Species-specific detection of Cronobacter Genus in infant formula using gyrB-targeted TaqMan real-time PCR

Cronobacter , a genus of opportunistic pathogens comprising seven species is capable to cause severe neonatal infections, despite variations in their individual pathogenicity. Currently, the identification of Cronobacter are restricted to the Cronobacter genus or Cronobacter sakazakii , necessitating more accurate methods to differentiate the full spectrum of species within this genus. In present study, specific primers and probes targeting the conserved region of the gyrB gene of Cronobacter strains are developed and evaluated to detect Cronobacter spp in infant formula milk powder. The results showed that the TaqMan real-time PCR assay could specifically detect all seven Cronobacter spp with no cross-reactivity against non-target strains. The minimum concentration detected by our method was estimated to be 10 3 CFU/mL, and analytical sensitivity was significantly enhanced to 10° CFU/mL following enrichment step. Crucially, the assay exhibited robust anti-interference capabilities, accurately identifying Cronobacter even in the presence of high concentrations of background flora. The practical utility of the method was validated through the testing of 60 commercial and artificially contaminated milk powder samples, where the TaqMan real-time PCR results were in full agreement with the national method. These results suggested that this TaqMan real-time PCR targeting gyrB gene is a rapid, accurate, and reliable method to detect Cronobacter in food matrices.