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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 |
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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.
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Received: 07 July 2021
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Corresponding Authors:
**huangyu_815@163.com; fuyuan163@163.com
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