猪流行性腹泻病毒微滴式数字PCR定量检测方法的建立及初步应用

Establishment and Preliminary Application of a Quantitative Droplet Digital PCR Assay for Porcine Epidemic Diarrhea Virus

旨在建立一种敏感性高、特异性强、重复性好的猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)微滴式数字PCR(droplet digital PCR,ddPCR)定量检测方法。本研究根据GenBank登录的PEDV(MK862249.1)M基因序列保守区设计合成特异性引物对和探针;通过对反应体系和反应条件的优化,成功建立了可用于检测PEDV的荧光定量PCR(fluorescent quantitative real-time PCR,FQ-PCR)方法,将自行建立的FQ-PCR方法的引物对/探针用来确定ddPCR方法;对ddPCR方法进行敏感性、特异性、重复性和初步应用试验。结果显示:自行建立的FQ-PCR方法在敏感性、重复性等方面均优于行标FQ-PCR方法;根据自行设计的FQ-PCR方法建立的ddPCR方法最低检测极限为0.15 copies·μL^(-1),敏感性高于行业标准(SN/T 1699—2017)FQ-PCR方法(1.0×10^(1)copies·μL^(-1));模板浓度为1.0×10^(0)~1.0×10^(4)copies·μL^(-1)时,具有良好的线性关系(R 2>0.99);批内、批间变异系数(CV%)为1.52%~7.40%;对猪丁型冠状病毒、猪伪狂犬病毒等11种对照病毒核酸检测结果均为阴性;分别用建立的ddPCR方法、行业标准FQ-PCR方法对150份临床样品进行PEDV核酸检测,ddPCR方法对行业标准FQ-PCR方法检测阳性样品的检测符合率为10^(0)%。本研究成功建立的PEDV ddPCR检测方法可用于临床上PEDV感染的早期检测和定量检测,为PEDV核酸标准物质的研制提供了定量手段。

The aim of this study was to develop a highly sensitive,specific and reproducible method for the quantitative detection of porcine epidemic diarrhea virus(PEDV)by droplet digital PCR(ddPCR).In this study,we designed and synthesized specific primer pairs and probes based on the conserved region of the M gene sequence of PEDV(MK862249.1)registered in GenBank,and successfully established a fluorescent quantitative real-time PCR(FQ-PCR)for the detection of PEDV by optimizing the reaction system and reaction conditions.By optimizing the reaction system and reaction conditions,a fluorescent quantitative real-time PCR(FQ-PCR)method for PEDV detection was successfully established,and the primer pairs/probes of the self-established FQ-PCR method were used to determine the ddPCR method;sensitivity,specificity,reproducibility and preliminary application tests of the ddPCR method were performed.The results showed that the self-established FQ-PCR method outperformed the industrial standard FQ-PCR method(SN/T 1699—2017)in terms of sensitivity and reproducibility;the lowest detection limit of the ddPCR method based on the self-designed FQ-PCR method was 0.15 copies·μL^(-1),which was more sensitive than the industrial standard FQ-PCR(1.0×10^(1)copies·μL^(-1));The linearity was good(R 2>0.99)at template concentrations of 1.0×10^(0)to 1.0×10^(4)copies·μL^(-1).The intra-and inter-batch coefficients of variation(CV%)ranged from 1.52%to 7.40%.The results were negative for 11 control viruses including porcine delta coronavirus and porcine pseudorabies virus.The PEDV nucleic acid test was performed on 150 clinical samples by the established ddPCR and FQ-PCR methods,and the coincidence rate of the ddPCR method with the FQ-PCR method in detecting the positive samples was 10^(0)%.The PEDV ddPCR assay successfully established in this study can be used for early detection and quantitative detection of PEDV infection in clinical settings,and provides a quantitative means for the development of PEDV nucleic acid standards.