Isothermal Self-Primer EXPonential Amplification Reaction (SPEXPAR) for Highly Sensitive Detection of Single-Stranded Nucleic Acids and Proteins

Development of versatile sensing methods for sensitive and specific detection of clinically relevant nucleic acids and proteins is of great value for disease monitoring and diagnosis. In this work, we propose a novel isothermal Self-primer EXPonential Amplification Reaction (SPEXPAR) strategy based on a rationally engineered structure-switchable Metastable Hairpin template (MH-template). The MH-template initially keeps inactive with its self-primer overhanging a part of target recognition region to inhibit polymerization. The present targets can specifically compel the MH-template to transform into an "activate" conformation that primes a target-recyclable EXPAR. The method is simple and sensitive, can accurately and facilely detect long-chain single-stranded nucleic acids or proteins without the need of exogenous primer probes, and has a high amplification efficiency theoretically more than 2n. For a proof-of-concept demonstration, the SPEXPAR method was used to sensitively detect the characteristic sequence of the typical swine fever virus (CSFV) RNA and thrombin, as nucleic acid and protein models, with limits of detection down to 43 aM and 39 fM, respectively, and even the CSFV RNA in attenuated vaccine samples and thrombin in diluted serum samples. The SPEXPAR method may serve as a powerful technique for the biological research of single-stranded nucleic acids and proteins.