中国人兽共患病学报2013，Vol.29Issue(4)：380-384,407,6.DOI:10.3969/cjz.j.issn.1002-2694.2013.04.013
口蹄疫病毒一步法TaqMan-MGB荧光定量RT-PCR检测方法的建立
Development of TaqMan-MGB fluorescence quantitative RT-PCR assay for detection of foot-and-mouth disease virus
摘要
Abstract
In this paper, a one-step real-time quantitative RT-PCR (rRT-PCR) method based on a TaqMan-MGB probe was developed to detect three major serotypes of FMDV in China (A, O and Asia 1). A conserved region in the 3D gene of FMDV was chosen as the target for primers and TaqMan probe, and then the optimization of rRT-PCR, sensitivity test, specificity test and repeatability test were carried out. Results showed that the MGB rRT-PCR could specifically detected A, O, and Asia I serotypes of FMDV and had negative results for detection CSFV, HPRRSV, PRRSV, PRV, PPV, PCV2 and JEV. The standard curves established showed fine linear relationship between threshold cycle (Ct) and template concentration. The sensitivity of this assay was 10 times higher than that of the multiplex RT-PCR and its minimum detectable concentration were 83. 4 copies/μL of recombinant plasmid and 7. 1 fg/μL of template RNA. The variation coefficients were less than 2% based on 5 replications of 4 samples. These results showed that the MGB rRT-PCR assay established in this study was sensitive, specific and stable, and it could be used for FMD diagnosis in clinical samples, epidemiological investigation, and livestock safety monitoring.关键词
口蹄疫病毒/TaqMan- MGB/荧光定量RT-PCRKey words
foot-and-mouth virus/ TaqMan-MGB/ real-time quantitative RT-PCR分类
医药卫生引用本文复制引用
李金海,李兴玉,曹三杰,文心田,陈斌,张毅,周哲学..口蹄疫病毒一步法TaqMan-MGB荧光定量RT-PCR检测方法的建立[J].中国人兽共患病学报,2013,29(4):380-384,407,6.基金项目
公益性行业(农业)科研专项(No.201203056) （农业）
