多重逆转录交叉置换扩增结合测流生物传感器技术检测H5N1型禽流感病毒  

Detection of avian influenza virus-H5N1 by multiplex reverse transcription and multiple cross displacement amplification combined with lateral flow biosensor technology

作　　者：王鹏飞 赵帆 闫琳琳 王静 杨瑞锋 胡守奎 Wang Pengfei;Zhao Fan;Yan Linlin;Wang Jing;Yang Ruifeng;Hu Shoukui(Department of Clinical Laboratory,Aerospace Center Hospital,Beijing100049,China;Department of Clinical Laboratory,Peking University Shougang Hospital,Beijing100144,China;Department of Clinical Laboratory,Zhengzhou Central Hospital affiliated to Zhengzhou University,Zhengzhou 450051,China)

机构地区：[1]航天中心医院临床检验科,北京100049 [2]北京大学首钢医院临床检验科,北京100144 [3]郑州大学附属郑州中心医院临床检验科,郑州450051

出　　处：《中华检验医学杂志》2024年第11期1326-1331,共6页Chinese Journal of Laboratory Medicine

基　　金：首都卫生发展科研专项项目(首发2022-2-6042);石景山区医学重点扶持专科建设项目(石卫健医发[2020]21号)。

摘　　要：目的将逆转录(RT)、多重交叉置换扩增(MCDA)以及基于纳米颗粒的测流生物传感器(LFB)结合在一起,建立一种快速检测H5N1禽流感病毒的新方法。方法根据H5N1禽流感病毒中血凝素(HA)和神经氨酸酶(NA)的特异性基因序列分别设计一套MCDA引物组。实现在一个反应体系中对目的基因HA和NA的逆转录和MCDA扩增,最终检测结果通过LFB显示。此方法被命名为H5N1-RT-MCDA-LFB。优化H5N1-RT-MCDA-LFB方法的最佳反应条件,同时评价此方法的敏感度和特异性。结果H5N1-RT-MCDA-LFB方法在恒温65℃下孵育40 min便可实现良好的扩增效果。该方法的检测下限为100 fg/反应,检测敏感度比实时荧光定量逆转录PCR(RT-qPCR)(检测下限为10 pg/反应)高100倍。该方法在对除H5N1禽流感病毒以外的其他28种常见病毒、支原体、衣原体、细菌和真菌的检测中均为阴性。除此之外,H5N1-RT-MCDA-LFB方法在模拟临床样本中得到较好的验证,其检测下限为1×10^(2)拷贝/ml。结论H5N1-RT-MCDA-LFB方法具有简便、快速、敏感度和特异性高等特点,是一种有效检测H5N1型禽流感病毒的分子诊断技术。Objective To establish of a new method for the rapid detection of H5N1-Avian Influenza virus by combining reverse transcription(RT),multiple cross displacement amplification(MCDA)and nanoparticle-based lateral flow biosensor(LFB).Methods MCDA primers were designed based on gene sequences specific to Hemagglutinin(HA)and Neuraminidase(NA)in H5N1 avian influenza virus.The target genes HA and NA were amplified through reverse transcription and MCDA in one reaction system.Results were displayed by LFB.The assay was named as H5N1-mRT-MCDA-LFB.The reaction conditions of the H5N1-RT-MCDA-LFB method were optimized,and the sensitivity and specificity were also assessed.Results The H5N1-RT-MCDA-LFB assay could achieve good amplification effect at a constant temperature of 65℃for 40 minutes.The method had a lower limit of detection of 100 fg per reaction with 100-fold higher sensitivity than that of the RT-qPCR(lower limit of detection 10 pg per reaction).The assay was negative in detecting 28 common viruses,mycoplasmas,chlamydias,bacterias and funguses,except for H5N1.In addition,the H5N1-RT-MCDA-LFB method showed better validation in simulated clinical samples with a lower limit of detection at 1×10^(2)copies/ml.Conclusion The H5N1-RT-MCDA-LFB assay is a valuable molecular diagnostic technique for detecting H5N1 avian influenza virus due to its simplicity,rapidity,sensitivity and specificity.

关 键 词：流感病毒A型 H5N1亚型 逆转录 恒温扩增法 测流生物传感器 诊断方法 

分 类 号：R446.5[医药卫生—诊断学]