猪细小病毒实时荧光定量LAMP检测方法的建立与初步应用  

Development and preliminary application of real-time fluorescent quantitative loop-mediated isothermal amplification method for porcine parvovirus

作　　者：曹志 郑佳 尹德华 杨瑞梅[1] 黄娟[1] 单虎[1] CAO Zhi;ZHENG Jia;YIN De-hua;YANG Rui-mei;HUANG Juan;SHAN Hu(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266109

出　　处：《中国兽医科学》2022年第10期1238-1244,共7页Chinese Veterinary Science

基　　金：山东省生猪产业技术体系项目(SDAIT-08-07);青岛农业大学高层次人才启动基金项目(663-1120018)

摘　　要：为了建立猪细小病毒(PPV)核酸环介导等温扩增(LAMP)定量检测方法,选择PPVNS1基因序列为靶序列,设计3对特异性引物,通过优化反应体系和反应条件,筛选用于提高反应体系稳定性的化学制剂,建立了一种PPV的实时荧光定量LAMP(qLAMP)检测方法。结果显示,添加Eva Green的反应体系的扩增效果明显好于SYBR GreenⅠ和钙黄绿素;体系中加入的10 g/L聚丙烯酰胺使质粒标准品在1.39×10^(5)~1.39×10^(1)copies/μL和C_(t)值之间的线性关系良好,标准曲线方程为Y=-2.9435X+44.354,相关系数R^(2)为0.9849;该方法不与其他病毒的核酸样品发生交叉反应,检测灵敏度达13.9 copies/μL,变异系数≤1.08%;对200份流产猪胎儿的样品进行检测,阳性率为18%,与实时定量PCR的检测结果一致。本试验中建立的qLAMP检测方法为PPV的临床快速检测提供了新方法。To develop loop-mediated isothermal amplification(LAMP)assays for quantitative detection of porcine parvovirus(PPV),a real-time fluorescence quantitative LAMP(q LAMP)method for PPVdetection was established.Three pairs of specific primers were designed based on the nucleotide sequence of the PPV NS1gene,reaction system was optimized and program were determined,while special reagents were also applied to stabilize the LAMP detection system.Results showed that the amplification efficiency of Eva Green was significantly higher than that of SYBR Green I and Calcein.Moreover,under the treatment of stabilizing reagent polyacrylamide(10 g/L),t he t hreshold cycle(C_(t))values and copy numbers of t he st andard recombinant plasmid(over the range of 1.39×10^(5)copies/μL—1.39×10^(1)copies/μL)displayed a good liner relationship with a correlation coefficient(R^(2))of 0.9849 and the equation Y=-2.9435X+44.354.The established method was less likely to cross-react with other viruses,with a detection sensitivity of 13.9 copies/μL and a coefficient of variation less than 1.08%.Furthermore,the established qLAMP was used to detect PPV in 200 clinical samples of aborted pig fetuses,detected a positive rate of 18%,which was consistent with the result from a normal qPCR.In conclusion,the qLAMP established in this experiment provides a new method for rapid quantitative detection of PPV.

关 键 词：猪细小病毒 环介导等温扩增 实时荧光 定量检测 

分 类 号：S852.65[农业科学—基础兽医学]