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
10.16303/j.cnki.1005-4545.2024.06.06
- VernacularTitle:鸭星状病毒1型SYBR Green Ⅰ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
- Author:
Yu TAO
1
;
Xudong FENG
;
Yanli FAN
;
Yan WANG
;
Ziliang ZHAO
;
Xiaowei YANG
;
Liwu ZHANG
;
Xiang CHEN
;
Guangwei ZHAO
Author Information
1. 西南大学动物医学院,重庆荣昌 402460
- Keywords:
duck astrovirus type 1;
isolation and identification;
diagnostic method;
SYBR green Ⅰdye
- From:
Chinese Journal of Veterinary Science
2024;44(6):1127-1132,1139
- CountryChina
- Language:Chinese
-
Abstract:
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.
