猪圆环病毒2型荧光定量PCR检测方法的建立

doi:10.7668/hbnxb.2014.02.012

华北农学报 ›› 2014, Vol. 29 ›› Issue (2): 66-70. doi: 10.7668/hbnxb.2014.02.012

所属专题： 畜牧；

猪圆环病毒2型荧光定量PCR检测方法的建立

李鹏^1,2, 郭军庆¹, 金前跃¹, 李青梅¹, 李清州³, 万博¹, 王选年², 王川庆⁴, 张改平⁴

1 河南省农业科学院农业部动物免疫学重点实验室河南省动物免疫学重点实验室河南郑州 450002;
2 新乡学院生命科学与技术系生物技术研究中心河南新乡 453003;
3 河南省农业科学院农业经济与信息研究所河南郑州 450002;
4 河南农业大学牧医工程学院河南郑州 450002

收稿日期:2013-08-19 出版日期:2014-04-28
通讯作者: 张改平(1960-),男,河南内黄人,中国工程院院士,博士生导师,主要从事动物免疫学及动物病毒分子致病机制研究。
作者简介:李鹏(1976-),男,河南新乡人,讲师,博士后,主要从事动物病毒学与分子免疫学研究。
基金资助:
中国博士后科学基金项目(2011M501179);河南省教育厅科学技术研究重点项目(13A230838)

Rapid Detection of Porcine Circovirus Type 2 Using a SYBR Green Ⅰ Real-time PCR

LI Peng^1,2, GUO Jun-qing¹, JIN Qian-yue¹, LI Qing-mei¹, LI Qing-zhou³, WAN Bo¹, WANG Xuan-nian², WANG Chuan-qing⁴, ZHANG Gai-ping⁴

1 Henan Provincial Key Laboratory of Animal Immunology, Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
2 Biotechnology Research Center, Department of Life Sciences and Technology, Xinxiang University, Xinxiang 453003, China;
3 Institute of Agricultural Information and Economy, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
4 College of Animal Science and Veternary Medicine, Henan Agricultural University, Zhengzhou 450002, China

Received:2013-08-19 Published:2014-04-28

摘要/Abstract

摘要： 根据PCV2 ORF1设计1对特异性引物,建立了SYBR GreenⅠReal-time PCR检测方法。该方法灵敏度可达10～100拷贝/μL,比常规PCR检测方法灵敏度高10倍,而与猪繁殖和呼吸综合征病毒(PRRSV)、猪伪狂犬病毒(PRV)、猪瘟病毒(CSFV)以及猪细小病毒(PPV)没有交叉反应,利用该方法从50份临床病料中检测出43份阳性PCV2病毒,阳性检出率为94%。与常规PCR比较结果显示,该方法具有较高的灵敏度和特异性,能够快速检测和监控PCV2的感染流行。

关键词: 猪圆环病毒2型, SYBR, GreenⅠ, 荧光定量PCR

Abstract: Porcine circovirus type 2 (PCV2) is the causative factor of Post-weaning multi-systemic wasting syndrome (PMWS) in pigs. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS. To establish a sensitive, specific assay for the detection and quantitation of PCV2, we designed and synthe- sized specific primers in the open reading frame 1 (ORF1 ), and to develop a SYBR Green Ⅰ Real-time PCR. The results indicated that the Real-time PCR assay could detect 10-100 copies of the genomic DNA per reaction, and its sensitivity was 10 times of the conventional PCR. The assay did not cross-react with classical swine fever virus (CSFV), porcine parvovirus(PPV), porcine reproductive and respiratory syndrome virus (PRRSV) and pseudora- bies virus (PRV) . The limits of detection and quantitation were 10 and 100 copies, respectively. Using the estab- lished Real-time PCR system,43 of the 50 samples we tested were detected as positive. In conclusion, the Real-time PCR assay is sensitive, specific, accurate and can be used for the monitoring of PCV2 infection.

Key words: Porcine circovirus type 2, SYBR Green Ⅰ, Real-time PCR

中图分类号:

李鹏, 郭军庆, 金前跃, 李青梅, 李清州, 万博, 王选年, 王川庆, 张改平. 猪圆环病毒2型荧光定量PCR检测方法的建立[J]. 华北农学报, 2014, 29(2): 66-70. doi: 10.7668/hbnxb.2014.02.012.

LI Peng, GUO Jun-qing, JIN Qian-yue, LI Qing-mei, LI Qing-zhou, WAN Bo, WANG Xuan-nian, WANG Chuan-qing, ZHANG Gai-ping. Rapid Detection of Porcine Circovirus Type 2 Using a SYBR Green Ⅰ Real-time PCR[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2014, 29(2): 66-70. doi: 10.7668/hbnxb.2014.02.012.

http://www.hbnxb.net/CN/Y2014/V29/I2/66

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