| ObjectiveStaphylococcus aureus has been considered one of the most important bacteria causing food poisoning. The percentage of food poisoning caused by S. aureus is round about 33% of bacterial food poisoning in America, 45% in Canada and more than 50% in some Europe countries such as Hungary, Finland. In our country, there are many cases that are caused by S. aureus.The reason of food poisoning is that some strains are capable of producing a highly heat - stable toxin. So far, a family of fourteen major serological types of SES has been described, of which the Staphylococcus enterotoxins A and D are i-solaled most often from outbreaks of food poisoning. The nature of the disease is often mild, but as little as l00ng of enterotoxin(s) can cause symptoms ( nause-a, vomiting, abdominal pain, and diarrhea) after 1 -6h in sensitive persons. The primary reservoirs for S. aureus are the skin and mucous membranes, especially of the nasopharyngeal region. The pathogen is founded in 30 - 80% of the human population and one - to two - thirds of theses carriers harbour enterotoxi-genic strains. Thus, unhygienic treatment of food has to be considered as a major risk of contamination, and staphylococcal food poisoning is often associated with highly manually handled food. Foods that are frequently incriminated in staphylococcal food poisoning include meat and meat products, milk and dairy products,residue rice and cool powder.The conventional method of inspecting food for possible contamination by coagulase positive S. aureus is too time - consuming, which can take 3 to 5 days to complete. And it is not enough sensitive to detect S. aureus. In othercountries, a rapid polymerase chain reaction (PCR) method for detection of S. aureus has been established, but there is no one to apply the method for detecting S. aureus in food in our country. In this study, specifically primers has been designed and a rapid method has been established to detect S. aureus in food to shorten time of detection, increase the sensitivity of method and provide a technical help.Methods1. Establishment of PCR methods1.1 DNA extraction and quantificationExtract DNA according operation of DNA extraction kit from all bacterial strains. S. aureus strains should be disposed by two methods ( acetone and lyso-staphin) before DNA extraction. Detect DNA concentration by ultraviolet spec-trophotometer.1. 2 Optimization of the PCR reactionThe optimization included the DNA concentration, the primers concentration, MgCl2 concentration and annealing temperature.1. 3 Detection of amplification productsThe PCR products were analyzed by agarose gel electrophoresis using 1.2% agarose. Gels were visualized and photographed using a gel documentation systems. To verify the identity of each amplification products, cloned fragments were sequenced.1.4 Specificity of amplification of primers by PCRThere were 83 bacterial strains to be detected including 52 S. aureus strains and 31 other bacterial strains except for S. aureus strains.1.5 Sensitivity of detection of S. aureusSerial decimal dilutions were prepared from the initial broth that cells were cultured in nutrient broth for 12h. DNA should be extracted from different dilution using two methods (acetone and lysostaphin) , preparing for amplification.2. Artificially contamination in food by S. aureus 2.1 Preparation of samplesA 25ml(g) samples was removed under aseptic conditions and added into 225ml of 7.5% sodium chloride broth and homogenized. The samples included ready - to - eat food ( aseptic milk and ice - cream) and none ready - to - eat food (milk exposed in air and meat).2.2 Artificially contamination by S. aureus in broth, ready - to - eat food and none ready - to - eat foodS. aureus were cultured at 371 for overnight. Prepare Serial decimal dilutions from this initial solution and count the colonies. According to estimation, three dilutions were selected and added into broth (7.5% NaCl broth: food =9; 1). The cells were cultured with shakin... |
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