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猪圆环病毒2型/3型双重荧光定量PCR检测方法
王林青,樊霖,赵宇,田润博,崔建涛,韩昊莹,赵丽,陈红英
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(郑州师范学院分子生物学郑州市重点实验室,郑州 450044; 河南农业大学动物医学院,郑州 450002;河南牧业经济学院动物医药学院,郑州 450008;河南农业大学动物医学院,郑州 450002; 郑州市猪重大疫病防控重点实验室,郑州450002)
摘要:
为了能够同时快速地检测和鉴别猪圆环病毒2型和3型(PCV2和PCV3),参考GenBank中已发表的PCV2和PCV3基因组序列,针对其保守区分别设计了2对特异性引物,经优化反应体系和条件,建立了能快速检测和鉴别PCV2/PCV3双重荧光定量PCR方法。结果表明,PCV2和PCV3的R2分别为0.999、0.999 3,E值分别为3.573 1、3.373 4。该方法能同时特异地检测PCV2和PCV3,而对其他5种猪病原均未检测到荧光信号;PCV2和PCV3的最低检测值分别为41.1 copies·μL-1、27.0 copies·μL-1;批内和批间变异系数均小于1%。临床样本检测结果显示,PCV2和PCV3的阳性率分别为62.12%(41/66)、48.48%(32/66),二者混合感染的阳性率为46.96%(31/66)。表明该方法具有敏感、特异和可靠等特点,该方法为PCV2和PCV3单独或者混合感染的早期诊断、定量检测及其流行病学调查提供了可行的技术支持。
关键词:  猪圆环病毒  PCV2和PCV3  双重荧光定量PCR  检测
DOI:10.13610/j.cnki.1672-352x.20210909.007
基金项目:河南省高校科技创新团队支持计划(19IRTSTHN007), 河南省科技开放合作项目(182106000048), 河南省高等学校青年骨干教师培养计划(2018GGJS161)和河南省科技攻关项目(192102110184)共同资助。
Duplex real-time fluorescence quantitative PCR method for detection of porcine circovirus Types 2 and 3
WANG Linqing,FAN Lin,ZHAO Yu,TIAN Runbo,CUI Jiantao,HAN Haoying,ZHAO Li,CHEN Hongying
(Zhengzhou Key Laboratory of Molecular Biology, Zhengzhou Normal University, Zhengzhou 450044; College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002;College of Veterinary Medicine, Henan University of Animal Husbandry and Economy, Zhengzhou 450008; College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002; Zhengzhou Key Laboratory of Prevention and Control of Major Epidemics of Pigs, Zhengzhou 450002)
Abstract:
In order to quickly detect and differentiate porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3) , two pairs of specific primers were designed based on the conserved of PCV2 and PCV3 published in GenBank, a duplex real-time fluorescence quantitative PCR for simultaneous detection and differentiation of PCV2/PCV3 was established by optimizing the reaction system and conditions. The results showed that R2 of PCV2 and PCV3 were 0.999 and 0.999 3, and the E values were 3.573 1 and 3.373 4, respectively. The assay could detect specifically PCV2 and PCV3, whereas no fluorescence was detected for other five porcine pathogens. The detection limits of PCV2 and PCV3 were 41.1 copies·μL-1 and 27 copies·μL-1, respectively. The intra- and inter-assay coefficients of the method were all less than 1%. The detection results for 66 clinical samples showed that the positive rates of PCV2 and PCV3 were 62.12%(41/66)and 48.48%(32/66), respectively, and their co-infection rate was 46.96% (31/66).The results reveal that the assay was sensitive, specific, reliable, and feasible for clinical samples, providing strong technical support for the early diagnosis and quantitative detection of PCV2 and PCV3.
Key words:  porcine circovirus  PCV2 and PCV3  duplex fluorescent quantitative PCR  detection

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