新城疫强毒特异性反转录荧光定量PCR检测方法的建立与应用  

Establishment and Application of Reverse Transcription Fluorescence Quantitative PCR Method for Virulent NDV Detection

作　　者：徐敏丽[1] 于江[1] 逯璐 张玉玉[1] 任素芳[1] 陈智[1] 孙文博[1] 郭立辉[1] 吴家强[1] 张琳[1] Xu Minli;Yu Jiang;Lu Lu;Zhang Yuyu;Ren Sufang;Chen Zhi;Sun Wenbo;Guo Lihui;Wu Jiaqiang;Zhang Lin(Institute of Animal Science and Veterinary Medicine,Shandong Academy of Agricultural Sciences/Shandong Key Laboratory of Animal Disease Control and Breeding,Jinan 250100,China;Emerging Economic Formats Research Institute of Shandong Management University,Jinan 250357,China)

机构地区：[1]山东省农业科学院畜牧兽医研究所/山东省畜禽疫病防治与繁育重点实验室,山东济南250100 [2]山东管理学院新兴业态发展研究所,山东济南250357

出　　处：《山东农业科学》2021年第8期112-118,共7页Shandong Agricultural Sciences

基　　金：泰山学者特聘专家工程经费(ts201511069);山东省农业科学院农业科技创新工程项目“主要畜禽重要疫病防控关键技术与产品创制”(CXGC2021A12)。

摘　　要：为建立一种敏感性高、特异性强、成本更低的新城疫病毒(Newcastle disease virus,NDV)特异性反转录荧光定量PCR(reverse transcription fluorescence quantitative PCR,RT-qPCR)检测方法,基于对所有已知NDVF基因序列特征的分析,设计了一对强毒特异性引物,以体外转录制备的RNA标准品为阳性模板,构建了标准曲线,并对该方法的敏感性、特异性和准确性进行了验证。结果表明,基于SYBRGreenⅠ嵌合荧光技术建立的检测方法能够特异性扩增NDV强毒,最低可检测1拷贝/μL的RNA,用于临床毒株的检测结果与序列测定一致,可作为适合大规模监测NDV强毒的方法和工具。In order to develop a high-sensitive,strong-specific and low-cost reverse transcription fluorescence quantitative PCR(RT-qPCR)method for virulent Newcastle disease virus(NDV),a pair of primers was designed according to the F gene,and the standard curve was established using RNA standard prepared in vitro transcription as positive template,and related indexes were further tested.The results showed that this assay based on SYBR GreenⅠbedded fluorescence technology could differentially detect virulent NDV with sensitivity low to 1 copy/μL RNA.When used in clinical NDV-infected samples,this assay could only amplified virulent NDV,which was consistent with the sequencing results.This method was a more suitable method for mass surveillance of virulent NDV.

关 键 词：新城疫病毒 强毒 反转录荧光定量PCR 监测 

分 类 号：S852.657[农业科学—基础兽医学] Q503[农业科学—兽医学]