A new application of a sodium deoxycholate-propidium monoazide-quantitative PCR assay for rapid and sensitive detection of viable Cronobacter sakazakii in powdered infant formula.

Abstract

A rapid, reliable, and sensitive method for the detection of Cronobacter sakazakii, a common foodborne pathogen that may cause serious neonatal disease, has been developed. In this study, a rapid real-time quantitative PCR (qPCR) assay combined with sodium deoxycholate (SD) and propidium monoazide (PMA) was developed to detect C. sakazakii contamination in powdered infant formula (PIF). This method could eliminate the interference from dead or injured bacteria. Optimization studies indicated that SD and PMA at 0.08% (wt/vol) and 5µg/mL, respectively, were the most appropriate. In addition, qPCR, PMA-qPCR, SD-PMA-qPCR, and plate count assays were used to account for the number of viable bacteria in cell suspensions that were exposed to a 55°C water bath at different length of time. As a result, the viable number by PMA-qPCR showed significantly higher than of the number from SD-PMA-qPCR or plate counts. The number of viable bacteria was consistent between SD-PMA-qPCR and traditional plate counts, which indicated that SD treatment could eliminate the interference from dead or injured cells. Using the optimized parameters, the limit of detection with the SD-PMA-qPCR assay was 3.3×102 cfu/mL and 4.4×102 cfu/g in pure culture and in spiked PIF, respectively. A similar detection limit of 5.6×102 cfu/g was obtained in the presence of the Staphylococcus aureus (107 cfu/mL). The combined SD-PMA-qPCR assay holds promise for the rapid detection of viable C. sakazakii in PIF.