RNA恒温扩增实时检测技术快速鉴定偶发分枝杆菌的研究

Rapid identification for Mycobacterium fortuitum with RNA isothermal transcription-mediated amplification and real-time detection assay

目的建立RNA恒温扩增实时检测技术快速鉴定偶发分枝杆菌(RIARD-MF)的方法,评估其在鉴定分枝杆菌临床分离株中的应用效果。方法将偶发分枝杆菌的16SrRNA特异序列作为目的靶标进行检测,设计含T7启动子逆转录扩增引物和RNA探针,分别对5种非分枝杆菌细菌、20种分枝杆菌标准株和259株临床分枝杆菌分离株进行42℃恒温扩增实时检测,参考标准为PCR基因测序结果。结果RIARD-MF方法学检测灵敏度可达60CFU/mL。在25种细菌的RIARD-MF特异性检测中:只有偶发分枝杆菌检测结果为阳性,其余24种细菌均为阴性,与测序检测结果一致。在检测分枝杆菌临床分离菌株时,RIARD-MF鉴定偶发分枝杆菌5株,其余为非偶发分枝杆菌,与测序结果一致,检测灵敏度和特异度均为100%。结论 RIARD-MF鉴定偶发分枝杆菌具有较高的特异度、灵敏度和准确性,且检测快速,有望成为一种新的偶发分枝杆菌临床分离菌株鉴定方法。

The aim of the study is to establish and evaluate a RNA isothermal transcription-mediated amplification and realtime detection assay （RIARD-MF） for the identification of Mycobacterium fortuitum in clinical isolates. RNA probes and spe- cific primers of reverse transcription and amplification for T7 promoter were designed based on the sequence of M. fortuitum 16S rRNA. The isothermal successive cycles of amplification were performed for real-time detection by using T7 RNA polymer- ase at 42 ~C. Five non-mycobacterium strains, 20 Mycobacterium strains and 259 clinical strains were detected by the estab- lished assay to evaluate the specificity and sensitivity, and the results were compared with those of PCR sequencing. In the test of 5 non-mycobacterium strains and 20 Mycobacterium strains, only M. fortuitum was positive, and the remaining 24 strains of bacteria were negative, which was consistent with PCR gene sequencing. The sensitivity and specificity of RIARD-MF reached 60 CFU/mL and 100%. In the test of 259 strains of clinical isolates, 5 strains were identified to be M. fortuitum, the remai- ning 254 strains were not identified to be M. Fortuitum, which was also consistent with PCR gene sequencing. Both the speci- ficity and sensitivity reached up to 100% in the detection of clinical isolates. It suggested that the RIARD-MF established in this study is a specific, sensitive, accurate and rapid method for the identification of M. Fortuitum and it may be hopeful for rapid identification of M. fortuitum in clinical isolates.