PCR-Based Detection of Foodborne Pathogens from Enriched Fly Samples

Place the polymerase chain reaction or PCR tubes containing PCR-ready tablets specific to the target foodborne pathogens onto a chilled PCR tube rack.

Each tablet includes target-specific primers, DNA polymerase, dNTPs, and a fluorescent dye used in the PCR reaction.

Add enriched fly lysate containing a high amount of DNA from pathogenic bacteria to form the reaction mixture.

Seal the tubes with optical caps to prevent evaporation.

Load the PCR tubes into the thermal cycler and perform separate PCR runs using pathogen-specific programs for each target pathogen.

During PCR, the DNA denatures, primers bind to the target sequence, and amplification produces double-stranded DNA that binds the dye and emits fluorescence.

Repeated cycles result in exponential DNA amplification and increased fluorescence.

The instrument tracks fluorescence in real time to detect the presence of the target foodborne pathogens in the sample.

To begin PCR-based detection, first, chill a PCR tube rack on a cooling block. Then, place the corresponding PCR tubes containing PCR-ready tablets for the target foodborne pathogen into the holder.

Carefully remove the caps from the PCR tubes using the decapping tool and discard them. Verify that each tube contains a tablet. Then, transfer 50 microliters of lysate to the appropriate PCR tubes.

Secure new optical caps onto the PCR tubes using the capping tool. After assembling the reactions, load the PCR tubes into the PCR cycler detection system. Then, run the program and view the results as described in the manufacturer's protocol.