RNA恒温扩增联合熔解曲线分析鉴定胞内分枝杆菌的应用研究

RNA isothermal transcription-mediated amplification and real-time detection combined melt curve analysis for the identification of Mycobacterium intracellulare

目的建立RNA恒温扩增联合熔解曲线分析技术(RIARD-MCA)鉴定胞内分枝杆菌的方法,评估其应用效果。方法以胞内分枝杆菌的16SrRNA的特异序列为检测靶标设计RNA探针和带有T7启动子的逆转录扩增引物,42℃恒温扩增实时检测后进行熔解曲线分析,对21种分枝杆菌标准株、5种非分枝杆菌和229株分枝杆菌临床分离株进行鉴别检测;同时以16SrRNA及hsp65基因PCR测序结果为金标准对照。结果 RIARD-MCA在21种分枝杆菌标准株及5种非分枝杆菌检测中只有胞内分枝杆菌阳性,其他阴性,与测序结果一致;在分枝杆菌临床分离株检测中,鉴定胞内分枝杆菌63株,其余为非胞内分枝杆菌,PCR测序为胞内分枝杆菌63株,其余166株为非胞内分枝杆菌,其灵敏度为100%(63/63),特异度为100%(166/166)。结论 RIARD-MCA鉴定胞内分枝杆菌具有较高的灵敏度、特异度和准确性,且检测快速,有望作为一种新的胞内分枝杆菌临床分离株鉴定方法。

The aim of the study is to establish and evaluate a RNA isothermal transcription-mediated amplification and real-time detection combined melt curve analysis method （RIARD-MCA） for the identification of Mycobacterium intracellulare in clinical isolates. Specific primers incorporating the T7 promoter sequence, and RNA probes was designed based on M. intra- cellular 16S rRNA gene and undergone successive cycles of amplification using T7 RNA polymerase at 42 ℃. Then we used 21 reference strains, 5 non-mycobacterium strains and 229 clinical strains to evaluate the specificity and sensitivity of the new as- say. At the same time, we compared the results with the 16S rRNA and hsp65 gene sequencing. In this test, only M. intracel lular was positive, with the sensitivity of 100% （63/63） and specificity of 100% （166/166）, comparing to PCR gene sequen- cing. It＇s suggested that RIARD-MCA is a sensitive, specific and rapid method in identification of M. intracellular, which may be used for rapid identification of M. intracellular in clinical isolates.