畜牧兽医学报2018，Vol.49Issue(4)：852-858,7.DOI:10.11843/j.issn.0366-6964.2018.04.025
猪丁型冠状病毒与猪流行性腹泻病毒双重实时荧光定量RT-PCR方法的建立和初步应用
Establishment and Application of the Real-time Reverse Transcription Quantitative PCR Assay for Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus
摘要
Abstract
Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) are enterpathogenic coronavirus which cause acute diarrhea,vomiting,dehydration and mortality in neonatal piglets.In order to establish a real-time reverse transcription quantitative PCR (RT-qPCR) assay to detect PDCoV and PEDV,two pairs of specific primers were designed according to the conservative sequence of N gene (PDCoV) and M gene (PEDV) registered in GenBank.The genes of the conservative region were amplified and cloned into pMD19-T vector.Taking Mix 10 times the gradient dilution of recombinant plasmid as a standard template,a RT-qPCR to PDCoV and PEDV was established.The sensitivity,specificity and repeatability were tested.Results showed that the sensitivity of this RT-qPCR assay was 51 and 32 copies · μL 1 for PDCoV and PEDV,respectively.No cross-reaction was detected to PBoV,TGEV,PRV and PCV2.The RT-qPCR assay was applied to the detection of 130 clinical samples collected from Hebei province during 2016-2017.PDCoV positive rate was 16.9％,PEDV positive rate was 66.2％,and co-infection was 2.3 ％.The sensitivity of fluorescence quantitative PCR was significantly higher than that of ordinary RT-PCR.These results indicated that the detection assay could be applied for rapid and high through-put diagnosis of PDCoV and PEDV.关键词
猪丁型冠状病毒/猪流行性腹泻病毒/实时定量PCR/临床检测Key words
porcine deltacoronavirus/porcine epidemic diarrhea virus/real-time quantitative PCR/clinical detection分类
农业科技引用本文复制引用
罗尚星,范京惠,刘宝京,师乾凯,侯林杉,左玉柱..猪丁型冠状病毒与猪流行性腹泻病毒双重实时荧光定量RT-PCR方法的建立和初步应用[J].畜牧兽医学报,2018,49(4):852-858,7.基金项目
河北农业大学百名青年学术带头人培养计划(0318011) （0318011）
河北省研究生创新能力培养资助项目(CXZZSS2017068) （CXZZSS2017068）
河北省自然科学基金课题(C2015204121) （C2015204121）
