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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 5 μg/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-PMAqPCR 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. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a spherical, gram-positive, pathogenic bacterium commonly associated with bovine mastitis and clinical infections. It is also recognized as a pathogen that causes outbreaks of food poisoning. The objective of this study was to develop and evaluate a rapid and reliable technique that combines propidium monoazide (PMA) staining with real-time quantitative (q)PCR to detect and quantify viable cells of Staph. aureus in milk powder and meat products. The inclusivity and exclusivity of the assay were evaluated using 58 strains belonging to 14 species. Serial dilutions of Staph. aureus cells were used to establish a standard curve and to confirm the effect of PMA treatment. Milk powder and meat products were used as the spiked foods, and the ability of PMA-qPCR to eliminate nonviable cells was determined in milk powder. Furthermore, meat products were inoculated with different concentrations of Staph. aureus and 105 cfu/g of Bacillus cereus and Salmonella enterica to test the interference by nontarget microorganisms. When PMA treatment was applied before DNA extraction, we were able to eliminate false-positive results with little effect on viable cells. The PMA-qPCR assay was specific and more sensitive than conventional PCR, and the level of detection was 3.0 x 10² cfu/g in spiked milk powder. Additionally, we observed no significant interference for the detection of viable Staph. aureus from other nontarget bacteria. The PMA-qPCR protocol is an effective and rapid method to quantify viable cells of Staph. aureus in food samples. The PMA-qPCR assay is specific and reliable, offering a valuable diagnostic tool for routine analysis in food and clinical diagnostic research at a reasonable cost. [ABSTRACT FROM AUTHOR]