犬细小病毒、犬腺病毒Ⅰ型和犬腺病毒Ⅱ型三联PCR诊断方法的建立  

Establishment of triple PCR diagnostic techniques for canine parvovirus(CPV),canine adenovirus typeⅠ(CAV-Ⅰ)and typeⅡ(CAV-Ⅱ)

作　　者：于永乐 王吉贵[2] 苏俊 杨双双 刘莹 李沛然 李兆达 刘维全[2] YU Yongle;WANG Jigui;SU Jun;YANG Shuangshuang;LIU Ying;LI Peiran;LI Zhaoda;LIU Weiquan(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao,Shandong 266109,China;State Key Laboratory of Agrobiotechnology,College of Biological Sciences,China Agricultural University,Beijing 100193,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266109 [2]中国农业大学,生物学院,农业生物技术国家重点实验室,北京100193

出　　处：《中国兽医学报》2021年第12期2340-2344,共5页Chinese Journal of Veterinary Science

基　　金：“十三五”国家重点研发计划资助项目(2016YFD0501001);青岛农业大学高层次人才科研基金资助项目(1120015/663)。

摘　　要：旨在建立可同时检测犬细小病毒(canine parvovirus,CPV)、犬腺病毒I型(canine adenovirus typeⅠ,CAV-Ⅰ)和犬腺病毒Ⅱ型(canine adenovirus typeⅡ,CAV-Ⅱ)的PCR诊断方法,分别将3种病毒基因组序列进行同源性比对,选择高同源区段,应用Primer Primier 5.0计算机软件设计并合成了2对特异性扩增引物,目的片段大小分别为670 bp(CPV)、497 bp(CAV-Ⅰ)和1019 bp(CAV-Ⅱ)。分别提取CPV、CAV-Ⅰ和CAV-Ⅱ细胞培养物的DNA,进行三联PCR扩增试验。结果表明,所建立的单项PCR及二联PCR扩增良好,其产物经测序比对与引物扩增序列完全一致。在此基础上,优化三联PCR扩增试验条件,成功建立了可同时检测CPV、CAV-Ⅰ和CAV-Ⅱ的三联PCR诊断方法。并对其特异性和灵敏度进行检测,三联PCR最低检测限度为0.16 pg总DNA,而对犬副流感病毒和犬瘟热病毒的PCR检测结果均为阴性。对送检的20份病料进行检测,结果显示15份为CPV阳性,5份为CAV-Ⅰ阳性,1份为CAV-Ⅱ阳性。The aim of this study is to establish the triple PCR diagnostic methods for canine parvovirus(CPV),canine adenovirus typeⅠ(CAV-Ⅰ)and canine adenovirus typeⅡ(CAV-Ⅱ).The published gene sequences in GenBank,was analyzed and compared with each other,respectively.According to the conservative sequences of VP2 gene for CPV,E3 gene for CAV-Ⅰand CAV-Ⅱ,two pairs of specific primers were designed with the Primer Primier 5.0 software for PCR amplification.The results showed that three specific fragments of the two viruses were simultaneously amplified and the length of amplified fragments were as follows:670 bp for CPV,497 bp for CAV-Ⅰand 1019 bp for CAV-Ⅱ.The sensitivity and specificity tests showed that the assay could detect at least 0.16 pg of target DNA,and there was no cross-reactions with those clinically relevant pathogens,i.e.,canine distemper virus and canine parainfluenza virus.The detection results for 20 clinical samples with the triple PCR showed that 15 samples were positive for CPV,5 for CAV-Ⅰand 1 for CAV-Ⅱ.

关 键 词：犬细小病毒 犬腺病毒Ⅰ型 犬腺病毒Ⅱ型 三联PCR 

分 类 号：S855.3[农业科学—临床兽医学]