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

Establishment and application of TaqMan-MGB multiple fluorescence quantitative PCR for detecting PCV2, Hps, and Mhp

作　　者：迟玉华 金风英 韩勇 CHI Yuhua;JIN Fengying;HAN Yong(Animal Disease Control and Prevention Center of Donggang District,Rizhao City,Rizhao 276800,China;Animal Husbandry Development Service Station at Rizhao City,Rizhao 276800,China;Animal Disease Prevention and Control Center of Rizhao City,Rizhao 276800,China)

机构地区：[1]山东省日照市东港区动物疫病预防控制中心,山东日照276800 [2]山东省日照市畜牧开发服务站,山东日照276800 [3]山东省日照市动物疫病预防控制中心,山东日照276800

出　　处：《养殖与饲料》2023年第6期17-21,共5页Animals Breeding and Feed

摘　　要：[目的]建立一种同时鉴别猪圆环病毒2型(PCV2)、副猪嗜血杆菌(Hps)、猪支原体肺炎(Mhp)的检测方法,为PCV2、Hps和Mhp的净化提供技术支撑。[方法]针对PCV2的ORF2基因、Hps的tbpA基因、Mhp的p36基因保守序列,设计3对引物和TaqMan-MGB探针。人工合成包含了荧光PCR扩增目的基因在内的序列,并与载体连接,构建重组质粒PCV2-ORF2、Hps-tbpA、Mhp-p36,测序正确后作为标准质粒。条件优化后建立PCV2、Hps和Mhp的TaqMan-MGB多重荧光定量PCR检测方法。[结果]该方法具有良好的特异性,与其他常见猪病原不发生交叉反应。PCV2、Hps和Mhp敏感性最低检出量分别达到1.33×10、1.77×10、1.86×10 copies/μL。[结论]TaqMan-MGB多重荧光定量PCR检测法具有良好的敏感性、特异性、重复性,而且快速、准确。[Objectives] To establish a method for simultaneously detecting Porcine circovirus type 2(PCV2), Haemophilus parasuis(Hps), and Mycoplasma hyopneumoniae(Mhp) and to provide support for the purification of PCV2, Hps, and Mhp. [Methods] Three pairs of primers and TaqMan-MGB probe were designed for the ORF2 gene of PCV2,tbpA gene of Hps, and p36 gene of Mhp to establish a method for rapidly diagnosing PCV2,Hps and Mhp. A TaqMan-MGB multiple fluorescent quantitative PCR assay was developed for detecting PCV2,Hps, and Mhp. [Results] The results of validation test showed that the method established had good specificity and did not cross-react with other common swine pathogens.The lowest sensitivity of PCV2,Hps, and Mhp was 1.33×10 copies/uL,1.77×10 copies/uL and 1.86×10 copies/uL,respectively. [Conclusions] TaqMan-MGB Multiple fluorescent quantitative PCR has good sensitivity, specificity and repeatability,and is fast and accurate.

关 键 词：猪圆环病毒2型 副猪嗜血杆菌 猪肺炎支原体 荧光定量聚合酶链式反应 防控 

分 类 号：S852.65[农业科学—基础兽医学]