RT-LAMP与RT-PCR法快速检测脑脊液结核分枝杆菌的效果比较

摘要

目的 探讨逆转录(RT)-环介导等温扩增(RT-LAMP)法、RT-聚合酶链反应(PCR)法检测脑脊液结核分枝杆菌(MTB)的效果,为其快速诊断及临床药效评估提供依据.方法 选取2015年12月至2017年4月解放军白求恩国际和平医院就诊的85例患者脑脊液标本进行研究,并将其分为 TBM 组(46例)、疑似TBM组(25例)和对照组(16例),根据结核分枝杆菌16S rRNA区段,设计特异性引物,建立RT-LAMP和RT-PCR检测技术体系,对两种方法的检测结果进行分析.结果 TBM 组阳性检出率分别为97.8% 和75.0%,疑似TBM 组阳性检出率分别为76.0% 和40.0%,对照组阳性检出率分别是0.0% 和12.0%,各组RT-LAMP法阳性检出率高于RT-PCR法,差异有统计学意义(P<0.01);对照组中,采用RT-PCR法检测时发现非特异性扩增,采用RT-LAMP法检测时未见非特异性扩增,RT-LAMP法检测特异性强于RT-PCR法;RT-PCR法检测灵敏度为100 CFU /mL,高于RT-LAMP法的1 CFU /mL.结论 RT-LAMP法检测MTB时具有简便、灵敏快速、可检测活菌特点,与PCR法比较,特异性强、操作简便、检测成本低、耗时短,有望成为基层、野战医疗单位及发展中国家等的常规检查工具.%Objective To investigate the effects of reverse transcription loop-mediated isothermal amplifica-tion(RT-LAMP)method and RT-PCR method for detecting Mycobacterium tuberculosis(MTB)in cerebro-spinal fluid(CSF)to provide a basis for its rapid diagnosis and clinical pharmacodynamic evaluation.Methods Eighty-five cases of CSF sample in the Bethune International Peace Hospital of PLA from December 2015 to April 2017 were selected for conducting the study and divided into the tuberculous meningitis(TBM)group(46 cases),suspected TBM group(25 cases)and control group(16 cases).The 16S rRNA region of MTB was used to design the specific primers.Then RT-LAMP and RT-PCR detection technological systems were estab-lished.Then the detection results by using the these two methods was analyzed.Results The positive detec-tion rates of the TBM group were 97.8% and 75.0% respectively,which of the suspected TBM group were 76.0% and 40.0% respectively,and which of the control group were 0.0% and 12.0% respectively,the posi-tive detection rate of each group in the RT-LAMP method was higher than that in the RT-PCR method,the difference was statistically significant(P<0.01);in the control group,adopting RT-PCR detection found non-specific amplification,while which was not found by adopting RT-LAMP method,indicating that the specifici-ty of RT-LAMP method was stronger than that of RT-PCR;the sensitivity of RT-PCR was 10.0 CFU/mL, which was higher than 1 CFU /mL of RT-LAMP.Conclusion RT-LAMP has the advantages of simpleness,sensitivity,rapidness and detecting viable bacteria,compared with PCR,which has strong specificity,easy op-erating,low cost and short time-consuming,is expected to be a routine detection tool of basic level and field medical institutions and developing countries.