水产学报2011，Vol.35Issue(5)：774-779,6.DOI:10.3724/SP.J.1231.2011.17358
草鱼呼肠孤病毒TaqMan real-time PCR检测方法的建立
Establishment of a TaqMan real-time PCR assay for detecting the grass carp reovirus
摘要
Abstract
A 1 250 bp coding region of grass carp reovirus(GCRV) VP6 protein was amplified by RT-PCR and cloned into pEGFP-N1 vector for the construction of recombinant plasmid pEGFP-N1-VP6.After being identified and confirmed by PCR reaction with specific primer paires, 10-fold serial dilutions of plasmid pEGFP-N1-VP6 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 GCRV was developed.It had a good linear relationship between the initial templates and Ct values with a detection range from 1 × l01 copies/μL to 1 × 106 copies/μL,the correlation coefficient( R2) was 0.998 09 and the slope value of standard curve was -3.373.The assay for specificity of the method established revealed that the TaqMan real-time PCR had a specific detection of GCRV, but had no detection signals to spring viremia carp virus(SVCV) and infectious hematopoietic necrosis virus(IHNV).The TaqMan real-time RT-PCR assay described here with high sensitivity and accuracy is considered to be a powerful tool for the rapid detection and quantification of GCRV both in vivo and in vitro.关键词
草鱼呼肠孤病毒/VP6蛋白编码基因/TaqMan real-time PCR/检测Key words
grass carp reovirus(GCRV)/ VP6 protein coding gene/ TaqMan real-time PCR/ detection分类
农业科技引用本文复制引用
周勇,曾令兵,范玉顶,徐进,马杰,罗晓松,肖艺..草鱼呼肠孤病毒TaqMan real-time PCR检测方法的建立[J].水产学报,2011,35(5):774-779,6.基金项目
国家现代农业产业技术体系建设专项资金资助(nycytx-49-16) （nycytx-49-16）
农业部公益性行业科研专项(200803013) （200803013）
