鸭星状病毒1型SYBR GreenⅠ荧光定量PCR检测方法的建立及其在病毒分离鉴定中的应用  

Establishment of SYBR GreenⅠdye fluorescence quantitative PCR(qPCR)method for detection of duck astrovirus type 1 and its application in virus isolation and identification

作　　者：陶禹 冯旭东 樊彦丽 王艳 赵自亮 杨晓伟 张立武 陈翔 赵光伟 TAO Yu;FENG Xudong;FAN Yanli;WANG Yan;ZHAO Ziliang;YANG Xiaowei;ZHANG Liwu;CHEN Xiang;ZHAO Guangwei(College of Veterinary Medicine,Southwest University,Chongqing 402460,China;Shanghai Customs,Shanghai 200135,China;Chongqing Sanjiezhongxin Bioengineering Co.Ltd.,Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆荣昌402460 [2]上海海关,上海200135 [3]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》2024年第6期1127-1132,1139,共7页Chinese Journal of Veterinary Science

基　　金：农牧高新技术产业研发专项资助项目;西南大学实验技术研究资助项目。

摘　　要：为实现鸭星状病毒1型(Duck astrovirus type 1,DAstV-1)的快速检测,本试验针对DAstV-1(WF1202株)保守区域设计特异性引物,建立了基于染料SYBR GreenⅠ的实时荧光定量PCR(qPCR)方法,利用所建方法对临床样本进行检测,并将其应用于阳性样本病毒的分离和鉴定。结果表明所建qPCR检测方法的检测下限为4.64×10^(3)拷贝/μL,高于普通RT-PCR方法10倍;对禽类其他常见病原均为阴性,特异性良好;批内、批间变异系数(Cv)分别为0.85%~2.85%和0.21%~2.94%,均低于3.00%,表明其重复性良好。利用该方法对2020-2022年间10省份不同鸭场的35份组织病料进行检测,阳性率为25.71%(9/35),与普通RT-PCR检测结果的符合率为94.29%。任取1份阳性病料接种健康鸭胚进行病毒的分离,收集尿囊液利用所建qPCR方法进行检测,确定为阳性后接种1日龄健康雏鸭进行动物回归试验,感染雏鸭呈现一过性发病但未出现死亡,qPCR检测DAstV-1均为阳性,剖检可见肝脏有轻微出血,病理组织学观察发现肝细胞空泡变性,说明所分离的DAstV-1毒株致病性较弱。综上,本试验成功建立了DAstV-1的荧光定量PCR检测方法,可为DAstV-1的临床诊断、分离鉴定及分子流行病学监测等提供技术支撑。In order to achieve the purpose of rapid detection of duck astrovirus type 1(DAstV-1),specific primers were designed based on the conservative region of ORFla which belonged to DAstV-1(WF1202 strain).A real-time fluorescent quantitative PCR(qPCR)detective method for DAstV-1 was established.Clinical samples were detected by the qPCR method and the positive samples were used for virus isolation and identification.Results showed that the detection limit of the established method was 4.64×10^(3)copies/μL,which was 10 times higher than the normal RTPCR method.In addition,no cross-reactions were found with other common infectious disease pathogens in poultry,indicating that the qPCR method had good specificity.What's more,the coefficient of variations(C_v)in intra-and inter-assays were 0.85%-2.85%and 0.21%-2.94%,respectively,both less than 3%,indicating that the qPCR method had a good repeatability.Using this method,35 tissue samples from different duck farms in 10 provinces from 2020 to 2022 were detected for DAstV-1.Results showed that the positive rate was 25.71%(9/35),and the coincidence rate was 94.29%when compared with the normal RT-PCR method.A positive sample randomly taken for the virus isolation through duck embryo passage,and the allantoic fluid was collected and then was verified by the qPCR method and inoculated with 1-day-old healthy ducklings for the animal regression experiment.The infected ducklings suffered from transient disease but did not die.The liver tissues were all positive with DAstV-1 when detected by qPCR.Meanwhile,autopsy showed that there were slight changes in the livers,and the histopathological observation showed that the liver cells were steatosis.These findings indicated that the isolated DAstV-1 strain had weak pathogenicity and might be a low virulent strain.To sum up,the qPCR detection method of DAstV-1 was successfully established in this work,and could provide technical support for clinical diagnosis,isolation and identification,and molecular epidemiology monitoring of DAstV-1.

关 键 词：鸭星状病毒1型 分离鉴定 诊断方法 SYBR GreenⅠ染料 

分 类 号：S852.65[农业科学—基础兽医学]