禽流感病毒H10N3亚型一步法双重RT-PCR检测方法的建立  

Establishment of a one-step duplex RT-PCR assay for detection of H10N3 subtype avian influenza viruses

作　　者：杨芷翊 王新凯 柳洋 付思源 张钰炘 史玉婷 曹琛福 贾伟新[1] YANG Zhi-yi;WANG Xin-kai;LIU Yang;FU Si-yuan;ZHANG Yu-xin;SHI Yu-ting;CAO Chen-fu;JIA Wei-xin(National Avian Influenza Para-Reference Laboratory(Guangzhou),Guangdong Engineering Laboratory for Medicament of Zoonosis Prevention and Control,National Local Joint Engineering Laboratory of Zoonosis Prevention and Control Agents,Key Laboratory of Zoonoses of Ministry of Agriculture and Rural Affairs,Guangdong Key Laboratory for Prevention and Control of Zoonotic Diseases,College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Customs District,Shenzhen 518045,China)

机构地区：[1]华南农业大学兽医学院,国家禽流感专业实验室(广州)广东省人兽共患病防控制剂工程实验室,人兽共患病防控制剂国家地方联合工程实验室,农业农村部人畜共患病重点实验室,广东省动物源性人兽共患病防控重点实验室,广东广州510642 [2]深圳海关动植物检验检疫技术中心,广东深圳518045

出　　处：《中国兽医科学》2023年第7期811-815,共5页Chinese Veterinary Science

基　　金：国家现代农业产业技术体系项目(CARS-41);广东省科技计划项目(2021B1212030015)。

摘　　要：2021年6月初,我国报道了全球首例H10N3亚型禽流感病毒感染人的事件,该亚型病毒具有跨宿主感染人类的能力,对社会公共卫生安全形成潜在的重大危害,因此,亟须建立一种快速、简便、适用于实验室的H10N3亚型禽流感病毒核酸检测方法。将自GISAID下载的H10N3亚型禽流感病毒HA基因和NA基因序列进行分析比对后,根据其保守区域设计并筛选出2对特异性检测引物,分别对引物浓度、核酸浓度、退火温度、循环数等进行优化,以确定最佳反应条件,建立禽流感病毒H10N3亚型一步法双重RT-PCR检测方法。结果显示,该方法可分别扩增出407 bp和602 bp禽流感病毒H10基因和N3基因的特异性条带,且不与H1N1、H3N2、H6N6、H7N9、H9N2亚型禽流感病毒以及NDV、IBV、IBDV、DTMUV发生交叉反应。该方法灵敏度高,最低检出限为900 pg。在对36份已知的临床样品的检测中,H10N3亚型禽流感病毒阳性率为2.78%,与禽流感H10Nx基因型、Hx N3基因型一步法RT-PCR检测方法以及病毒分离鉴定结果一致,符合率均为100%。上述结果表明,本研究建立的一步法双重RT-PCR方法可以在一次PCR反应中检出H10与N3亚型禽流感病毒混合感染或者单一感染样品,为兽医工作者开展H10N3、H10Nx和Hx N3亚型禽流感的临床诊断和流行病学监测提供了一种简单、有效的分子生物学快速检测方法。In early June 2021,China reported the world's first case of human infection with H10N3 subtype avian influenza virus.The H10N3 virus has the ability to spread across hosts and infect humans,posing a potential threat to human public health.Therefore,it is necessary to establish a rapid,simple,sensitive,and specific nucleic acid detection method for H10N3 subtype avian influenza virus.After analyzing and comparing with the HA and NA gene sequences of H10N3 subtype avian influenza virus downloaded from GISAID,two pairs of specific detection primers were designed based on their conservative regions.We optimized the reaction system such as primer concentration and nucleic acid concentration,and optimized the reaction procedures such as annealing temperature and cycle number to determine the best reaction conditions.The specificity test results indicate that this method can amplify 407 bp and 602 bp specific bands of the H10 and N3 genes of avian influenza virus,respectively,and does not cross react with H1N1,H3N2,H6N6,H7N9,H9N2 subtypes avian influenza virus,as well as NDV,IBV,IBDV,DTMUV.The detection sensitivity of this method is high,with a minimum detection limit of 900 pg.Three methods were used to detect 36 clinical samples.The detection result using the method established in this study showed that the positive rate of H10N3 subtype avian influenza virus was 2.78%,which was consistent with the results of two-step RT-PCR detection and virus isolation.The coincidence rate of the three methods was 100%.The one-step dual RT-PCR nucleic acid detection assay established in this study can detect mixed or single infection of H10 and N3 subtype avian influenza virus in a single PCR reaction.It provides a simple and effective rapid molecular biological detection method for veterinary workers to carry out clinical diagnosis and epidemiological surveillance of H10N3,H10Nx and HxN3 subtypes of avian influenza.

关 键 词：禽流感病毒 H10N3 双重RT-PCR 检测方法 

分 类 号：S852.659.5[农业科学—基础兽医学]