Development of a novel rapid visual LAMP platform for detection of methicillin-resistant Staphylococcus aureus (MRSA) in food.

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) has been increasingly identified as a significant foodborne pathogen that can causes severe infections in humans. Furthermore, MRSA has become a pervasive presence in both food products and healthcare environments. In order to address the lack of rapid, on-site detection methods capable of producing visual readouts for MRSA, a novel detection system targeting the nuc (S. aureus-specific) and mecA (methicillin-resistance) genes was developed using an enzyme-primed probe-based loop-mediated isothermal amplification (EP-LAMP) method. This method builds upon conventional loop-mediated isothermal amplification (LAMP) by incorporating a circular probe and the RNase H2 enzyme. The process of hybridization of the ribonucleotide within the probe to the complementary deoxyribonucleotide sequence of the target gene is followed by RNase H2 cleavage of the ribonucleotide site. This results in the separation of the fluorescent reporter from its quencher and subsequent signal generation. The results can be visually interpreted using a smartphone interface. For the nuc gene, the limit of detection (LOD) of the EP-LAMP method was 5.94 × 10¹ copies per μL for plasmid DNA and 4.17 × 10¹ CFU mL^-1 for genomic DNA. Similarly, the LOD for the mecA gene was 6.53 × 10¹ copies per μL for plasmid DNA and 4.17 × 10¹ CFU mL^-1 for genomic DNA. All reactions were completed within 30 minutes. In clinical sample testing, EP-LAMP exhibited 100% concordance with quantitative PCR (qPCR) results. The method's efficacy as a tool for MRSA detection in clinical settings is attributable to its high sensitivity, excellent specificity, and rapid turnaround time. Moreover, the visual readout facilitates straightforward result interpretation, enabling point-of-care diagnostics in resource-limited environments.