畜牧兽医学报2024，Vol.55Issue(1)：413-418,6.DOI:10.11843/j.issn.0366-6964.2024.01.040
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
摘要
Abstract
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 × 101 copies·μL-1);The linearity was good(R2＞0.99)at template concentrations of 1.0×100 to 1.0 × 104 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 100％.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.关键词
猪流行性腹泻病毒/微滴式/数字PCR/方法/建立Key words
porcine epidemic diarrhea virus/droplet/digital PCR/method/establishment分类
农业科技引用本文复制引用
周建浩,康台生,胡煜锋,李博文,闫若潜,王东方,刘影,王淑娟,马震原,谢彩华,赵雪丽,杨海波,冯桂丹..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[J].畜牧兽医学报,2024,55(1):413-418,6.基金项目
河南省重大科技专项课题(221100110600) （221100110600）
河南省重点研发专项(231111111300) （231111111300）
河南省现代农业产业技术体系(HARS-22-12-T) （HARS-22-12-T）
河南省重点研发与推广专项(232102111053) （232102111053）
