猪流行性腹泻病毒N基因克隆及实时荧光定量PCR检测方法的建立  

Cloning of N Gene of Porcine Epidemic Diarrhea Virus and Establishment of Fluorescent Real-time Quantitative PCR Detection Method

作　　者：李丹[1] 付强[1] 塞力克·杰恩斯 王万顺 刘芯怡 史慧君[1] LI Dan;FU Qiang;Selik Jens;WANG Wan-shun;LIU Xin-yi;SHI Hui-jun(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China)

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052

出　　处：《新疆农业大学学报》2021年第5期361-366,共6页Journal of Xinjiang Agricultural University

基　　金：自治区天山创新团队(2020D14005);新疆农业大学研究生科研创新项目(XJAUGRI2021030)。

摘　　要：猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)感染引起5~8周龄断奶仔猪出现顽固性腹泻,给养猪业造成巨大的经济损失。本研究基于PEDV核衣壳蛋白(N,nucleocapsid)基因建立实时荧光定量PCR检测方法,将为PEDV诊断提供重要的方法。采集猪流行性腹泻新鲜粪便样品,提取总RNA后反转录成cDNA,使用PCR扩增PEDV N基因,并克隆至pMD19-T载体,经测序后获得N基因序列,提交NCBI Blast完成基因序列分析和遗传进化树构建;设计实时荧光定量PCR引物,以pMD19-T-N质粒为模板,将其稀释成不同浓度,使用SYBR Green I实时荧光定量PCR测定Ct值,并建立标准曲线,分析其线性回归系数和扩增效率,确定最低检测下限。研究结果表明,成功克隆PEDV N基因,大小为750 bp,并成功构建pMD19-T-N载体,经测序后分析发现PEDV N基因与GenBank数据库中多个PEDV毒株同源性高达98%以上,其中与KU847996.1亲缘关系最近,高达98.93%,属于同一个遗传进化分支;基于N基因建立SYBR Green I实时荧光定量PCR,线性回归系数和扩增效率分别为0.994和97.57%,最低检测下限为10 copies/μL。综上表明成功克隆PEDV N基因,并基于N基因成功建立SYBR Green I实时荧光定量PCR检测PEDV方法,为今后PEDV的有效防控提供参考。Porcine epidemic diarrhea virus(PEDV)infection causes intractable diarrhea in weaned piglets aged 5-8 weeks,resulting in huge economic losses to the pig industry.This study aims to established a real-time fluorescence quantitative PCR detection method based on the nucleocapsid protein(N)gene of PEDV,which will provide an important method for the diagnosis of PEDV.The N gene of PEDV was amplified by PCR,and cloned into pMD19-T vector.Then N gene was sequenced and submitted to NCBI blast for gene homology analysis and phylogenetic tree construction.The primer for fluorescent real-time quantitative PCR were designed,and pMD19-T-N plasmid served as the template was diluted into serial different concentrations.SYBR Green I fluorescent real-time quantitative PCR was used to determine Ct value,and the standard curve was established and its linear regression and amplification efficiency,and the lowest detection limit were determined.The results showed that the N gene of PEDV,750 bp in size was successfully cloned,and pMD19-T-N vector was successfully constructed.After sequencing,PEDV N gene shared more than 98%homology with several PEDV strains in GenBank database,among which the closest genetic relationship with KU847996.1 was 98.93%.The linear regression R2 and amplification efficiency E were 0.994 and 97.57%,respectively.The lowest detection limit was 10 copies/μL.In conclusion,the N gene of PEDV is cloned successfully,and the SYBR Green I fluorescent real-time quantitative PCR method for detection of PEDV is successfully established based on the N gene,which provides a reference for the effective prevention and control of PEDV in the future.

关 键 词：猪流行性腹泻病毒 N基因 遗传进化树 实时荧光定量PCR 

分 类 号：S188[农业科学—农业基础科学]