EvaGreen实时荧光定量PCR检测猪圆环病毒2型方法的建立

生物技术通报 ›› 2014, Vol. 0 ›› Issue (6): 75-80.

EvaGreen实时荧光定量PCR检测猪圆环病毒2型方法的建立

蔡晔, 饶品彬, 吴海港, 姜永厚

（浙江理工大学生命科学学院，杭州 310018）

收稿日期:2014-01-18 出版日期:2014-06-25 发布日期:2014-06-25
作者简介:蔡晔，女，研究方向：动物病毒学；E-mail：meviky_cy@sina.cn；饶品彬同为本文第一作者
基金资助:
浙江省科技攻关项目（2008C22081）

Development of EvaGreen Real-time Fluorescence Quantitative Polymerase Chain Reaction to Detect Porcine Circovirus Type 2

Cai Ye, Rao Pinbin, Wu Haigang, Jiang Yonghou

（College of Life Science，Zhejiang Sci-Tech University，Hangzhou 310018）

Received:2014-01-18 Published:2014-06-25 Online:2014-06-25

摘要/Abstract

摘要： 根据GenBank中的猪圆环病毒2型（PCV2）ORF4基因序列设计一对特异性引物，通过常规PCR扩增PCV2的ORF4基因，并将其纯化的PCR产物克隆入pMD18-T载体中，构建重组质粒。应用该重组质粒进行EvaGreen实时荧光定量PCR，建立了PCV2 DNA的标准曲线，并进行熔解曲线分析。结果显示：建立的EvaGreen实时荧光定量PCR特异性强，与其他非靶标病毒基因不发生交叉反应；灵敏度高，最高可检测到10拷贝/μL的病毒量；重复性好，3个浓度的批间和批内的变异系数均小于2.5%；对118份临床样品进行检测，阳性样品25份，阳性率为21.2%，检测结果与普通PCR相符率为99.2%，其中一份样品经荧光PCR检测为PCV2阳性，普通PCR检测为阴性。结果表明，建立的EvaGreen实时荧光定量PCR方法具有特异性强、敏感度高、重复性好、简便快捷等特点，可用于临床PCV2感染的早期诊断以及分子流行病学调查。

关键词: 猪圆环病毒2型, EvaGreen, 实时荧光定量PCR, 检测

Abstract: According to genome sequences of ORF4 of porcine circovirus type 2（PCV2）published in GenBank, a pair of primers was designed. The ORF4 gene was amplified with traditional PCR. The PCR product was cloned into pMD18-T vector and sequenced to construct positive recombinant plasmid. The recombinant plasmid was used as template for EvaGreen real-time PCR to generate standard curve and melt curve. The results showed that EvaGreen real-time PCR in the present study was highly specific while used to detect other nontarget viruses, and its sensitivity was proved to be 10 copies/μL and reproducibility for both intra-assay and inter-assay were less than 2.5%. Then the established method was utilized to test 118 clinical samples. The result indicated that the PCV2 positive rate was 25/118, which was 99.2% consistent with that of conventional PCR tests, except one sample that was positive for PCV2 by real-time PCR but negative by conventional PCR. Thus, this method in the current study showed the characteristics of specificity, sensitivity, reproducibility and simple operation, which could be used in subclinical diagnosis and epidemiological investigation of PCV2.

Key words: Porcine circovirus type 2, EvaGreen, Real-time, PCR, Detection

蔡晔, 饶品彬, 吴海港, 姜永厚. EvaGreen实时荧光定量PCR检测猪圆环病毒2型方法的建立[J]. 生物技术通报, 2014, 0(6): 75-80.

Cai Ye, Rao Pinbin, Wu Haigang, Jiang Yonghou. Development of EvaGreen Real-time Fluorescence Quantitative Polymerase Chain Reaction to Detect Porcine Circovirus Type 2[J]. Biotechnology Bulletin, 2014, 0(6): 75-80.

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