Development and application of a real-time fluorescent RAA assay for H9 subtype avian influenza viruses.

IF 1.9 4区医学 Q3 GENETICS & HEREDITY

Virus Genes Pub Date : 2025-08-01 Epub Date: 2025-06-11 DOI:10.1007/s11262-025-02167-x

Wanting Zhou, Xiaoqi Li, Jie Tian, Shuo Liu, Cheng Peng, Jinping Li, Guangyu Hou, Jizhe Yang, Wenming Jiang, Hualei Liu

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引用次数: 0

Abstract

The H9N2 subtype of avian influenza virus (AIV) is a critical pathogen responsible for avian infectious diseases, inducing respiratory symptoms in poultry and exhibiting high susceptibility to coinfections, which complicates clinical diagnosis. In this study, we designed specific primers and probes targeting the hemagglutinin (HA) gene of H9N2 AIV and developed a real-time fluorescent reverse transcription recombinase-aided isothermal amplification (RT-RAA) assay through systematic optimization of reaction components and conditions. The established method demonstrated exclusive reactivity with the H9N2 subtype, yielding negative results for all other tested viral pathogens, thereby showcasing high specificity. Analytical sensitivity testing revealed the capability to detect as low as 13.5 copies/μL of H9N2 viral RNA, indicating superior sensitivity. To further validate the practical application of this method, a total of 48 clinical samples, comprising oropharyngeal and cloacal swabs, were tested using the developed RT-RAA assay. The results revealed a positivity rate of 79%, reflecting strong diagnostic performance. This study presents a rapid RT-RAA assay characterized by high specificity and sensitivity, offering a robust technical platform for the immediate identification of H9N2 AIV and facilitating epidemiological surveillance in avian populations.

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H9亚型禽流感病毒实时荧光RAA检测方法的建立与应用。

H9N2亚型禽流感病毒（AIV）是禽类传染病的重要病原体，可引起家禽呼吸道症状，并对共感染具有高度易感性，使临床诊断复杂化。本研究设计了针对H9N2 AIV血凝素（HA）基因的特异性引物和探针，通过系统优化反应组分和条件，建立了实时荧光逆转录重组酶辅助等温扩增（RT-RAA）检测方法。所建立的方法对H9N2亚型具有排他性，对其他所有病毒病原体检测结果均为阴性，具有较高的特异性。分析灵敏度测试表明，该方法可检测低至13.5拷贝/μL的H9N2病毒RNA，具有较高的灵敏度。为了进一步验证该方法的实际应用，使用所开发的RT-RAA检测方法对总共48份临床样本（包括口咽和肛肠拭子）进行了检测。结果显示阳性率为79%，反映出较强的诊断性能。本研究提出了一种特异性和敏感性高的快速RT-RAA检测方法，为H9N2 AIV的即时鉴定和鸟类种群的流行病学监测提供了强有力的技术平台。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Virus Genes 医学-病毒学

CiteScore

3.30

自引率

0.00%

发文量

审稿时长

3 months

期刊介绍： Viruses are convenient models for the elucidation of life processes. The study of viruses is again on the cutting edge of biological sciences: systems biology, genomics, proteomics, metagenomics, using the newest most powerful tools. Huge amounts of new details on virus interactions with the cell, other pathogens and the hosts – animal (including human), insect, fungal, plant, bacterial, and archaeal - and their role in infection and disease are forthcoming in perplexing details requiring analysis and comments. Virus Genes is dedicated to the publication of studies on the structure and function of viruses and their genes, the molecular and systems interactions with the host and all applications derived thereof, providing a forum for the analysis of data and discussion of its implications, and the development of new hypotheses.