PCR-Based Detection of Foodborne Pathogens from Enriched Fly Samples

Abstract

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 ca....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.