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猪伪狂犬病病毒和猪圆环病毒3型双重SYBR GreenⅠ 荧光定量PCR方法的建立
金钺,陈曦艋,李洪炫,赵宇,李新生,陈红英,王林青
0
(河南农业大学动物医学院,郑州 450002;河南农业大学动物医学院,郑州 450002; 郑州师范学院分子生物学郑州市重点实验室,郑州 450044)
摘要:
为了同时检测猪伪狂犬病病毒(PRV)野毒和猪圆环病毒3型(PCV3),基于PRV gE基因和PCV3 Rep基因保守序列各合成了1对引物,在优化反应体系和扩增条件的基础上,建立了检测PRV和PCV3的双重SYBR Green Ⅰ荧光定量PCR方法。结果显示:PRV和PCV3的Tm值分别为87和81.5 ℃,能特异地检测PRV和PCV3,而对其他5种猪病原均未检测到荧光信号;PRV和PCV3的最低检测值分别为37.0和33.8 copies·μL-1;批内批间变异系数均小于2%;对2017—2019年期间采自河南地区的78份猪临床样品进行检测,PRV和PCV3的阳性率分别为19.23%(15/78)和41.03%(32/78),二者混合感染的阳性率为8.97%(7/78)。表明该方法具有高度的敏感性、特异性和稳定性,可用于监测PRV野毒和PCV3及其流行病学调查。
关键词:  猪伪狂犬病病毒  猪圆环病毒3型  荧光定量PCR
DOI:10.13610/j.cnki.1672-352x.20220907.008
基金项目:中原知名专家项目(204200510015), 河南省高校科技知名专家支持计划(21HASTIT039)和河南省自然科学基金(222300420586)共同资助。
Double SYBR GreenⅠfluorescence quantitative PCR for detecting porcine pseudorabies virus and porcine circovirus type 3
JIN Yue,CHEN Ximeng,LI Hongxuan,ZHAO Yu,LI Xinsheng,CHEN Hongying,WANG Linqing
(College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002;College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002; Zhengzhou Key Laboratory of Molecular Biology, Zhengzhou Normal University, Zhengzhou 450044)
Abstract:
In order to detect porcine pseudorabies virus (PRV) and porcine circovirus type 3 (PCV3) simultaneously, two pairs of specific primers were synthesized based on the conservative region of the gE gene of PRV and Rep gene of PCV3.A dual SYBR GreenⅠfluorescent quantitative PCR method for the detection of PRV and PCV3 was developed based on the optimization of reaction system and amplification conditions. The results showed that: the Tm values of PRV and PCV3 were 87 and 81.5 ℃, respectively, which could specifically detect PRV and PCV3, while no fluorescent signals were detected for the other five porcine pathogens; The lowest detection values of PRV and PCV3 were 37.0 and 33.8 copies·μL-1 f, respectively. The coefficients of variation in intra-batch and inter-batch assays were all less than 2.0%. The SYBR GreenⅠ-based duplex real-time RT-PCR assay was then used to detect 78 clinical samples of porcine collected from 2017 to 2019 in Henan Province, and the positive rates of PRV and PCV3 were 19.23% (15/78) and 41.03% (32/78), respectively, while the positive rate of co-infection for PCV3 and PRV was 8.97%(7/78). It showed that the method has high sensitivity, specificity and stability for monitoring PRV, PCV3 and their epidemiological investigation.
Key words:  porcine pseudorabies virus  porcine circovirus type 3  real-time fluorescence quantitative PCR

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