The invention relates to a method for simultaneously detecting two types of
pathogenic bacteria based on two-color time-resolved
fluorescence labeling-
magnetic separation aptamer recognition; the method is used for detecting
salmonella typhimurium and
staphylococcus aureus in milk and dairy products. The method comprises the steps of enabling an
aptamer of the
salmonella typhimurium and an
aptamer of the
staphylococcus aureus to be respectively coupled with NaYF4: Ce / Tb time
fluorescence resolution nanomaterial and NaGdF4: Eu time
fluorescence resolution nanomaterial to form
signal probes; meanwhile, enabling the two aptamers to be coupled with Fe3O4 magnetic nanomaterial to form capture probes; when a target material exists, finally forming a
sandwich compound due to the specific recognition of the aptamer for the target material. The
salmonella typhimurium and the
staphylococcus aureus can be quantitatively detected by monitoring the intensities of fluorescence signals at the parts of 544nm and 615nm, the linear ranges of the salmonella typhimurium and the
staphylococcus aureus are respectively 10<2>-10<5>cfu ml<-1> and 10<2>-10<5>cfu ml<-1>, and the lowest detection limits of the salmonella typhimurium and the
staphylococcus aureus are respectively 15cfu ml<-1> and 20cfu ml <-1>. The method can be used for detecting the two types of
pathogenic bacteria at the same time, and has the advantages of being high in sensitivity, rapid, simple and convenient. The method is applied to the detection of the milk and the dairy products, and is accurate and reliable in results.