Duplex Real-Time PCR Method for the Differentiation of Cronobacter sakazakii and Cronobacter malonaticus

Cronobacter sakazakii and Cronobacter malonaticus are the most common species of Cronobacter , so it is necessary to detect the two species as soon as possible in surveillance programs. We developed a real-time PCR method for identifying C. sakazakii and C. malonaticus from the genus Cronobacter . In this study, the two pairs of primers and probes were designed, targeting 16S rRNA and fusA, respectively. The specificity of the real-time PCR assay was validated with 112 strains of Cronobacter , including 56 C. sakazakii , 32 C. malonaticus , 16 Cronobacter dublinensis , 6 Cronobacter turicensis , and 2 Cronobacter muytjensii . The results showed that C. sakazakii and C. malonaticus were all correctly identified, consistent with the results of another method by analyzing the clustering of the fusA sequence. The detection limit for pure culture was 10² CFU/ml and 10³ CFU/g for artificially contaminated rehydrated powdered infant formula. Therefore, the developed real-time PCR was a rapid, sensitive, and reliable method for the identification of C. sakazakii and C. malonaticus .