摘要
为建立一种快速且准确检测鸭源鹅细小病毒(D-GPV)的实时荧光定量PCR方法,本研究通过分析D-GPV基因序列的保守区设计并合成一对特异性引物,构建携带NS1基因的重组质粒作为标准品,绘制实时荧光定量PCR标准曲线,并进行敏感性、特异性、重复性试验和临床样品的检测.结果显示,该方法标准曲线的的线性关系良好,相关系数为0.999;灵敏度高,检测底限为10拷贝;特异性与重复性好,与其他病毒无交叉反应,批间和批内变异系数均低于1％.该方法对人工感染D-GPV鸭泄殖腔拭子和咽拭子样品的检测结果显示,鸭在攻毒后第1d即可以通过口腔和泄殖腔向外排毒,排毒量在第3d达到高峰,排毒持续期可达到42d.本研究所建立的实时荧光定量PCR方法灵敏度高、特异性和稳定性好,可用于临床样品中的D-GPV检测.
Abstract
To develop a qPCR assay for rapid and accurate detection of Duck-origin goose parvovirus(D-GPV),we designed and synthesize specific primers based on the sequences of D-GPV gene.A recombinant plasmid was then constructed and verified.Subsequently,we used it as the positive template to draw the standard curve of this assay and then tested its sensitivity,specificity,repeatability and infectious samples.The results showed that the linear relationship of the standard curve of this method was good with the correlation coefficient at 0.999 and the sensitivity at 10 copies.Its specificity was also good as there was no cross-reactivity with other viruses.In the repeatability test,the inter-assay and intra-assay coefficients of variation were both lower than 1％with good stability.Examination of oropharyngeal and cloacal swabs from ducks infected with D-GPV showed that ducks shed viruses through mouths and cloacae at 1 dpi till 42 dpi.The viral loads reached the peak at 3 dpi.The qPCR method developed in this research had high sensitivity,specificity and stability,and provided a fast and accurate method for clinical detection of D-GPV.
维普
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