摘要
针对猪脑心肌炎病毒(EMCV)基因组保守区域,设计并合成了特异性引物和TaqMan探针,建立了EMCV的TaqMan实时荧光定量RT-PCR方法。通过常规RT-PCR方法扩增EMCV保守序列并将其连入pMD19-T载体,制备阳性标准质粒,优化反应条件,以10倍梯度稀释的标准品绘制标准曲线,EMCV标准曲线的相关系数为0.999。结果显示,该方法检测灵敏度达到10拷贝/μL,对猪捷申病毒、猪繁殖与呼吸综合征病毒、猪瘟病毒、乙型脑炎病毒、猪传染性胃肠炎病毒检测结果均为阴性。方法重复性好,批内和批间变异系数均小于2%。检测采集自上海供港猪场、进境美国种猪、加拿大、法国种猪的血清样本145份,EMCV核酸的检出率国内猪场53.6%,美国猪检出率为27.6%,加拿大猪检出率为20%,法国猪检出率为26.7%。EMCV实时荧光RT-PCR方法的建立为该病的快速诊断和流行病学调查提供了有效手段。
A Taqman real-time fluorescence quantitative PCR was established using primers and probe based on the conserved region of encephalomyocarditis virus （EMCV） genomes. The amplification sequence of EMCV was cloned into the pMD 19-T vector and a series of diluted recombinant plasmid was used to generate standard curves. The real-time quantitative PCR assay could detect as less as 10 copies of target cDNA of EMCV. The assay showed good specificity with negative reactions for other porcine viruses （PTV, PRRSV, CSFV, JEV and TGEV） . The variation coefficient of intra-batch or inter-batch was below 2%, indicating good reproducibility. 145 sermn samples of pigs from domestic farms and USA, Canada, France were detected by the real-time PCR, and the results showed that 53.6%, 27.6%, 20%and 26.7% of the sample were positive for EMCV respectively. The method can be used for EMCV infection investi- gation.
出处
《中国动物检疫》
CAS
2014年第5期81-84,共4页
China Animal Health Inspection
基金
上海检验检疫局科技项目(HK012-2010)
