Detection of Staphylococcus aureus in Processed Meat Using PCR-Based Detection Kit

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.