Wang Zhiyun, Zhao Qiuzi, Huang Mengqian, Duan Yuqin, Li Feifei, Wang Tao
School of Environmental Science and Engineering, Tianjin University, Tianjin, China.
School of Life Sciences, Tianjin University, Tianjin, China.
Front Microbiol. 2022 Jun 30;13:934475. doi: 10.3389/fmicb.2022.934475. eCollection 2022.
Influenza A viruses (IAV) are classified based on their surface proteins hemagglutinin (HA) and neuraminidase (NA). Both pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses pose a significant threat to public health. Effective methods to simultaneously distinguish H1N1 and H5N1 are thus of great clinical value. In this study, a protocol for detection of HA proteins of both H1N1 and H5N1 was established. Specifically, we designed an aptasensor for HA using fluorescence resonance energy transfer (FRET) strategy combined with DNase I-assisted cyclic enzymatic signal amplification. HA aptamers of H1N1 and H5N1 IAVs labeled with various fluorescent dyes were used as probes. Graphene oxide (GO) acted as a FRET acceptor for quenching the fluorescence signal and protected aptamers from DNase I cleavage. The fluorescence signal was recovered owing to aptamer release from GO with HA protein. DNase I-digested free aptamers and HA proteins were able to further interact with more fluorescent aptamer probes, resulting in increased signal amplification. The limits of detection (LOD) of H5N1 HA and H1N1 HA were 0.73 and 0.43 ng/ml, respectively, which were 19 and 27 times higher than LOD values obtained with the DNase I-free system. The recovery rate of HA protein in human serum samples ranged from 88.23 to 117.86%, supporting the accuracy and stability of this method in a complex detection environment. Our rapid, sensitive, and cost-effective novel approach could be expanded to other subtypes of IAVs other than H1N1 and H5N1.
甲型流感病毒（IAV）是根据其表面蛋白血凝素（HA）和神经氨酸酶（NA）进行分类的。大流行H1N1（pH1N1）病毒和高致病性禽流感（HPAI）H5N1病毒均对公众健康构成重大威胁。因此，能够同时区分H1N1和H5N1的有效方法具有重要的临床价值。在本研究中，建立了一种检测H1N1和H5N1 HA蛋白的方案。具体而言，我们设计了一种基于荧光共振能量转移（FRET）策略并结合DNase I辅助循环酶信号放大的HA适配体传感器。用各种荧光染料标记的H1N1和H5N1 IAVs的HA适配体用作探针。氧化石墨烯（GO）作为FRET受体，用于淬灭荧光信号并保护适配体不被DNase I切割。由于适配体与HA蛋白从GO上释放，荧光信号得以恢复。经DNase I消化的游离适配体和HA蛋白能够进一步与更多荧光适配体探针相互作用，从而增强信号放大。H5N1 HA和H1N1 HA的检测限（LOD）分别为0.73和0.43 ng/ml，分别比无DNase I系统获得的LOD值高19倍和27倍。人血清样本中HA蛋白的回收率在88.23%至117.86%之间，证明了该方法在复杂检测环境中的准确性和稳定性。我们这种快速、灵敏且经济高效的新方法可扩展至H1N1和H5N1以外的其他IAV亚型。
