Integrating Helicase-Dependent and Rolling Circle Amplification in a Single Tube for Colorimetric Detection of Staphylococcus aureus

Background/Objectives: Rapid, equipment-free molecular detection of bacterial pathogens at the point of care (POC) remains a critical challenge. Staphylococcus aureus is a leading cause of severe infections, necessitating simple and sensitive diagnostic tools. Methods: We developed an integrated assay combining helicase-dependent amplification (HDA) and rolling circle amplification (RCA) in a sequential ‘one-pot’ format. Asymmetric HDA generates short, single-stranded amplicons from S. aureus DNA, enabling specific padlock probe ligation and subsequent exponential RCA. For equipment-free visual detection, biotin-labeled nucleotides are incorporated during RCA, and products are captured on a silica membrane and detected using a streptavidin-HRP conjugate with 3,3′,5,5′-tetramethylbenzidine substrate, producing an unambiguous blue color. Results: The assay detected as few as 101 genome copies of S. aureus per reaction. Evaluation against a panel of nine non-target respiratory pathogens and human genomic DNA demonstrated 100% specificity, with no cross-reactivity. The entire procedure is performed isothermally at 65 °C in a single tube with a total assay time of approximately 90 min. Conclusions: This ‘one-pot’ HDA-RCA colorimetric assay combines high sensitivity and specificity for S. aureus in a user-friendly, almost equipment-free format. Its simplicity and robust visual readout make it a promising tool for POC diagnostics in resource-limited settings, enabling rapid clinical decisions without specialized instrumentation.