鸭呼肠孤病毒TaqMan与SYBR Green荧光定量检测方法的建立及比较  

Establishment and comparison of TaqMan and SYBR Green real-time fluorescence quantitative PCR detection methods for novel duck reovirus

作　　者：陆婧 李敏[1] 王艳 朱盈名 赵自亮 邓欣竹 刘霞[3] 张立武 程方俊[1] 赵光伟[1] LU Jing;LI Min;WANG Yan;ZHU Yingming;ZHAO Ziliang;DENG Xinzhu;LIU Xia;ZHANG Liwu;CHENG Fangjun;ZHAO Guangwei(Immunology Research Center,Medical Research Institute,Southwest University,Chongqing 4024609 China;Shanghai Customs,Shanghai 200135,China;Guizhou Provincial Center for Animal Disease Control and Prevention,Guiyang 550008,China;Chongqing Sanjiezhongjcin Bioengineering Co.,Ltd.,Chongqing 402460,China)

机构地区：[1]西南大学动物医学免疫研究中心,重庆402460 [2]上海海关,上海200135 [3]贵州省动物疫病预防控制中心,贵州贵阳55OOO8 [4]重庆三捷众新生物工程有限公司,重庆402460

出　　处：《中国兽医学报》2022年第2期237-243,共7页Chinese Journal of Veterinary Science

基　　金：贵州省科技支撑计划资助项目(黔科合支撑[2020]1Y032)。

摘　　要：本研究旨在针对新型鸭呼肠孤病毒(NDRV)建立两种快速、敏感、特异的荧光定量PCR诊断方法并对其临床实用性进行比较。试验根据呼肠孤病毒S1基因序列设计合成1对特异性引物和1条MGB探针,分别建立TaqMan和SYBR GreenⅠ两种荧光定量PCR检测方法,并对两种方法的特异性、灵敏性、重复性进行比较。结果显示,两种检测方法标准曲线的相关系数均为0.999,对鸭细小病毒(DPV)、A型鸭甲肝病毒(DHV-1)、C型鸭甲肝病毒(DHV-3)、鸭传染性支气管炎(IBV)、鸭坦布苏病毒(DTMUV)、鹅星状病毒(JSHV)的检测结果均为阴性,特异性良好。SYBR GreenⅠ法的最低检测限度为10;拷贝/μL,TaqMan法的最低检测限度为10;拷贝/μL,前者的灵敏度比后者高10倍。SYBR GreenⅠ法和TaqMan法重复性试验的组内和组间变异系数均分别小于2.1%和1.8%。利用这两种方法对临床30份疑似病料进行检测,其中TaqMan法检出29份,SYBR GreenⅠ法检出30份,符合率为97%。综上,两种荧光定量方法均可用于临床NDRV的检测,尤以SYBR GreenⅠ法灵敏度更高。To develop a fast, sensitive, specific TaqMan and SYBR GreenⅠfluorescent quantitative PCR diagnostic methods for duck reovirus(NDRV) and compare their clinical practicability, a pair of specific primers and a MGB probe were designed and synthesized according to the S1 gene sequence of reovirus.The results showed that the correlation coefficients of the standard curves of the two detection methods were all 0.999,and the detection results of duck parvovirus(DPV),duck hepatitis A virus(DHV-1),duck hepatitis A virus(DHV-3),duck infectious bronchitis(IBV),duck Tembusu virus(DTMUV) and goose astrovirus(JSHV) were all negative.The lowest detection concentration of SYBR GreenⅠmethod was 10;copies/μL,and the TaqMan method was 10;copies/μL.The coefficient of variation of SYBR GreenⅠand TaqMan methods less than 2.1% and 1.8%.The two methods were used to detect 30 suspected clinical samples, of which 29 were positive by TaqMan method and 30 were positive by SYBR GreenⅠmethod, with a coincidence rate of 97%.In conclusion,both fluorescence quantitative methods can be used to detect clinical NDRV,and SYBR GreenⅠmethod has higher sensitivity.

关 键 词：新型鸭呼肠孤病毒 荧光定量PCR TaqMan探针法 SYBR Green法 

分 类 号：S852.65[农业科学—基础兽医学]