A novel double-stranded probe minimizes false-positive and false-negative LAMP results with ultrasensitivity and high...

Loop-mediated isothermal amplification (LAMP) is one of the most widely used molecular diagnostic methods and is considered a promising tool for point-of-care testing (POCT). However, because LAMP requires multiple complex primers, false-positive and false-negative results can occur, thereby limiting its clinical applicability. Herein, we propose a novel chemically modified double-stranded probe (CDP) to reduce false-positive and false-negative LAMP results, thereby developing CDP-assisted LAMP (CLAMP). The CDP consists of a recognition strand (RS) and a 2'-fluoro (2'-F)-modified signal strand (SS). We found that the 2'-F-modified SS has a significantly higher melting temperature than its natural counterpart, providing excellent thermostability and a high signal-to-noise ratio. During the CLAMP reaction, the CDP specifically binds to LAMP amplicons via its RS, and the SS is efficiently displaced by Bst DNA polymerase, enabling specific fluorescence generation. With the assistance of CDP, CLAMP effectively reduces false-positive and false-negative results and tolerates common interfering compounds. Furthermore, CLAMP was 100-fold more sensitive than conventional LAMP and exhibited 100% accuracy in clinical validation. In summary, a novel CDP-based strategy was established to enhance the performance of LAMP, thereby improving its applicability and offering a promising tool for POCT molecular diagnosis.