PCR-Based Detection of Foodborne Pathogens from Enriched Fly Samples

Samenvatting

Source: Pava-Ripoll, et al. Detection of Foodborne Bacterial Pathogens from Individual Filth Flies. J. Vis. Exp. (2015)

This video demonstrates a PCR-based method that amplifies target bacterial DNA from enriched fly samples using pathogen-specific tablets, enabling real-time fluorescence detection and melting curve analysis to confirm the presence of foodborne pathogens in individual insects.

Protocol

1. Hydrate PCR (polymerase chain reaction)-Ready Tablets

1. Select a chilled (4 °C) PCR cooling block and place a PCR tube rack over the insert.
2. Place corresponding PCR tubes containing the PCR-ready tablets (included with each kit) for the target foodborne pathogen in the holder, according to the rack file.
3. Using the decapping tool, carefully remove the caps from PCR-tubes. Discard the caps and verify that each tube contains a tablet.
4. Transfer 50 µl (for Salmonella and Cronobacter) or 30 µl (for L. monocytogenes) of lysate to specific PCR tubes. Use new optical caps and secure them tightly onto the PCR tubes using the capping tool.
   NOTE: After adding the lysate to PCR-ready tablets, samples must remain chilled at 2 - 8 °C until loaded into the PCR-based detection system. The PCR tubes can be centrifuged at 2,500 x g for a few seconds to ensure that the full volume is at the bottom of the tube.
5. Load the PCR tubes into the PCR cycler/detection system instrument by opening the instrument drawer.
6. Place the rack of PCR tubes into the wells in the drawer and check that the tubes are seated correctly.
7. Close the drawer and initiate the program as described by the manufacturer’s protocol.
   NOTE: The PCR-based instrument has preset cycling parameters for each foodborne pathogen.
8. Verify that the PCR cycling status bar displays a blue bar indicating that the amplification portion of the program is running.
   NOTE: For standard PCR assays, the processing time of the full program (amplification and detection) takes approximately 3 - 3.5 hr to complete.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Capping tool	DuPont	D11677028
Decapping tool	DuPont	D11134095
PCR tube rack/holder	DuPont	D12701663
Fine point, straight tip forceps	BioQuip	4731	Sterilize before use. Multiple suppliers.
Cooling block			Multiple suppliers.
Centrifuge			Multiple suppliers.