猪繁殖与呼吸综合征病毒PCR-DHPLC检测新技术的建立及应用

应用PCR结合变性高效液相色谱（DHPLC）技术检测猪繁殖与呼吸综合征病毒（PRRSV）,根据PRRSV GP5基因的序列特点设计特异性引物,PCR扩增产物经DHPLC技术进行快速检测。以猪细小病毒、猪圆环病毒Ⅱ型、猪流感病毒、猪瘟病毒和猪伪狂犬病毒进行特异性试验,无交叉反应,具有较好的特异性和重复性;对阳性标准品的检测结果表明,所建立的PCR-DHPLC法灵敏度可达1.0×101拷贝/μL。对16份疑似病料分别应用本试验所建立的PCR-DHPLC法与SYBR GreenⅠ实时荧光PCR法、PCR-凝胶电泳法、病毒培养法进行检测,发现有15份荧光定量PCR阳性,15份PCR-DHPLC阳性,12份PCR-凝胶电泳阳性。结果表明,建立的PCR-DHPLC法具有特异、敏感、快速、重复性好等优点,可用于临床PRRSV感染的早期诊断以及分子流行病学调查。

Development and application of PCR-DHPLC assays for detection of the porcine reproductive and respiratory syndrome virus

To identify the porcine reproductive and respiratory syndrome(PRRS),a PCR-DHPLC assay was performed in this study.Primers specific for the partial region of the PRRSV GP5 gene were selected to conduct the PCR-DHPLC assays.The specific testing was performed with PRRS and PPV,PCV-Ⅱ,SIV,CSFVand PRV,without cross reation,with good specification and nice repeatability.Sensitivity analysis showed that the developed PCR-DHPLC could detect 1.0×101 copy/μL.Then the established method was used to detect the clinical samples,15 positives could be observed by PCR-DHPLC for 16 suspicious positive samples,it is consistent with SYBR GreenⅠreal-time PCR test and 12 positive by normal RT-PCR.The method could be used in clinical diagnosis and epidemiological investigation.