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

Abstract

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 2 n . 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.

Mass Spectrometric Multiplex Detection of MicroRNA and Protein Biomarkers for Liver Cancer

“…Xu et al effectively realized the simultaneous detection of nucleic acid and protein, and the protein was recognized by the aptamer and converted into fluorescence detection of specific released DNA sequence . Chen et al employed an isothermal self-priming exponential amplification reaction (SPEXPAR) to achieve their simultaneous detection …”

“…12 Chen et al employed an isothermal self-priming exponential amplification reaction (SPEXPAR) to achieve their simultaneous detection. 13 However, there are only a few reports on the multiplex detection of nucleic acid and protein at present, mainly for the following three reasons. First, the properties and content distribution of nucleic acid and protein are different, which determines their different detection schemes.…”

Cas12a-assisted RTF-EXPAR for accurate, rapid and simple detection of SARS-CoV-2 RNA

“…Notably, Carter et al recently described a new isothermal method (called RTF-EXPAR) by combining exponential amplification reaction (EXPAR) with reverse transcription-free (RTF) step to convert RNA to DNA to achieve indirectly detection of the Orf1ab gene, allowing complete test within 10 min ( Carter et al, 2021 ). Despite great progress, these isothermal amplification diagnostics often suffer from problems associated with nonspecific DNA amplification, followed by high rates of false-positive diagnosis ( Chen et al, 2021b ; Feng et al, 2021 ; Reid et al, 2018 ; Schneider et al, 2019 ; Tomita et al, 2008 ). Moreover, they are much less sensitive than RT-qPCR ( Glökler et al, 2021 ).…”

Self-Customized Multichannel Exponential Amplifications Regulate Powered Monitoring of Terminal Deoxynucleotidyl Transferase Activity

“…With the expeditious development of nucleic acid biochemistry in the field of biomedical study, numerous excellent isothermal nucleic acid amplifications (e.g., cyclical strand-displacement amplification, − rolling circle amplification, − and nicking-assisted exponential amplification − ) have been developed as useful analytical tools for biochemical analysis and disease diagnosis. Due to the structural and functional information encoded in nucleic acids and the predictivity, programmability, and design flexibility of oligonucleotide probes, isothermal nucleic acid biosensing has dramatically broadened the scope of analytical science and sensing technology for realizing the sensitized detection of various interested target analytes (e.g., proteins, metal ions, nucleic acids, organics, viruses, and cells).…”