Abstract

Diarrhea caused by bacterial and viral pathogens remains a major cause of childhood morbidity and mortality, leading to dehydration, malnutrition, and substantial healthcare burdens; rapid and accurate diagnosis is therefore essential for timely treatment. In clinical practice, detection still commonly relies on traditional methods such as stool culture and enzyme-linked immunosorbent assays. These approaches are not only time-consuming and lack sensitivity but also typically require separate, pathogen-specific tests, complicating comprehensive diagnosis. While quantitative PCR (qPCR) offers speed and sensitivity, dye-based methods lack specificity, and fluorescent probe-based assays are costly. To address these limitations, we developed an integrated assay that combines PCR with a sequence-specific invasive reaction (Invader) in a single closed tube. This method detects six major pediatric enteric pathogens: Shigella spp., Vibrio cholerae, non-typhoidal Salmonella, Campylobacter jejuni, rotavirus, and enteric adenovirus (types 40/41). The assay demonstrated high sensitivity, with detection limits as low as 2 copies per reaction for each target and exhibited no cross-reactivity. The entire process was completed within 45 minutes. By employing universal Invader probes, we reduced the cost per test to approximately 2 RMB. Validation using both simulated and clinical stool samples suggested the assay’s potential reliability and consistency with standard methods. This closed-tube PCR-Invader assay provides a rapid, cost-effective, and unified platform for the parallel detection of six major diarrheal pathogens in a single test run. It shows promise as a practical alternative to current disparate and pathogen-specific testing strategies, with considerable potential for use in primary healthcare settings and large-scale screening to guide the management of childhood infectious diarrhea.