PCR and multiplex PCR assays for the detection of Cronobacter species using specific targets obtained by a bioinformatics approach

Abstract Cronobacter species are opportunistic pathogens associated with severe infections in immunocompromised individuals. Species differentiation is essential as the sensitivity of different species to chemical reagents, antibiotics, and virulence factors may be diverse. This study aimed to mine new molecular targets for the detection of Cronobacter strains at the species level. Using a bioinformatics approach and the online BLAST software, we obtained serial Cronobacter species-specific target genes. Primers for the selected genes were constructed, and the PCR products were evaluated using various Cronobacter species and non-Cronobacter strains. As a result, 2, 5, 7, and 5 specific target genes were found for C. sakazakii, C. malonaticus, C. dublinensis, and C. turicensis, respectively. Thereafter, 4- and 5-multiplex PCR (mPCR) was successfully developed to identify these four species based on the validated species-specific primers. Overall, our data indicated that PCR and mPCR assays have excellent specificity and sensitivity for the detection of Cronobacter species, suggesting the potential of their use in food inspection and clinical diagnosis.