致病性猪圆环病毒基因分型三重荧光定量PCR检测方法的建立  

Establishment of triple fluorescent quantitative PCR methods for genotyping pathogenic porcine circovirus

作　　者：金玉兰[1] 李寒影 董伟仁[1] 颜焰[1] 周继勇[1] JIN Yu-lan;LI Han-ying;DONG Wei-ren;YAN Yan;ZHOU Ji-yong(Zhejiang University Center for Veterinary Sciences,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学动物医学中心,浙江杭州310058

出　　处：《中国兽医科学》2022年第11期1360-1366,共7页Chinese Veterinary Science

基　　金：浙江省重点研发计划项目(2020C02011)。

摘　　要：为建立一种同时鉴别致病性猪圆环病毒(PCV)不同型的检测方法,本研究对PCV2、PCV3、PCV4各自Rep基因进行特异性保守序列分析,建立了致病性PCV的TaqMan单重和三重荧光定量PCR检测方法。该方法能特异性区分PCV2、PCV3和PCV4,与PCV1及PRV等临床上常见的猪传染性病原均无交叉反应。建立的单重及三重PCR方法的最低检测限度均为101copies∕μL,组内及组间的变异系数均小于1%,表明重复性良好。对354份猪临床组织病料进行三重荧光定量PCR检测,结果显示,PCV2、PCV3和PCV4的阳性率分别为66.7%、26.8%和0;PCV2和PCV3共感染率为15.8%。结果表明,本研究建立的三重荧光定量PCR方法能准确鉴别致病性PCV,灵敏性优于普通PCR法,检测过程更高效、便捷,为致病性PCV的临床诊断及流行病学调查提供了检测工具。To establish a detection method for simultaneous identification of pathogenic porcine circovirus(PCV),the specific conserved sequences of Rep gene of PCV2,PCV3 and PCV4 were analyzed,respectively.Both the single and triple Taq Man fluorescence real-time PCR detection methods were established to specifically distinguish PCV2,PCV3 and PCV4,and no cross-reaction occurred with PCV1 and common pig infectious diseases such as PRV.The detecting limits of both single and triple detection methods were 101copies/μL,and the intra-group and inter-group variation coefficients were less than1%.Results showed that the positive rates of PCV2,PCV3 and PCV4 of 354 pig tissue samples were 66.7%,26.8%and 0 respectively,while the co-infection rate of PCV2 and PCV3 was 15.8%.The established triple Taq Man PCR method could accurately identify pathogenic PCV,and its sensitivity was better than that of traditional PCR method.Accordingly,the established triple Taq Man fluorescence real-time PCR method in this study was an efficient and convenient detection tool for clinical diagnosis and epidemiological investigation of pathogenic PCV.

关 键 词：猪圆环病毒 分型 三重荧光定量PCR 

分 类 号：S852.659.2[农业科学—基础兽医学]