鸭坦布苏病毒TaqMan探针实时荧光定量PCR检测方法的建立

Establishment of TaqMan Probe Real-time Quantitative PCR Detection Method for Duck Tembusu Virus

为了建立一种有效、快速、准确的鸭坦布苏病毒(DTMUV)TaqMan探针实时荧光定量PCR(qPCR)检测方法,本试验针对NCBI发布的DTMUV E基因保守序列设计特异性引物和探针,建立qPCR反应体系。通过制备的标准质粒建立标准曲线,评估该方法的特异性、敏感性和重复性,并使用该方法对临床样本进行检测。结果显示,所建立的标准曲线相关系数为0.999 5,有良好的线性关系;该方法可特异检测DTMUV;对标准质粒的最低检测限为2.72×10^(2) copies/μL,是普通PCR的100倍;CT值组内变异系数为0.97%~1.32%,组间变异系数为1.24%~1.79%;人工感染DTMUV的20份鸭胚成纤维细胞样本以及12份人工攻毒2 d的雏鸭脾脏组织样本和12份泄殖腔棉拭子样本阳性率为100%(44/44);疑似感染的12份活鸭泄殖腔棉拭子阳性率为25.00%(3/12),14份病死鸭脾脏组织样本阳性率为28.57%(4/14)。结果表明,本试验建立的TaqMan探针qPCR方法有良好的敏感性、准确性和重复性,能特异检测DTMUV,适用于临床快速检测,为DTMUV的分子流行病学调查和临床疾病诊断提供了技术支撑。

To establish an efficient,rapid,and accurate TaqMan probe real-time quantitative PCR(qPCR)detection method for Duck tembusu virus(DTMUV),this study designed specific primers and a probe targeting the conserved sequence of the DTMUV E gene published by NCBI.qPCR reaction system was developed,and the standard curve was established using prepared standard plasmid to evaluate the method's specificity,sensitivity,and repeatability.This method was also applied to detect clinical samples.The results showed that the established standard curve had a correlation coefficient of 0.9995,indicating a good linear relationship.The method specifically detected DTMUV,with a minimum detection limit for the standard plasmid of 2.72×10^(2) copies/μL,100 times more sensitive than conventional PCR.The intra-group coefficient of variation for the CT values ranged from 0.97%to 1.32%,and the inter-group coefficient of variation ranged from 1.24%to 1.79%.Positive detection rates were 100%(44/44)for 20 DTMUV-infected duck embryo fibroblast samples,12 spleen tissue samples from ducklings artificially infected for two days,and 12 cloacal swab samples.Among 12 cloacal swab samples from suspected infected live ducks,the positive rate was 25.00%(3/12),and for 14 spleen tissue samples from dead ducks,the positive rate was 28.57%(4/14).The results demonstrated that the TaqMan probe qPCR method established in this study has good sensitivity,accuracy,and repeatability,and it can specifically detect DTMUV.It is suitable for rapid clinical detection and provides technical support for molecular epidemiological surveys and clinical diagnosis of DTMUV infections.