传染性喉气管炎病毒RAA检测方法的建立  

Establishment of RAA detection method for infectious laryngotracheitis virus

作　　者：冯婉莹 王转转 刘一宁 陈光明 郭霄慧 李伟欣 李卫晴 张志强[1] 李佩国[1] 张召兴 吴同垒[1] 贾青辉[1] FENG Wanying;WANG Zhuanzhuan;LIU Yining;CHEN Guangming;GUO Xiaohui;LI Weixin;LI Weiqing;ZHANG Zhiqiang;LI Peiguo;ZHANG Zhaoxing;WU Tonglei;JIA Qinghui(Hebei Provincial Key Laboratory of Preventive Veterinary Medicine,Hebei Normal University of Science and Technology,Qinhuangdao,Hebei 066604,China;Hebei Tourism Vocational and Technical College,Chengde,Hebei 067000,China)

机构地区：[1]河北科技师范学院河北省预防兽医学重点实验室,河北秦皇岛066604 [2]河北旅游职业学院,河北承德067000

出　　处：《中国兽医学报》2025年第2期212-218,共7页Chinese Journal of Veterinary Science

基　　金：河北省重点研发计划资助项目(21326614D);河北省现代农业产业体系蛋禽创新团队资助项目(HBCT2024260209);河北省现代农业产业体系肉禽创新团队资助项目(HBCT2024270207)。

摘　　要：为建立一种快速、高效、灵敏的传染性喉气管炎病毒(infectious laryngotracheitis virus, ILTV)检测方法。提取ILTV的DNA作为模板,通过条件优化、敏感性和重复性分析,建立针对ILTV的重组酶介导的等温扩增(RAA)荧光检测方法,同时使用禽流感病毒(avian influenza virus, AIV)、传染性支气管炎病毒(infectious bronchitis virus, IBV)、新城疫病毒(Newcastle disease virus, NDV)的核酸进行检测,验证此方法的特异性;最后利用此方法对采集自河北省多家规模化养鸡场的59份临床样本进行检测,并与国家标准中的实时荧光定量(qPCR)和PCR方法进行对比分析。结果显示,本研究建立的RAA检测方法,其反应体系为缓冲液25.0μL、引物2.1μL、探针0.6μL、乙酸镁5.0μL、模板5.0μL,反应温度为39℃,扩增时间为20 min以内;该方法的灵敏度为10^(1) copies/μL,特异性检测100%;对59份临床样本检测结果显示,RAA荧光法与qPCR法均检出17份阳性,PCR法检出12份,RAA(荧光法)检出率与实时荧光定量与qPCR一致,高于PCR检测法。结果表明:RAA荧光法检测时间短,并有较好的特异性与灵敏度,可用于ILTV的快速检测。The aim of this study is to establish a rapid,efficient,and sensitive method for detecting the infectious laryngotracheitis virus(ILTV).The DNA of ILTV was extracted and used as a tem-plate to develop a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence de-tection method for ILTV through optimization of conditions,sensitivity analysis,and repeatability assessment.Additionally,the nucleic acids of avian influenza virus(AIV),IBV,and Newcastle dis-ease virus(NDV)were detected to verify the specificity of this method.Finally,this method was applied to analyze 59 clinical samples collected from multiple large-scale chicken farms in Hebei Province,and the results were compared with those obtained from real-time fluorescence quantifi-cation(qPCR)and PCR methods according to national standards.The results showed that the RAA detection method established in this study had a reaction system of 25.0μL buffer,2.1μL primer,0.6μL probe,5.0μL magnesium acetate,and 5.0μL template.The reaction temperature was 39℃and the amplification time was within 20 minutes.The sensitivity of this method was 10^(1) copies/μL,and the specificity detection was 100%.Testing of 59 clinical samples showed that 17 were detected positive by both RAA fluorescence and qPCR,and 12 were detected by PCR,and the detection rate of RAA(fluorescence)was consistent with real-time fluorescence quantification and qPCR,which was higher than that of the PCR assay.The research results indicate that the RAA fluorescence method has a short detection time,good specificity and sensitivity,and can be used for rapid detection of ILTV.

关 键 词：传染性喉气管炎 重组酶 RAA 

分 类 号：S854.43[农业科学—临床兽医学]