This method can be used to detect Salmonella from food samples and identify the pathogen to the strain level through genetic fingerprinting. The main advantage of this technique is that it combines Salmonella detection and subtyping into a single workflow and substantially shortens the turnaround time from Salmonella-contaminated food sample to fingerprint of the pathogen. To begin, aseptically place a 25 gram sample of food inside a sterile laboratory blender bag with a built-in filter or optionally prepare an environmental swab by aseptically moistening a sponge with enrichment broth, dragging it across a pre-determined surface and placing it inside a blender bag.
Using a laboratory blender, thoroughly mix each sample with 225 milliliters of RV enrichment broth. Then incubate the mixture at 42 degrees Celsius for four to 21 hours. After this, collect a 50 milliliter subsample of enrichment broth from the filtered side of the bag.
Then centrifuge the subsample at 100 times g for 10 minutes. Carefully transfer the supernatant to a new 50 milliliter centrifuge tube and centrifuge at 3, 000 to 6, 000 times g for 10 minutes. Discard the supernatant and wash the pellet in five milliliters of BPW.
Resuspend the pellet in five milliliters of BPW. First, thaw the sample buffer and the reaction buffer from the MDA kit on ice. Place one milliliter of resuspended cells in BPW and 20 microliters of anti-Salmonella beads in a microcentrifuge tube.
Place the microcentrifuge tube on a rotating mixer for 30 minutes at room temperature. After this, place the tube on a magnetic stand for three minutes. Invert the magnetic rack several times to concentrate the beads into a pellet.
Keeping the tube on the magnetic rack, aspirate and discard the supernatant. Next, add one milliliter of wash buffer to the tube. Remove the tube holder from the rack and invert the tube holder several times to remove any non-specifically binding bacteria from the complex.
After the third wash, aspirate the supernatant from the tube and discard it. Then remove the tube from the magnetic rack and centrifuge it for one second. Then place the tube on the magnetic rack for three minutes before removing any residual wash buffer.
Resuspend the bead Salmonella complexes in nine microliters of sample buffer and incubate the tube at 95 degrees Celsius for three minutes. After cooling the complexes on ice, add nine microliters of reaction buffer and one microliter of enzyme mix. Incubate the tube in a thermal cycler according to the text protocol and cool the tube on ice.
Next, use a fluorospectrometer instrument to measure the DNA concentration and purity of the sample. Store the final products at negative 20 degrees Celsius for future experiments. Prepare 18 microliters of PCR mixture per sample according to the text protocol.
Then mix the MDA product by gently pipetting up and down. Add two microliters of the MDA product suspension to the PCR mixture. Using an optimized real-time PCR protocol, run the real-time PCR according to the text protocol.
Add buffer solutions and reagents to MDA product as specified in the text protocol and transfer the resulting 40 microliters of the sequencing prepped MDA product to a new plate and add 20 microliters of PCR purification beads to each well. Next, incubate the plate at room temperature for 10 minutes. Then wash the beads with 80%ethanol and dry them for 12 minutes.
Resuspend the dried beads in 53 microliters of resuspension buffer and incubate the beads for two minutes at room temperature. Finally, dilute and pool libraries according to the manufacturer's instructions. In this protocol, targeted quantity assessment of Salmonella DNA was performed by real-time PCR.
Representative results from two brands of Salmonella-contaminated chicken breast ranged from 701.54 to 945.86 nanograms per microliter indicating that the sample DNA was effectively amplified. While attempting this procedure, it is important to remember to wash and handle IMS beads carefully, keep MDA reagents and products free from contaminants and maintain a clean hood. After its development, this technique paved the way for researchers in the field of food safety and public health to explore rapid and high-resolution tracking of Salmonella contaminants in food samples, product environments and supply chains.
Don't forget that working with foodborne pathogens can be extremely hazardous and precautions such as wearing personal protective equipment and following good laboratory practices should always be taken while performing this procedure.
