Investigation on the multiplex PCR in detection of five types of bacteria

OBJECTIVE To establish a multiplex polymerase chain reaction(PCR)assay in detection of Salmonella,Shigella,Vibrio parahaemolyticus,Staphylococcus aureus and Escherichia coli O157.METHODS Based on the gene sequences of hilA gene in Salmonella,ipaH gene in Shigella,TDH gene in Vibrio parahaemolyticus,pSa-442 Sau3AI fragment gene Staphylococcus aureus and rfbE gene Escherichia coli O157,three pairs of primers were designed.Genomic DNA was extracted by metal bath after shaking culture for 4 hours,and the PCR-amplified products were analyzed by automatic capillary electrophoresis.RESULTS The predicted specific DNA amplication bands were demonstrated at sequences of 580 bp,423 bp,257 bp,245 bp and 194 bp.Assay on sensitivity of this multiplex PCR revealed that it could be detected as little as 101-2 cfu/ml from salmonella,101 cfu/ml from shigella,102 cfu/ml from vibrio parahaemolyticus,102 cfu/ml from Staphylococcus aureus and 101 cfu/ml from Escherichia coli O157 in simulation experiments.CONCLUSION The multiplex PCR method is a method with the aspects of rapid,specific and sensitive as well as high distinguishability,which could be useful for the rapid detection of food-borne bacterial pathogens.