Development of a novel rapid genome exponential amplification reaction (GEAR) assay for high-sensitivity detection of...

Campylobacter coli is a major zoonotic foodborne pathogen, but its detection through conventional culture-based methods is challenged by intricate taxonomy, fastidious growth requirements, and auxotrophic characteristics. Prompt and precise detection of C. coli is therefore crucial for effective outbreak control and robust food safety monitoring and recall. This study describes the development and evaluation of a GEAR (genome exponential amplification reaction) test for the precise detection of C. coli. The assay combined three key elements: a simplified centrifugation-assisted DNA extraction protocol, thermal cycler-free amplification, and SYBR Green I dye-mediated visualization. Targeting the glyA gene of C. coli, the assay demonstrated high specificity, distinguishing C. coli from a non-coli strain and fourteen other bacterial species associated with foodborne diseases. GEAR amplification was performed at 62 °C for 60 min, achieving an analytical sensitivity of 50 fg/reaction, which was 100-fold higher than real-time PCR (5 pg/reaction) and 1,000-fold higher than end-point PCR (50 pg/reaction) using purified genomic DNA. In artificially contaminated pork samples, the assay detected as low as 250 CFU/g without prior enrichment and 25 CFU/g following a 12-hour enrichment, representing a 10-fold lower detection limit than real-time PCR in the food matrix. With rapid results, operational simplicity, low cost, high sensitivity, and minimal instrumentation requirements, the GEAR assay offers a promising tool for routine C. coli detection in resource-limited food testing laboratories. This represents the first reported application of GEAR for C. coli screening.