A Duplex Isothermal RPA–Lateral Flow Platform for Concurrent Detection of Staphylococcus Aureus and mecA-Mediated Methicillin Resistance
- 1Department of Clinical Pathology, Faculty of Medicine, , Navamindradhiraj University, Bangkok 10300, Thailand;
- 2Department of Pathology, Faculty of Medicine, , Nakhon Nayok 26120, Thailand; (S.S.); (K.O.)
- 3Center of Excellence in Medical and Environmental Innovation Research (CEMEIR), , Bangkok 10110, Thailand
Abstract
Background/Objectives: Rapid identification of Staphylococcus aureus and differentiation of methicillin-resistant S. aureus (MRSA) remains critical to infection control and antimicrobial stewardship, particularly where rapid screening is desirable. In this study, we developed and evaluated a duplex recombinase polymerase amplification coupled with lateral flow dipstick (RPA–LFD) platform enabling simultaneous detection of the species-associated spa locus and the methicillin resistance determinant mecA within a single isothermal reaction. Methods: Primer–probe architecture and reaction parameters were systematically adjusted, with 39 °C selected as the operating temperature, to enable concurrent visual detection of both targets within 15 min. Results: In a preliminary ten-fold serial dilution experiment, visible detection was observed down to 10¹ CFU/mL for both targets, whereas conventional PCR produced visible amplification down to 10² CFU/mL under the respective assay conditions. This difference represents an assay-specific observation and was not used to establish a statistically validated detection limit. No cross-reactivity was observed against a panel of non-target bacterial species. A preliminary evaluation of 25 nasal swab-derived DNA specimens showed complete concordance with reference PCR within this limited sample set. Conclusions: These findings demonstrate the feasibility of concurrent visual detection of species-associated and resistance targets within a single RPA–LFD configuration. Although further analytical and clinical validation is required, the platform provides a technical framework for simplified duplex isothermal detection of S. aureus and mecA.
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BibTeX
@article{ckumdee2026duplex,
title = {{A Duplex Isothermal RPA–Lateral Flow Platform for Concurrent Detection of Staphylococcus Aureus and mecA-Mediated Methicillin Resistance}},
author = {Jutturong Ckumdee and Somchai Santiwatanakul and Kwanchai Onruang and Thongchai Kaewphinit},
journal = {Diagnostics (Basel, Switzerland)},
year = {2026},
month = oct,
doi = {10.3390/diagnostics16203295},
url = {https://europepmc.org/article/PMC/PMC13703673},
note = {CC BY},
}
RIS
TY - JOUR
TI - A Duplex Isothermal RPA–Lateral Flow Platform for Concurrent Detection of Staphylococcus Aureus and mecA-Mediated Methicillin Resistance
AU - Jutturong Ckumdee
AU - Somchai Santiwatanakul
AU - Kwanchai Onruang
AU - Thongchai Kaewphinit
PY - 2026
DA - 2026/10/01
JO - Diagnostics (Basel, Switzerland)
DO - 10.3390/diagnostics16203295
UR - https://europepmc.org/article/PMC/PMC13703673
AB - Background/Objectives: Rapid identification of Staphylococcus aureus and differentiation of methicillin-resistant S. aureus (MRSA) remains critical to infection control and antimicrobial stewardship, particularly where rapid screening is desirable. In this study, we developed and evaluated a duplex recombinase polymerase amplification coupled with lateral flow dipstick (RPA–LFD) platform enabling simultaneous detection of the species-associated spa locus and the methicillin resistance determinant mecA within a single isothermal reaction. Methods: Primer–probe architecture and reaction parameters were systematically adjusted, with 39 °C selected as the operating temperature, to enable concurrent visual detection of both targets within 15 min. Results: In a preliminary ten-fold serial dilution experiment, visible detection was observed down to 10¹ CFU/mL for both targets, whereas conventional PCR produced visible amplification down to 10² CFU/mL under the respective assay conditions. This difference represents an assay-specific observation and was not used to establish a statistically validated detection limit. No cross-reactivity was observed against a panel of non-target bacterial species. A preliminary evaluation of 25 nasal swab-derived DNA specimens showed complete concordance with reference PCR within this limited sample set. Conclusions: These findings demonstrate the feasibility of concurrent visual detection of species-associated and resistance targets within a single RPA–LFD configuration. Although further analytical and clinical validation is required, the platform provides a technical framework for simplified duplex isothermal detection of S. aureus and mecA.
SN - 2075-4418
ER -