(2.青岛农业大学动物医学院, 青岛 266109)
(3.扬州大学, 扬州 225009)
【摘要】基于国内流行的新城疫病毒强毒株F蛋白裂解位点分子特征设计特异性引物及Taqman探针,建立了一种可检测新城疫强毒株的一步法实时荧光定量RT-PCR方法。通过体外转录法制备cRNA标准品作为阳性模板制作标准曲线以及临床样品的检测绘制出ROC曲线,对诊断指标进行系统评价。结果显示:该方法的标准曲线为Y=-3.390X+38.23,相关系数R2为0.9999;灵敏度试验显示,该方法的最低检测限为2拷贝/μL,比常规RT-PCR高10倍;特异性试验显示该方法与常见禽病病毒无交叉反应;重复性试验的组内和组间的变异系数分别低于1%和1.5%。通过检测1 974份临床样品绘制ROC曲线显示,ROC曲线下面积为0.9861,当Youden Index为93.12时,cutoff值设为35,诊断的敏感性和特异性分别为94.82%、98.3%,与病毒分离方法的Kappa系数为0.919。本研究建立的方法敏感性高、特异性强、重复性好、诊断准确性极好,给实验室提供一种快速、实用的检测临床样品的方法。
【基金资助】 国家重点研发计划(项目号:2016YFD0500800)，题目:家禽重要疫病诊断与检测新技术研究; 公益性行业(农业)科研专项(项目号:201303033)，题目:鸡新城疫防控技术研究与示范;
Establishment and Application of Fluorescence Quantitative RT-PCR for Detection of the Velogenic Newcastle Disease Virus
(2.College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China 266109)
(3.Yangzhou University, Yangzhou, China 225009)
【Abstract】The primers and probe of a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were designed according to the characteristics of fusion protein cleavage sites of velogenic Newcastle disease viruses (NDVs). The analytical sensitivity of qRT-PCR was evaluated by standard curves using cRNA standards in vitro as positive controls. Analytical specificity was identified by detection of the common avian viruses and lentogenic NDV. Diagnostic sensitivity and specificity were validated based on receiver-operating characteristic (ROC) curves through detection of clinical samples. The limit of detection limit of this assay reached ≥ 2 copies and no cross-reaction was found. The coefficient of variation (CV) for intra-assay and inter-assay repeatability was < 1% and 1.5% respectively. The area under the ROC curve of 1974 clinical samples was 0.986 1. According to the Youden Index, the Cutoff value was 35. The sensitivity and specificity of this diagnosis was 94.82% and 98.3% respectively. The Kappa coefficient for isolation of the virus was 0.919. Our study demonstrated that a one-step qRT-PCR was highly sensitive, specific, reproducible and accurate, thereby providing a fast and practical method to detect the NDV in clinical samples.
【Keywords】 Newcastle disease virus (NDV); Virulent virus; Fluorescence quantitative RT-PCR; ROC curve;
【Funds】 National Key Research and Development Plan (2016YFD0500800); Public Welfare Industry (Agriculture) Research Project (201303033);
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