Wang Weifeng, Jiang Min, Liu Ying, Wu Xuan, Chen Yan, Zhang Xiaoyun, Liu Weiwei
Department of Laboratory Medicine, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Department of Laboratory Medicine, Shanghai Tenth People's Hospital, Tongji University of Medicine, Shanghai, China.
Front Microbiol. 2025 Jan 29;16:1528094. doi: 10.3389/fmicb.2025.1528094. eCollection 2025.
The persistent infection of high-risk HPV (HR-HPV) is intricately linked to the onset and progression of cervical cancer. This research endeavored to develop a high-throughput 2D PCR method for closed-tube genotyping of 11 HR-HPVs.
Base-quenched probes were specifically designed for FAM, VIC, and CY5 channels. The 2D PCR system underwent optimization, with its detection performance assessed in terms of specificity and sensitivity. Plasmid mixtures was used to simulate multiple infections of HPV, providing preliminary insights into the detection efficacy and throughput of the 2D PCR technology. Ultimately, the detection capability of this method was assessed using clinical samples.
The sequenced tags, when paired with primers, could generate Tm differences exceeding 3°C. These were then integrated with a fluorescent channel and Tm to differentiate and identify target genes upon detection. The refined 2D PCR system was confirmed to be free from cross-reactions and exhibited high specificity, capable of detecting 12 target genes within a single tube. A total of 294 cervical exfoliated cell samples were tested using 2D PCR and flow fluorescence hybridization method. The overall concordance between the two detection methods was 96.17% (Kappa = 0.910).
The 2D PCR method, which integrates asymmetric PCR amplification with melting curve analysis, has the capacity to detect 11 types of HR-HPVs across three channels. This closed-tube detection approach offers several benefits including high throughput, straightforward operation, and low detection cost. Consequently, it can be effectively utilized for early screening and prevention of cervical cancer.
高危型人乳头瘤病毒（HR-HPV）的持续感染与宫颈癌的发生和发展密切相关。本研究致力于开发一种用于11种HR-HPV封闭管基因分型的高通量二维聚合酶链反应（2D PCR）方法。
针对FAM、VIC和CY5通道专门设计了碱基淬灭探针。对2D PCR系统进行了优化，并从特异性和灵敏度方面评估了其检测性能。使用质粒混合物模拟HPV的多重感染，初步了解2D PCR技术的检测效果和通量。最终，使用临床样本评估该方法的检测能力。
测序标签与引物配对时，可产生超过3°C的熔解温度（Tm）差异。然后将这些差异与荧光通道和Tm整合，以便在检测时区分和鉴定靶基因。优化后的2D PCR系统被证实无交叉反应，具有高特异性，能够在单管内检测12个靶基因。使用2D PCR和流式荧光杂交法对294份宫颈脱落细胞样本进行了检测。两种检测方法的总体一致性为96.17%（Kappa = 0.910）。
将不对称PCR扩增与熔解曲线分析相结合的2D PCR方法能够通过三个通道检测11种HR-HPV。这种封闭管检测方法具有高通量、操作简单和检测成本低等优点。因此，它可有效地用于宫颈癌的早期筛查和预防。
