QuantiTect Multiplex PCR Kit (1000)

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Publication protocol

The EDSP’s Salmonella/Shigella/Campylobacter real-time, multiplex PCR assay was based on that published by Wiemer, D et al. but modified to exclude the Yersinia primer/probe set and include the exogenous, spike-in green fluorescent protein (GFP, IDT) IAC.5 Total reaction volumes of 25-μL contained Qiagen QuantiTect Multiplex PCR Kit (with ROX dye) reagents, with final concentrations as follows: 0.4 μM each primer and 0.2 μM each probe with the exception of the GFP probe, which was 0.04 μM; 0.1 nanograms GFP DNA; and 0.5-2.0 μg (2.5 μL) sample DNA. The thermal cycling program was as follows: 1 cycle of 15 min at 95 °C; 45 cycles of 1 min at 94 °C, 30 sec at 50 °C, and 30 sec at 72 °C; and 1 cycle of 10 min at 72 °C.

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