摘要
为快速鉴别检测H3、N2和N8亚型禽流感病毒(Avian influenza virus,AIV),建立了一种可同时检测H3、N2、N8亚型AIV的三重RT-LAMP(triplex reverse transcript loop-mediated isothermal amplification,tRT-LAMP)方法。根据NCBI中H3、N2和N8亚型AIV的NA基因保守序列分别设计3套LAMP引物,同时还设计了与FIP的F1c反向互补的荧光探针(FD),并通过退火的方式将FD结合到标记淬灭基团的FIP(FIP)上,形成FIP-FD复合探针后的荧光被淬灭,当FD与FIP分离后,可检测到荧光信号。通过优化反应温度、特异性、敏感性、干扰性和临床样品检测等成功建立了同时检测H3、N2、N8亚型AIV的tRT-LAMP方法。该方法只需要67℃恒温条件就能准确检测和鉴别AIV的3种不同目的基因,且检测其他相关的禽类病原时不产生非特异性扩增;每反应管检测H3、N2和N8亚型AIV最低限度分别为756、735、790 copies/μL,不同浓度模板之间互不干扰;临床样品检测结果显示,tRT-LAMP结果与RT-qPCR及测序结果完全一致。结果表明该方法具有快速、经济、灵敏和特异的优点,扩增结果还可直接肉眼观察,可应用于临床同时检测H3、N2和N8亚型禽流感病毒。
In order to rapidly identify and detect H3,N2 and N8 subtypes of avian influenza virus(AIV),a triplex reverse transcript loop-mediated isothermal amplification(tRT-LAMP)method was established to detect H3,N2 and N8 subtypes of AIV at the same time.Three sets of LAMP primers were designed according to the conserved sequences of HA gene of H3 AIV,NA gene of N2 isotype AIV and NA gene of N8 isoform AIV in NCBI,and fluorescent probes(FD)that were inversely complementary to F1c of FIP were also designed,and FD was combined to FIP(FIP)labeled with quenching group by annealing,and the fluorescence of FD was quenched after the FIP-FD composite probe was formed,and the fluorescence signal could be detected when FD and FIP were separated.In this study,tRT-LAMP for the simultaneous detection of H3,N2 and N8 isoforms of AIV was successfully established by optimizing the reaction temperature,specificity,sensitivity,interference and clinical sample detection.The results showed that the method only required 67℃constant temperature to accurately detect and identify three different target genes of AIV,and did not produce non-specific amplification with other related avian pathogens,and the minimum levels of H3,N2 and N8 subtype AIV were 756,735 and 790 copies/μL per reaction tube,respectively,and the different concentrations of templates did not interfere with each other,and the detection results of clinical samples showed that the results of tRT-LAMP were 100%consistent with the results of qRT-PCR and sequencing.The above results show that the method has the advantages of rapidity,economy,sensitive and specificity,and the amplification results can be directly observed by the naked eye,which can be widely used in primary clinical practice.
作者
黄青红
谢芝勋
李孟
李丹
罗思思
张民秀
谢丽基
余丹
HUANG Qing-hong;XIE Zhi-xun;LI Meng;LI Dan;LUO Si-si;ZHANG Min-xiu;XIE Li-ji;YU Dan(College of Animal Science and Technology,Guangxi University,Nanning,Guangxi,530004,China;Guangxi Veterinary Research Institute,Guangxi Key Laboratory of Veterinary Biotechnology,Key Laboratory of China(Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control,Ministry of Agriculture and Rural Affairs of China,Nanning,Guangxi,530001,China)
出处
《动物医学进展》
北大核心
2025年第2期1-9,共9页
Progress In Veterinary Medicine
基金
广西重点研发计划项目(桂科AB21076004)
广西自然科学基金项目(2022GXNSFAA035445,2021GXNSFBA196031)
广西八桂学者专项(2019-A50)
广西基本科研业务费(桂科专项24-8)。
