Teste rápido para detecção de bactérias resistentes a antibióticos baseado em Loop Mediated Isothermal Amplification (LAMP) colorimétrica e ensaio de fluxo lateral

Abstract

Bacterial resistance to antibiotics represents one of the major threats to global public health, and addressing this issue requires the development of rapid, sensitive, and reliable diagnostic methods for the identification of pathogens and antimicrobial resistance-associated genes. The increasing prevalence of bacteria resistant to clinically used antibiotics has exacerbated the impact of bacterial infections and significantly limited the available therapeutic options. In this context, pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus spp., and macrolide-resistant bacteria are of particular concern, and their identification based on genetic determinants of resistance is essential to guide clinical decision-making and control strategies. In this regard, the present study developed and optimized a molecular system based on isothermal DNA amplification, namely Loop-Mediated Isothermal Amplification (LAMP), combined with colorimetric detection using a pH indicator, targeting the genes nuc, mecA, vanA, and ermB, which are associated with the identification of S. aureus and resistance to methicillin, vancomycin, and erythromycin, respectively. Additionally, for the mecA and vanA genes, the LAMP reaction was coupled to a lateral flow assay for the development of a multiplex detection system. LAMP reaction parameters were optimized with respect to temperature, incubation time, Bst 2.0 DNA polymerase concentration, and primer concentrations, resulting in an efficient and reproducible protocol. Assay specificity was confirmed against different bacterial species, with no cross-reactivity observed for any of the evaluated targets. The limit of detection determined by colorimetric reading, estimated through probit regression analysis, was 48 DNA copies/µL of reaction for both targets. When combined with lateral flow detection, the system allowed detection down to 30 DNA copies/µL of reaction for the mecA gene and 10 DNA copies/µL of reaction for the vanA gene. Furthermore, the feasibility of simultaneous detection of mecA and vanA in a single reaction was demonstrated, highlighting the potential of this approach for the rapid identification of multiple clinically relevant bacterial resistance markers.