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

猪丁型冠状病毒(PDCoV)与猪流行性腹泻病毒(PEDV)均为致病性冠状病毒,可以引起猪呕吐、腹泻脱水和新生仔猪死亡。为了建立一种快速检测PDCoV和PEDV的双重SYBR Green I荧光定量RT-PCR方法,根据GenBank登录的2种病毒基因的保守序列,设计了两对特异性引物,分别扩增其N、M基因保守区,并将其克隆至pMD19-T载体;将10倍梯度稀释的混合重组质粒作为标准品模板,建立了一种检测PDCoV和PEDV的双重的SYBR Green I荧光定量RT-PCR方法,并对其反应的敏感度、特异性和重复性进行了检测。结果表明,本试验所建立的双重荧光定量RT-PCR检测方法对PDCoV和PEDV的最低检测量分别为51和32拷贝·μL-1,且与PBoV、TGEV、PRV、PCV2无交叉反应,特异性较好;对2016年至2017年在河北省收集的130份仔猪腹泻样本检测,结果显示,PDCoV的检出率为16.9%,PEDV的检出率为66.2%,二者同时感染的检出率为2.3%,与普通RT-PCR检测方法相比,敏感性更高。本研究为PDCoV和PEDV的诊断及分子流行病学调查提供了一种快速、定量检测方法。

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 andPEDV,respectively.No cross-reaction was detected to PBoV,TGEV,PRV and PCV2.The RTqPCR assay was applied to the detection of 130 clinical samples collected from Hebei province during2016-2017.PDCoV positive rate was 16.9%,PEDV positive rate was 66.2%,and co-infection was2.3%.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.