Non-spectroscopic multiplex molecular diagnosis for simultaneous detection of virulence and antibiotic resistance genes from pathogenic bacteria.

The widespread use of antibiotics has accelerated the emergence of antibiotic-resistant bacteria, posing a significant threat to global public health. This resistance is often mediated by plasmids that transfer antibiotic resistance genes between bacteria, converting susceptible strains into resistant ones. Accordingly, there is an urgent need for diagnostic platforms capable of simultaneously identifying both the genomic DNA of pathogenic bacteria and plasmid-encoded resistance genes to guide effective treatment and limit the spread of resistant infections. While conventional multiplex molecular diagnostic tools, such as quantitative real-time polymerase chain reaction (qPCR), offer high sensitivity, they require thermocyclers and complex fluorescence optics. To overcome these limitations, we developed a simplified platform that integrates loop-mediated isothermal amplification (LAMP) with a retroreflective Janus microparticle (RJP)-based non-spectroscopic optical detection approach. Under isothermal conditions, dual-labeled DNA amplicons are generated with an antigenic small molecule on one end and biotin on the other. These amplicons are selectively captured via antigen-antibody interactions on a sensing surface, and the signal is transduced through binding to avidin-coated RJPs, which are visualized using only an LED and a standard camera. This approach enables multiplexed detection of distinct DNA targets using a single type of RJP probe. As a model assay, we simultaneously detected the invA gene of Salmonella typhimurium, a major foodborne pathogen, and the plasmid-encoded tetA gene responsible for tetracycline resistance. Both genes similarly obtained LOD values at the level of 0.59 CFU/reaction (equivalent to 196 CFU/mL), with detection ranging from 1-104 CFU/reaction (equivalent to 3.3 × 101 - 3.3 × 105 CFU/mL). The platform is especially valuable in field applications where rapid identification and resistance profiling are critical, such as pre-screening in the food supply chain or during outbreak response. Our system offers robust, multiplex molecular diagnostics on a single chip, with strong potential for point-of-care use in both food safety and clinical settings.