新城疫强毒株荧光定量RT-PCR检测技术的建立及应用

罗瑶瑶1,2 王静静1 吕艳1 赵云玲1 郑东霞1 魏润宇1,3 于松梅1 左媛媛1 张乐萃2 单虎2 刘华雷1,2 王志亮1

(1.中国动物卫生与流行病学中心, 青岛 266032)
(2.青岛农业大学动物医学院, 青岛 266109)
(3.扬州大学, 扬州 225009)
【创新点】基于国内流行毒株,建立了一种可检测所有流行强毒的荧光定量RT-PCR方法,结果证实本方法敏感性高、特异性强、重复性好、诊断准确性极好。通过对1974份临床样品检测,证实ROC曲线下面积为0.9861,诊断的敏感性和特异性分别为94.82 %、98.3 %,适用于新城疫大规模病原学主动监测和流行病学调查,具有显著的推广前景。

【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。

【关键词】 新城疫病毒(NDV); 强毒; 荧光定量RT-PCR; ROC曲线;

【DOI】

【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 Funding:The present work was supported by the National Key Research and Development Plan(2016YFD0500800) 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 and the Public Welfare Industry(Agriculture)Research Project(201303033)

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This Article

ISSN:1000-8721

CN: 11-1865/R

Vol 34, No. 04, Pages 541-549

June 2018

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Article Outline

创新点

摘要

  • 材料与方法
  • 结果
  • 7 诊断性能评价
  • 8 试剂盒的比对试验
  • 讨论
  • 参考文献