Persistent infection with high-risk human papillomavirus (HR-HPV) is the primary and initiating factor for cervical cancer. With over 200 identified HPV types, including 14 high-risk types that integrate into the host cervical epithelial cell DNA, early determination of HPV infection type is crucial for effective risk stratification and management. Presently, on-site immediate testing during the HPV screening stage, known as Point of Care Testing (POCT), remains immature, severely limiting the scope and scenarios of HPV screening. This study, guided by the genomic sequence patterns of HPV, established a multiplex recombinase polymerase amplification (RPA) technology based on the concept of “universal primers.” This approach achieved the multiple amplification of RPA, coupled with the CRISPR/Cas12a system serving as a medium for signal amplification and conversion. The study successfully constructed a POCT combined detection system, denoted as H-MRC12a (HPV—Multiple RPA—CRISPR/Cas12a), and applied it to high-risk HPV typing detection. The system accomplished the typing detection of six high-risk HPV types (16, 18, 31, 33, 35, and 45) can be completed within 40 min, and the entire process, from sample loading to result interpretation, can be accomplished within 45 min, with a detection depth reaching 1 copy/μL for each high-risk type. Validation of the H-MRC12a detection system’s reproducibility and specificity was further conducted through QPCR on 34 clinical samples. Additionally, this study explored and optimized the multiplex RPA amplification system and CRISPR system at the molecular mechanism level. Furthermore, the primer design strategy developed in this study offers the potential to enhance the throughput of H-MRC12a detection while ensuring sensitivity, providing a novel research avenue for high-throughput detection in Point-of-Care molecular pathogen studies.

中文翻译：

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基于多重RPA-CRISPR/Cas12a的POCT技术及其在人乳头瘤病毒（HPV）分型检测中的应用

高危型人乳头瘤病毒（HR-HPV）持续感染是宫颈癌的首要和始发因素。已识别的 HPV 类型超过 200 种，其中包括 14 种融入宿主宫颈上皮细胞 DNA 的高危类型，早期确定 HPV 感染类型对于有效的风险分层和管理至关重要。目前，HPV筛查阶段的现场即时检测，即护理点检测（POCT）尚不成熟，严重限制了HPV筛查的范围和场景。本研究以HPV基因组序列模式为指导，建立了基于“通用引物”概念的多重重组酶聚合酶扩增（RPA）技术。该方法实现了RPA的多重扩增，并结合CRISPR/Cas12a系统作为信号放大和转换的介质。该研究成功构建了POCT联合检测系统，命名为H-MRC12a（HPV—Multiple RPA—CRISPR/Cas12a），并将其应用于高危HPV分型检测。系统可在40分钟内完成6种高危HPV型别（16、18、31、33、35、45）的分型检测，从样本加载到结果判读整个过程可在5分钟内完成。 45 min，每种高危类型检测深度达到1 copy/μL。通过 QPCR 对 34 个临床样本进一步验证了 H-MRC12a 检测系统的重现性和特异性。此外，本研究还从分子机制层面对多重RPA扩增系统和CRISPR系统进行了探索和优化。此外，本研究开发的引物设计策略有可能在确保灵敏度的同时提高 H-MRC12a 检测的通量，为床旁分子病原体研究中的高通量检测提供了新的研究途径。