期刊文献+

猪丁型冠状病毒TaqMan实时定量RT-PCR检测方法的建立和应用

Development and application of a real-time TaqMan fluorescent quantitative RT-PCR assay for detection of porcine deltacoronavirus
分享 导出
摘要 根据GenBank中登录的猪丁型冠状病毒(PDCoV)N基因序列在保守区设计合成特异性引物和TaqMan探针,构建含有PDCoVN基因的重组质粒作为阳性标准品,通过优化各项反应条件,建立了检测PDCoV的TaqMan实时荧光定量RT-PCR方法,并对该方法进行特异性、定量线性范围、敏感性和重复性等试验。结果显示,该方法仅对PDCoV出现特异性扩增反应,与猪流行性腹泻病毒、猪传染性胃肠炎病毒、A群猪轮状病毒、猪嵴病毒、猪繁殖与呼吸综合征病毒、伪狂犬病病毒、猪瘟病毒及猪细小病毒均无交叉反应。该方法的标准曲线Ct值与2.2×10 9-2.2×10 1copies/L之间的质粒浓度具有良好的线性关系,标准曲线方程为Ct=-3.539×lg X+38.95,线性相关系数(R2)为1.0,检测下限为2.2 copies/L。对3个不同浓度(2.2×10 2、2.2×10 4、2.2×10 6copies/L)的pMD18-PDCoV-N进行2次重复检测,每个浓度重复试验的Ct值的变异系数均小于1.0%,表明该方法具有良好的重复性。应用建立的方法对64份广西临床腹泻样品进行检测,结果从其中18份样品中检出PDCo V,样品阳性率为28.1%,说明广西猪群存在PDCoV感染。结果表明,建立的TaqMan实时荧光定量RT-PCR方法为PDCoV的检测和定量分析提供了一种快速、敏感和特异的技术手段。 Primers and TaqMan fluorescent probe were designed according to the conserved regions of N gene sequences of porcine deltacoronavirus(PDCo V),a recombinant plasmid containing N gene of PDCo V was constructed as the positive standard sample,then a real-time TaqMan fluorescent quantitative RT-PCR(FQ-RT-PCR) assay was developed by optimization of reacting conditions.Furthermore,the sensitivity,specificity and repetition assay of the FQ-RT-PCR were tested.The results showed that there was no cross-reaction with porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus,porcine rotavirus group A,porcine kobuvirus,porcine reproductive and respiratory syndrome virus,pseudorabies virus,classical swine fever virus and porcine parvovirus.The assay was linear for the template plasmid pMD18-PDCoV-N in the range from 2.2×109—2.2×101 copies/L,the standard equation was Ct=-3.539×lg X+38.95,correlation coefficient R2=1.0,and detection limit was low to 2.2 copies/L.Also,the assay showed the excellent reproducibility with the coefficient of variation less than 1.0%.Eighteen of 64 clinical fecal samples collected from different areas in Guangxi were positive for PDCoV tested by the assay developed in this study,and the positive rate was 28.1%,indicating there was PDCoV infection in Guangxi swine herds.In conclusion,the development of this assay provides a rapid,sensitive and specific diagnostic method for PDCoV detection and quantitative analysis.
作者 秦毅斌 何苹萍 卢冰霞 韦嫔媛 何颖 李斌 苏乾莲 段群棚 周英宁 赵武 QIN Yi-bin1, HE Ping-ping2, LU Bing-xia1, WEI Pin-yuan2, HE Ying1, LI Bin1, SU Qian-lian1, DUAN Qun-peng1, ZHOU Ying-ning1, ZHAO Wu1 (1. Guangxi Key Laboratory of Veterinary Biatechnology/Guangxi Veterinary Research Institute, Nanning 530001, China; 2. Guangxi Key Laboratory of A quatie Genetic Breeding and Healthy A quaculture/Guangxi Academy of Fishery Sciences ,Nanning 530021, China)
出处 《中国兽医科学》 CSCD 北大核心 2018年第6期692-699,共8页 Veterinary Science in China
基金 广西创新驱动发展专项资金项目(桂科AA17204057) 广西自然科学基金项目(2017GXNSFBA198092) 广西兽医生物技术重点实验室开放基金课题(16-380-45-B-3) 广西水产畜牧科技项目(桂渔牧科201633041,201633044,201633034) 广西基本科研业务费专项(桂科专项16-2,17-2) 南宁市西乡塘区科技开发项目(2017-2-10310,201710317)
关键词 猪丁型冠状病毒 荧光定量RT-PCR TAQMAN探针 porcine deltacoronavirus real-time TaqMan fluorescent quantitative PCR TaqMan fluorescence probe
作者简介 秦毅斌(1983-),男,广西临桂人,助理研究员,硕士,研究方向:动物传染病病原与分子生物学,Tel:0771-3120372,E-mail:qinyibin5188@163.com。;通信作者:赵武,主要从事动物传染病防控研究.E-mail:zhaowu168866@163.com。
  • 相关文献

参考文献4

二级参考文献38

共引文献48

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部 意见反馈