猪萨佩罗病毒TaqMan实时荧光定量PCR检测方法的建立

Establishment of a TaqMan fluorescent real-time quantitative PCR for detection of porcine sapelovirus

【目的】建立快速、特异、灵敏的猪萨佩罗病毒(porcine sapelovirus,PSV)TaqMan实时荧光定量(RT-qPCR)检测方法,为临床快速检测PSV提供新方法。【方法】参照GenBank中PSV的全基因序列,针对PSV 5′端保守区设计1对特异性引物,PCR扩增目的片段,将目的片段克隆至pMD-18T载体,构建阳性重组质粒,并以阳性质粒标准品为模板,建立标准曲线。在此基础上,设计特异性引物与探针,进行反应体系和反应条件优化,建立PSV TaqMan RT-qPCR检测方法,并对方法的敏感性、特异性和重复性进行验证。应用建立的方法对2018-2019年在河南不同地区猪场收集的83份样品(粪便样品39份,肠道样品44份)进行检测。【结果】建立了PSV TaqMan RT-qPCR检测方法,该方法的灵敏度为3.88×10^(1)拷贝/μL;与猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪δ冠状病毒和猪繁殖与呼吸综合征病毒均无交叉反应,特异性良好;批内与批间变异系数均小于1.0%,重复性较好。临床样品检测结果表明,TaqMan RT-qPCR检测PSV呈阳性的样品有31份(22份粪便样品,9份肠道样品)检出率为37.3%(31/83),显著高于普通PCR检测结果(检出率12.0%(10/83))。【结论】建立了TaqMan RT-qPCR检测方法,该方法灵敏度高,特异性好。

【Objective】This study established a rapid,efficient and stable TaqMan fluorescent real-time quantitative PCR(RT-qPCR)method for the detection of the porcine sapelovirus(PSV),which provide a new method for clinically rapid detection of PSV.【Method】A pair of specific primers were designed by selecting conservative region of 5′UTR based on complete sequences of PSV from GenBank.The target fragment was amplified by PCR and cloned into pMD-18T vector to construct a positive standard plasmid.Taking the positive plasmid as the template,the standard curve was established.Another pair of primers and probe were designed,and reaction system and conditions were optimized.The sensitivity,specificity and reproducibility of the assay were verified.This method was used to test 83 samples(39 feces samples and 44 intestinal samples)collected from swine farms in different regions of Henan from 2018 to 2019.【Result】The TaqMan RT-qPCR method for detection of PSV was established with sensitivity of 3.88×10^(1) copies/μL.There was no cross-reaction with porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),porcine delta coronavirus(PDCoV)and porcine reproductive and respiratory syndrome virus(PRRSV),indicating this method had good specificity.The coefficients of variation(CV)of intra-assay and inter-assay were less than 1.0%,which indicated that this method had good repeatability.The results of clinical samples showed that 31 samples(22 feces samples and 9 intestinal samples)were positive for PSV by TaqMan RT-qPCR with detection rate of 37.3%(31/83),which was significantly higher than 12.0%(10/83)by conventional PCR.【Conclusion】The TaqMan RT-qPCR assay established in this study had high sensitivity and specificity.