Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, PR China.
Anhui Provincial Center for Animal Disease Control and Prevention, Hefei 230000, PR China.
Avian Dis. 2021 Jun;65(2):281-286. doi: 10.1637/aviandiseases-D-20-00115.
Duck viral hepatitis (DVH) mainly affects ducklings under 1 month of age, causes liver necrosis, enlargement, and hemorrhage, and is highly lethal, seriously jeopardizing the duck industry. The prevalence of duck hepatitis A virus (DHAV-1) and duck astrovirus type 3 (DAstV-3) is increasing, and coinfection is common. Moreover, the similar clinical characteristics of the DHAV-1 and DAstV-3 infections and the high frequency of coinfection make diagnosis difficult. In this study, to establish a method for the rapid, simultaneous detection of DHAV-1 and DAstV-3, two pairs of specific primers were designed according to their conserved gene regions. An SYBR Green I-based qPCR assay was successfully established that can quickly and differentially detect the two viruses. Moreover, the assay is highly specific and does not show cross-reaction with other common viruses. The detection limit of the method is 7.34 × 10 copies/µl and 3.78 × 10 copies/µl for DHAV-1 and DAstV-3, respectively, indicating high sensitivity. A total of 34 clinical samples were tested using the established method; the positive rates for DHAV-1 and DAstV-3 were 14.71% and 8.82%, respectively, and that for coinfection was 2.94% (1/34), which was better than that obtained with conventional PCR. In summary, the SYBR Green I-based qPCR assay established in this study has high specificity, good sensitivity and accuracy, high feasibility, and is rapid. Thus, it can be a powerful tool for the coinfection detection of DHAV-1 and DAstV-3 and for future epidemiologic studies.
鸭病毒性肝炎（DVH）主要影响 1 月龄以下雏鸭，引起肝脏坏死、肿大和出血，致死率高，严重危害养鸭业。鸭甲肝病毒（DHAV-1）和鸭星状病毒 3 型（DAstV-3）的流行率不断增加，且常发生混合感染。此外，DHAV-1 和 DAstV-3 感染的临床症状相似，且混合感染率高，这使得诊断变得困难。本研究旨在建立一种快速、同时检测 DHAV-1 和 DAstV-3 的方法，根据其保守基因区域设计了两对特异性引物。成功建立了基于 SYBR Green I 的 qPCR 检测方法，可快速且特异性地检测两种病毒。此外，该方法特异性高，与其他常见病毒无交叉反应。该方法的检测限分别为 DHAV-1 和 DAstV-3 的 7.34×10 拷贝/μl 和 3.78×10 拷贝/μl，表明具有较高的灵敏度。使用建立的方法检测了 34 份临床样本，DHAV-1 和 DAstV-3 的阳性率分别为 14.71%和 8.82%，混合感染率为 2.94%（1/34），优于常规 PCR。综上所述，本研究建立的 SYBR Green I 基 qPCR 检测方法具有特异性好、灵敏度高、准确性高、可行性高、快速等特点，可用于 DHAV-1 和 DAstV-3 的混合感染检测和未来的流行病学研究。
