Double special bodyguard: Selenium-enhanced loop-mediated isothermal amplification combined with CRISPR/Cas12a biosensing strategy

Abstract

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.

Keywords: CRISPR; Detection; Isothermal amplification; Selenium.