Development of a novel genome exponential amplification reaction (GEAR) assay for rapid and ultrasensitive detection of Salmonella.

Abstract

Salmonella is a major contributor to foodborne disease outbreaks worldwide, making its surveillance crucial from both public well-being and food safety perspectives. Rapid and sensitive methods for monitoring Salmonella contamination in food stuffs are especially essential in low-resource settings to help avert outbreaks and enable timely product recalls. In the present work, a novel genome exponential amplification reaction (GEAR) based detection assay was developed for Salmonella by amplifying the invA gene. The inclusive and exclusive specificity of the test was verified to be 100% employing reference strains of Salmonella and other non-target pathogenic bacteria. The reaction sensitivity of the GEAR was determined to be 32.4 fg/µL or fg/reaction, making it 100 times more sensitive than conventional molecular techniques such as PCR and real-time PCR. The method sensitivity of the developed GEAR test was evaluated using experimentally contaminated pork samples, showing a detection limit of 212 CFU/g for culture independent direct detection and 2.12 CFU/g when combined with 6 h of enrichment. The assay was further validated on 140 real-world pork samples and was evaluated alongside real-time PCR and culture methods. It successfully detected five culture-positive samples identified by the ISO 6579:2002 isolation method, demonstrating 100% accuracy. This ultrasensitive, rapid assay, which does not require sophisticated instrumentation, presents a viable alternative to time-consuming microbiological methods and resource-intensive molecular techniques. To our knowledge, this is the first report of a GEAR assay developed and validated for the detection of Salmonella.