Identifying a novel Listeria innocua subgroup and enhancing Listeria detection in grain: A duplex real-time PCR approach informed by comparative genomic analysis

This study focuses on enhancing the detection accuracy of Listeria species in the food industry, particularly addressing the challenges posed by closely related strains that complicate pathogen identification. Detecting Listeria monocytogenes, a key psychrotolerant foodborne pathogen, is crucial in the food industry. Although real-time PCR-based methods targeting the iap gene are commonly employed for identification, our investigation of L. innocua strains isolated from wheat in South Korea revealed false positives, indicating an imperative for more accurate detection approaches. We used comparative genomic analysis to identify a novel subgroup of L. innocua with closer genomic affiliations to L. monocytogenes. Consequently, we developed new primer and probe sets for more accurate detection of both L. monocytogenes and L. innocua, demonstrating high specificity and selectivity. Additionally, we revealed the importance of food matrix effects, particularly color pigments, in the design of diagnostic assays. These advances provide insight into genomic differences between L. monocytogenes and L. innocua, and pioneer new detection techniques for Listeria species.