尼帕病毒N基因逆转录微滴式数字PCR检测方法建立  

Establishment of a Reverse Transcription Droplet Digital PCR Detection Method for the Nipah Virus N Gene

作　　者：王世伟 刘晶晶[3] 徐东甫 牛春艳[2] WANG Shiwei;LIU Jingjing;XU Dongfu;NIU Chunyan(Jilin Institute of Chemical Technology,School of Information and Control Engineering,Jilin 132022,China;National Institute of Metrology,Beijing 100029,China;Northeast Electric Power University,Faculty of Automation Engineering,Jilin 132011,China)

机构地区：[1]吉林化工学院信息与控制工程学院,吉林132022 [2]中国计量科学研究院,北京100029 [3]东北电力大学自动化工程学院,吉林132011

出　　处：《计量科学与技术》2025年第1期62-68,共7页Metrology Science and Technology

基　　金：国家重点研发计划(2023YFF0724501)。

摘　　要：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基因的保守序列为靶基因构建重组质粒,利用该质粒作为模板得到体外转录RNA,同时设计并合成五套特异性引物探针进行数字PCR扩增。通过对比不同引物探针,并优化反应条件,初步建立了能够检测NIV的RT-ddPCR方法。随后对方法的线性和重复性进行考察,并利用该方法在不同平台进行比较,同时与两步法进行了比较,并评估了所建立方法的检测特异性。最后利用建立的方法对体外转录RNA标准物质候选物进行了定量测量。结果显示,所建立的方法具有良好的特异性和重复性,RT-ddPCR方法与重量法之间的相关系数为0.999 9,在不同平台得到的定量结果并无系统误差,一步法定量结果优于两步法。建立的RT-ddPCR方法特异性强、重复性好,能够用于NIV标准物质定值,为NIV定量检测提供支持。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.

关 键 词：计量学 尼帕病毒 数字PCR 核酸检测 标准物质 

分 类 号：TB99[一般工业技术—计量学]