{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:35:53Z","timestamp":1785404153705,"version":"3.56.0"},"reference-count":27,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,4,9]],"date-time":"2026-04-09T00:00:00Z","timestamp":1775692800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012190","name":"Ministry of Science and Higher Education of the Russian Federation","doi-asserted-by":"crossref","award":["075-15-2025-582"],"award-info":[{"award-number":["075-15-2025-582"]}],"id":[{"id":"10.13039\/501100012190","id-type":"DOI","asserted-by":"crossref"}]},{"award":["075-15-2025-582"],"award-info":[{"award-number":["075-15-2025-582"]}],"id":[{"id":"https:\/\/ror.org\/00ghqgy32","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>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 \u2018one-pot\u2019 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\u2032,5,5\u2032-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 \u00b0C in a single tube with a total assay time of approximately 90 min. Conclusions: This \u2018one-pot\u2019 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.<\/jats:p>","DOI":"10.3390\/diagnostics16081131","type":"journal-article","created":{"date-parts":[[2026,4,9]],"date-time":"2026-04-09T13:09:31Z","timestamp":1775740171000},"page":"1131","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Integrating Helicase-Dependent and Rolling Circle Amplification in a Single Tube for Colorimetric Detection of Staphylococcus aureus"],"prefix":"10.3390","volume":"16","author":[{"given":"Polina","family":"Chirkova","sequence":"first","affiliation":[{"name":"Engelhardt Institute of Molecular Biology (EIMB), Russian Academy of Sciences, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3183-318X","authenticated-orcid":false,"given":"Dmitry","family":"Gryadunov","sequence":"additional","affiliation":[{"name":"Engelhardt Institute of Molecular Biology (EIMB), Russian Academy of Sciences, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5468-4119","authenticated-orcid":false,"given":"Alexander","family":"Chudinov","sequence":"additional","affiliation":[{"name":"Engelhardt Institute of Molecular Biology (EIMB), Russian Academy of Sciences, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9011-134X","authenticated-orcid":false,"given":"Sergey","family":"Lapa","sequence":"additional","affiliation":[{"name":"Engelhardt Institute of Molecular Biology (EIMB), Russian Academy of Sciences, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"100363","DOI":"10.1016\/j.snr.2025.100363","article-title":"A universal fluorescent lateral flow strip approach based on molecular beacon-assisted rolling circle amplification for point-of-care nucleic acid detection","volume":"10","author":"Chen","year":"2025","journal-title":"Sens. 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