禽流感病毒H5、H9亚型RT-RAA-LFD检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.12.033

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (12): 6308-6315.doi: 10.11843/j.issn.0366-6964.2025.12.033

禽流感病毒H5、H9亚型RT-RAA-LFD检测方法的建立

蔺雅婷¹(), 刘书怡¹(), 仇德洋¹, 杨大为¹, 陈虎¹, 李桂梅¹^,²^,³^,*(), 邵迪⁴

1. 青岛农业大学动物医学院, 青岛 266109
2. 山东省新兽药创制协同创新中心, 青岛 266109
3. 山东省兽药诊断试剂工程技术研究中心, 青岛 266109
4. 青岛海关技术中心, 青岛 266000

收稿日期:2025-03-05 出版日期:2025-12-23 发布日期:2025-12-24
通讯作者: 李桂梅 E-mail:664825313@qq.com;lsydyx0910@163.com;201201054@qau.edu.cn
作者简介:蔺雅婷(1998-)，女，山东东营人，硕士，主要从事预防兽医学研究，E-mail：664825313@qq.com
刘书怡(1999-)，女，山东济南人，硕士，主要从事预防兽医学研究，E-mail：lsydyx0910@163.com
第一联系人：
蔺雅婷和刘书怡为同等贡献作者
基金资助:
山东省重大科技创新工程项目(2024CXGC010910)；海关总署科研项目(2023HK039)

Establishment of RT-RAA-LFD Detection Method for Avian Influenza Virus H5 and H9 Subtypes

LIN Yating¹(), LIU Shuyi¹(), QIU Deyang¹, YANG Dawei¹, CHEN Hu¹, LI Guimei¹^,²^,³^,*(), SHAO Di⁴

1. College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China
2. Novel Veterinary Pharmacy Innovation Center of Shandong Province, Qingdao 266109, China
3. Research Center for Engineering Technology in Veterinary Medicine and Veterinary Diagnostic Reagent of Shandong Province, Qingdao 266109, China
4. Technology Center of Qingdao Customs, Qingdao 266000, China

Received:2025-03-05 Online:2025-12-23 Published:2025-12-24
Contact: LI Guimei E-mail:664825313@qq.com;lsydyx0910@163.com;201201054@qau.edu.cn

摘要/Abstract

摘要：

禽流感(avian influenza，AI)是威胁我国养禽业健康发展的重要疫病，其感染传播迅速、范围广泛且具有跨物种传播特性，对动物和人类健康均构成严重威胁。尤其是人畜共患的流感病毒感染危害性更大，可能引发巨大的经济损失和社会影响。因此，快速、灵敏地检测常见禽流感病毒对于控制疾病的传播具有重要意义。本研究旨在建立一种高效、便捷的禽流感检测方法。根据禽流感病毒H5N1 HA和H9N2 HA保守序列设计引物和探针，结合重组酶介导等温扩增(recombinase-aid amplification，RAA)技术和侧向流试纸条(lateral flow dipstick，LFD)技术，建立了一种禽流感病毒的RT-RAA-LFD检测方法。能够在37 ℃下15 min完成检测。结果显示：AIV H5亚型和AIV H9亚型之间，以及与其他常见禽病的核酸均无交叉反应，特异性良好。RT-RAA-LFD检测灵敏度可达每个反应100个拷贝，比普通RT-PCR检测的灵敏度高约100倍。此外，在测试120份临床标本后，RT-RAA和商品化RT-qPCR以及RT-PCR检测之间达到了99%的一致性。综上，该方法能够快速、准确、简便、直观地检测出禽流感病毒H5和H9亚型，为禽流感的检测提供了一种直观有效的方法。

关键词: 禽流感病毒, H5亚型, H9亚型, 重组酶介导等温扩增

Abstract:

Avian influenza (AI) is a significant threat to the healthy development of the poultry industry in China. It spreads rapidly and widely, with the potential for cross-species transmission, posing a severe threat to the health of both animals and humans. In particular, zoonotic influenza virus infections are highly hazardous and can cause substantial economic losses and social impacts. Therefore, the rapid and sensitive detection of common avian influenza viruses is of great significance for controlling the spread of the disease. This study aims to develop an efficient and convenient detection method for avian influenza, addressing the actual needs of prevention and control. Based on the conserved sequences of the H5N1 HA and H9N2 HA genes of the avian influenza virus (AIV), primers and probes were designed. A detection method for AIV using reverse transcription recombinase polymerase amplification (RT-RAA) combined with lateral flow dipstick (LFD) was established. This method can complete the detection in just 15 minutes at 37℃. There was no cross-reactivity between the AIV H5 subtype and the AIV H9 subtype, as well as between AIV and other viruses such as Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and infectious bursal disease virus (IBDV), indicating good specificity. The sensitivity of the RT-RAA-LFD method can reach 100 copies per reaction, which is approximately 100 times higher than that of conventional RT-PCR. Furthermore, after testing 120 clinical samples, a 99% concordance rate was achieved between RT-RAA and commercialised RT-qPCR as well as conventional RT-PCR. The results demonstrate that this method can rapidly, accurately, conveniently, and intuitively detect AIV H5 and H9 subtypes, providing an effective and intuitive approach to the detection of avian influenza.

Key words: avian influenza virus, H5 subtype, H9 subtype, recombinase-aid amplification

中图分类号:

S852.659.5

蔺雅婷, 刘书怡, 仇德洋, 杨大为, 陈虎, 李桂梅, 邵迪. 禽流感病毒H5、H9亚型RT-RAA-LFD检测方法的建立[J]. 畜牧兽医学报, 2025, 56(12): 6308-6315.

LIN Yating, LIU Shuyi, QIU Deyang, YANG Dawei, CHEN Hu, LI Guimei, SHAO Di. Establishment of RT-RAA-LFD Detection Method for Avian Influenza Virus H5 and H9 Subtypes[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6308-6315.

图/表 5

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引物/探针 Primer/Probe	序列(5′→3′) Sequences(5′→3′)	片段长度/bp Fragment length
H5N1-F	ATGAATGTATGGAAAGTGTAAGAAACGGGACG	168
H5N1-R	BIOTIN-TGATTGCCAGCACTAGGGAACTCGCCAC
H5N1-P	FAM-TAAGTGGAGTAAAACTGGAATCAATAGGAAT[THF]TACCAAATACTGTCA-C3
H9N2-F	ACAGTGGAACAAGCAATGCATGTTCAGATTCA	148
H9N2-R	BIOTIN-GGGTGATTTATTCCCCACATGAAAAGAATGC
H9N2-P	FAM-GACTCAAAAGAACAACGATTACCCTATTC[THF]AGACGCCCAATACACA-C3

病毒亚型 The virus subtype	检测方法 Detection method	阳性/份 Positive/part	阴性/份 Negative/part	阳性检出率/% Positive detection rate/%	符合率/% Compliance rate/%
H5	RT-RAA-LFD	10	110	8.3	99
	RT-PCR	9	111	7.5
	RT-qPCR试剂盒	10	110	8.3
H9	RT-RAA-LFD	10	110	8.3	99
	RT-PCR	9	111	7.5
	RT-qPCR试剂盒	10	110	8.3

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禽流感病毒H5、H9亚型RT-RAA-LFD检测方法的建立

Establishment of RT-RAA-LFD Detection Method for Avian Influenza Virus H5 and H9 Subtypes

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