犬细小病毒、犬冠状病毒和犬轮状病毒三重PCR方法的建立及初步应用  

Establishment and application of triple PCR method for CPV,CCoV and CRV

作　　者：沙聪 唐川杰 何雨薇 李妍 黄坚 SHA Cong;TANG Chuan-jie;HE Yu-wei;LI Yan;HUANG Jian(School of Animal and Veterinary Sciences,Southwest Minzu University,Chengdu 610041,China;Chengdu Minda Animal Hospital,Chengdu 610041,China)

机构地区：[1]西南民族大学畜牧兽医学院,四川成都610041 [2]成都民大动物医院,四川成都610041

出　　处：《西南民族大学学报（自然科学版）》2023年第2期142-148,共7页Journal of Southwest Minzu University(Natural Science Edition)

基　　金：四川省科技厅项目(2019YFN0133);西南民族大学中央高校基本科研业务费专项(ZYN2023046)。

摘　　要：犬细小病毒(Canine parvovirus, CPV)、犬冠状病毒(Canine coronavirus, CCoV)和犬轮状病毒(Canine rotavirus, CRV)是引起犬腹泻的主要病原.3种病原引发的疾病临床症状相似,难以诊断,因此需要建立可以高效鉴别这3种病原的分子检测方法.通过对引物浓度、退火温度的条件优化,建立了可同时检测CPV、CCoV和CRV 3种病毒的三重PCR方法,并进行特异性、敏感性和重复性试验.结果显示,该方法可特异性扩增CPV VP2基因(253 bp)、CCoV ORF-1b基因(379 bp)和CRV VP6基因(852 bp)的目的片段,未检出其他相关病原;对CPV、CCoV和CRV的最低检测浓度分别为6.44×10^(-1)pg/μL、8.72×10^(-1)pg/μL和8.35×10^(-1)pg/μL,方法的灵敏度高;重复性试验显示该方法具有良好的稳定性.应用建立的三重PCR方法对2019~2020年成都市区采集的135份犬腹泻粪便样本进行了检测,并与单重PCR检测结果相比较.结果显示CPV、CCoV和CRV的检出率分别为16.30%、20.74%和4.44%;总感染率为51.11%(65/135)),其中多病原混合感染率为20.00%(13/65),检测结果与单重PCR相符.建立的三重PCR方法,可以用于相关病原的临床检测和流行病学调查.Canine parvovirus(CPV),canine coronavirus(CCoV)and canine rotavirus(CRV)are the three main causative vi⁃ruses of diarrhea in dogs with similar clinical symptoms;thereby it is necessary to establish a high effective molecular detection method for differentiating the above pathogens.By optimizing the primer concentration and annealing temperature,a triple PCR method was established for simultaneous detection of CPV,CCoV and CRV,and then the specificity,sensitivity and repeatability of the method were tested.The results showed that the target fragments of CPV VP2 gene(253 bp),CCoV ORF⁃1b gene(379 bp)and CRV VP6 gene(852 bp)could be accurately amplified by the triple PCR method with high specificity,the detection limits of CPV,CCOV and CRV were 6.44×10^(-1)pg/μL,8.72×10^(-1)pg/μL and 8.35×10^(-1)pg/μL respectively with high sensi⁃tivity,and the method had good stability.Using this triple PCR method,135 canine diarrhea fecal samples collected in Chengdu region from 2019 to 2020 were detected,and compared with those of single PCR method.The detection rates of CPV,CCoV and CRV were 16.30%,20.74%and 4.44%,respectively,and the total infection rate was 51.11%(65/135)with 20.00%(13/65)co⁃infection rate.The detection results were consistent with three single PCR methods.In conclusion,CPV/CCoV/CRV tri⁃ple PCR method successfully established in this paper can be applied as an effective molecular method to detection of related pathogens and to the epidemiological investigation.

关 键 词：犬细小病毒(CPV) 犬冠状病毒(CCoV) 犬轮状病毒(CRV) 三重PCR 腹泻 

分 类 号：S829.2[农业科学—畜牧学] S852.65[农业科学—畜牧兽医]