三重PCR和双重荧光探针法检测ALV-A/J亚群方法的建立与应用  

Establishment and Application of a Triple PCR and a Double Fluorescence Probe Method for Detection of ALV-A/J Subgroup

作　　者：沈艳[1] 周香 何长生[1] 朱良强[1] 李宏伟 陈芳芳[2] Shen Yan;Zhou Xiang;He Changsheng;Zhu Liangqiang;Li Hongwei;Chen Fangfang(Anhui Provincial Center for Animal Disease Control and Prevention,Hefei,Anhui 230091,China;Laboratory of Veterinary Pathology and Disease Control,Anhui Agricultural University,Hefei,Anhui 230036,China)

机构地区：[1]安徽省动物疫病预防与控制中心,安徽合肥230091 [2]安徽农业大学兽医病理学与疫病防控实验室,安徽合肥230036

出　　处：《中国动物检疫》2023年第5期88-96,共9页China Animal Health Inspection

基　　金：国家动物疫病监测与防治项目。

摘　　要：为快速检测禽白血病病毒(avian leukemia virus,ALV)并同时鉴别A亚群(ALV-A)和J亚群(ALV-J),根据ALV不同亚群基因设计特异性引物和TaqMan荧光探针,经优化反应条件,建立通用型、A亚群与J亚群三重PCR以及A亚群与J亚群双重荧光探针检测方法。结果显示:三重PCR检测方法能特异性扩增ALV所有亚群以及A亚群和J亚群,检测灵敏度达1×103拷贝/μL;建立的ALV-A/J双重荧光探针检测方法特异性强,可检测的最低拷贝浓度为40拷贝/μL。本研究建立的三重PCR方法与商品化ALV实时荧光PCR检测试剂盒均有较高的符合率(Kappa系数>0.75);针对24份发病鸡精液,ALV-A/J双重荧光探针检测方法与商品化ALV实时荧光PCR检测试剂盒检测结果一致;本研究建立的两种检测方法均能同时鉴别出ALV A亚群和J亚群。利用本研究建立方法对安徽省394份临床样本进行检测,发现ALV总阳性率为38.58%,以J亚群单一感染为主,同时伴有A亚群单一感染以及A/J亚群混合感染。结果表明,本研究建立的ALV-通用型/A亚群/J亚群三重PCR方法和ALV-A/J亚群双重荧光探针检测方法特异性强,检测快速,具有临床样本检测的应用潜力,为临床上快速诊断及有效防控ALV感染及鉴别A/J亚群的单一或混合感染提供了技术支持。In order to rapidly detect avian leukemia virus(ALV)and simultaneously identify its subgroup A(ALV-A)from J(ALV-J),specific primers and TaqMan fluorescent probes were designed based on different subgroup genes,followed by optimizing reaction conditions,a triple PCR for ALV universal type,subgroups A and J as well as a double fluorescent probe method for subgroups A and J were established.The results showed that the triple PCR could be used to specifically amplify all subgroups including A and J,with a sensitivity of 1×103 copies/μL;the double probe method,with strong specificity,could be used to detect the minimum copy concentration of 40 copies/μL.The established triple PCR was highly consistent(Kappa coefficient>0.75)with the commercial real-time fluorescence PCR kit;for 24 semen samples from infected chickens,the results by the double probe method were the same as those by commercial real-time fluorescence PCR kit;subgroups A and J could be simultaneously identified by the two methods established in the paper.394 clinical samples collected in Anhui Province were detected by the established methods,it was found that the total positive rate of ALV infection was 38.58%,mainly due to single infection with subgroup J,accompanied by single infection with subgroup A and mixed infection with subgroup A/J.In conclusion,the two established methods,with strong specificity,rapidity and application potential for clinical samples,could technically support rapid diagnosis and effective prevention and control of ALV in clinical practice as well as identification of single or mixed infection with subgroup A/J.

关 键 词：禽白血病 A亚群 J亚群 鉴别诊断 

分 类 号：S855.3[农业科学—临床兽医学]