Introduction: Infectious diarrhea remains a leading cause of childhood morbidity . Current diagnostic approaches—including stool culture and immunoassays—are time-consuming, lack sensitivity , or require separate tests for different pathogens. Although qPCR offers speed and sensitivity , dye-based methods lack specificity , while probe-based assays are costly for multiplex detection. Objectives: To develop a novel, closed-tube assay that integrates PCR amplification with Invader-based sequence-specific detection for rapid, low-cost identification of six major pediatric diarrheal pathogens. Methods: We designed a two-stage, high-annealing-temperature PCR protocol that seamlessly couples amplification with Invader reaction in a single closed tube, eliminating the need for probe modifications. The assay targets Shigella spp., Vibrio cholerae, non-typhoidal Salmonella, Campylobacter jejuni, rotavirus, and enteric adenovirus (types 40/41). Analytical performance was evaluated using plasmid DNA and reference strains; clinical validation was performed on simulated and actual stool samples. Results: The assay achieved a detection limit of 2 copies per reaction for each target, with no cross-reactivity . The entire process—from amplification to detection—was completed within 45 minutes in a closed-tube format on a standard qPCR instrument. By employing universal Invader probes, the per-test cost was reduced to approximately 2 RMB. Validation confirmed concordance with reference methods. Conclusions: This PCR-Invader assay provides a rapid, low-cost, and contamination-free platform for the parallel detection of six major diarrheal pathogens. It offers a practical alternative to current disparate testing strategies, with strong potential for deployment in primary healthcare settings and large-scale screening to guide targeted therapy and reduce antibiotic misuse in childhood infectious diarrhea.