水产学报2013，Vol.37Issue(4)：607-613,7.DOI:10.3724/SP.J.1231.2013.38441
鲤疱疹病毒Ⅱ型TaqMan real-time PCR检测方法的建立及应用
Establishment of a TaqMan real-time PCR assay for detecting the Cyprinid herpesvirus Ⅱ
摘要
Abstract
A 1 446 bp coding region of Cyprinid herpesvirus 0 (CyHV-2)DNA helicase gene was amplified by PCR and cloned into pMD19T vector for the construction of recombinant plasmid. After being identified and confirmed with PCR reaction, 10-fold serial dilutions of recombinant plasmid were used as standard templates for TaqMan real time PCR to quantify the virus genomic copy number and generate standard curve. Herein, a TaqMan real-time PCR of detecting CyHV-2 was developed. It had a good linear relationship between the initial templates and Ct values with a detection range from 1 x 101 copies/μL to 1 × 107 copies/μL, the correlation coefficient ( R2 ) was 0. 999 1 , and the slope value of standard curve was -3.412. The detection results showed that the specificity of this assay was high for CyHV-2 without cross reactions with DNA templates from KHV and GSIV. The diseased crucian carp from Sheyang and Baoying, Jiangsu Province, were detected with the established method and the results showed that the content of CyHV-2 were 6. 89 × 104 copies/μL and 3. 02 × 102 copies/μL, respectively. The real-time PCR assay described here with high sensitivity and accuracy is considered to be a powerful tool for the rapid detection and quantification of CyHV-2 in fish.关键词
鲫/造血器官坏死症/鲤疱疹病毒Ⅱ型/TaqMan real-time PCR/检测方法Key words
crucian carp/hematopoietic necrosis disease/Cyprinid herpesvirus Ⅱ (CyHV-2)/TaqMan real-time PCR/detection method分类
农业科技引用本文复制引用
周勇,曾令兵,张辉,范玉顶,徐进..鲤疱疹病毒Ⅱ型TaqMan real-time PCR检测方法的建立及应用[J].水产学报,2013,37(4):607-613,7.基金项目
现代农业产业技术体系建设专项(CARS-46-11) （CARS-46-11）
中国水产科学研究院基本科研业务费(2013A0606) （2013A0606）
