基于ORF3基因检测猪流行性腹泻病毒荧光定量RT-PCR方法的建立与应用  

Development and preliminary application of ORF3-based fluorescent quantitative RT-PCR method for detection of porcine epidemic diarrhea virus

作　　者：冉伟[1] 田宇 李梓健 张金悦 赵飞宇 李璐 孔凡志[1] 孙东波[1] RAN Wei;TIAN Yu;LI Zi-jian;ZHANG Jing-yue;ZHAO Fei-yu;LI Lu;KONG Fan-zhi;SUN Dong-bo(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国预防兽医学报》2021年第4期394-398,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：十三五国家重点研发计划(2017YFD0501604-5);黑龙江省农垦总局重点科研项目(宋军);黑龙江省杰出青年基金(JC2017007);科研团队支持计划猪病防制创新团队(TDJH201804);黑龙江省博士后科研启动基金项目(LBH-Q16188);兽医生物技术国家重点实验室开放课题(SKLVBF201908);黑龙江省博士后资助经费(LBH-Z18259)。

摘　　要：为建立猪流行性腹泻病毒(PEDV)的荧光定量RT-PCR检测方法,本研究参照已有的研究报告,对PEDV ORF3基因序列进一步比较优化,选择PEDV ORF3更为保守的序列设计引物,经PCR扩增目的片段后构建了PEDV ORF3基因的重组质粒标准品pMD18-T-ORF3,以其为模板,经反应条件优化,建立了基于PEDV ORF3基因的SYBR Green I荧光定量RT-PCR检测方法。结果显示,以重组质粒标准品构建的标准曲线的相关系数为0.9982,Ct值与质粒标准品在102拷贝/μL~1010拷贝/μL范围内具有良好的线性关系。该方法除对PEDV检测结果为阳性外,对猪传染性胃肠炎病毒、猪轮状病毒、猪德尔塔冠状病毒、猪圆环病毒3型和猪繁殖与呼吸道综合征病毒核酸扩增结果均为阴性,特异性强;该方法对重组质粒标准品的检测下限为102拷贝/μL,比普通RT-PCR灵敏100倍,敏感性高;批内和批间重复试验的变异系数均小于5%,重复性好。临床样品的检测结果显示,本研究建立的荧光定量RT-PCR检出阳性样品22份,而常规RT-PCR仅检出16份阳性样品,二者的符合率为82.86%。本研究建立的荧光定量RT-PCR方法特异性强、灵敏度高、重复性好,对PEDV的快速及定量检测具有重要意义。In order to develop a real-time reverse transcription quantitative PCR(RT-PCR) assay to detect porcine epidemic diarrhea virus(PEDV), the conserved sequence of PEDV ORF3 gene was amplified using the designed specific primers and inserted into pMD18-T. Using the constructed pMD18-T-ORF3 plasmid as the template, SYBR Green I real-time fluorescence quantitative RT-PCR was developed after optimizing conditions. Results showed that correlation coefficient of the standard curve was 0.9982 with a linear relationship when the amount of template was in the range of 102 copies/μL to 1010 copies/μL. This method was specific for detecting PEDV, and had no cross-amplifications with TGEV, PRoV, PDCoV, PCV3 or PRRSV. This method also had high sensitivity with a limit detection of 102 copies/μL, and was 100 times more sensitive than that of the regular RT-PCR. In addition, the coefficient of variation was less than 5% for both intra-and inter-assays. Clinical samples were detected using this method, results showed that 22 samples were positive, while only 16 samples were positive using regular PCR method with coincidence rate 82.86%.The method developed in this study is considered to be a high sensitive, specificity and repeatability tool for the rapid detection and quantification of PEDV.

关 键 词：猪流行性腹泻病毒 荧光定量RT-PCR ORF3基因 

分 类 号：S852.65[农业科学—基础兽医学]