猪细小病毒7型SYBR Green Ⅰ荧光定量PCR检测方法的建立  

Development of a SYBR Green Ⅰ real-time PCR method for detection of PPV7

作　　者：孙文超[1,2,3] 韩继成[2] 解长占 张萍[2,4] 张金勇[2,4] 曹亮[2,4] 崔卓栋 靖杰[2] 温树波[2] 肖朋朋[2] 南福龙 张赫[2] 庄忻雨 鲁会军[2] 金宁一[2] 

机构地区：[1]广西大学动物科技学院,广西南宁530004 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]温州大学病毒学研究所,浙江温州325035 [4]吉林农业大学动物科技学院,吉林长春130118 [5]吉林大学动物科技学院,吉林长春130062

出　　处：《中国预防兽医学报》2017年第8期641-644,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家高技术研究与中国发展计划(863计划)(2011AA10A208)

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7型 荧光定量PCR 检测方法 

分 类 号：S852.65[农业科学—基础兽医学]