| Staphylococcus aureus has been considered one of the most important bacteria causing food poisoning. In the word,there are many cases that are caused by S.aureus. Foods that are frequently incriminated in staphylococcal food poisoning include meat and meat products,milk and dairy products and so on.The conventional method of inspecting food for possible contamination by coagulase positive Staphylococcus 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 this study specifically primers has been designed and a rapid method has been established to detect Staphylococcus aureus in food to shorten time of detection increase the sensitivity of method and provide a technical help.In this study,DNA concentration, MgCl2 concentration,,annealing temperature and PCR circles are optimized to determine the optimal PCR.The reaction mixture consisted of 5μl of 10xPCR 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-μl of dNTPs(1mM each in solution),0.5μl of each primer(40uM stock solution),0.25m of Taq(5U ?μl-1 of stock solution),2μl of DNA,and 37.75μl 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 52℃ 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 PCR products 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 80 bacterial strains to be detected including 60 S.aureus strains and 20 other bacterial strains except for S.aureus strains in order to determine specificity of amplification of primers.The results of 60 strains of Staphylococcus aureus were positive and those of 20 other strains were negative.With the isolated target the lower detection limit was 0.675 pg of DNA.The effect of 6 methods of extracting DNA from Staphylococcus aureus in dairy products were compared.A efficient extraction procedure was confirmed for extraction of Staphylococcus aureus DNA from dairy products. Staphylococcus aureus DNA was directly extracted from dairy products without enrichment. The extracted DNA was suitable for PCR detection.A solvent extraction procedure was successfully modified for extraction of Staphylococcus aureus DNA from artifically contaminated whole milk ,skim milk and cheese.The sensitivity of the uniplex PCR is 10cfu? ml-1of whole milk,skim milk and 55cfu· g-1 cheese.The developed methodology allows detection of Staphylococcus aureus...
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