TaqMan-MGB多重荧光定量PCR检测PCV2、PCV3和PCV4方法的建立和应用  

Establishment and Application of TaqMan-MGB Multiplex Fluorescent Quantitative PCR for Detection of PCV2,PCV3 and PCV4

作　　者：韩勇 刘玉洁 吕桂霞 马珍驰 任增超 范承祥 HAN Yong;LIU Yu-jie;LYU Gui-xia;MA Zhen-chi;REN Zeng-chao;FAN Cheng-xiang(Rizhao Animal Disease Prevention and Control Center,Rizhao 276800,China;Dongying Modern Animal Husbandry Development Center,Dongying 257000,China;Zaozhuang Animal Husbandry and Fishery Development Center,Zaozhuang 277000,China)

机构地区：[1]山东省日照市动物疫病预防控制中心,山东日照276800 [2]山东省东营市现代畜牧业发展中心,山东东营257000 [3]山东省枣庄市畜牧渔业事业发展中心,山东枣庄277000

出　　处：《中国兽医杂志》2023年第8期29-34,共6页Chinese Journal of Veterinary Medicine

基　　金：山东省动物疫情监测与防治项目(201811-1);日照市动物疫病监测项目(日牧动卫发[2020]10号,日牧动卫发[2021]9号)。

摘　　要：为建立一种可同时鉴别致病性猪圆环病毒(PCV)不同基因型的检测方法,本试验针对PCV2、PCV3和PCV4的ORF2基因保守区域设计引物和探针,将人工合成目的基因与质粒连接,构建阳性质粒PCV2-ORF2、PCV3-ORF2和PCV4-ORF2。通过优化引物浓度、探针浓度、退火温度等参数,建立PCV2、PCV3和PCV4 TaqMan-MGB多重荧光定量PCR检测方法,并验证该方法的特异性、敏感性和重复性。应用建立的方法对38份临床样品进行应用检测,对24个养猪场(户)进行流行病学调查。结果显示,建立的方法能特异性区分PCV2、PCV3和PCV4,且与其他常见猪病病原不发生交叉反应;对PCV2、PCV3和PCV4的最低检出浓度分别为1.56×10、1.28×10和1.76×10拷贝/μL;批内和批间变异系数均小于2%,重复性好;38份临床样品中PCV2、PCV3和PCV4阳性样品分别为17、3和1份,从24个养猪场(户)采集的689份样品中PCV2、PCV3和PCV4阳性率分别为33.33%、12.50%和8.33%。结果表明,本试验建立的PCV2、PCV3和PCV4 TaqMan-MGB多重荧光定量PCR检测方法特异性强、敏感性高、重复性好,可用于PCV的快速分型检测,为PCV的临床诊断和流行病学调查提供了检测工具。In order to establish a detection method capable of simultaneously identifying different genotypes of pathogenic porcine circovirus(PCV),this study designed primers and probes targeting the conserved region of ORF 2 gene in PCV2,PCV3,and PCV4.Synthetic target genes were ligated with plasmids to construct positive plasmids PCV2-ORF2,PCV3-ORF2,and PCV4-ORF2.By optimizing parameters such as primer and probe concentrations,annealing temperature,etc.,a TaqMan-MGB multiplex fluorescent quantitative PCR method for the detection of PCV2,PCV3,and PCV4 was established,and the specificity,sensitivity,and repeatability of the method were validated.The established method was applied to detect 38 clinical samples and conduct epidemiological investigations in 24 pig farms.The results showed that the method could specifically differentiate PCV2,PCV3,and PCV4 without cross-reactivity with other common swine pathogens.The minimum detectable amounts of PCV2,PCV3,and PCV4 were 1.56×10,1.28×10,and 1.76×10 copies/μL,respectively.The intra-and inter-assay coefficients of variation were both less than 2%,indicating good repeatability.Among the 38 clinical samples,17,3,and 1 samples were positive for PCV2,PCV3,and PCV4,respectively.The positive rates of PCV2,PCV3,and PCV4 among 689 samples of the 24 pig farms were 33.33%,12.50%,and 8.33%,respectively.These results indicate that the TaqMan-MGB multiplex fluorescent quantitative PCR method established in this study is highly specific,sensitive,and repeatable,and can be used for rapid genotyping of PCV,providing a valuable tool for clinical diagnosis and epidemiological investigations of PCV.

关 键 词：猪圆环病毒2型 猪圆环病毒3型 猪圆环病毒4型 荧光定量PCR 净化 

分 类 号：S855.1[农业科学—临床兽医学]