作　　者： (孙文超）; (韩继成）; (解长占）; (张萍）; (张金勇）; (曹亮）; (崔卓栋）; (靖杰）; (温树波）; (肖朋朋）; (南福龙）; (张赫）; (庄忻雨）; (鲁会军）; (金宁一）;

机构地区： 广西大学动物科技学院,广西南宁530004 军事医学科学院军事兽医研究所,吉林长春130122 温州大学病毒学研究所,浙江温州325035

出　　处： 《中国预防兽医学报》 2017年第8期641-644,共4页

摘　　要： 为建立猪细小病毒7型(PPV7)的快速定量检测方法,本研究根据PPV7(KU563733.1)基因序列设计一对特异性引物,扩增155 bp的PPV7衣壳蛋白基因,构建重组质粒pClon007-PPV7,以其作为标准品建立了PPV7的SYBR GteenⅠ荧光定量PCR检测方法。结果显示,该方法在5.00×10~9拷贝/μL^5.00×10~3拷贝/μL范围内呈良好的线性关系。该方法对猪圆环病毒2型、猪繁殖与呼吸综合征病毒、伪狂犬病毒、乙脑病毒、口蹄疫病毒常见病原的检测均无特异性扩增,仅PPV7出现特异性扩增;该方法检测灵敏度可达5.00×10~1拷贝/μL;批内和批间变异系数均低于1%,具有较高的重复性和稳定性。对临床样品的检测结果显示,所建立的荧光定量PCR阳性检测率为71.88%,比普通PCR法检测的阳性率提高了约4.55%。该研究方法的建立为PPV7提供了一种新的快速定量检测技术。 A SYBR Green I real-time PCR method for porcine parvovirus 7（PPV7） was established using primers derived from the published sequences.A plasmid containing PPV7 capsid gene was constructed and served as a standard template to generate the standard curve.The standard curve produced a good linear relationship between Ct value and concentrations of the standard template at a range of 5.00×10~9 copies/μL to 5.00×10~3 copies/μL.The specificity assay showed no amplification of PRRSV,PCV2,JEV,PRV and FMDV except PPV7.The sensitivity of this method was 5.00×10~1 copies/μL.The reproducibility was high,and the variation coefficients of intra-assay and inter-assay were less than 1%.The positive rate was 71.88% of samples collected from Guangxi province decected by this method,which was 4.55%higher than the conventional PCR assay.This study could provide a new rapid quantitative method to detect PPV7.

关 键 词： 猪细小病毒 型 荧光定量 检测方法