禽腺病毒I群和禽腺病毒4型双重TaqMan探针荧光定量PCR检测方法的建立与应用  

Establishment and application of a duplex TaqMan real-time PCR for Fowl adenovirus serotype I and Fowl adenovirus serotype 4 detection

作　　者：薛晓岩 张振兴 杨钦鸿 王位 张伟[3] 李素华 宋建领[2] XUE Xiao-yan;ZHANG Zhen-xing;YANG Qin-hong;WANG Wei;ZHANG Wei;LI Su-hua;SONG Jian-ling(College of Life Sciences,Southwest Forestry University,Kunming 650224,China;Yunnan Academy of Animal Husbandry and Veterinary,Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory,Kunming 650224,China;Xishuangbanna Prefecture Animal Epidemic Disease Prevention Center,Xishuangbanna 650224,China)

机构地区：[1]西南林业大学生命科学学院,云南昆明650224 [2]云南省畜牧兽医科学院云南省热带亚热带动物病毒病重点实验室,云南昆明650224 [3]西双版纳州动物疫病预防制中心,云南西双版纳650224

出　　处：《中国预防兽医学报》2025年第2期152-158,184,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：禽腺病毒4型抗原表位定位和AS-ONs抑制感染分子机制研究(202301AS070006)。

摘　　要：为建立能快速检测禽腺病毒I群(FAd V-I)和禽腺病毒4型(FAd V-4)的双重Taq Man探针荧光定量PCR(qPCR)方法,本研究根据禽腺病毒(FAd V)的Hexon基因,设计针对FAd V-I和FAd V-4的通用型与特异型引物与探针。采用上述引物经PCR扩增靶基因并克隆至p MD19-T载体中,构建重组质粒标准品p MD19-T-FAd V-I和p MD19-T-FAd V-4,并均经PCR和测序鉴定。将两种重组质粒标准品分别10倍倍比稀释后等体积混合作为模板,采用棋盘法对反应体系和反应条件进行优化,建立了能检测上述病原的双重Taq Man探针q PCR方法,同时对该方法的特异性、敏感性、重复性及对临床样品的适用性进行评价。结果显示,两种重组质粒标准品标准曲线的相关系数(R2)均大于0.996,表明两种质粒标准品混合物的拷贝数与其Ct值具有良好的线性关系;该方法能够特异性检测和区分FAd V-I和FAd V-4,与其他禽类传染病病原核酸不发生交叉反应,具有较强的特异性;该方法对FAd V-I和FAd V-4重组质粒标准品的检测限分别为4.6×10~2拷贝/μL和2.01×10~2拷贝/μL,比常规PCR方法敏感性高10倍,具有较高的敏感性;批内和批间重复性试验的变异系数均小于1.0%,具有较好的重复性和稳定性。采用该方法和常规PCR法对32份疑似感染FAd V-I的家禽组织样品、8份疑似感染FAd V-4的家禽粪便样品进行检测,结果显示二者检测结果的符合率均为100%。结果表明,本研究建立的FAd V-I/FAd V-4双重Taq Man探针q PCR检测方法特异性强、敏感性高、准确性好,为快速诊断FAd V-I的同时鉴别FAd V-4提供了技术支持。To establish a dual TaqMan probe fluorescence quantitative PCR method for rapid detection of avian adenovirus groupⅰ(FAdV-I)and avian adenovirus type 4(FAdV-4),universal and specific primer probessets for were designed based on Hexon gene of avian adenovirus(FAdV).Using these primers,the target gene was amplified by PCR and cloned into the pMD19-T vector to construct recombinant plasmid standards pMD19-T-FAdV-I and pMD19-T-FAdV-4,which were identified by PCR and sequencing.Two recombinant plasmid standards were then serially diluted at a 10-fold ratio and used as templates.The reaction system and conditions were optimized using a chessboard method to establish the dual TaqMan probe fluorescence quantitative PCR method for detecting these pathogens.Additionally,the specificity,sensitivity repeatability and applicability of this method to clinical samples were evaluated.The results showed that the standard curves generated from the two plasmid standards exhibited excellent linearity,with correlation coefficients(R2)exceeding 0.996.This method could specifically detect and distinguish FAdV-I and FAdV-4 without cross-reactivity with nucleic acids of other avian infectious disease pathogens hedetection limit of this method was as low as 4.6×102 copies/μL for FAdV-I and 2.01×102 copies/μL for FAdV-4,which is 10 times more sensitive than the conventional PCR detection methods and indicating high sensitivity.The intra-batch and inter-batch repeatability assay coefficient of variation were less than 1.0%,highlighting the high specificity,repeatability and stability of this method.The coincidence rate of 32 suspected FAdV-I group and 8 suspected FAdV-4 poultry tissue samples was 100%by using this method and conventional PCR.The results showed that the dual TaqMan probe fluorescence quantitative PCR method developed in this study for detecting FAdV-I/FAdV-4 exhibited high accuracy,specificity and sensitivity,providing arobust technical support for rapid diagnosis of FAdV-I and simultaneous identification of FAdV-4.

关 键 词：禽腺病毒I群 禽腺病毒4型 HEXON TAQMAN探针 荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]