鸡传染性喉气管炎病毒微滴式数字PCR定量检测方法的建立  

Establishment of Droplet Digital PCR Quantitative Detection Method forDetection of Infectious Laryngotracheitis Virus

作　　者：谢志勤 谢芝勋 张艳芳 范晴 谢丽基 万丽军 罗思思 李孟 张民秀 曾婷婷 黄娇玲 王盛 李丹 韦悠 李小凤 任红玉 阮志华 XIE Zhiqin;XIE Zhixun;ZHANG Yanfang;FAN Qing;XIE Liji;WAN Lijun;LUO Sisi;LI Meng;ZHANG Minxiu;ZENG Tingting;HUANG Jiaoling;WANG Sheng;LI Dan;WEI You;LI Xiaofeng;REN Hongyu;RUAN Zhihua(Guangxi Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary Research Institute,Nannning,Guangxi 530001,China)

机构地区：[1]广西壮族自治区兽医研究所,广西兽医生物技术重点实验室,南宁530001

出　　处：《西北农业学报》2024年第3期381-388,共8页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：广西重点研发计划(桂科AB16380003);广西基地与人才专项(AD17195083);广西创新驱动专项专项(AA17204057);国家“万人计划”领军人才专项(W02060083);“广西八桂学者”专项(2019A50)。

摘　　要：旨在建立检测鸡传染性喉气管炎病毒(infectious laryngotracheitis virus, ILTV)绝对定量方法。根据已发表的鸡ILTV的TK基因序列,针对其保守区域分别设计1对特异引物和1条探针,建立检测鸡ILTV的微滴式数字PCR(droplet digital PCR,ddPCR)方法,并评价其特异性、敏感性和重复性。结果显示,建立方法的最佳引物浓度为20μmol/μL,最佳探针浓度为10μmol/μL,最佳退火温度为56℃。特异性检测结果显示,建立的方法只检出ILTV,没有检出其他病原株。敏感性检测结果显示,采用建立的方法定量检出ILTV重组质粒标准品的最低限为4.6拷贝/μL。对3个连续稀释的pMD18-ILTV重组质粒DNA进行检测,3次重复检测结果的变异系数均小于5%。对83份病鸡喉拭子、肺及脾组织样品进行检测,采用建立的ddPCR检出ILTV阳性样品10份,荧光定量PCR检出ILTV阳性样品9份,ddPCR的阳性检出率(12.05%)高于荧光定量PCR的阳性检出率(10.84%)。结果表明,建立的ddPCR方法定量检测ILTV特异性强、敏感性高、重复性好,为绝对定量检测ILTV提供更好的技术支撑。The aim of present study is to develop and evaluate a droplet digital PCR(ddPCR)for detection of infectious laryngotracheitis virus(ILTV).Specific oligonucleotide primers and probe were designed and synthesized to recognize the genomic sequences of the thymidine kinase(TK)gene of ILTV based on published reference sequences of ILTV.After the reaction conditions were optimized,the ddPCR was used to detect ILTV with the selected primers and probe.The specificity,sensitivity and reproducibility of ddPCR were subsequently evaluated.The results showed that the optimal concentrations of primers and probe were 20μmol/μL and 10μmol/μL,respectively.The annealing temperature was 56℃.Only ILTV strains were dectected with the established ddPCR,but other avian pathogens in the specificity tests were not detect.The minimum limit for quantitative detection of pMD18-ILTV recombinant plasmid DNA was 4.6 copies/μL.Three independently repeated experiments showed that the coefficients of variation were all less than 5%for detection of the three serially diluted pMD18-ILTV recombinant plasmid.Eighty three samplessuch as throat swabs,lungs and spleens collected from sick chickens were examined.Ten samples were tested positive for ILTV by ddPCR and the positive detection rate was 12.05%.Nine samples were tested positive for ILTV by fluorescent quantitative PCR and the positive detection rate was 10.84%.The sensitivity of ddPCR for the tested samples was slightly higher compared with that of the fluorescent quantitative PCR.The ddPCR described in this study was highly specific,sensitive and reproducible for detection of ILTV.The established ddPCR in our lab may provide an alternative means for absolute quantitative detection of ILTV.

关 键 词：鸡传染性喉气管炎病毒 微滴式数字PCR 定量检测 

分 类 号：S858.31[农业科学—临床兽医学]