Portable biochip-based qPCR for the simultaneous detection of Pediococcus with novel genetic markers identified via genomic analysis

The genus Pediococcus plays a significant role in the fermentation of products such as fermented vegetables, meats, and dairy, contributing to flavor development, preservation, and probiotic benefits. The functional contributions of Pediococcus species vary, making species-level differentiation crucial for optimizing fermentation processes and ensuring product quality and safety. However, their high genetic similarity makes species-level differentiation challenging with conventional methods, which struggle to distinguish closely related species. To overcome this, a portable quantitative polymerase chain reaction (qPCR) system integrated with a biochip was developed to simultaneously detect 10 Pediococcus species. Through bioinformatics, novel genetic markers for each species were identified. Portable qPCR demonstrated high sensitivity, detecting down to 10¹ colony-forming units/ml for all target species, with clear Ct values and no cross-reactivity. Compared to conventional methods (16S rRNA gene sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) that face limitations due to genetic homogeneity or database gaps, the portable qPCR assay offered enhanced accuracy and specificity. The 10-well biochip design allowed the rapid detection of all species in real time, completing a single run in <20 min, and was successfully applied to the on-site monitoring of various food products, providing significant benefits for quality control in different fermentation environments. This system is a reliable, field-deployable solution for microbial monitoring, addressing the limitations of traditional methods. It can improve quality control and optimize fermentation processes, ensuring safety and consistency in food production.