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

摘要：

目的 建立一种快速、特异检测Nam Dinh病毒(NDiV)的实时荧光PCR方法。方法 根据GenBank和本实验室分离的NDiV进行序列比对, 找出保守序列(RdRp)并设计特异引物和TaqMan-MGB 探针, 通过调整引物、探针浓度, 优化其反应条件, 对方法做灵敏度、特异性、稳定性实验, 以评价反应体系。结果 NDiV的TaqMan-MGB 实时荧光PCR检测方法用时短, 灵敏度高, 最低检测下限为0.1 PFU。与登革热1～4血清型病毒、流行性乙型脑炎、人呼吸道合胞病毒、轮状病毒、星状病毒、腺病毒无交叉反应, 特异性良好;将4份核酸含量不同的标准样品重复检测5次, 平均Ct值变异系数范围为1.67%～3.68%, 稳定性较高。通过监测, 龙岗区蚊虫携带NDiV概率为18.00%。结论 NDiV的TaqMan-MGB实时荧光PCR方法是一种快速、特异、灵敏、稳定性好的方法, 可应用于流行病学环境监测, 提高病毒的快速检测能力。

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