口蹄疫病毒一步法TaqMan-MGB荧光定量RT-PCR检测方法的建立

摘要

目的 建立一种能快速、高效、敏感、特异地检测口蹄疫病毒(FMDV)的一步法荧光定量RT-PCR检测方法.方法 根据FMDV聚合酶3D基因序列比对结果,设计特异性引物和MGB探针;通过优化,得到最佳反应体系和反应条件;分别以质粒和病毒RNA为模板,构建标准曲线;并进行特异性、敏感性、重复性试验.结果 该方法能特异性检测A型、O型、Asia 1型FMDV,而对猪瘟、猪蓝耳病、猪圆环病毒、猪细小病毒、猪狂犬病毒、猪乙型脑炎等病原检测结果均为阴性;构建的荧光定量标准曲线Ct值与模板浓度呈良好的线性关系,检测质粒的敏感性可达83.4copies/μL,检测病毒RNA的敏感性可达7.1 fg/μL,比多重RT-PCR敏感性高10倍;对4份样品进行5次批内和批间重复检测,检测结果变异系数均小于2%.结论 建立的口蹄疫病毒一步法TaqMan-MGB荧光定量RT-PCR检测方法特异性强、敏感性高、重复性好,可用于口蹄疫临床样品诊断、流行病学调查和畜产品安全监测.%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.