Fig. 4 shows the amplification results from the DNA engine
tetrad using DNA prepared from1ml enriched samples spikedwith
1.6 0.3 102
to 1.6 0.3 105
wild-type S. flexneri cells. Although
most DNA samples contained Shigella DNA, we noticed differences depending on the produce types, the method used to prepare the
DNA, and even the polymerase used in themultiplex reaction.Most
InstaGene- and DNeasy-prepared DNA samples could be used un-
diluted, improving the sensitivity of the multiplex assay (Fig. 4;
lanes 2 and 3, respectively). On the other hand, most DNA samples
collected with the PrepMan Ultra method were strongly inhibitory
to the multiplex PCR reaction and therefore needed to be diluted
1:10 with sterile water, as per the manufacturer’s recommenda-
tions (Fig. 4.; lanes 4 and 4*). This was seen with the HotStarTaq
Plus DNA polymerase (Fig. 4AeF1) and the fast-cycling PCR master
mix although, for the sweet pepper DNA samples only, the fast-
cycling master mix relieved the inhibition observed with the un-
diluted PrepMan Ultra DNA samples (Fig. 4F1 and F2). For the other
samples, we did not notice any differences in the amplification
outcome using the HotStarTaq Plus DNA polymerase or the fast-
cycling PCR master mix on the DNA engine Tetrad (data not
shown). PCR inhibitionwas also observedwith both polymerases in
the DNeasy cilantro DNA samples and the InstaGene green onion
DNA samples (Fig. 4C and D).