中文摘要：

为建立猪萨佩罗病毒（PSV）的检测方法,根据GenBank中PSV的5′端非编码区基因的保守序列设计并合成1对针对PSV的特异性引物,建立了基于SYBR GreenⅡ检测PSV的实时荧光定量RT-PCR方法。结果显示,该方法能很好地将PSV与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪嵴病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒、A群猪轮状病毒区分开,特异性强。检测下限为6.37×10^2 copies/μL,比普通PCR敏感100倍,敏感性高。扩增产物的熔解曲线分析只有1个单特异峰。所建立的实时荧光定量RTPCR的批内变异系数为0.44%～1.14%,批间变异系数为0.70%～1.32%,重复性较好。应用该方法对采集自四川省部分地区的58份有腹泻症状的猪粪便进行检测,实时荧光定量RT-PCR检测阳性率为56.9%,与常规PCR检测方法的阳性符合率为100%。结果表明,本研究建立的实时荧光定量RT-PCR特异性较强、灵敏度高,可用于该病毒的临床检测、定量分析及流行病学调查。

英文摘要：

A TaqMan real-time RT-PCR assay was established for the detection of porcine sapelovirus.A pair of special primers was designed according to the highly conserved fragment of the 5′-UTR of gene in GenBank and a real-time reverse transcription polymerase chain reaction（RRT-PCR）based on SYBR GreenⅡ was developed for quantization of porcine sapelovirus.The results indicated that no amplification was detected from other DNA samples by this method,such as CSFV,PRRSV,PKoV,PEDV,TGEV and ProV A,respectively.The detection limit of the method was 6.37×10^2 copies/μL of initial templates,which was100-fold more sensitive than that of the conventional PCR.And the melting curve analysis using SYBR GreenⅡ dye showed one specific peak with no primer-dimer peak.Excellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0.44% to 1.14% and inter-assay of0.70%to 1.32%.A total of 58 clinical specimens collected from Sichuan Province were detected by the established RT-PCR and the positive rate is 56.9%（33/58）.The results of the quantitative RT-PCR were the same as that of the conventional RT-PCR.The results proved that the RRT-PCR was specific and sensitive to detect porcine sapelovirus,which can support the clinic detection,quantitative analysis and molecular epidemiological investigation.