犬瘟热病毒、犬细小病毒和犬冠状病毒多重PCR方法的建立及应用

Establishment and Application of A multiplex PCR Assay for Detection of CPV,CDV,CCoV-Ⅰand CCoV-Ⅱ

为建立一种特异、高通量同时检测犬细小病毒(CPV)、犬瘟热病毒(CDV)、Ⅰ型及Ⅱ型犬冠状病毒(CCoV)的多重PCR方法,根据GenBank相关病毒基因序列,选取CPV VP2基因、CDV H基因和CCoV M基因高度保守序列区域,设计引物分别用于扩增长度为573 bp(CPV)、410 bp(CDV)、289 bp(CCoV-Ⅰ)、105 bp(CCoV-Ⅱ)的4条特异性目的片段。经反应条件优化,利用建立的多重PCR进行特异性、敏感性及重复性试验。结果表明,在同一PCR反应体系中可以同时检测到以上4种病毒,而大肠埃希氏菌、沙门氏菌、多形拟杆菌、犬副流感病毒(CPIV)及犬腺病毒(CAV)均未检测到,CPV、CDV、CCoV-Ⅰ及CCoV-Ⅱ的最低检测限分别为1×10^(7)copies/μL、1×10^(3)copies/μL、1×10^(6)copies/μL和1×10^(4)copies/μL,与单项PCR敏感性差异较小;3个不同浓度模板于不同时间、不同PCR仪器扩增,结果重复稳定性良好;使用该方法对50份临床病例进行检测,CPV、CDV、CCoV-Ⅰ及CCoV-Ⅱ的感染阳性率分别为32%(16/50)、24%(12/50)、18%(9/50)、10%(5/50),发现有CPV+CCoV-Ⅰ、CCoV-Ⅰ+CCoV-Ⅱ及CDV+CCoV-Ⅰ+CCoV-Ⅱ混合感染病例。证明建立的多重PCR具有高效、敏感、特异性强、重复性好的特点,并能够实际应用于临床样品检测中,为今后犬重要疫病的流行病学调查及病原学研究提供帮助。

This study was aimed to establish a multiplex PCR assay to simultaneously and specifically detect canine parvovirus(CPV),canine distemper virus(CDV)and Canine coronavirus typeⅠandⅡ(CCoV-Ⅰand CCoV-Ⅱ).According to the gene sequences in GenBank,the highly conserved sequence regions of CPV VP2 gene,CDV H gene,CCoV M gene were selected to amplify specifically.Four pairs of primers were designed and amplified the fragments of 573 bp(CPV),410 bp(CDV),289 bp(CCoV-Ⅰ)and 105 bp(CCoV-Ⅱ)respectively.The specificity,sensitivity and repetition of the multiplex PCR were tested.The results indicated that the templates of CPV,CDV,CCoV-Ⅰand CCoV-Ⅱwere detected and other pathogens and negative controls had no amplification;The sensitivity of CPV,CDV,CCoV-Ⅰand CCoV-Ⅱwere 1×10^(7) copies/μL,1×10^(3) copies/μL,1×10^(6) copies/μL and 1×10^(4) copies/μL respectively,the sensitivity was not different from the single PCR.Among 50 clinical samples,CPV 16 positive samples(32%),CDV 12 positive samples(24%),CCoV-Ⅰ9 positive samples(18%)and CCoV-Ⅱ5 positive samples(10%)were detected,and the mixed infection cases CPV+CCoV-Ⅰ,CPV+CCoV-Ⅰand CDV+CCoV-Ⅰ+CCoV-Ⅱalso been detected.The results indicated that the multiplex PCR method established in this study had the advantages of high efficiency,sensitivity,specificity and repeatability,and can be applied to detect the clinical samples,which can provide help for the epidemiological investigation and etiological research of canine viruses in the future.