SPECIAL: Phosphorothioate dNTP assisted RPA equipped with CRISPR/ Cas12a amplifier enables high-specific nucleic acid testing

Recombinase polymerase amplification (RPA) is one of the most widely used isothermal amplification methods and considered to be a promising tool for point-of-care testing (POCT) molecular diagnosis. However, RPA is prone to have nonspecific amplification occur, due to the poor recognition accuracy of polymerase and recombinase, which severely hindered its clinical application. It is important to improve the specificity of RPA further. Herein, we developed a novel nucleic acid testing method termed phosphorothioate dNTP (dNTP alpha S) assisted RPA (S-RPA) that employs dNTP alpha S as substrates to suppress nonspecific amplification effectively. We found that dNTP alpha S could improve the recognition accuracy of Bsu polymerase and recombinase, thereby enhancing their amplification specificity. Our S-RPA provided much higher specificity (approximately 40 % improvement compared to classical RPA), realizing detection target with single nucleotide mutation. Based on its outstanding performance, we further combined the S-RPA with CRISPR/Cas12a to achieve highly specific and sensitive fluorescence detection, namely S-RPA equipped with CRISPR/Cas12a amplifier (SPECIAL). Our SPECIAL was more sensitive (10-fold higher) than the classical RPA-CRISPR/Cas12a assay, offering 100 % agreement with the qPCR during clinical validation. In summary, a strategy based on dNTP alpha S was established to enhance the specificity of RPA, thereby improving its practicability and providing a potential POCT tool for molecular diagnosis.