鸭冠状病毒实时荧光定量PCR检测方法的建立及应用  

Development and Application of SYBR Green Real-time Fluorescent Quantitative PCR Method for Detection of Coronaviruses in Duck

作　　者：高月花[1] 朱彤[1] 刘丽萍 郭效珍 马芹 胡峰[1] 汪建华 于可响[1] 黄兵[1] 宋敏训[1] 李玉峰[1] GAO Yuehua;ZHU Tong;LIU Liping;GUO Xiaozhen;MA Qin;HU Feng;WANG Jianhua;YU Kexiang;HUANG Bin;SONG Minxun;LI Yufeng(The Poultry Institute of Shandong Academy of Agricultural Sciences Poultry Disease Diagnosis and Immunology,Jinan 250023,China;Shandong Hekangyuan Biological Breeding Co.Ltd,Jinan 250101,China)

机构地区：[1]山东省农业科学院家禽研究所禽病诊断与免疫重点实验室,济南250023 [2]山东和康源生物育种股份有限公司,济南250101

出　　处：《病毒学报》2023年第6期1633-1641,共9页Chinese Journal of Virology

基　　金：济南市农业应用技术创新计划(项目号:202018),题目:肉鸭新发疫病监测与生物安全防控技术集成;山东省农业科学院科技创新工程(项目号:CXGC2022A25),题目:家禽主要疫病病原监测、诊断和防控暨净化技术研究;山东省重点研发计划(项目号:2022CXGC010606),题目:畜禽重要病原监测预警与防控关键技术研发与应用;山东省家禽产业技术体系(项目号:SDAIT-11-01);国家自然基金(项目号:32002286),题目:禽网状内皮组织增生症病毒蛋白酶切割波形蛋白的分子机制;韩国农林部国际合作(项目号:122014-2),题目:鸭病毒病国际联合监测与控制技术体系的建立。

摘　　要：为建立一种鸭冠状病毒(Duck coronaviruses,DuCoV)的临床快速诊断方法,根据鸭冠状病毒1b基因保守区域设计特异性引物,成功建立了用于检测鸭冠状病毒的特异性SYBR GreenⅠ荧光定量PCR方法。该方法特异性强、敏感性高、重复性好,对鸭冠状病毒有特异性扩增,最低检测限为8.04×100拷贝/μL,比普通PCR方法敏感10倍,批内变异系数与批间变异系数分别为0.28%~0.34%、0.25%~0.36%,均小于1%。对临床可疑鸭泄殖腔拭子进行检测,本方法与常规PCR方法的检测结果阳性符合率为100%,阴性符合率为96.43%,样本总符合率为97.62%。本研究建立的鸭冠状病毒SYBR GreenⅠ实时荧光定量PCR方法,可用于鸭冠状病毒的临床快速诊断及流行病学监测。To establish a rapid diagnostic method for duck coronavirus(DuCoV),a real-time quantitative PCR(qRT-PCR)method was developed by utilizing specific primers designed according to the conserved region of the 1b gene sequence.The method showed highly sensitive,specific and reproducible for identifying DuCoV,and negative for other duck pathogens,with a sensitivity of 8.04 copies/μL which was 10 times higher than that of conventional PCR.The coefficients of variation within and between batches were 0.28%~0.34% and 0.25%~0.36%,respectively,both were less than 1%.Total of 42 clinical suspicious anal swabs were analyzed by qRT-PCR and conventional RT-PCR,the positive coincidence rate of qRT-PCR was 100%,while the negative coincidence rate was 96.43%,and the total coincidence rate of samples was 97.62%.The results showed that the qRT-PCR method developed in this study can be used for rapid clinical diagnosis and epidemiological monitoring of duck coronavirus.

关 键 词：鸭冠状病毒 检测 SYBR GreenⅠ 荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]