Propidium monoazide combined with real-time PCR for selective detection of viable Staphylococcus aureus in milk powder and meat products

Abstract

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 10(5) 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 3.0×10(2) 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.

The Core and Seasonal Microbiota of Raw Bovine Milk in Tanker Trucks and the Impact of Transfer to a Milk Processing Facility

“…For the purpose of distinguishing living and dead cells, several studies have applied propidium monoazide (PMA) to measure the living fraction of cells in processed or pasteurized milk (5, 34, 35). However, because raw milk has not undergone thermal processing and therefore likely contains a preponderance of living cells, we did not use PMA treatment.…”

Advances and Challenges in Viability Detection of Foodborne Pathogens

“…It has also been recognized as a pathogen that causes outbreaks of food poisoning (Zhang et al, 2015b). S. aureus can contaminate a variety of foods such as salad, cheese, milk, fish, and meat (Alarcon et al, 2006; Vazquez-Sanchez et al, 2014).…”

“…This conventional method takes from 5 to 6 days and has low sensitivity and specificity (Alarcon et al, 2006). Hence, conventional method may underestimate the level of contamination (Zhang et al, 2015b). …”

“…The authors believed that the PMA-qPCR results may reflect the real bacterial number in light of the presence of VBNC among bacterial populations (Martinon et al, 2012a). Zhang et al (2015b) combined PMA with qPCR for selective detection of viable S. aureus in milk power and meat products and found that the PMA-qPCR assay was more specific and sensitive than conventional PCR, and the limit of detection was 3.0 × 10 2 CFU/g in spiked milk powder. The data indicated that the PMA treatment effectively eliminated the DNA amplification signals from dead cells but had little effect on viable cells (Zhang et al, 2015b).…”

In vitro gastrointestinal resistance of Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 in soy and/or milk-based synbiotic apple ice creams

“…Several studies have suggested the application of the qPCR method combined with a propidium monoazide (PMA) sample pre-treatment, which allows the exclusion of the dead cells in PCR amplification (García-Cayuela et al, 2009;Gensberger et al, 2014;Nocker et al, 2009;Zhang et al, 2015). The qPCR enables a more rapid and specific detection of the targeted organisms and PMA treatment facilitates the exclusion of false positive results caused by DNA from dead cells (Gensberger et al, 2014).…”