Department of Bioengineering, Rice University, Houston, TX, USA.
Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, MD, USA.
Sci Rep. 2023 Nov 21;13(1):20397. doi: 10.1038/s41598-023-47582-y.
Cervical cancer is a leading cause of death for women in low-resource settings despite being preventable through human papillomavirus (HPV) vaccination, early detection, and treatment of precancerous lesions. The World Health Organization recommends high-risk HPV (hrHPV) as the preferred cervical cancer screening strategy, which is difficult to implement in low-resource settings due to high costs, reliance on centralized laboratory infrastructure, and long sample-to-answer times. To help meet the need for rapid, low-cost, and decentralized cervical cancer screening, we developed tailed primer isothermal amplification and lateral flow detection assays for HPV16, HPV18, and HPV45 DNA. We translated these assays into a self-contained cartridge to achieve multiplexed detection of three hrHPV genotypes in a disposable cartridge. The developed test achieves clinically relevant limits of detection of 50-500 copies per reaction with extracted genomic DNA from HPV-positive cells. Finally, we performed sample-to-answer testing with direct lysates of HPV-negative and HPV-positive cell lines and demonstrated consistent detection of HPV16, HPV18, and HPV45 with 5000-50,000 cells/mL in < 35 min. With additional optimization to improve cartridge reliability, incorporation of additional hrHPV types, and validation with clinical samples, the assay could serve as a point-of-care HPV DNA test that improves access to cervical cancer screening in low-resource settings.
尽管人乳头瘤病毒(HPV)疫苗接种、早期发现和癌前病变治疗可预防宫颈癌,但宫颈癌仍是资源匮乏地区女性死亡的主要原因之一。世界卫生组织建议采用高危型 HPV(hrHPV)作为宫颈癌筛查的首选策略,但由于成本高、依赖集中式实验室基础设施以及样本周转时间长,该策略在资源匮乏地区难以实施。为满足对快速、低成本和分散式宫颈癌筛查的需求,我们开发了用于 HPV16、HPV18 和 HPV45 DNA 的长尾引物等温扩增和侧向流检测分析。我们将这些分析转化为一个独立的试剂盒,以在一次性试剂盒中实现三种 hrHPV 基因型的多重检测。该检测方法在提取 HPV 阳性细胞的基因组 DNA 时,可达到 50-500 拷贝/反应的临床相关检测限。最后,我们直接对 HPV 阴性和 HPV 阳性细胞系的裂解物进行了样本至答案的测试,并证明在<35 分钟内可稳定检测到 5000-50000 个细胞/mL 的 HPV16、HPV18 和 HPV45。通过进一步优化以提高试剂盒的可靠性、纳入更多的 hrHPV 类型以及对临床样本进行验证,该检测方法可作为一种即时护理 HPV DNA 检测方法,改善资源匮乏地区宫颈癌筛查的可及性。