The multiplex PCR assay ensured a sensitive and specific
tool for the detection of C. botulinum in the inoculated food
and fecal samples. The optimal enrichment time varied from 1
to 5 days, depending on the inoculated C. botulinum strain and
sample material (Table 4). In general, by extending the enrichment
time by up to 5 days, the assay had an increased sensitivity
for the types A and F strains, whereas the type B strains
in all samples and type E strain in beef and fish were easily
detected in 1 to 3 days. The optimal enrichment temperatures
seemed to vary by the sample material rather than by the
inoculated group I and II strains; optimal enrichment of the
beef was obtained at 37°C, while that of the fish and feces was
at 30°C. The lowest detection limits were observed in minced
beef and hot-smoked fish, where 102 to 101 spore/g of sample
material was detected. These spore counts correspond to
the natural contamination level of C. botulinum in foods (7).
For feces, the detection limit was higher; 101 to 103 spores/g
of feces were found, with the highest detection level being that
for the type E strain. A number of sample materials, including
feces and fatty foods, are generally known to cause failure in
the PCR detection of microorganisms due to the inhibition of
the DNA polymerase enzyme. However, it seems that this is
not the case in this study, as the concentrations of all sample
materials in the PCR were shown to be below the inhibitory
level (Table 3).
The multiplex PCR assay ensured a sensitive and specifictool for the detection of C. botulinum in the inoculated foodand fecal samples. The optimal enrichment time varied from 1to 5 days, depending on the inoculated C. botulinum strain andsample material (Table 4). In general, by extending the enrichmenttime by up to 5 days, the assay had an increased sensitivityfor the types A and F strains, whereas the type B strainsin all samples and type E strain in beef and fish were easilydetected in 1 to 3 days. The optimal enrichment temperaturesseemed to vary by the sample material rather than by theinoculated group I and II strains; optimal enrichment of thebeef was obtained at 37°C, while that of the fish and feces wasat 30°C. The lowest detection limits were observed in mincedbeef and hot-smoked fish, where 102 to 101 spore/g of samplematerial was detected. These spore counts correspond tothe natural contamination level of C. botulinum in foods (7).For feces, the detection limit was higher; 101 to 103 spores/gof feces were found, with the highest detection level being thatfor the type E strain. A number of sample materials, includingfeces and fatty foods, are generally known to cause failure inthe PCR detection of microorganisms due to the inhibition ofthe DNA polymerase enzyme. However, it seems that this isnot the case in this study, as the concentrations of all samplematerials in the PCR were shown to be below the inhibitorylevel (Table 3).
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