappa为0.885,mFLOS-LAMP方法与Xpert MTB/RIF检测具有较高的一致。mFLOS-LAMP方法和mFLOS-PCR方法的一致率为93.2%(82/88),Kappa值为0.861。结论mFLOS-LAMP方法具有检测快速、特异性强、灵敏性好、准确性高和仪器设备适用范围广等优点,可作为新型MTBC检测方法。

Objective To establish the mFLOS-LAMP method and evaluate its clinical value for rapid detection of Mycobacterium tuberculosis complex(MTBC).Methods February 2022 to December 2023,42 cases of pulmonary tuberculosis(TB)and 46 patients suffering from other respiratory diseases were selected from the First Affiliated Hospital and the Eighth Affiliated Hospital of Xinjiang Medical University,collect their morning sputum specimens and extract sample DNAUsing the MTBC insertion sequences IS1081 and IS6110 genes as detection targets,a set of specific primers were designed respectively.Based on the design principle of FLOS probes,the ring primers of each set of genes were labeled with corresponding fluorophores,the mFLOS-LAMP method containing dual targets was established,and the detection results of the method were analyzed.Results Using the optimized reaction system,a series of dilutions of genomic DNA of Mycobacterium tuberculosis standard strain H37Rv was used to detect the lower limit of detection of mFLOS-LAMP at 500 fg/μL and the lower limit of detection of mFLOS-PCR at 5 pg/μL and the lower limit of detection of mFLOS-LAMP was 10-fold lower than that of mFLOS-PCR.The mFLOS-LAMP method was non-cross-reactive and highly specific for 24 microbial strains.Clinical specimen testing showed that,using clinical diagnosis results as the reference standard,the sensitivity and specificity of the mFLOS-LAMP method were 92.9%and 100.0%,respectively;the Kappa value was 0.931;and the area under the ROC curves was 0.964.Using the culture method as the gold standard,the sensitivity of the mFLOS-LAMP method was 92.6%.The consistency between the mFLOS-LAMP method and the Xpert MTB/RIF assay was 94.3%(83/88);the Kappa was 0.885,indicating the mFLOS-LAMP method had a high consistency with Xpert MTB/RIF assay.The consistency between the mFLOSLAMP method and the mFLOS-PCR method was 93.2%(82/88),with a Kappa value of 0.861.Conclusions Compared with the mFLOS-PCR method,the mFLOS-LAMP method has the advantages of rapid detection,high specificity,good sensitivity,high accuracy,and a wider applicability of instrumentation.It can be used as a new type of MTBC detection method.