貂肠炎病毒SYBR Green I 荧光定量PCR检测方法的建立

兽类学报 ›› 2015, Vol. 35 ›› Issue (1): 102-109.

貂肠炎病毒SYBR Green I 荧光定量PCR检测方法的建立

于永乐张传美杨海燕杨瑞梅张洪亮单虎

青岛农业大学山东省预防兽医学重点实验室

出版日期:2015-02-07 发布日期:2015-05-18
通讯作者: 张传美 zhangchuanmei100@163.com; 单虎 shanhu67@163.com
基金资助:
科技基础性工作专项：畜禽重要疫病流行病学调查（2012FY111000）；公益性行业（农业）科研专项：宠物疫病快速诊断与疫苗研究与示范（201303042）；山东省农业重大应用技术创新项目：水貂犬瘟热、细小病毒防制技术研究

Development of a SYBR Green I real-time quantitative PCR assay for the detection of Mink enteritis virus

YU Yongle, ZHANG Chuanmei, YANG Haiyan, YANG Ruimei, ZHANG Hongliang, SHAN Hu

Shandong Key Laboratory of Preventive Veterinary Medicine, Qingdao Agricultural University

Online:2015-02-07 Published:2015-05-18
Contact: ZHANG Chuanmei：zhangchuanmei100@163.com；SHAN Hu：shanhu67@163.com

摘要/Abstract

摘要： 根据GenBank 登录的水貂肠炎病毒（ＭＥＶ）ＶＰ２蛋白基因的保守序列设计了１对特异性引物，经ＰＣＲ扩增出１４６ｂｐ的目的片段，并克隆到ｐＭＤ１８－Ｔ载体上，提取重组质粒经ＰＣＲ及测序鉴定正确后，以１０倍梯度稀释质粒作为阳性标准品，绘制荧光定量标准曲线，以此建立一种基于ＳＹＢＲ Green 荧光染料的定量ＰＣＲ特异性检测水貂肠炎病毒的方法。结果表明：该方法对ＭＥＶ有很好的特异性，检测灵敏度为３４拷贝／ μＬ，且重复性试验变异系数均小于２％。临床检测中，在８０份样品中检测到３０份阳性样品，高于普通ＰＣＲ和电镜观察方法的检出率。该方法的建立实现了对ＭＥＶ临床早期快速诊断和定量分析，为毛皮动物ＭＥＶ的防控提供了可靠的检测方法。

关键词: 水貂肠炎病毒, SYBR Green I, 荧光定量PCR

Abstract: A quantitative PCR assay was developed for specific and sensitive detection of mink enteritis virus. The primer pair targeting a 146 bp fragment of the conserved VP2 gene was selected based on alignment of viral sequences available at GenBank. The analytic sensibility of this assay was 34 copies of plasmid DNA,and it had high repeatability with CV <2% ． The assay was evaluated by testing 80 fecal samples collected from suspicious cases. The results showed that 30 samples were found positive for the virus infection,which was higher than that of routine PCR and electron microscopy. Therefore the new assay provides a useful method for the rapid diagnosis of mink enteritis and the prevention and control of the disease.

Key words: Fluorescence quantitative PCR, Mink enteritis virus, SYBR Green I

于永乐张传美杨海燕杨瑞梅张洪亮单虎. 貂肠炎病毒SYBR Green I 荧光定量PCR检测方法的建立[J]. 兽类学报, 2015, 35(1): 102-109.

YU Yongle, ZHANG Chuanmei, YANG Haiyan, YANG Ruimei, ZHANG Hongliang, SHAN Hu. Development of a SYBR Green I real-time quantitative PCR assay for the detection of Mink enteritis virus[J]. ACTA Theriologica Sinica, 2015, 35(1): 102-109.