Simultaneous Visual Detection of 12 Pathogenic Bacteria Based on Multiplex PCR-Gene Membrane Chip Technology

Rapid and accurate detection of pathogenic bacteria is of critical concern in the food and pharmaceutical sectors. In this study, a gene membrane chip detection method combining multiplex PCR with reverse dot blot hybridization was developed to enable visual, high-throughput simultaneous identification of 12 common pathogenic bacteria. Specific primers and probes were designed targeting Klebsiella pneumoniae, Proteus mirabilis, Vibrio parahaemolyticus, Enterobacter cloacae, Pseudomonas putida, Listeria monocytogenes, Salmonella spp., Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Clostridium perfringens, and Staphylococcus aureus. The multiplex PCR reaction system, hybridization temperature, and color development conditions were systematically optimized to construct the gene membrane chip detection platform. The specificity, limit of detection, and stability of the method were rigorously evaluated. Results demonstrated that the 12 selected specific primer pairs effectively amplified the corresponding targets, yielding amplicon lengths ranging from 81 to 298 bp, with no nonspecific amplification observed. Under the optimized detection system, each probe produced clear, visually distinct color spots exclusively for its target, with no cross-reactivity. The method achieved a limit of detection as low as 0.01 ng/μL for mixed templates, and exhibited excellent intra- and inter-assay reproducibility. The established visual gene membrane chip assay successfully enables simultaneous differentiation of 12 control bacteria in a single reaction, providing a robust technical foundation and promising potential for rapid screening in food and drug safety, but its practical application requires further validation through authentic or spiked food samples.