title: 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.
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China.
authors.forename: Wenting
authors.initials: W
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China.
authors.forename: Hengyi
authors.initials: H
authors.lastname: Xu
authors.affiliation: Zystein LLC, Fayetteville, AR 72704.
authors.forename: Zoraida P
authors.initials: ZP
authors.lastname: Aguilar
authors.affiliation: Food and Nutritional Science, School of Biological Science, The University of Hong Kong, Pokfulam Road, Hong Kong, China. Electronic address: npshah@hku.hk.
authors.forename: Nagendra P
authors.initials: NP
authors.lastname: Shah
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China. Electronic address: weihua114@live.cn.
authors.forename: Hua
authors.initials: H
authors.lastname: Wei
mesh_terms: D000818:Animals; D001386:Azides; D002417:Cattle; D003470:Culture Media; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000302:isolation & purification; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D008461:Meat Products / Q000382:microbiology*; D008892:Milk / Q000382:microbiology*; D011419:Propidium / Q000031:analogs & derivatives*; D060888:Real-Time Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000302:isolation & purification*
chemical_list: D001386:Azides; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium
keywords: Staphylococcus aureus; milk powder; propidium monoazide; real-time PCR