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 Green Ⅰ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 × 103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.
Establishment of SYBR Green Ⅰ dye fluorescence quantitative PCR(qPCR)method for detection of duck astrovirus type 1 and its application in virus isolation and identification
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 ORF1a 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 4.64×103 copies/μL,which was 10 times higher than the normal RT-PCR 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 coef-ficient of variations(Cv)in intra-and inter-assays were 0.85％-2.85％and 0.21％-2.94％,re-spectively,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 coinci-dence rate was 94.29％when compared with the normal RT-PCR method.A positive sample ran-domly taken for the virus isolation through duck embryo passage,and the allantoic fluid was col-lected 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 clini-cal diagnosis,isolation and identification,and molecular epidemiology monitoring of DAstV-1.
duck astrovirus type 1isolation and identificationdiagnostic methodSYBR green Ⅰdye
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