中国兽医科学2017，Vol.47Issue(5)：557-562,6.DOI:10.16656/j.issn.1673-4696.2017.05.004
猪Delta冠状病毒和猪流行性腹泻病毒双重RT-PCR诊断方法的建立及初步应用
Establishment and preliminary application of a duplex reverse transcription-PCR for the detection of porcine deltacoronavirus and porcine epidemic diarrhea virus
摘要
Abstract
Since the first report of porcine deltacoronavirus(PDCoV) in USA in 2014,it has also been detected in some pig farms in China.Due to the clinical and pathological symptoms are very similar to that of porcine epidemic diarrhea(PED),it is difficult to distinguish them by clinical diagnosis.Moreover,the co-infection of PDCoVand porcine epidemic diarrhea virus(PEDV) is common clinically,which causes huge economic losses to the swine industry.So it has important significance to establish a rapid diagnostic method for the simultaneous detection of PDCoV and PEDV.According to the GenBank sequences of PDCoVand PEDV,two pairs of primers were designed to amplify the PDCoV N gene and PEDVM genefragments,respectively.By optimizing the PCR reaction conditions,aduplexRT-PCR detecting method was established for the simultaneous detection of PDCoV and PEDV,and then the specificity,sensitivity and repeatability of this method were analyzed.The results showed that the duplex RT-PCR method had a sensitivity of 3.14× 103 copies/μ L for PDCoV and 3.88 × 104 copies/μ L for PEDV,as well as a good specificity,which was only positive for PDCoVand PEDV,and negative for porcine transmissible gastroenteritis virus(TGEV),porcine bocavirus(PBoV),porcine reproductive and respiratory comprehensive syndrome virus(PRRSV) and porcine pseudorabies virus(PRV).Further more this duplexRT-PCR method was used to test 111 clinical samples that collected from the diarrheic pigs.PDCoV positive rate was 19.82％(22/111),PEDV positive rate was 24.32％ (27/111),and PDCoV and PEDVco-infection positive rate was 9.01％(10/111).Therefore,the duplex RT-PCR method in this experiment was established and can be used for quick and accurate diagnosis of PDCoV and PEDV infection clinically,especially for the PEDV and PDCoV mixed-infection,which provides a new technical method for the clinical identification of these two diseases.关键词
猪Delta冠状病毒(PDCoV)/猪流行性腹泻病毒(PEDV)/双重RT-PCRKey words
porcine deltacoronavirus(PDCoV)/porcine epidemic diarrhea virus(PEDV)/duplex RT-PCR分类
农业科技引用本文复制引用
韩丽,周雍,李炳晓,曹贝贝,梁青青,张利卫,胡慧..猪Delta冠状病毒和猪流行性腹泻病毒双重RT-PCR诊断方法的建立及初步应用[J].中国兽医科学,2017,47(5):557-562,6.基金项目
国家重点研发计划项目(2016YFD0500102) （2016YFD0500102）
河南省科技开放合作项目(152106000047) （152106000047）
