Detection of Salmonella spp.,Escherichia coli and Staphylococcus aureus by Multiplex PCR / 微生物学通报

ABSTRACT

According to DNA sequences of the invA gene of Salmonella spp.,the phoA gene of Escherichia coli and the nuc gene of Staphylococcus aureus,three pairs of oligonucleotide primers were designed and synthesized to amplify the special DNA sequences by multiplex PCR. Moreover,the reaction conditions of multiplex PCR were optimized. The results showed the multiplex PCR using the three pairs of primers produced specific amplicons of expected sizes,284bp for Salmonella spp.,622bp for Escherichia coli,484bp for Staphylococcus aureus. The optimized reaction conditions followed as the concentration of primer 40nmol/L for Salmonella spp.,40nmol/L for Escherichia coli,80nmol/L for Staphylococcus aureus,2.4mmol/L Mg 2+ ,200?mol/L dNTP,1.5U Taq DNA polymerase,anneal temperature from 55.0℃ to 57.4℃. Under the condition,the detection limits for DNA template were 10.2pg,10.2pg and 102.0pg for Salmonella spp.,Escherichia coli and Staphylococcus aureus,respectively. The whole process could be completed within 4h. The multiplex PCR assay was a specific,sensitive,rapid and reliable method for detecting Salmonella spp.,Escherichia coli and Staphylococcus aureus,which establish important foundation for simultaneous detection for these three bacteria in food.