Procedure 32 In situ DNA amplification of Salmonella spp. with magnetic primers for the real-time electrochemical detection based on m-GEC electrodes

This chapter presents a procedure to perform an in situ polymerase chain reaction (PCR) amplification of the IS200 insertion sequence of Salmonella spp. with double labeling of the amplicon on magnetic bead primers and to quantify the double-labeling amplification products growth on magnetic beads by using an electrochemical strategy based on magnetic beads and magneto graphite–epoxy composite (m-GEC) electrodes. In situ salmonella genome amplification with magnetic bead primers include two steps: DNA extraction, magnetic bead primers preparation, and PCR reaction. For extraction overnight culture of Salmonella enteric is centrifuged. DNA is extracted twice with phenol–chloroform–isoamyl alcohol and DNA is precipitated with isopropanol. For preparation of magnetic beads primer streptavidin-modified magnetic beads are immobilized and put on the magnetic separator. Further, a washing step is performed with 140 μL Milli-Q water for 10 min at 42°C. The PCR is performed according to the kit manufacturer.