犬细小病毒高效纳米PCR检测方法的建立及初步应用  

Establishment and Application of Nano PCR Assay for Detection of the Canine Parvovirus

作　　者：秦彤[1,2] 周灵 由欣月[1,2] 梁琳 史利军 张建伟[3] 李金祥 崔尚金[1,2] QIN Tong;ZHOU Ling;YOU Xinyue;LIANG Lin;SHI Lijun;ZHANG Jianwei;LI Jinxiang;CUI Shangjin(Institute of Animal Sciences (IAS),Chinese Academy of Agricultural Sciences (CAAS),Beijing 100193,China;Scientific Observing and Experiment Station of Veterinary Drugs and Diagnostic Technology of Ministry of Agriculture,Beijing 100193,China;Beijing Animal Husband Service,Beijing 100107,China;Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]农业部兽用药物与诊断技术北京科学观测实验站,北京100193 [3]北京市畜牧总站,北京100107 [4]中国农业科学院,北京100081

出　　处：《畜牧兽医学报》2019年第6期1268-1274,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：中国农业科学院创新工程项目(ASTIP-IAS15);中国农业科学院基本科研业务费(2018-YWF-YTS-16);“十三五”国家重点研发计划(2016YFD0501003;2017YFD0501603)

摘　　要：为建立灵敏、快速的犬细小病毒(CPV)检测方法,本研究针对CPVVP2基因保守序列设计一对能扩增574bp片段的特异性引物,对纳米PCR的退火温度、引物浓度等反应条件进行了优化,并对其特异性和灵敏度进行了评估。结果表明,所建立的纳米PCR特异性和灵敏性良好,最低核酸检出量为2拷贝·μL^-1,其敏感性比常规PCR高1000倍。分别采用纳米PCR和常规PCR,对广西、北京、吉林送检的75份疑似CPV的临床样品进行检测,CPV阳性率分别为89.3%(67/75)和70.7%(53/75),表明该方法具有更高的敏感性,适用于CPV低含量临床样品的检测。本方法的建立为CPV感染的早期快速、灵敏、准确的诊断提供了新方法,为CPV的防控奠定基础,具有重大的临床应用意义和价值。In order to establish a rapid and sensitive nanoparticle-assisted PCR (nano PCR) assay for detection of canine parvovirus (CPV),a pair of primers was designed based on the conserved region of the VP2 gene to amplify 574 bp fragment. The annealing temperature,primer concentration and other reaction conditions of nano PCR were optimized,and its specificity and sensitivity were evaluated. The results showed that the established nano PCR had good specificity and sensitivity,with a minimum nucleic acid detection amount of 2 copies·μL^-1 ,and its sensitivity was 1 000 times higher than that of conventional PCR. Seventy-five clinical samples collected from Beijing,Guangxi and Jilin were tested,the positive rate of CPV was 89.3%(67/75) and 70.7%(53/75) by nano PCR and conventional PCR,respectively. The results showed that the method had higher sensitivity and was suitable for the detection of low amount of CPV in clinical samples. The established nano PCR in this study provided a new method for rapid,sensitive and accurate diagnosis of CPV infection in the early stage,which laid a foundation for the prevention and control of CPV and had significant value in clinical application.

关 键 词：犬细小病毒 VP2基因 纳米PCR 临床检测 

分 类 号：S852.659.2[农业科学—基础兽医学]