PDCoV和PEAV二重RT-PCR检测方法的建立及初步应用

猪德尔塔冠状病毒(PDCoV)和猪肠道α冠状病毒(PEAV)是两种新发现的猪传染性冠状病毒,临床上均以腹泻症状为主,症状相似,临床上难以快速诊断。为建立一种快速、准确区分PDCoV和PEAV的二重RT-PCR方法,本研究根据PDCoV和PEAV N基因序列,分别设计2对特异性引物,以阳性质粒为模板,对二重RT-PCR反应条件进行优化,并进行特异性、敏感性试验。结果显示,所建立的方法能够扩增出PDCoV和PEAV的特异性片段,大小分别为690 bp和208 bp,且对猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(PRV)和猪圆环病毒2型(PCV2)无扩增条带;对PDCoV和PEAV的最低检测量分别为5.13×102copies/μL、4.73×101copies/μL。对60份临床腹泻样本病料进行检测,结果PDCoV的检出率为21.6%,未检测出PEAV。本研究所建立的二重RT-PCR方法可用于临床流行病学监测,为快速诊断PDCoV和PEAV提供理论依据和技术支持。

Establishment and preliminary application of double RT-PCR for detection of PDCoV and PEAV

Porcine deltacoronavirus(PDCo V)and porcine enteric alphacoronavirus(PEAV)are two newly discovered porcine infectious coronaviruses.The clinical symptoms are similar to diarrhea symptoms,which is difficult to quickly diagnose.To establish a rapid and sensitive double RT-PCR method for distinguishing PDCo V and PEAV,two pairs of specific primers were designed based on PDCo V and PEAV N gene sequences,respectively.By optimizing the PCR reaction conditions,a double RT-PCR detection method was established for the simultaneous detection of them.The results showed that the method was able to amplify the specific fragment sizes of PDCo V and PEAV to 690 bp and 208 bp,respectively.PEDV,TGEV,PRV and PCV2 had no amplified bands.The minimum detection amounts for PDCo V and PEAV were 5.13×102 copies/μL and 4.73×101 copies/μL,respectively.Sixty cases of clinical diarrhea samples were tested,and the detection rate of PDCo V was 21.6%,PEAV was negative.The double RT-PCR detection method in this study can be used for clinical epidemiological monitoring.This study provides theoretical basis and technical support for the rapid diagnosis of PDCo V and PEAV.