摘要
为建立检测多种犬病的多重PCR方法,本研究根据GenBank登录犬瘟热病毒(CDV)的N基因序列、犬细小病毒(CPV)的NS基因序列和犬腺病毒(CAV)的E3基因序列,设计合成3对特异性引物。通过优化反应条件,建立同时扩增CDV(507 bp)、CPV(287 bp)、CAV-Ⅰ(590 bp)和CAV-Ⅱ(1 027 bp)的多重PCR方法。实验结果表明:在同一PCR反应体系中可以同时检测以上4种病毒,而狂犬病病毒检测为阴性;CDV、CPV、CAV-Ⅰ和CAV-Ⅱ的最低检出限分别为101.9TCID50、101.7TCID50、101.7TCID50和102.2TCID50。利用该方法对由黑龙江省不同地区所采集的30份犬病料样品进行检测,CDV阳性率为23.33%(7/30)、CPV阳性率为20%(6/30)、CAV-Ⅰ和CAV-Ⅱ的阳性率均为3.33%(1/30),证明建立的PCR方法可以用于临床诊断。
To establish a method for simultaneous detection of canine distemper virus （CDV）, canine parvovirus （CPV）, canine adenovirus-I （CAV-I） and canine adenovirus-II （CAV-II）, a multiplex PCR was developed with a set of specific primers designed based on the conserved sequences within N gene of CDV, NS gene of CPV and E3 gene of CAV. Under the optimized conditions of multiplex PCR, four special fragments of 507 bp （CDV）, 287 bp （CPV）, 590 bp （CAV-I） and 1,027 bp （CAV-II） were amplified with a detection limit of 10^19 TCID50 （CDV）, 10^17 TCID50 （CPV）, 10^17 TCID50 （CAV-I） and 10^2.2 TCIDs0 （CAV-II）, respectively. The results of specificity showed that no PCR product was detected for rabies virus （RV）. Thirty clinical samples of affected dogs from different areas of Heilongjiang province were detected by the multiplex PCR, and the detection rate were 23.33% （7/30） for CDV, 20% （6/30） for CPV, 3.33% for CAV-I and CAV-II, which indicated that the multiplex PCR for detecting CDV, CPV, CAV-I and CAV-II was rapid, specific and sensitive, and could be used in clinic diagnoses.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2012年第11期911-914,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
横向课题[犬瘟热、细小病毒病、腺病毒病(Ⅰ型)三联活疫苗]
关键词
犬瘟热病毒
犬细小病毒
犬腺病毒
canine distemper virus
canine parvovirus
canine adenovirus
