猪瘟病毒TaqMan实时荧光定量PCR检测方法的建立及初步应用

中国畜牧兽医

猪瘟病毒TaqMan实时荧光定量PCR检测方法的建立及初步应用

岳锋¹^，2，朱艳平^1，2，银梅³，王选年¹^，2

（1.新乡学院生命科学与技术系，河南新乡 453003；2.新乡学院生物技术研究所，河南新乡 453003；3.河南科技学院动物科学学院，河南新乡 453003）

修回日期:2014-03-06 出版日期:2014-06-20 发布日期:2014-07-24
通讯作者: 王选年（1969—），男，河南人，博士，教授，研究方向：动物病理与分子免疫学。E-mail：wangxuannian@163.com；Tel：0373-3682111
作者简介:岳锋（1983—），男，河南人，博士生，助教，研究方向：动物病理与分子免疫学。
基金资助:
国家自然科学基金（31291877、31272539）；河南省科技厅基础与前沿技术研究项目（122300410150）；河南省教育厅科学技术研究重点项目（13A230837）。

Development and Application of TaqMan Fluorescence Quantitative PCR Assay for Detecting Classical Swine Fever Virus

YUE Feng¹^，2, ZHU Yan-ping¹^，2, YIN Mei³, WANG Xuan-nian¹^，2

(1. Department of Life Science and Technology, Xinxiang University, Xinxiang 453003, China；2. Institute of Biotechnology, Xinxiang University, Xinxiang 453003, China；3. College of Animal Science, Henan Institute of Science and Technology, Xinxiang 453003, China)

Revised:2014-03-06 Online:2014-06-20 Published:2014-07-24

摘要/Abstract

摘要： 根据GenBank中猪瘟病毒（classical swine fever virus，CSFV）5′NTR的保守序列，设计1对特异性引物和TaqMan探针，以CSFV全长基因重组质粒pGEM-CSFV为标准品，建立TaqMan实时荧光定量PCR标准曲线，进行特异性、敏感性和重复性试验，并检测人工感染CSFV后不同时期猪血浆中CSFV的载量。结果表明，标准曲线循环阈值与模板浓度具有良好的线性关系，R²=0.999；敏感性高，最低检测限为1×10¹拷贝/μL；特异性强，与猪圆环病毒2型、猪细小病毒、猪伪狂犬病病毒和猪繁殖与呼吸综合征病毒无交叉反应性；重复性好，组内和组间变异系数均小于2%。人工感染CSFV后第7天猪血浆中CSFV载量达到峰值，之后逐渐降低。结果表明，建立的TaqMan实时荧光定量PCR方法具有特异、敏感、重复性好等优点，为CSFV的定量分析及临床诊断奠定基础。

关键词:

猪瘟病毒; 实时荧光定量PCR; TaqMan探针

Abstract: According to the conserved sequence of 5′nontranslated region (5′NTR)of classical swine fever virus (CSFV) in GenBank, a pair of primers and a TaqMan probe were designed. The recombinant plasmid pGEM-CSFV containing the complete gene of CSFV was as a standard control to establish fluorescence quantitative PCR standard curve. Specificity, sensitivity and reproducibility of the method were determined. CSFV load in plasma was measured at different time points after experimental infection of CSFV in pigs. The results showed that standard curve established had a good linear relationship between threshold cycle and template concentration, R²=0.999. The assay had high sensitivity and could detect 1×10¹copies/μL. The specificity of the method was high， there was no cross reaction between CSFV and other pathogens containing porcine circovirus 2, porcine parvovirus, porcine pseudorabies virus and porcine reproductive and respiratory syndrome virus. The method showed good reproducibility， there was a coefficient of variations less than 2% for both inter-assay and intra-assay. CSFV load in plasma increased to peak value at 7 days after experimental infection of CSFV in swine and gradually decreased later. These results indicated that the developed TaqMan fluorescence quantitative PCR assay had the advantages of good specificity, sensitivity and reproducibility. It provided a basis for quantification analysis and clinical diagnosis of CSFV.

Key words:

classical swine fever virus; fluorescence quantitative PCR; TaqMan probe

岳锋，朱艳平，银梅，王选年. 猪瘟病毒TaqMan实时荧光定量PCR检测方法的建立及初步应用[J]. 中国畜牧兽医.

YUE Feng, ZHU Yan-ping, YIN Mei, WANG Xuan-nian. Development and Application of TaqMan Fluorescence Quantitative PCR Assay for Detecting Classical Swine Fever Virus[J]. .