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

Abstract

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

CLC Number:

S852.659.1

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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Establishment and Application of TaqMan Fluorescent Quantitative PCR Detection Method for Duck Plague Virus

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