An APE1-assisted cyclic amplification biosensor based on rolling circle amplification for highly sensitive quantitative and qualitative detection of nucleic acids

Sensitive, specific and simple quantitative and qualitative detection of nucleic acids is crucial for clinical diagnosis, especially in resource-limited settings. Therefore, in this study, an apurinic/apyrimidinic endonuclease 1 (APE1)-assisted cyclic amplification biosensor based on rolling circle amplification (RCA), referred to as A-RCA, was developed using molecular beacons as signal probes for highly sensitive and specific quantitative and qualitative detection of nucleic acids. The experimental results demonstrated that A-RCA exhibits exceptional sensitivity, with a detection limit of 19.5 aM and 14.3 aM for Mycoplasma pneumoniae ( M. pneumoniae ) DNA and M. pneumoniae RNA, respectively. In addition, A-RCA exhibits a broad linear range extending from 0.1 fM to 0.1 nM, and high specificity, enabling it to distinguish M. pneumoniae from other common respiratory pathogens. More importantly, a fluorescent test strip approach based on A-RCA (A-RCA-strip) was developed, enabling sensitive and specific point-of-care testing for the qualitative detection of nucleic acids. Similarly, the A-RCA-strip demonstrates high sensitivity, capable of detecting target signals as low as 0.1 fM, and exhibits high specificity in distinguishing M. pneumoniae from other common respiratory pathogenic microorganisms. Clinical detection showed that both A-RCA and A-RCA-strip could accurately identify M. pneumoniae , with 100 % concordance to established clinical methods. As a proof of concept, A-RCA and A-RCA-strip can detect other nucleic acid targets by simply changing the target sequence, providing a robust and versatile strategy for developing new clinical detection methods.