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当前位置: 首页 > 文章 > 牛副流感病毒3型TaqMan实时荧光定量RT-PCR检测方法的建立及应用 中国兽医科学 2014 (6) 617-623
Position: Home > Articles > Establishment and application of TaqMan real-time fluorescence quantitative RT-PCR for detection of bovine parainfluenza virus type 3 Chinese Veterinary Science 2014 (6) 617-623

牛副流感病毒3型TaqMan实时荧光定量RT-PCR检测方法的建立及应用

作  者：
董秀梅;朱远茂;蔡虹;王姝;吕闯
单  位：
北京市水产技术推广站;东北农业大学动物医学学院中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室大动物传染病研究室;中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/大动物传染病研究室;中国农业科学院哈尔滨兽医研究所;东北农业大学动物医学学院
关键词：
实时荧光定量RT-PCR;TaqMan探针;牛副流感病毒3型
摘  要：
根据牛副流感病毒3型(bovine parainfluenza virus type 3,BPIV3)膜蛋白M基因设计引物及探针,以体外转录法制备的cDNA标准品为模板,建立了TaqMan实时荧光定量RT-PCR方法,对其特异性、敏感性、重复性进行了测定,并对人工感染BPIV3的牛群临床样本进行了检测。结果显示,建立的TaqMan实时荧光定量RT-PCR的最低检测限为17.6copies/μL,同时进行批内、批间5次重复性试验,变异系数均小于2.5%。应用建立的方法检测牛传染性鼻气管炎病毒、牛腺病毒3型、牛呼吸道合胞体病毒、牛病毒性腹泻-黏膜病病毒1型,结果均为阴性,说明建立的方法特异性较强。用建立的方法检测人工感染BPIV3的犊牛鼻拭子样本,检测结果与样本TCID50的测定结果相符合,但比其更敏感。结果表明,本试验中建立的TaqMan实时荧光定量RT-PCR可以作为BPIV3早期诊断及定量分析的有效技术手段。
译  名：
Establishment and application of TaqMan real-time fluorescence quantitative RT-PCR for detection of bovine parainfluenza virus type 3
作  者：
DONG Xiu-mei;ZHU Yuan-mao;CAI Hong;WANG Shu;L Chuang;MA Lei;XUE Fei;College of Veterinary Medicine,Northeast Agricultural University;State Key Laboratory of Veterinary Biotechnology/Division of Livestock Infectious Diseases,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences;
关键词：
real-time fluorescence quantitative RT-PCR;;TaqMan probe;;bovine parainfluenza virus type 3
摘  要：
According to the sequence of bovine parainfluenza virus type 3(BPIV3)M gene,the primers and probe were designed.The cDNA was prepared by in vitro transcription and used as standard positive template.TaqMan real-time fluorescence quantitative RT-PCR was established.The specificity test,sensitivity test,and reproducible detection were assessed,and the method was applied to detection of BPIV3infected cattle.In result,the developed TaqMan real-time fluorescence quantitative RT-PCR was much more sensitive than the conventional RT-PCR and could detect the template as low as 17.6copies/μL.The intraand inter-batch repeatability tests were conducted five times using the standard plasmid and the coefficients of variation were less than 2.5%.There were no cross reactions with the other respiratory pathogens,such as infectious bovine rhinotracheitis virus,bovine adenovirus type 3,bovine respiratory syncytial virus,and bovine viral diarrhea virus type 1.The results of fluorescence quantitative RT-PCR detection for clinical samples were correlated well with those of median tissue culture infective dose measurement,but more sensitive.The above results showed that the developed TaqMan real-time fluorescence quantitative RT-PCR could be used as an effective tool for detection and quantification of BPIV3.

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