Study On Polymerase Chain Reaction Assay For Detection Of Staphylococcus Aureus In Meat Products

Staphylococcus aureus is a widespread bacterial pathogen that can infect animinals and human beings. It exists in most foods, such as meat, fermented meat, milk, dairy products and vegetables. The foods most prone to contamination by Staphylococcus aureus are meat and meat products. Moreover, some strains of Staphylococcus aureus are capable of producing highly heat-stable toxins, thus presenting a widespread danger to society. In recent years, there have been increasing reports of Staphylococcus aureus related food poisoning. Staphylococcus aureus poisoning accounts for 33% of all bacterial food poisoning in United States, 45% in Canada and more than 50% in some Europe countries.Increasing rates of infection warrant a greater emphasis on the development of detection methods that are faster, more specific and highly sensitive. Current standard detection methods require lengthy culture enrichment steps to increase target bacterial numbers before isolation and identification can be performed. These conventional food microbiological techniques often require several days to detect bacterial pathogens present in food at low levels. Molecular biology-based techniques such as PCR may offer distinct advantages in terns of sensitivity and specificity. However, a critical factor in the use of PCR is the ability to provide a quality nucleic acid template, free of any inhibitory substances. The use of PCR in the detection of Staphylococcus aureus in meat has been hampered by many factors, including the presence of fat, protein and iron. Thus the key to detection appears to be the extraction of pure DNA template.In present study, MgCl₂ concentration, annealing temperature and PCR circles are optimized to determine the optimal PCR. The reaction mixture consisted of 5 uL of 10×PCR amplification buffer (200 mM Tris-HCl[pH 8.3], 500 mM KC1, 15 mM MgCl₂, 0.1%[wt/vol] gelatin, 0.5% Tween 20), 4 uL of dNTPs(1mM each in solution), 1 uL of each primer(20 μM stock solution), 0.25 uL of Taq(5 U/μL of stock solution), and 38.75 uL of double-distilled water. The reaction was run under the following conditions: Cool start;DNA pre-denaturation at 94℃ for 4 min;DNA denaturation at 94℃ for 1 min, primer annealing at 58℃ for 0.5 min, and DNA extension at 72℃ for 1.5 min for 35 cycles;the last extension was performed at 72℃ for 3.5 min. The PCRproducts were examined by electrophoresis with 2% agarose gel. Synthetic oligonucleotide primers of 21 and 24 bases, respectively, were used in the polymerase chain reaction to amplify a sequence of the nuc gene, which encodes the thermostable nuclease of Staphylococcus aureus. A DNA fragment of 279 bp was amplified. PCR products were confirmed by DNA sequencing. There were 54 bacterial strains to be detected including 33 S. aureus strains and 21 other bacterial strains except for S. aureus strains in order to determine specificity of amplification of primers. The results of 33 strains of Staphylococcus aureus were positive and those of 21 other strains were negative.The detection limit of PCR-based assays with FTA filters as templates was 20 cfu per PCR reaction. It is well known that cell wall of S. aureus are very thick, so it can not be easily lysed, but in this study S. aureus were easily lysed by FTA filter, inhibitors that may affect PCR in here, such as culture medium, cell debris were removed by washing with FTA purification buffer.In the present study, we developed a protocol that uses FTA filter to resolve the problems associated with DNA template preparation. The methods of extracting DNA from Staphylococcus aureus in meat with FTA filter were studied in here. According to the result of agarose- electrophoresis, an efficient deal procedure was confirmed for extraction of Staphylococcus aureus DNA from meat with FTA filter. The sample was pplied to the FTA filter and allowed to dry: at this point, the cellular material lyses and the nucleic acid become entangled in the paper. A boiling procedure with 10% SDS were taken and washed several times to remove contaminants and DNA binding. Staphylococcus aureus DNA was directly extracted from meat without enrichment. The extracted DNA was suitable for PCR detection.An assay using Polymerase Chain Reaction PCR) was developed for the detection of Staphylococcus aureus in meat. Based on flotation and solvent extraction technology, FTA filter was used to extract S. aureus DNA from artificially contaminated meat. Primers targeting the thermostable nuclease gene(nuc) were used to amplify a 279 bp DNA fragment which was confirmed by DNA sequencing. The detection limit of PCR was20 cfu.g"1 meat of S. aureus. This novel FTA-PCR assay allows for detection of Staphylococcus aureus in meat in less than 6h, which is 12 - 24 h less than that of conventional PCR with enrichment method.The effect of seven methods of extracting DNA from Staphylococcus aureus in meat were compared. An efficient extraction procedure was confirmed for extraction of Staphylococcus aureus DNA from meat without enrichment. Staphylococcus aureus DNA was directly extracted using FTA filter. The extracted DNA was suitable for PCR detection. The sensitivity of the PCR are 20cfu/g of pork, beef, mutton, chicken and sausage.Seventy-two samples were analyzed and the detection rate using the standard cultivation method was 70.8%, detection time was 5d. The detection rate of PCR amplification using filters was 73.6%, detection time was 6h. The detection rate of Baird-Parker R.P.F method was 69.4%, detection time was 18h. The detection rate of petrfilm RSA method was 61.1%, detection time was 18h. Thus PCR amplification using filters provides a faster and more sensitive method of S. aureus detection than the standard cultivation method.An efficient buffer that was used to washing FTA filter was invented, it is better and cheaper than the specific FTA filter buffer that is purchased in Whatman company.It provides a universal process for preparing DN A template...