While it is clear that this multiplex assay could expedite the
analysis of food for Shigella contamination, success is linked to the
ability of the pathogen to compete with resident microflora. Food
legislation in many countries requires the absence of Shigella in
25 g sample sizes. Since it is not possible to directly carry out PCR
on 25 g of produce, an enrichment step is necessary. The current
FDA bacteriological analytical method for Shigella recommends a
1:9 ratio of grams of produce to mls of enrichment media,
meaning that 25 g of commodities are tested in 225 ml of
enrichment medium and 1 g of food is tested in 9 ml of enrich-
ment medium. Because the LOD of the multiplex assay lies be-
tween 1.6 102
and 1.6 103
CFU per ml, 9 times more cells
(between 1400 and 14,000) and 9*25 times more Shigella cells
(between 3.5 104
and 3.5 105
) need to arise during enrichment
from 1 g and 25 g of produce, respectively (Table 4). Consequently,
if one Shigella cell is present in 25 g of produce pre-enrichment,
the multiplex assay will show presence of the pathogen if that
cell underwent 18.45 doublings during enrichment (Table 4). If the
ratio of produce (g) to enrichment media (mL) was 1:4.5, the
doubling requirement will not vary much (Table 4). More infor-
mation is needed on the growth of Shigella during enrichment to
evaluate the risk, if any, of using molecular assays to support the
food legislation for Shigella.