Modified Most Probable Number Assay to Quantify Salmonella in Raw and Ready-to-Cook Chicken Products

Our research develops rapid detection, quantification, and characterization technologies for foodborne pathogens, focusing on sensitive methods that can improve food safety by identification of a contamination in real world settings. Key challenges include achieving rapid sensitive detection and complex matrices with low cost, yet scalable solutions. We also address difficulties in reducing false positives and improving compatibility with diverse testing environments.

For food safety, this means accurately detecting pathogens at low concentrations in a variety of food matrices. Our protocol addresses the gap between prevalence detection and quantification of salmonella to provide deterministic values for the level of contamination in food products. The process is minimally disruptive as it incorporates validated assays used in FSIS protocols and flexible enough to accommodate additional pathogens and food matrices.

To begin, place the ground chicken acquired from the fresh meat department of local retailers on a working platform. Aseptically divide the meat into 25 gram samples. Acquire ready to cook chicken products from the frozen food section of local retailers and aseptically divide them into 25 gram samples.

Streak salmonella enterica serovar Typhimurium ATCC 14028 on a brain heart infusion auger plate. Incubate the plate overnight at 37 degrees Celsius. The next day, inoculate 25 milliliters of brain heart infusion broth with one colony of freshly grown salmonella.

Aerobically grow culture overnight at 37 degrees Celsius with shaking at 100 RPM. To prepare a series of tenfold dilution, transfer 0.5 milliliters of culture into 4.5 milliliters of buffered peptide water or BPW and mix thoroughly. Then transfer 0.5 milliliters from the prepared dilution into 4.5 milliliters of BPW for each subsequent dilution.

Next, aseptically transfer 25 grams of irradiated ground chicken into sterile stomacher bags. Inoculate the sample with one milliliter of the desired concentration of the culture dilution. Using a sterile cell spreader, gently distribute the inoculum evenly over the surface of the chicken and allow them to stand at four degrees Celsius for one hour.

After incubating irradiated ground chicken samples with salmonella culture for one hour, add 225 milliliters of BPW to each sample to achieve the desired sample to media ratio. Homogenize the chicken samples using the stomacher at a normal speed for 120 seconds. Using a 50 milliliter pipette, carefully remove the liquid from the filtered side of the stomacher and split it into two sterile centrifuge bottles.

Centrifuge the samples at 10, 000 G for 10 minutes and discard the supernatant. Resuspend the cell pellet in three milliliters of BPW using a sterile spatula. Add 12 milliliters of BPW to the resuspended pellet and mix thoroughly by stirring with a sterile spatula.

Combine the contents of both centrifuge bottles into one bottle. Add three milliliters of the resuspended sample to each well in column one of the 48 well block to create eight replicates. To prepare a series of tenfold dilution across columns one to six using an eight channel pipette, add 0.3 milliliters of the sample from each well into 2.7 milliliters of BPW in the next column ensuring thorough mixing.

Incubate blocks overnight at 37 degrees Celsius with shaking at 100 RPM. The next day, using a multi-channel pipette, plate seven microliters of culture from each dilution onto brain heart infusion auger plates in a four by six grid. Allow the plates to dry for 10 minutes before incubating them at 37 degrees Celsius for 18 to 24 hours.

After incubation, examine auger plates, marking at least one colony as positive and no growth as negative. Enter the number of dilution and dilution factor in a worksheet. Input the appropriate volume or weight of the sample.

Then specify the number of replicates and enter the number of samples that tested positive. Click on calculate results to analyze annotated positive and negative results using the simple maximum probability resolution method. To begin, set up the 48 well MPN block for salmonella culture inoculated with chicken samples.

Mix cultures in a 48 well block by pipetting up and down several times. Transfer 200 microliters of each culture into a 96 well PCR plate. Seal the plate and centrifuge at 6, 600 G for 10 minutes.

Remove the supernatant and add 20 microliters of the DNA extraction kit reagent to the pellet. Mix the pellet by pipetting up and down. Again, seal the plate and heat it at 99 degrees Celsius for 10 minutes, followed by cooling to 20 degrees Celsius.

After centrifugation, use two microliters of the supernatant to prepare the QPCR mixture according to established protocol. Using the following conditions, perform the real-time PCR. After PCR, export the results and consider the wells with cycle threshold values less than or equal to 30 as positive and greater than 30 as negative.

Mix cultures in a 48 well block by pipetting up and down several times. Transfer 20 microliters of each sample into the molecular detection assay salmonella kit provided lysis tube. Heat the samples at 100 degrees Celsius for 15 minutes.

Then incubate the samples for 10 minutes at room temperature. Transfer 20 microliters of lysate from each lysis tube into a reagent tube and load the reagent tubes into the holder. Add the holder with reagent tubes to the molecular detection system instrument.

Configure the software settings for the molecular detection system assay. Run the assay and export the report for analysis. The MDS Run report provides detailed output information, which is positive or negative.