Long Keyi, Han Tiao, Hu Wenxi, Yang Mei, Huo Danqun, Huang Zhen, Hou Changjun
Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College of Chongqing University, Chongqing, 400044, PR China.
Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College of Chongqing University, Chongqing, 400044, PR China.
Anal Chim Acta. 2025 Oct 1;1369:344353. doi: 10.1016/j.aca.2025.344353. Epub 2025 Jun 20.
Cervical cancer is closely associated with human papillomavirus (HPV) infection, underscoring the necessity for effective HPV detection. Various biosensors utilizing isothermal amplification techniques have been developed for HPV DNA detection. These methods offer high sensitivity and specificity, making them suitable for point-of-care testing. However, the in vitro DNA polymerases used in these isothermal amplification methods lack a repair system, may leading to polymerization errors. This limitation highlights the need for improved detection methods that can enhance accuracy and reliability in HPV testing. Here, a sensing strategy called selenium nucleic acid-enhanced loop-mediated isothermal amplification (Se-LAMP) combined with CRISPR/Cas12a (SLC) was developed. The SLC has two specific bodyguards, which ensure the accuracy and specificity of biosensors through two aspects. One bodyguard is dNTPαSe, a supplement added to the initial Se-LAMP step, which guarantees the accuracy of DNA polymerase and reduces background signals. The other bodyguard is CRISPR system, integrating three functions of specific verification, signal amplification and signal output. CRISPR activation can only rely on Se-LAMP products successfully rechecked by crRNA. As a readable signal of SLC, the detection limit was as low as 0.38 copies/μL (0.64 fM) within 65 min, which was one order of magnitude lower than that of non-selenium-modified methods and two orders of magnitude lower than that of qPCR. The SLC is the first to use CRISPR system as the output mode of Se-LAMP, proving the CRISPR/Cas12a system has good recognition ability for selenium-modified nucleic acid. The ultrasensitive dual-specificity guard enables SLC, ensuring accuracy in biodetection. This innovation offers novel clinical testing approaches and holds significance for diagnosing and monitoring disease progression.
宫颈癌与人乳头瘤病毒（HPV）感染密切相关，这凸显了有效检测HPV的必要性。已经开发了各种利用等温扩增技术的生物传感器用于HPV DNA检测。这些方法具有高灵敏度和特异性，适用于即时检测。然而，这些等温扩增方法中使用的体外DNA聚合酶缺乏修复系统，可能导致聚合错误。这一局限性突出了需要改进检测方法，以提高HPV检测的准确性和可靠性。在此，开发了一种名为硒核酸增强环介导等温扩增（Se-LAMP）与CRISPR/Cas12a相结合的传感策略（SLC）。SLC有两个特定的“保镖”，从两个方面确保生物传感器的准确性和特异性。一个“保镖”是dNTPαSe，它是在初始Se-LAMP步骤中添加的一种补充物，可保证DNA聚合酶的准确性并降低背景信号。另一个“保镖”是CRISPR系统，它整合了特异性验证、信号放大和信号输出这三种功能。CRISPR激活只能依赖于被crRNA成功复检的Se-LAMP产物。作为SLC的可读信号，在65分钟内检测限低至0.38拷贝/μL（0.64 fM），比非硒修饰方法低一个数量级，比qPCR低两个数量级。SLC首次将CRISPR系统用作Se-LAMP的输出模式，证明CRISPR/Cas12a系统对硒修饰核酸具有良好的识别能力。超灵敏的双特异性保护使SLC得以实现，确保了生物检测的准确性。这一创新提供了新的临床检测方法，对疾病的诊断和病情监测具有重要意义。
