关键词: Nam Dinh病毒, 实时荧光定量PCR, TaqMan-MGB 探针, 应用

Abstract:

Objective To establish a rapid and specific real-time fluorescent PCR method for Nam Dinh virus (NdiV). Methods According to the sequence alignment of GenBank and NDiV isolated by our laboratory, we found out the conservative sequence (RdRp) and design specific primers and TaqMan-MGB probe. In order to evaluate the reaction system, the concentration of primers and probe were adjusted to optimize the reaction conditions, and the sensitivity, specificity, stability tests for our method were also conducted. Results TaqMan-MGB real-time fluorescence PCR detection method of NDiV were less time-consuming and highly sensitive, and the low detection limit was 0.1 PFU. It had a good specificity characteristic for having no cross reaction with dengue serotype 1-4, epidemic encephalitis b virus, respiratory syncytial virus, rotavirus, stellate virus, and adenovirus; The five times repeated testing of four nucleic acid content in different standard samples revealed that the average coefficient of variation range of Ct was 1.67%-3.68%, and thus it had high stability. Through monitoring, the positive probability of mosquitoes collected from Longgang district for NDiV was 18.00%. Conclusion NDiV TaqMan-MGB real-time fluorescent PCR method is a rapid, specific, sensitive and stable method, it can be applied to epidemiological monitoring in order to improve the ability of rapid detection of viruses.

Key words: Nam Dinh virus, Real-time fluorescence PCR, TaqMan-MGB, Application