尼帕病毒 N 基因逆转录微滴式数字 PCR 检测方法建立

王世伟 ¹，刘晶晶 ²，徐东甫 ²，牛春艳 ³

Abstract

为建立尼帕病毒 (NIV) 逆转录微滴式数字 PCR (RT-ddPCR) 检测方法，以 NIV-N 基因保守序列为靶基因构建重组质粒，以此质粒为模板获取体外转录 RNA，设计合成 5 套特异性引物探针开展数字 PCR 扩增。比对筛选引物探针并优化反应条件，初步构建尼帕病毒 RT-ddPCR 检测体系。考察方法线性范围与重复性，开展不同检测平台、一步法与两步法比对试验，评价检测特异性，最终对体外转录 RNA 标准物质候选样品定量测定。结果显示，该方法特异性与重复性良好，RT-ddPCR 方法与重量法相关系数为0.9999；不同平台定量结果无系统偏差，一步法检测效果优于两步法。该 RT-ddPCR 方法特异稳定，可用于尼帕病毒标准物质定值，为病毒定量检测提供技术支撑。

To establish a reverse transcription droplet digital PCR (RT-ddPCR) detection method for Nipah virus (NIV),
a recombinant plasmid was constructed using the conserved sequence of the NIV-N gene as the target gene. This plasmid served as a template to produce in vitro transcribed RNA. Five sets of specific primer-probe pairs were designed and synthesized for digital PCR amplification. By comparing different primer-probe sets and optimizing reaction conditions, a preliminary RT-ddPCR method capable of detecting NIV was established. Subsequently, the linearity and repeatability of the method were evaluated, and the method was compared across different platforms as well as with a two-step method to assess detection specificity. Finally, the established method was used to quantify candidate reference materials of in vitro transcribed RNA. The results demonstrated that the established method possesses excellent specificity and repeatability, with a correlation coefficient of 0.9999 between the RT-ddPCR method and the weight-based method. Quantitative results
obtained on different platforms showed no systematic errors, and the one-step method yielded superior quantitative results compared to the two-step method. The established RT-ddPCR method exhibits strong specificity and good repeatability, making it suitable for the quantification of NIV reference materials and providing robust support for the quantitative detection of NIV.