Early and sensitive detection of avian influenza virus (AIV) such as H9N2 is needed as AIV threatens human health and the food industry. Here, we developed an ultrasensitive and naked-eye counting strategy for quantifying AIV by two probes. One probe was a complex of M13 phage and gold nanoparticles (GNPs) modified with antiphage and anti-AIV antibody. The other probe was magnetic nanoparticles modified with the same anti-AIV antibody. The two probes cocaptured target AIV to form a sandwich structure. Because each M13-GNP probe contains multiple phages and each phage could be plated into an eye-visible plaque, the phages acted as a signal amplifier. After the phages from the sandwich structure were plated, the copy numbers of resultant plaques and target AIV were numerically correlated. Consequently, counting the number of the plaques with the naked eyes resulted in the quantification of the target AIV. The sensitivity of the naked-eye counting strategy for quantification of AIV was determined to be down to 50 PFU/mL, close to the sensitivity of digital polymerase chain reaction. With the feature of ultrasensitivity, the phage-GNP probe–mediated naked-eye counting method holds a great promise for early visualized quantification of pathogenic viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

由于禽流感病毒威胁着人类健康和食品工业，因此需要尽早而灵敏地检测出禽流感病毒（H9N2）。在这里，我们开发了一种超灵敏的肉眼计数策略，用于通过两种探针定量AIV。一种探针是用抗噬菌体和抗AIV抗体修饰的M13噬菌体和金纳米颗粒（GNP）的复合物。另一个探针是用相同的抗AIV抗体修饰的磁性纳米颗粒。两种探针共同捕获靶标AIV以形成夹心结构。因为每个M13-GNP探针都包含多个噬菌体，并且每个噬菌体都可以铺在肉眼可见的噬菌斑中，所以这些噬菌体可以充当信号放大器。将来自夹心结构的噬菌体铺板后，将所得噬菌斑的拷贝数与靶标AIV进行数值关联。所以，用肉眼计数斑块的数量导致目标AIV的定量。肉眼计数策略对AIV定量的灵敏度被确定为低至50 PFU / mL，接近数字聚合酶链反应的灵敏度。噬菌体-GNP探针介导的裸眼计数方法具有超高灵敏度的特征，可用于早期可视化定量病原病毒，例如严重急性呼吸系统综合症冠状病毒2（SARS-CoV-2）。