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Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. are common food-borne pathogens in milk that may cause serious diseases. In the present study, we established a simple, rapid, and specific method to simultaneously detect viable E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk. Three specific genes, fliC from E. coli O157:H7, cgcA from Cronobacter spp., and invA from Salmonella spp., were selected and used to design primers and probes. False-positive results were eliminated with the use of a combined sodium deoxycholate (SD) and propidium monoazide (PMA) treatment. Using the optimized parameters, this SD-PMA treatment combined with multiplex real-time PCR (SD-PMA-mRT-PCR) detected E. coli O157:H7, Cronobacter spp. and Salmonella spp. respectively, at 10² cfu/mL in pure culture or artificially spiked skim milk samples. A reasonable recovery rate (from 100 to 107%) for detection of viable bacteria using the SD-PMA-mRT-PCR assay was obtained in the presence of dead bacteria at 107 cfu/mL. The SD-PMA-mRTPCR method developed in this study can accurately detect and monitor combined contamination with E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk and milk products. [ABSTRACT FROM AUTHOR]

Bacillus cereus is a causative agent of an emetic food-borne disease. In this study, visual detection of viable emetic B. cereus was developed using a propidium monoazide (PMA)-asymmetric polymerase chain reaction (asPCR) and unmodified gold nanoparticles (AuNPs). To detect only the viable emetic B. cereus , PMA treatment was selected before DNA extraction to eliminate the false-positive results from dead bacteria. In the presence of viable target bacteria, the long genomic DNA fragments from the cereulide synthetase gene ( cesB ) were produced by asPCR, which could be effectively absorbed onto naked AuNPs via coordination between Au and the nitrogen atoms of the exposed bases. After adding NaCl solution, visual detection with the naked-eye or with UV–vis spectrophotometer was possible within a few minutes. Under the optimum conditions, the limit of detection (LOD) for viable emetic B. cereus reached as low as 9.2 × 10 1 CFU/mL in 0.01 M phosphate-buffered saline and 3.4 × 10 2 CFU/mL in milk, which was adequate to meet the maximum limit imposed by the Commission Regulation (EC) No 2073/2005 (500 CFU/mL). The proposed method also exhibited excellent discrimination against 10 common pathogenic bacteria in milk. The PMA-asPCR-AuNPs colorimetric assay offers a promising application in the detection of low concentrations of food-borne pathogens. [ABSTRACT FROM AUTHOR]

Salmonella Enteritidis, Eschericha coli O157:H7 and Listeria monocytogenes are important pathogens contaminating fresh vegetables. The aim of this study was to develop a rapid and accurate approach for simultaneous detection of viable S. Enteritidis, E. coli O157:H7 and L. monocytogenes in fresh vegetables. To detect viable cells, propidium monoazide (PMA) was applied to eliminate the false-positive results. In addition, an internal amplification control (IAC) was added to the quadruplex PCR as indicator of false negative results that are mainly caused by the PCR inhibitors. The limit of detection (LOD) for the viable cells with or without PMA treatment was 10 2 CFU/mL for S. Enteritidis, 10 3 CFU/mL for E. coli O157:H7 and 10 4 CFU/mL for L. monocytogenes , showing that the PMA treatment did not influence the sensitivity. After 12 h enrichment, the PMA-mPCR-IAC combination assay could detect as low as 10 1 CFU/g for viable S. Enteritidis, E. coli O157:H7 and L. monocytogenes in spiked vegetables. The PMA-mPCR-IAC combination assay holds promise for the simultaneous detection of viable S. Enteritidis, E. coli O157:H7 and L. monocytogenes in fresh vegetables. [ABSTRACT FROM AUTHOR]

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]

Cronobacter sakazakii , Staphylococcus aureus , and Bacillus cereus are important pathogens contaminating infant food products. In this study, we developed a specific, sensitive, and accurate technique for the simultaneous detection of viable C. sakazakii , S. aureus , and B. cereus in infant food products. An internal amplification control (IAC) was added to the multiplex PCR (mPCR) as indicator of false negative results that are mainly caused by the PCR inhibitors in food products. In addition, to detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the PCR signal from dead cells. The limit of detection (LOD) for the viable cells with or without PMA treatment was 10 4 CFU/mL for C. sakazakii and B. cereus , and 10 2 CFU/mL for S. aureus in pure culture, showing that the PMA treatment did not influence the sensitivity. After 12 h enrichment, the PMA-mPCR-IAC assay could detect as low as 10 1 CFU/g for C. sakazakii and S. aureus , and 10 0 CFU/g for B. cereus in spiked infant food products (infant formula, noodles, milk and rice noodles). This PMA-mPCR-IAC assay we developed hold promise for the simultaneous detection of C. sakazakii , S. aureus , and B. cereus in infant food products. [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]

A rapid, reproducible, sensitive and specific combination assay was developed for Escherichia coli O157:H7 comprising sequential immunomagnetic separation (IMS), followed by propidium monoazide (PMA), exclusion by viable cells, internal amplification control (IAC) and polymerase chain reaction (PCR). IMS was used to improve sensitivity, reduce detection time and eliminate false positives. PMA was used to detect viable cells, and IAC was incorporated into the system to eliminate the inhibitors of PCR from the food matrix. In the presence of inhibitors, IAC produced no signal, thereby eliminating false negative results. The results indicated that the IMS cell capture efficiency was about 90% at a concentration of 1 × 106 cfu mL−1 with a detection limit of 2.1 × 102 cfu mL−1 for pure culture. In an unoptimised system, IMS–PMA–PCR with IAC showed a detection limit of 5 × 103 cfu mL−1 in spiked milk over a 240 min assay, faster than conventional methods of detection. [ABSTRACT FROM AUTHOR]

To improve the accuracy of current methods for the detection of Escherichia coli O157:H7 foodborne disease outbreaks, two reagents, propidium monoazide (PMA) and sodium deoxycholate (SD), were utilized to eliminate the interference of dead and injured cells for qPCR that was combined with immunomagnetic separation (IMS) for cell enrichment in an IMS-SD-PMA-qPCR assay. The optimal SD concentration and the optimal incubation time with SD were recorded at 0.1% and 20 min, respectively. The number of bacteria survivors was compared using plate counts, qPCR, PMA-qPCR, and SD-PMA-qPCR assays after the cell suspensions were heat treated at 63 °C or freeze stored at −20 °C. Cell suspensions treated or not treated with PMA and treated with SD resulted in significantly lower number of bacteria survivors than those analyzed without SD treatment which indicated that dead cell DNA was eliminated with SD. More importantly, the number of bacteria survivors from those analyzed with SD-PMA-qPCR showed a direct correlation with those analyzed using the plate counts methods. In addition, in order to improve the limit of detection (LOD) and shorten the detection time, immunomagnetic separation (IMS) was adopted to capture and enrich the target bacteria. Using spiked milk as a matrix, the IMS-SD-PMA-qPCR showed a detection limit of 102 CFU/mL. Significantly, even in the presence of 106 CFU/mL of non-target bacteria, the LOD for the SD-PMA-qPCR with IMS separation for E. coli O157:H7 in spiked milk matrix was recorded at 102 CFU/mL. This combination assay holds promise for the detection of foodborne E. coli O157:H7. [ABSTRACT FROM AUTHOR]

Bacillus cereus is an etiological agent of food-borne disease that can cause a type of emesis. To develop a sensitive and reliable diagnosis technique for detecting all the species of the B. cereus group, specific primers were designed to target a recently discovered part of the cereulide synthetase gene (cesB) for emetic B. cereus and 16S rRNA for non-emetic B. cereus. To detect PCR signals only from viable cells, propidium monoazide (PMA) was selected to eliminate the false-positive results. In addition, an internal amplification control (IAC) was applied to meet diagnostic multiplex PCR requirements that will prevent the occurrence of false-negative results. The inclusivity and exclusivity of the mPCR assay were estimated using a panel of 100 strains, including 2 emetic B. cereus, 77 non-emetic B. cereus and 21 non-Bacillus strains. The limit of detection (LOD) for dead B. cereus without PMA treatment in pure bacteria culture was 4.0 × 102 CFU/mL, as low as 7.5 × 100 CFU/mL for viable B. cereus without PMA treatment, and 7.5 × 101 CFU/mL for viable B. cereus with PMA treatment. B. cereus in spiked food produce was detected specifically and sensitively at 1.0 × 103 CFU/g which was the lowest concentration detected. This novel PMA-mPCR-IAC assay is rapid and reliable, providing an efficient diagnostic tool with promising application in monitoring food samples. [ABSTRACT FROM AUTHOR]

Abstract: We developed a rapid and reliable technique for simultaneous detection of Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes that can be used in food products. Magnetic nano-beads (MNBs) based immunomagnetic separation (IMS) was used to separate the target bacterial cells while multiplex PCR (mPCR) was used to amplify the target genes. To detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the DNA detection from dead cells. The results showed the detection limit of IMS-PMA-mPCR assay was about 102 CFU/ml (1.2 × 102 CFU/ml for S. Typhimurium, 4.0 × 102 CFU/ml for E. coli O157:H7 and 5.4 × 102 CFU/ml for L. monocytogenes) in pure culture and 103 CFU/g (5.1 × 103 CFU/g for S. Typhimurium, 7.5 × 103 CFU/g for E. coli O157:H7 and 8.4 × 103 CFU/g for L. monocytogenes) in spiking food products samples (lettuce, tomato and ground beef). This report has demonstrated for the first time, the effective use of rapid and reliable IMS combined with PMA treatment and mPCR assay for simultaneous detection of viable S. Typhimurium, E. coli O157:H7 and L. monocytogenes in spiked food samples. It is anticipated that the present approach will be applicable to simultaneous detection of the three target microorganisms for practical use. [Copyright &y& Elsevier]

Abstract: Salmonella is the leading pathogenic bacteria in food and contaminated water. The aim of this study was to develop a rapid and reliable technique for simultaneous detection of the main three serotypes (Salmonella enterica serovars Typhimurium, Paratyphi B and Typhi) of Salmonella. Primers were designed to amplify the genes specific to each of these three serotypes for simultaneous detection using polymerase chain reaction (PCR). To ensure the detection of only viable cells, propidium monoazide (PMA) was applied to selectively suppress the DNA signal from dead cells. Results showed that the PMA-multiplexed PCR (PMA-mPCR) assay always gave negative results for heat-killed Salmonella at concentrations up to 1 × 106 CFU/ml in pure culture or 1 × 106 CFU/g in spiked food products (tomato, chicken, beef and ham). Results showed that the detection limits of the PMA-mPCR assay were approximately 102 CFU/ml (4.3 × 102 CFU/ml for S. Typhimurium, 3.7 × 102 CFU/ml for S. Paratyphi B, 7.2 × 102 CFU/ml for S. Typhi) in pure culture and 103 CFU/g (4.3 × 103 CFU/g for S. Typhimurium, 3.7 × 103 CFU/g for S. Paratyphi B, 7.2 × 103 CFU/g for S. Typhi) in food produce. These results demonstrated that the PMA-mPCR assay can simultaneously detect and identify viable S. Typhimurium, Paratyphi B and Typhi in a short period of time, even in food produce. [Copyright &y& Elsevier]

Staphylococcus aureus is a common foodborne pathogenic microorganism. A modified propidium monoazide (PMAxx) dye combined with recombinase aided amplification (RAA) was proposed for the rapid and real-time detection of viable S. aureus. PMAxx, a kind of nucleic acid dye is used to distinguish viable bacteria from dead bacteria. Additionally, RAA technique has the advantage of a short detection time (20 min) and uniform temperature (39 °C), which is of great significance for the efficient detection of S. aureus. The S. aureus nuc gene was used as primer and probe in a microplate assay. The effect of PMAxx treatment exhibited permeation of the dead cell membranes that was observed under confocal laser scanning microscope (CLSM). After the enrichment of viable target bacteria ranging from 5.4 × 101 to 5.4 × 103 CFU/mL, the LOD for viable S. aureus was 102 CFU/mL under 3 h enrichment and 101 CFU/mL under 6 h enrichment in artificially contaminated milk, respectively. In the presence of nontarget bacteria at 106 CFU/mL, the system could detect 107 CFU/mL S. aureus in milk sample. The proposed PMAxx-RAA assay exhibited potential for the accurate and rapid viable S. aureus detection in milk. • Real-time fluorescence detection reactions of Staphylococcus aureus were reported. • PMAxx was selected to more accurately eliminate the interference from dead cells. • Verify the effect of PMAxx through confocal laser scanning microscope (CLSM). • The assay could detect 101 CFU/mL viable cells in milk sample after 6 h enrichment. [ABSTRACT FROM AUTHOR]

• LSCM and microplate reader were used to verify the effect of PMA treatment. • Addition of dead bacteria (˜104 CFU/mL) did not affect detection of viable bacteria. • Low concentration of viable bacteria (102 CFU/mL) is detectable in irrigation water. • The method is sensitive and accurate for bacteria detection in irrigation water. Escherichia coli O157:H7 is a kind of waterborne pathogen and recognized as a common pathogenic microorganism in irrigation water. In this study, a quantitative PCR (qPCR) assay with propidium monoazide (PMA) treatment called PMA-qPCR assay was developed to detect the presence of E. coli O157:H7 in natural irrigation water that was more specific, more sensitive, and was unaffected by dead cells compared with traditional and conventional methods. Specific gene, fliC from E. coli O157:H7 was selected to design the unique primer and probe for the assay while propidium monoazide (PMA) was used to effectively prevent the interference of DNA from dead cells. Evaluation of the effect of PMA treatment exhibited permeation of the dead cell membranes that was observed under confocal laser scanning microscope (CLSM). The PMA-qPCR assay efficiently detected viable cells even in the presence of 104 CFU/mL dead cells. Under optimized conditions the limit of detection for viable cells was 102 CFU/mL or 10 CFU/mL in pure culture and spiked irrigation water in a 3 h assay. The PMA-qPCR assay did not show significant difference in the absence of the dead bacteria indicating specificity and selectivity for viable E. coli O157:H7. The PMA-qPCR assay developed in this study has potential for precise and sensitive detection of viable E. coli O157:H7 in irrigation water. [ABSTRACT FROM AUTHOR]