Abstract:
Staphylococcus aureus is a ubiquitous pathogen found in man and
his environment. S. aureus can also be found in food such as raw and
processed meat, milk and dairy products, and bakery and confectionery
products. S. aureus produces heat-stable enterotoxins, which, when
present in large doses, can cause food poisoning. The detection of S.
aureus in the food sample can serve as an indicator of the presence of
enterotoxins. Therefore, it is relevant that food is tested for the presence of
S. aureus. The conventional culturing method can detect S. aureus to a
minimum limit of 102 CFU/gram. However, this method requires
approximately two weeks to confirm the presence of the microorganism.
In addition, due to great demands for processed meat, there is a need for
faster and more convenient methods for food handlers to maintain a
relatively safe level of S. aureus, and keep the consumer public healthy
and safe from S. aureus food poisoning. For these reasons, BIOTECH has
developed a PCR-based detection kit that would detect S. aureus in food
in a much more rapid pace than the conventional culturing method. To
gain an insight into the consistency of the PCR-based detection method
with the traditional method, 40 processed meat samples were examined for
the presence of S. aureus, using both methods. Samples were enriched,
eight methods of DNA extraction were tested, and PCR conditions were
optimized. More specifically, Mg++ concentration was increased from 2.5
mM to 3.0 mM, two BIOTECH primer sets (Sa 01/02 vs. Sa 03/04) were
compared, DNA template was diluted up to 10-2, total reaction volume
was increased from 25 μL to 50 μL, and annealing temperature was
decreased from 60°C to 55°C. It was observed that one-step sample
enrichment for 36 hours, DNA extraction by Boiling/NaOH, and PCR
conditions such as 2.5 mM Mg++ concentration, BIOTECH primer set Sa
03/04, one-fold dilution of DNA template, 50 μL total reaction volume,
and 60°C annealing temperature increase the detection of S. aureus in
processed meat products. From 108 CFU/gram, the BIOTECH kit can
detect to a minimum of 103 CFU/gram. In inoculated samples, the PCR based
detection kit was relatively consistent with the conventional method.
But in uninoculated samples, there was a low percent agreement.
Moreover, in comparison with the conventional method, it requires only a
week for detection of the microorganism.
