鸭瘟病毒TaqMan荧光定量PCR检测方法的建立及应用

doi:10.11843/j.issn.0366-6964.2025.02.026

摘要/Abstract

摘要：

为建立一种鸭瘟病毒(duck plague virus, DPV)TaqMan荧光定量PCR快速检测方法，基于DPV US6基因保守序列设计特异性引物和探针，构建重组质粒DPV-US6标准品，对方法敏感性、特异性和重复性进行评价，并应用于DPV在鸡胚成纤维细胞(CEF)上增殖及人工感染鸭器官组织上分布规律研究。结果显示，成功建立了DPV TaqMan荧光定量PCR检测方法，该方法最低检测限可以达到10 copies·μL^-1，与番鸭细小病毒、鸭圆环病毒、鸭坦布苏病毒、鸭呼肠孤病毒、H9N2亚型禽流感病毒、鸭甲型肝炎病毒(Ⅰ型和Ⅲ型)和鸭衣原体均无交叉反应，批间和批内变异系数均小于2.0%。DPV-AX株感染CEF细胞后，病毒核酸拷贝数检测结果表明，4~8 h病毒增殖缓慢，12~60 h迅速上升，60 h达到最高峰，72~144 h逐渐下降，与TCID₅₀法测定的病毒滴度相比，两种检测方法具有良好相关性，可实现拷贝数替代TCID₅₀。DPV人工感染鸭组织脏器的病毒载量分布检测表明，肝脏中的病毒载量最高。本试验建立的检测方法为研究DPV-AX株在CEF上的增殖规律提供工具。

关键词: 鸭瘟病毒, US6基因, TaqMan探针, 荧光定量PCR, 增殖规律, 人工感染

Abstract:

To establish a rapid TaqMan fluorescent quantitative PCR detection method for duck plague virus (DPV), specific primers and probes were designed based on the conserved sequence of the DPV US6 gene, and a recombinant plasmid DPV-US6 standard was constructed. The sensitivity, specificity, and reproducibility of the method were evaluated, and it was applied to study the proliferation of DPV in chicken embryo fibroblast (CEF) cells and the distribution pattern of virus load in the organs and tissues of artificially infected ducks. The results showed that the DPV TaqMan fluorescent quantitative PCR detection method was successfully established, with a minimum detection limit of 10 copies·μL^-1. There was no cross-reaction with Muscovy duck parvovirus, duck circovirus, duck Tembusu virus, duck reovirus, H9N2 subtype avian influenza virus, duck hepatitis A virus types Ⅰ and Ⅲ, or duck chlamydia. The inter-batch and intra-batch coefficient of variation were both less than 2.0%. After DPV-AX strain infected CEF cells, the viral nucleic acid copy number detection results indicated that the virus proliferated slowly from 4 to 8 hours, rapidly increased from 12 to 60 hours, peaked at 60 hours, and gradually declined from 72 to 144 hours. Compared with the virus titer determined by the TCID₅₀ method, the two detection methods showed good correlation, allowing the copy number to replace TCID₅₀. The virus load distribution detection in organs and tissues of ducks artificially infected with DPV showed the highest virus load in the liver. The detection method established in this experiment provides a tool for studying the proliferation pattern of the DPV-AX strain in CEF cells.

Key words: duck plague virus, US6 gene, TaqMan probe, fluorescence quantitative PCR, proliferation characteristics, artificial infection

中图分类号:

S852.659.1

于江玮, 程慧敏, 林健, 杨宝琳, 黄程, 杨志远, 胡格. 鸭瘟病毒TaqMan荧光定量PCR检测方法的建立及应用[J]. 畜牧兽医学报, 2025, 56(2): 765-773.

YU Jiangwei, CHENG Huimin, LIN Jian, YANG Baolin, HUANG Cheng, YANG Zhiyuan, HU Ge. Establishment and Application of TaqMan Fluorescent Quantitative PCR Detection Method for Duck Plague Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 765-773.

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引物名称 Primers	引物序列(5′→3′) Primer sequence (5′→3′)	扩增片段长度/bp Amplification fragment length
DPV-F	GCCACGTCCAAAACAGCAAT	514
DPV-R	AGTTCGGCCTGTGATCCAAG	514
DPV-q1-F	GGTATTTGTGGGTGGGTCATCTA	128
DPV-q1-R	CAGTCAAGCCTATCCTCTTCGTC
探针Probe	FAM-CAACCAAAGATGATAAGCGCCGCCAC-TAMRA
DPV-q2-F	GTATTTGTGGGTGGGTCA	123
DPV-q2-R	CAAGCCTATCCTCTTCGTC	123

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