二重微滴式数字PCR定量检测禽呼肠孤病毒和鸡滑液囊支原体方法的建立  

Establishment and application of duplex droplet digital PCR for quantitative detection of avian reovirus and Mycoplasma synoviae

作　　者：谢志勤 谢芝勋 张艳芳 李小凤 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 任红玉 阮志华 XIE Zhi-qin;XIE Zhi-xun;ZHANG Yan-fang;LI Xiao-feng;FAN Qing;XIE Li-ji;WAN Li-jun;LUO Si-si;LI Meng;ZHANG Min-xiu;ZENG Ting-ting;HUANG Jiao-ling;WANG Sheng;LI Dan;WEI You;REN Hong-yu;RUAN Zhi-hua(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 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：《中国兽医科学》2023年第10期1232-1241,共10页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31660715);广西重点研发专项项目(桂科AB16380003);中央引导地方发展专项项目(桂科ZY19183013);广西创新驱动专项项目(AA17204057);广西“八桂学者”专项项目(2019A50);国家“万人计划”领军人才专项项目(W02060083)。

摘　　要：为绝对定量检测禽呼肠孤病毒(avian reovirus,ARV)和鸡滑液囊支原体(Mycoplasma synoviae,MS),本研究建立了二重微滴式数字PCR(ddPCR)。根据已发表的ARV S1基因和MS的VlhA基因序列保守序列,分别设计了针对S1和VlhA基因的特异引物和探针,通过优化引物和探针的浓度及反应条件后,建立了二重ddPCR定量检测ARV和MS方法,并对建立方法的特异性、敏感性和重复性进行测试。结果显示,优化后最佳的引物和探针浓度分别为1.2和0.35μmol/L,最佳退火温度为55℃。特异性检测结果只检出ARV和MS,没有检出其他对照的禽病病原。敏感性检测结果显示,该方法检测ARV及MS重组质粒DNA的检出限分别为微滴数6.3和5.4 copies/μL,检测方法的检出限与单重ddPCR最低检出限相同,敏感性比荧光定量PCR方法高10倍。对3个连续稀释的ARV和MS重组质粒DNA检测结果的变异系数均小于5%,重复性好。对92份病鸡关节囊、喉拭子及肺样品进行检测,二重ddPCR方法检出12份样品呈ARV阳性,9份样品呈MS阳性,其中2份样品呈ARV和MS阳性,ARV阳性检出率为13.0%(12/92),MS阳性检出率为9.8%(9/92),混合感染阳性率为2.2%(2/92)。结果表明,本研究建立的二重ddPCR方法在定量检测ARV及MS时特异性强、敏感性高、重复性好,为绝对定量检测ARV及MS提供了更敏感的检测技术手段。The objective of this study was to establish and develop a duplex droplet digital PCR(dd PCR)method for absolute quantitative detection of avian reovirus(ARV)and Mycoplasma synoviae(MS).Two set of specific oligonucleotide primers and two probes were designed and synthesized to recognize the conserved gene sequences of the S1 and Vlh A gene based on published sequences separately.After optimizing the concentration of primers,probes and reaction conditions,the duplex dd PCR method was established for the quantitative detection of ARV and MS.Then,the specificity,sensitivity and repeatability were tested.The results showed that the final concentration of primers and probes were 1.2 and0.35μmol/L,respectively.The annealing temperature was 55℃.The method could detect all ARV and MS strains and the resucts of other avian pathogens were negative in the specificity test.The minimum limit for quantitative detection of ARV and MS recombinant plasmid DNA were 6.3 and 5.4 copies/μL,respectively.The detection limit was the same as the minimum detection limit of single dd PCR.The sensitivity of detection was 10 times higher than that of fluorescence quantitative PCR.The coefficients of variation were all less than 5%for the three successively diluted ARV and MS recombinant plasmid DNA,which indicated that the method had a good repeatability.Ninety-two samples of throat swabs,lungs and joint capsules collected from sick chickens were tested by duplex dd PCR.twelve of 92 samples were positive for ARV.Six of 92 samples were positive for MS.Two samples were positive for ARV and MS.The positive detection rate of ARV was 13.0%(12/92),the positive rate of MS was 9.8%(9/92)and the positive rate of ARV and MS mixed infection was 2.2%(2/92).In conclusion,this duplex dd PCR method had strong specificity,high sensitivity and good repeatability,which provides a more sensitive detection technique for absolute quantitative detection of ARV and MS.

关 键 词：二重微滴式数字PCR 定量检测 禽呼肠孤病毒 鸡滑液囊支原体 

分 类 号：S859.659.4[农业科学—临床兽医学]