Establishment of SYBR GreenⅠReal-time Quantitative PCR Assay for Detecting Duck astrovirus-3
FU Qiu-Ling1,2,*, ZHANG Rui1,*, FU Guang-Hua1,2,**, WAN Chun-He1,2,**, CHENG Long-Fei1,2, SHI Shao-Hua1,2, CHEN Hong-Mei1,2, LIU Rong-Chang1,2, HUANG Yu1,2,**
1 Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China; 2 Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention, Fuzhou 350013, China
Abstract：Duck astrovirus-3 (DAstV-3) is a newly emerging infectious disease pathogen in ducks (Anas platyrhynchos domestica) in recent years. In this study, specific primers were designed based on the RNA-dependent RNA polymerase gene (RDRP) sequences of DAstV-3 strains isolated and published in GenBank, and a SYBR Green Ⅰ real-time quantitative PCR assay for detecting DAstV-3 was developed. The results demonstrated that a good linear relationship with the standard curve cycle threshold (Ct) and the template concentrations in the range of 1.15×101~1.15×107 copies/μL, the correlation coefficient R2 and amplification efficiency were 0.999 and 2.08, respectively. Besides, the assay had good specificity for DAstV-3 and had no cross-reaction with other common infectious disease pathogens (H9 subtype Avian influenza virus, Avian tambusu virus, Duck reovirus, Muscovy parvovirus) in ducks. High sensitivity, minimum detection limit in each rection of the assay was 23 copies for DAstV-3. Good reproducibility, the coefficient of variations (CV) in intra- and inter-assays were 0.14%~0.65% and 0.53%~0.94%, , respectively, both less than 1%, indicating that the method had a good repeatability. 209 suspected DAstV-3 samples during June 2018 and March 2021 were detected by the established assay, and the DAstV-3 detection rate was 56.5% (118/209). SYBR GreenⅠ real-time PCR assay established had good specificity, sensitivity and repeatability, which could provide an effective technical support for detection and epidemiological investigation of DAstV-3.
[1] 刘俊斌, 王钜华, 朱国强. 2019. 禽星状病毒研究进展[J]. 中国家禽, 41(22): 58-62. (Liu J B, Wang J H, Zhu G Q.2019. Research progress on Avian astrovirus[J]. Chinese Poultry, 41(22): 58-62.) [2] 刘宁, 张大丙. 2015a. 鸭星状病毒2型和3型特异性RT-PCR的建立//[C]. 第三届水禽疫病防控研讨会论文集. 中国畜牧兽医学会, pp. 58. (Liu N, Zhang D B.2015. The development of specific RT-PCR assay for detecting Duck astrovirus-2 and astrovirus-3)//[C]. Proceedings of the Third Symposium on prevention and control of waterfowl diseases. Chinese Association of Animal Science and Veterinary Medicine, pp. 58.) [3] 刘宁, 张大丙. 2015b. 鸭星状病毒CPH株的分子检测:流行病学及致病性分析//[C]. 全国禽病分子生物技术青年工作者会议. 中国畜牧兽医学会, pp. 44. (Liu N, Zhang D B.2.15. The molecular detection of Duck astrovirus CPH strain: Epidemiology and pathogenic analysis//[C]. The youth worker meeting on molecular biotechnology of national avian diseases. Chinese Association of Animal Science and Veterinary Medicine, pp. 44.) [4] 师志海, 王文佳, 张彬,等. 2021. 牛星状病毒EvaGreen实时荧光定量PCR检测方法的建立及应用[J]. 中国兽医学报, 41(1): 50-55. (Shi Z H, Wang W J, Zhang B, et al.2021. Establishment and application of EvaGreen real-time PCR assay for detecting Bovine astrovirus[J]. Chinese Journal of Veternary Sciene, 41(1): 50-55. [5] 岳稳, 张瑞, 黄瑜, 等. 2020. 扬州鹅关节痛风病例的诊断及病原鉴定[J]. 福建畜牧兽医, 42(6): 25-26. (Yue W, Zhang R, Huang Y, et al.2020. Diagnosis and pathogen identification of joint gout in Yangzhou Geese[J]. Fujian Journal of Animal Husbandry and Veternary Medicine, 42(6): 25-26.) [6] 章丽娇, 黄欣梅, 刘飞, 等. 2019. 新型鹅星状病毒AHQJ18株的分离鉴定[J]. 江苏农业学报, 35(5): 260-262. (Zhang L J, Huang X M, Liu F, et al.2018. Isolation and identification of a novel goose astrovirus AHQJ18[J]. Jiangsu Journal of Agricultural Science, 35(5): 260-262.) [7] 张蕊, 傅光华, 傅秋玲, 等. 2018. 一株樱桃谷种鸭胚源鸭3型星状病毒分离鉴定与序列分析[J]. 中国家禽, 40(21): 66-68. (Zhang R, Fu G H, Fu Q L, et al.2018. Isolation, identification and gene sequence analysis of a Duck astrovirus-3 strain from the embryo of cherry valley breeding ducks[J]. China Poultry, 40(21): 66-68.) [8] Appleton H, Higgins P G, et al.1975. Letter: Viruses and gastroenteritis in infants[J]. Lancet, 1(7919): 1297. [9] Baxendale W, Mebatsion T.2004. The isolation and characterisation of astroviruses from chickens[J]. Avian Pathology, 33(3): 364-370. [10] Biain M, Lojkic I, Tisljar M, et al.2012. Astroviruses associated with stunting and pre-hatching mortality in duck and goose embryos[J]. Avian Pathology, 41(1): 91-97. [11] Carter M, Willcocks M.1996. The molecular biology of astroviruses[J]. Archives of Virology Supplementum, 12(12): 277-277. [12] Domańska-Blicharz K, Jacukowicz A, Bocian L, et al.2014. Astroviruses in Polish commercial turkey farms in 2009-2012[J]. Avian Diseases, 58(1): 158-64. [13] Fu Y, Pan M, Wang X, et al.2009. Complete sequence of a Duck astrovirus associated with fatal hepatitis in ducklings[J]. Journal of General Virology, 90(5): 1104-1108. [14] Liao Q, Liu N, Zhang D, et al.2015. Genetic characterization of a novel astrovirus in Pekin ducks[J]. Infection, Genetics and Evolution, 32: 60-67. [15] Liu N, Jiang M, Dong Y, et al.2018. Genetic characterization of a novel group of Avastroviruses in geese[J]. Transboundary & Emerging Diseases, 65(6): 1-6. [16] Liu N, Wang F, Zhang D.2014. Complete sequence of a novel Duck astrovirus[J]. Archives of Virology, 159(10): 2823-2827. [17] Matsui S, Kim J, Greenberg H, et al.1993. Cloning and characterization of human astrovirus immunoreactive epitopes[J]. Journal of Virology, 67(3): 1712-1715. [18] Mcneilly F, Connor T, Calvert V, et al.1994. Studies on a new enterovirus-like virus isolated from chickens[J]. Avian Pathology, 23(2): 313-327. [19] Narita M, Umiji S, Furuta K, et al.1991. Pathogenicity of Avian nephritis virus in chicks previously infected with infectious bursal disease virus[J]. Avian Pathology, 20(1): 101-111. [20] Niu X, Tian J, Jing Y, et al.2018. Novel goose astrovirus associated gout in gosling, China[J]. Veterinary Microbiology, 220: 53-56. [21] Nunez LFN, Parra S, Mettifogo E, et al.2015. Isolation of Chicken astrovirus from specific pathogen-free chicken embryonated eggs[J]. Poultry Science, 94(5): 947. [22] Pankovics P, Ákos B, Kiss T, et al.2015. Detection of a mammalian-like astrovirus in bird, European roller (Coracias garrulus)[J]. Infection, Genetics and Evolution, 34(18): 114-121. [23] Wohlgemuth N, Once R, Schultz-Cherry S.2019. Astrovirus evolution and emergence[J]. Infection, Genetics and Evolution, 69: 30-37. [24] Wunner W, Calisher C, Dietzgen R, et al.2014. Classification and nomenclature of viruses sixth report of the international committee on taxonomy of viruses[J]. International Union of Microbiological Society Virology Division, 61(12): 1326-1332.
