Circulating cell-free DNA (cfDNA) has attracted attention as a non-invasive biomarker for diagnosing and monitoring various cancers. Given that human papillomavirus (HPV) DNA integration and overexpression of E6/E7 oncogenes are pivotal events for carcinogenesis, we sought to determine if HPV E7 cfDNA could serve as a specific biomarker for cervical cancer detection. We applied droplet digital PCR (ddPCR) to quantify HPV16/18 E7 cfDNA from the serum of patients with cervical cancer, cervical intraepithelial neoplasia, and controls. HPV16/18 E7 cfDNA was highly specific for cervical cancer, displaying 30.77% sensitivity, 100% specificity, and an area under the curve of 0.65. Furthermore, we developed a sensitive isothermal detection of HPV16/18 E7 and the PIK3CA WT reference gene based on recombinase polymerase amplification combined with a lateral flow strip (RPA-LF). The assay took less than 30 min and the detection limit was 5–10 copies. RPA-LF exhibited 100% sensitivity and 88.24% specificity towards HPV16/18 E7 cfDNA in clinical samples. The agreement between RPA-LF and ddPCR was 83.33% (κ = 0.67) for HPV16 E7 and 100% (κ = 1.0) for HPV18 E7, indicating a good correlation between both tests. Therefore, we conclude that HPV E7 cfDNA represents a potential tumor marker with excellent specificity and moderate sensitivity for minimally invasive cervical cancer monitoring. Moreover, the RPA-LF assay provides an affordable, rapid, and ultrasensitive tool for detecting HPV cfDNA in resource-limited settings.

循环游离 DNA (cfDNA) 作为一种用于诊断和监测各种癌症的非侵入性生物标志物而引起了人们的关注。鉴于人乳头瘤病毒 (HPV) DNA 整合和 E6/E7 癌基因的过表达是致癌的关键事件，我们试图确定 HPV E7 cfDNA 是否可以作为宫颈癌检测的特异性生物标志物。我们应用液滴数字 PCR (ddPCR) 来量化宫颈癌、宫颈上皮内瘤变和对照患者血清中的 HPV16/18 E7 cfDNA。HPV16/18 E7 cfDNA 对宫颈癌具有高度特异性，敏感性为 30.77%，特异性为 100%，曲线下面积为 0.65。此外，我们开发了基于重组酶聚合酶扩增结合侧向流动条 (RPA-LF) 的 HPV16/18 E7 和 PIK3CA WT 参考基因的灵敏等温检测。检测时间不到 30 分钟，检测限为 5-10 个拷贝。RPA-LF 在临床样本中对 HPV16/18 E7 cfDNA 表现出 100% 的敏感性和 88.24% 的特异性。RPA-LF 与 ddPCR 的一致性为 83.33%（HPV16 E7 的κ  = 0.67) 和 HPV18 E7 的 100% ( κ  = 1.0)，表明两种测试之间具有良好的相关性。因此，我们得出结论，HPV E7 cfDNA 代表了一种潜在的肿瘤标志物，对于微创宫颈癌监测具有极好的特异性和中等敏感性。此外，RPA-LF 检测为在资源有限的环境中检测 HPV cfDNA 提供了一种经济实惠、快速且超灵敏的工具。