新发猪Delta冠状病毒和猪流行性腹泻病毒SYBR GreenⅠ双重荧光RT-PCR检测方法的建立及应用

Establishment and application of a duplex SYBR Green Ⅰ real-time RT-PCR assay for simultaneous detection of emerging PDCoV and PEDV

猪Delta冠状病毒（porcine delta coronavirus，PDCoV）是一种新发现的仔猪腹泻类冠状病毒，感染仔猪主要表现出水样腹泻、呕吐和脱水等临床症状，和猪流行性腹泻（porcine epidemic diarrhea，PED）临床症状极其类似，且I临床上混合感染病例较多，单靠临床症状和剖检变化很难辨别这2种疾病，因此建立同时检测且能区分PDCoV和PEDV的检测方法具有重要意义。参照GenBank登录的PDCoVM基因和PEDVORF3基因特异性序列设计引物，扩增相应的基因片段，克隆构建重组质粒pMD-18T—PDCovM与pMD-18T—PEDVORF8，测序验证后，分别以此重组质粒为标准品，建立了能同时检测PDCoV和PEDV的SYBRGreenI双重荧光RTPCR方法，并对其扩增体系和扩增条件等进行了优化，同时对所建立的双重荧光RT—PCR方法的特异性、灵敏性和重复性进行了检测。结果显示，该试验成功建立了能同时检测PDCov和PEDv的SYBRGreenI双重荧光RTPCR方法，最低能检测到PDCoV和PEDV分别是28．6拷贝/μL和99．6拷贝/μL的质粒浓度，显示出较好的敏感性；在同样扩增条件下该方法具有较好的重复性；特异性试验表明，该方法仅能检测出PEDV和PDCoV，而对猪传染性胃肠炎病毒（TGEV）、猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）和猪伪狂犬病毒（PRV）等均不产生特异性扩增曲线。将所建立的双重荧光RTPCR方法以及单重荧光定量RT—PCR方法分别对198份临床收集的仔猪腹泻样品进行PEDV和PD—CoV检测，结果显示，PDCoV阳性样品41份，阳性率为20．7％；PEDV阳性样品59份，阳性率为29．8％；同时感染这2种病毒的样品17份，阳性率为8．6％。与单重荧光定量RT—PCR方法对PEDV和PDCoV的检出率一样，符合率100．0％。本研究成功建立了PDCoV和PEDV的双重荧光RT—PCR方法，能同时准确地检测PDCoV和PEDV，这为PDCoV和PEDV的临床鉴别诊断和流行病学调查研究奠定基础。

Porcine delta coronavirus（PDCoV） was a novel coronavirus associated with swine intesti- nal diarrhea disease. It was difficult to distinguish clinically the infection of PDCoV from porcine epidemic diarrhea virus（PEDV） because they had similar clinical symptoms with watery diarrhea, vomiting, and dehydration in infected piglets and a high rate of PDCoV and PEDV co-infection ex-according to the sequences of PDCoV and PEDV in GenBank. Plasmids containing the M gene of PDCoV and the ORF3 gene of PEDV were respectively used to establish melting and standard curves of the duplex SYBR Green I real-time RT PCR. By optimizing the conditions, we estab- lished a duplex RT-PCR with high sensitivity and specificity for PDCoV and PEDV,limited detec- tion was 28.6 copies/μL and 99.6 copies/μL respectively, and no non-specific amplifications for TGEV,PRRSV,PCV2, PRV was found. 198 clinical samples collected from the clinical diarrhea pigs were tested by the duplex or single SYBR Green I real-time RT-PCR assays. The results showed that the positive rates of PDCoV and PEDV were 20.71%（41/198） and 29.8%（59/198） respectively,and co-infection of PDCoV and PEDV was 8. 6% （17/198）. The above data showed that the duplex SYBR Green I RT-PCR method established in this study could be used for the de- tection of PDCoV and PEDV,which lay a foundation for further clinical differential diagnosis and epidemiological investigation of PEDV and PDCoV.