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Use of the DCode System to Detect the Food-Borne Bacterial Pathogen Listeria monocytogenes

as Touchdown, was also used to reduce the formation of spurious byproducts. 8 This involved setting the annealing temperature (Ta) 10 C higher than the calculated primer melting temperature for the first cycle, then decreasing it by 0.5 C every cycle for 20 cycles, and finally annealing at the Tm during 9 subsequent cycles. When the reaction was carried out with primer pair 341F (with GC clamp) and 534R, the annealing temperature (Touchdown) was set at 60 C for the first cycle and decreased by 0.5 C each cycle thereafter for 20 cycles, with the final 9 cycles performed at Tm of 50 C as shown below.

When the reaction was carried out with primer set 1055F and 1406R (with GC-clamp), the annealing temperature was initially set at 65 C for the first cycle and decreased by 0.5 C each cycle thereafter for 20 cycles, with the final 9 cycles performed at Tm of 55 C as shown below.

In the final step of the reaction, the mixture was held at 75 C for 10 min to allow extension of incomplete products.9 Amplified products were analyzed by agarose gel electrophoresis as follows: 5 ml product was mixed with 1 ml gel loading buffer (40% sucrose, Mallinckrodt Inc., St. Louis, MO; 0.5% Brom Phenol Blue, Allied Signal Specialty Chemical, Michigan Center, MI) and the mixture applied to a 1% agarose low melt preparative grade gel (Bio-Rad Laboratories, Hercules, CA) in Tris-boric acid-EDTA buffer (89 mM Sigma 7-9 Tris[hydroxymethyl]aminomethane, Sigma Chemical Co, St. Louis, MO; 89 mM boric acid, Mallinckrodt Inc., St. Louis, MO; 2 mM ethylenediamine tetraacetic acid, Fisher Scientific, Pittsburgh, PA). Ethidium bromide (Bio-Rad Laboratori
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