微滴式数字PCR定量检测滑液囊支原体方法的建立及应用  

Establishment and Application of Droplet Digital PCR for Quantitative Detection of Mycoplasma synoviae

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(Key Laboratory of China(Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control,Ministry of Agriculture and Rural Affairs,Guangxi Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary Research Institute,Nannning,Guangxi 530001)

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,农业农村部中国-东盟(广西)跨境动物疫病防控重点实验室,广西南宁530001

出　　处：《中国家禽》2023年第2期39-45,共7页China Poultry

基　　金：广西重点研发计划(桂科AB16380003);国家“万人计划”领军人才专项(W02060083);“广西八桂学者”专项(2019A50)。

摘　　要：为绝对定量滑液囊支原体(Mycoplasma synoviae,MS),试验根据已发表的MS VlhA基因序列,针对其保守区域分别设计了1对特异引物和1条探针,建立了ddPCR定量检测MS的方法,并检测该方法的特异性、敏感性和重复性。结果显示:建立的方法最佳引物浓度为20μmol/μL,最佳探针浓度为10μmol/μL,最佳退火温度为54℃;该方法仅检测出MS毒株,不能检测出其他禽源病原,MS重组质粒标准品最低检测限为3.5拷贝/μL,3个连续稀释的MS重组质粒DNA检测的变异系数均小于5%;45份病鸡喉拭子、肺脏及关节囊样品检测结果显示,该方法的MS阳性检出率(11.1%)高于荧光定量PCR(8.9%)。结果表明,建立的ddPCR方法定量检测MS特异性强、敏感性高、重复性好,为绝对定量MS提供了适合的技术手段。To absolutely quantify of Mycoplasma synoviae(MS),a set of specific oligonucleotide primers and probe were designed and synthesized to recognize the distinct genomic sequences of VlhA gene based on published sequences of MS.As established to detect MS with this primers and probe,and specificity,sensitivity and repeatability of the established method were detected.The results showed that the optimizing reaction conditions of the established method were 20μmol/μL of primers,10μmol/μL of probe,54℃ for annealing temperature.All MS strains were amplified and did not cross-react with other avian pathogens by this method.The minimum limit for quantitative detection of MS recombinant plasmid DNA was 3.5 copies/μL.The coefficients of variation were all less than 5% for three consecutive dilution MS recombinant plasmids.Forty-five samples of throat swabs,lungs and joint capsules collected from sick chickens were tested,the the positive detection rate of the established ddPCR(11.1%)was higher than that of fluorescent quantitative PCR(8.9%).It's indicated that the ddPCR method had strong specificity,high sensitivity and good repeatability for MS detection,which supplied a suitable method for absolute quantitation of MS.

关 键 词：微滴式数字PCR 定量检测 鸡滑液囊支原体 

分 类 号：S852.62[农业科学—基础兽医学]