Detection of Cronobacter sakazakii in powdered infant milk formula using real-time PCR

摘要

Cronobacter sakazakii is a neonatal pathogen that has been found commonly incontaminated dried infant milk formula and milk powder. The fluorogenic selectivemarker, 4-Methylumbelliferyl-α-D-glucoside and secondary selective markers, sodiumthiosulfate & ferric citrate have been used in differential media to indicate the presence ofC. sakazakii based on α-D-glucosidase enzymes unique to this pathogen. This researchwill compare four enrichment broths for maximum recovery from powdered infant milkformula: C. sakazakii – Enterobacter sakazakii enrichment (ESE) broth, Tryptic SoyBroth (TSB), Enterobacteriaceae enrichment (EE) broth, and M-Coliform broth.Differential selective and nonselective agars including Trypticase Soy Agar (TSA),Violet red bile agar (VRBA), Violet red bile D-glucose agar (VRBDGA), and a newlydeveloped KJ medium will be compared for the efficacy of isolation for the species andfor optimal α-D-glucosidase activity with the fluorogenic selective marker, 4-Methylumbelliferyl-α-D-glucoside and secondary selective markers. C. sakazakii strains ATCC29544, ATCC 29004, ATCC 12868, and ATCC 51329 will be utilized as positivecontrols to run in artificially contaminated powder infant milk formula (PIMF) with eachenrichment broth. DNA will be extracted from enrichments, which will be examinedusing real-time TaqMan PCR in order to compare to culture-based detection to determinerelative sensitivities between the two approaches. The fluorogenic selective marker,secondary selective markers, and using a TaqMan probe PCR protocol will prove to be arapid and specific powerful tool for the detection of Cronobacter sakazakii in powdered infant milk formula.