High-risk human papillomavirus (HPV) testing can significantly decline the incidence and mortality of cervical cancer. Microfluidic technology provides an effective method for accurate detection of high-risk HPV by utilizing multiplex single-cell droplet polymerase chain reaction (PCR). However, current strategies are limited by low-integration microfluidic chip, complex reagent system, expensive detection equipment and time-consuming droplet identification. Here, we developed a novel multiplex droplet PCR method that directly detected high-risk HPV sequences in single cells. A multiplex microfluidic chip integrating four flow-focusing structures was designed for one-step and parallel droplet preparation. Using single-cell droplet PCR, multi-target sequences were detected simultaneously based on a monochromatic fluorescence signal. We applied machine learning to automatically identify the large populations of single-cell droplets with 97% accuracy. HPV16, 18 and 45 sequences were sensitively detected without cross-contamination in mixed CaSki and Hela cells. The approach enables rapid and reliable detection of multi-target sequences in single cells, making it powerful for investigating cellular heterogeneity related to cancer diagnosis and treatment.

高危人乳头瘤病毒 (HPV) 检测可显着降低宫颈癌的发病率和死亡率。微流控技术利用多重单细胞液滴聚合酶链反应 (PCR) 为准确检测高危 HPV 提供了一种有效方法。然而，目前的策略受到低集成度的微流控芯片、复杂的试剂系统、昂贵的检测设备和耗时的液滴识别的限制。在这里，我们开发了一种新型多重液滴 PCR 方法，可直接检测单细胞中的高危 HPV 序列。集成四个流聚焦结构的多重微流控芯片被设计用于一步和平行液滴制备。使用单细胞液滴 PCR，基于单色荧光信号同时检测多目标序列。我们应用机器学习以 97% 的准确率自动识别大量单细胞液滴。在混合的 CaSki 和 Hela 细胞中，可以灵敏地检测到 HPV16、18 和 45 序列，而不会发生交叉污染。该方法能够快速可靠地检测单个细胞中的多靶点序列，使其成为研究与癌症诊断和治疗相关的细胞异质性的有力工具。