鹅源星状病毒TaqMan探针和SYBR Green Ⅰ染料法荧光定量PCR检测方法的建立及其应用比较  

Establishment and application comparison of TaqMan probe and SYBR Green Ⅰ dye fluorescence quantitative PCR (qPCR) methods for the detection of goose astrovirus

作　　者：邓昕竹 余群[1] 朱盈名 赵自亮 陆婧 赵光伟[1,2] 张立武 程方俊 杨晓伟[1,2] DENG Xinzhu;YU Qun;ZHU Yingming;ZHAO Ziliang;LU Jing;ZHAO Guangwei;ZHANG Liwu;CHENG Fangjun;YANG Xiaowei(CollegeofVeterinaryMedicine,SouthwestUniversity,Rongchang,Chongqing 402460,China;Chongqing Sanjiezhongzin Bioengineering Co.,Ltd.,Rongchang,Chongqing402460,China)

机构地区：[1]西南大学动物医学学院,重庆荣昌402460 [2]重庆三杰众鑫生物工程有限公司,重庆荣昌402460

出　　处：《中国兽医学报》2022年第10期1970-1976,共7页Chinese Journal of Veterinary Science

基　　金：农牧高新技术产业研发专项基金资助项目;贵州省科技支撑基金资助项目(2020-1Y032)。

摘　　要：鹅源星状病毒(goose astrovirus, GoAstV)是国内新发雏鹅痛风病病原,为更好地应用于临床检测,本试验针对GoAstV分别建立了基于TaqMan探针和SYBR GreenⅠ染料的2种实时荧光定量PCR方法,并比较两者的优劣。首先根据GoAstV的ORF1b基因保守序列设计特异性引物和探针,通过优化反应条件和反应体系,分别建立TaqMan和SYBR GreenⅠ荧光定量PCR方法,并对其进行特异性、敏感性、重复性测定和临床样品检验。结果显示,2种方法均能特异性检测GoAstV,Ct值在质粒标准品浓度2.5×10~2~2.5×10~8 copies/μL范围内线性关系良好,相关系数均为0.999;探针法和染料法的最低检测限分别为2.5×10~1,2.5×10~2 copies/μL;2种方法重复性试验组内与组间变异系数均小于2.50%。对近1年收集的71份临床疑似样本进行检测,探针法和染料法的阳性检出率分别为49.30%和42.25%,探针法的检出率较高。结果表明,本试验建立的TaqMan探针和SYBR GreenⅠ染料荧光定量PCR方法均具有特异性强、敏感性高、重复性好优点,可应用于临床GoAstV的检测,其中尤以TaqMan探针法的灵敏度较高。Goose astrovirus(GoAstV) is an emerging causative agent of gosling gout in China.For better application in clinical detection, two qPCR methods based on TaqMan probe and SYBR GreenⅠdye were established.Specific primers and probes were designed according to the conserved sequence of ORF1 b gene of GoAstV.After optimizing the reaction conditions and system, specificity, sensitivity and reproducibility of these two methods were validated and compared.Also, utilizing these two methods, 71 samples collected during June 2020 to June 2021 were detected.Results showed that both methods had good linear relationships(R~2=0.999) at concentration 2.5×10~8 copies/μL to 2.5×10~2 copies/μL of standard plasmids.The minimum detection limits of probe method and SYBR GreenⅠdye method were 2.5×10~1 copies/μL and 2.5×10~2 copies/μL,respectively.The coefficients of variation within and between the two methods were less than 2.50%.Positive detection rates for the clinical samples of the two methods were 49.30% and 42.25%,respectively.All these results indicated that both qPCR methods were specific,sensitive and duplicable for the GoAstV detection and can be applied in the detection of clinical gosling gout.Especially,TaqMan probe method presented higher sensitivity than SYBR GreenⅠ dye method.

关 键 词：鹅源星状病毒 荧光定量PCR TAQMAN探针 SYBR GreenⅠ染料 

分 类 号：S852.65[农业科学—基础兽医学]