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The aim of the study was to perform in-house validation of the developed multiplex PCR (mPCR)-based alternative method to detect Shiga toxin-producing Escherichia coli (STEC), Listeria monocytogenes ( L. monocytogenes ) and Salmonella spp. in raw meats following the ISO 16140-2: 2016. A comparative study of the developed mPCR against the Bacteriological Analytical Manual (BAM) method was evaluated for inclusivity and exclusivity, sensitivity and the relative level of detection (RLOD). Inclusivity levels for each target bacterium were all 100%, while exclusivity for non-target bacteria was 100%. The sensitivity of the developed mPCR was calculated based on the analysis of 72 samples of raw meat. The sensitivity of the developed mPCR was 100%. The RLOD values of the developed mPCR for STEC, L. monocytogenes and Salmonella spp. were 0.756, 1.170 and 1.000, respectively. The developed mPCR showed potential as a tool for the fast, specific and sensitive detection of the three bacteria in the raw meat industry.

A colorimetric mixed-pH dye-based indicator with potential for the development of intelligent packaging, as a "chemical barcode" for real-time monitoring of skinless chicken breast spoilage, is described. Also investigated was the relationship between the numbers of microorganisms and the amount of volatile compounds. This on-package indicator contains two groups of pH-sensitive dyes, one of which is a mixture of bromothymol blue and methyl red, while the other is a mixture of bromothymol blue, bromocresol green and phenol red. Carbon dioxide (CO2) was used as a spoilage metabolite because the degree of spoilage was related to the amount of increased CO2, and which was more than the level of total volatile basic nitrogen (TVB-N) during the storage period. Characteristics of the two groups of indicator solutions were studied, as well as their response to CO2. A kinetic approach was used to correlate the response of the indicator label to the changes in skinless chicken breast spoilage. Color changes, in terms of total color difference of a mixed-pH dye-based indicator, correlated well with CO2 levels of skinless chicken breast. Trials on skinless chicken breast samples have verified that the indicator response correlates with microbial growth patterns, thus enabling real-time monitoring of spoilage either at various constant temperatures or with temperature fluctuation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A colorimetric mixed pH dye-based indicator with potential for the development of intelligent packaging, as a "chemical barcode" for real-time monitoring of intermediate-moisture dessert spoilage, is described. This on-package indicator contains mixed pH-sensitive dyes, bromothymol blue and methyl red, that respond through visible color change to carbon dioxide (CO(2)) as a spoilage metabolite. Both indicator solution and indicator label characteristics were studied, as well as their response to CO(2). A kinetic approach was used to correlate the response of the indicator label to the changes in intermediate-moisture dessert spoilage. Color changes, in terms of total color difference of a mixed pH dye-based indicator, correlated well with CO(2) levels of intermediate-moisture dessert. Trials on golden drop have verified that the indicator response correlates with microbial growth patterns in dessert samples, thus enabling the real-time monitoring of spoilage either at various constant temperatures or with temperature fluctuation.

Monoclonal antibodies (MAbs) raised against Escherichia coli O6:H16 were screened against 15 strains of E. coli and 19 non-E. coli bacteria. A MAb-luminescence assay using MAb-5.8, which shows no cross-reactions with non-E. coli bacteria, and a photon-counting television camera were developed for rapid enumeration of E. coli O6:H16 in water. The membrane filter that retained bacteria was boiled for 5 min in a buffer and incubated with biotinylated MAb-5.8. After incubation with streptavidin-peroxidase conjugate, it was reacted with luminol-based reaction mixture. Luminous image and light intensity of the filter was recorded with a Biocell Counter. Levels of E. coli O6 higher than 7 x 10(3) CFU were detected by the MAb-luminescence assay when E. coli O6 was spotted onto the membrane filter. The sample that contained E. coli O6:H16 was filtered through a membrane filter, and the filter that retained bacteria was incubated on a filter paper soaked with nutrient broth supplemented with 0.5% NaCl at 37 degrees C for 6 h. The number of light emission points on the filter correlated well with initial E. coli O6:H16 counts within the range of 1 to 3 x 10(2) CFU. The correlation coefficient was 0.89.

A bioluminescence assay carried out with a photon-counting TV camera was evaluated for rapid enumeration of viable bacterial counts. The test sample was filtered through a membrane filter, and the membrane filter retaining bacteria was incubated at 37 degrees C for 6 h on a filter paper soaked with nutrient broth supplemented with 0.5% NaCl. The membrane filter was then subjected to a bioluminescence reaction, and the intensity of light and numbers of light emission points on the filter were measured with a photon-counting TV camera. The light intensity measured on seven different bacteria correlated with initial viable counts; the correlation coefficient was calculated to be 0.89. The number of light emission points measured on Escherichia coli also correlated with the initial viable counts (r = 0.81) in a range from 1 to 100 CFU. Presumptive bacterial counts by the present bioluminescence assay determined on 79 samples of vegetables and 122 samples of environmental water correlated well with the viable counts obtained by the conventional plating method, with correlation coefficients of 0.87 and 0.82, respectively.

The random amplified polymorphic DNA (RAPD) band patterns from 23 Salmonella spp. produced by use of an oligonucleotide primer (called du primer) designed on the basis of the N-terminal sequence of dulcitol 1-phosphate dehydrogenase (5'-GTGGTGACCCAGGATGGCCAGGTG-3') were different from those from 16 non-Salmonella spp. The bands at 460 and 700 bp were produced in all Salmonella strains tested. These RAPD fragments obtained from Salmonella typhimurium strongly hybridized with the corresponding RAPD bands from the other strains of Salmonella, but not with those from non-Salmonella spp. in Southern blot analysis. The RAPD bands were detected by ethidium bromide staining even when genomic DNA prepared from as few as 2.8 x 10(3) cells was used. The minimum detectable cell number in the initial inoculum of S. typhimurium was 4 x 10(-1) CFU/25 g of raw beef after the preenrichment in Enterobacteriaceae enrichment mannitol (EEM) broth for 6 h and the selective enrichment in dulcitol-magnesium chloride-pyridinesulfonic acid-brilliant green-novobiocin (DMPBN) medium for 18 h at 42 degrees C. Seven raw foods inoculated with S. typhimurium at numbers from 4 x 10(-1) to 2.6 x 10(2) CFU/25 g of food were positive in both the RAPD analysis and the conventional culture method.