Development and application of a triplex ddPCR method for simultaneous detection of MLSB resistance genes in feces...

Macrolide-lincosamide-streptogramin B (MLS_B) resistance has emerged as a critical public health challenge, significantly reducing therapeutic efficacy and exacerbating healthcare burdens. Therefore, the establishment of advanced molecular diagnostic techniques for the identification of MLS_B resistance genes is critically important. Such techniques not only offer precise guidance for clinical therapy, but also provide a robust foundation for the development of effective epidemiological surveillance strategies. In this study, a novel droplet digital PCR (ddPCR) method was developed that enables the simultaneous detection and absolute quantification of three MLS_B resistance genes (ermB, ermF, and ermG). Through rigorous design and validation of primers and probes, combined with systematic optimization of ddPCR parameters, including annealing temperature, concentration of primer/probe sets and extension time, the optimized method was able to detect single-copy erm genes in samples. Additionally, the developed triple ddPCR method demonstrated excellent sensitivity, with limits of detection (LOD) ranging from 3.37 to 8.31 copies per reaction, and good repeatability (coefficient of variation < 20%), adequately meeting the requirements for accurate quantification of erm genes. Furthermore, this new method was applied to a total of 137 samples, including 18 human fecal samples, 40 animal-derived food samples, 20 sewage samples, 35 surface water samples and 24 farm-derived samples. Among them, 97.08% detected positive for ermB, 91.97% for ermF, and 88.32% for ermG, with erm gene concentrations ranging from undetectable to 4.79 × 10⁹ copies/g. In summary, this novel ddPCR method enables efficient detection and quantification of MLS_B resistance genes, providing valuable support for antimicrobial resistance surveillance and evidence-based infection control measures.