Universal all-in-one hyper-branched rolling circle amplification based biosensor for quantitative and qualitative detection of nucleic acids

Abstract

Universal, simple, sensitive, and specific approaches for the quantitative and qualitative detection of nucleic acids are urgently needed in clinical diagnosis and biological detection. Therefore, in this study, a universal all-in-one hyper-branched rolling circle amplification (HRCA) based biosensor (referred to as the all-in-one HRCA biosensor) was developed for highly sensitive and specific nucleic acid quantification and quantitative detection using an elaborately screened detection set, which includes a padlock probe, forward primer, reverse primer, and molecular beacon (MB). The developed all-in-one biosensor exhibited a broad linear dynamic range of 1 fM to 0.1 nM and high specificity, enabling discrimination of single base differences in the target sequence. The test results of real samples showed that the established all-in-one HRCA biosensor could be used for qualitative or quantitative detection of nucleic acids in cells, throat swabs, and plasma samples, demonstrating high accuracy. Therefore, we envision that the all-in-one HRCA biosensor can serve as a robust assay for universal, sensitive and specific detection of various nucleic acid targets, as well as clinical molecular diagnosis.