摘要
为了建立犬瘟热病毒(CDV)强弱毒株的鉴别PCR检测方法,根据犬瘟热病毒强弱毒株H基因的保守区域各设计了两对特异性引物,制备阳性对照模板,优化反应体系及反应程序;采集CDV自然感染犬只病料样本,利用该方法和胶体金试纸条进行对比检测。结果表明,PCR产物扩增判断为356 bp,最佳扩增体系20μL,最佳模板浓度为5ng/μL,最佳引物浓度6.25μmol/L,最佳退火时间为5 s,最佳延伸温度为15 s,最佳循环数40。CDV感染犬表现出明显的神经症状,对血液样本的检测结果表明,该方法和胶体金符合率100%;PCR检测结果显示心脏和脾脏为CDV核酸阳性,肝脏、肺脏、肾脏、十二指肠(含肠内容物)为CDV核酸阴性。该方法扩增效率高、特异性好,可区分疫苗毒株和野生型毒株,为有效诊断CDV并鉴别强弱毒株提供了保证和技术支撑。
In order to establish a differential PCR method for detection of CDV strong or weak strains, two pairs of specific primers were designed according to the conserved region of the H gene of CDV strong or weak strains, and the positive control template was prepared to optimize the reaction system and procedure. Samples of CDV naturally infected dogs were collected, and the method was compared with colloidal gold strip for detection.The results showed that the PCR product amplification was 356 bp, the optimal amplification system was 20 μL, the optimal template concentration was 5 ng/μL, the optimal primer concentration was 6.25 μmol/L, the optimal annealing time was 5 s, the optimal extension temperature was 15 s, and the optimal cycle number was 40. CDV infected dogs showed obvious neurological symptoms. The results of blood samples showed that the coincidence rate of this method with colloidal gold test strip was 100%. The PCR results showed that the heart and spleen were positive for CDV nucleic acid, while the liver, lung, kidney and duodenum(containing intestinal contents) were negative for CDV nucleic acid. This method has high amplification efficiency and good specificity, and can distinguish vaccine strains from wild-type strains, which provides a guarantee and technical support for the effective diagnosis of CDV and the identification of strong and weak strains.
作者
张可
薛姣雄
喻娇
唐青海
曾繁荣
刘雪晴
隆泽桧
侯鑫军
Zhang Ke;Xue Jiao-xiong;Yu Jiao;Tang Qing-hai;Zeng Fan-rong;Liu Xue-qing;Long Ze-hui;Hou Xin-jun(Hunan Key Laboratory for Conservation and Utilization of Biological Resources in the Nanyue Mountainous Region,College of Life Sciences and Environment Hengyang Normal University,Hengyang 421008,Hunan,PRC;Hengyang animal husbandry and aquatic aquaculture center,Hengyang 421008,Hunan,PRC)
出处
《湖南畜牧兽医》
2021年第4期45-50,共6页
Hunan Journal of Animal Science & Veterinary Medicine
基金
大学生科技创新项目([2021]1-9、S202110546001)
湖南省教育厅优秀青年项目(17B041)。
关键词
犬瘟热病毒
聚合酶链反应
强弱毒株
胶体金试纸条
鉴别
Canine distemper virus
polymerase chain reaction
wild-type and vaccine strain
colloidal gold test strip
Identification
