Visual Detection Method Of Salmonella And Staphylococcus Aureus Based On PCR-LFD

In this study,a visual detection method for Salmonella and Staphylococcus aureus based on PCR-LFD was established,which provided a reference for the quality control of dairy products and the development of related detection methods.（1）The PCR reaction system and conditions of Salmonella and Staphylococcus aureus were established.Two pairs of primers were designed for the inva gene of Salmonella and the nuc gene of Staphylococcus aureus.The inva-2 primer of Salmonella and the nuc-1 primer of Staphylococcus aureus were determined as the best primers for PCR.The optimal PCR reaction system of Salmonella was determined as follows:mix premix 12.5μL,upstream primer 0.5μL,downstream primer 0.5μL,DNA template 0.75μL,dd H₂O 10.75μL;The optimal PCR reaction system of Staphylococcus aureus was determined as follows:mix premix 12.5μL,upstream primer 1μL,downstream primer 1μL,DNA template 1μL,dd H₂O 9.5μL;the optimal PCR conditions were pre-denaturation at 94℃for 4 min,denaturation at 94℃for 30 s,annealing at 56℃for 30 s,extension at 72℃for 30 s,30 cycles,extension at 72℃for 10 min.Under the optimal PCR system and conditions,the detection limits of Salmonella and Staphylococcus aureus were 1.85×10⁴CFU/m L and 1.35×10⁴CFU/m L,respectively.The detection limits of Salmonella and Staphylococcus aureus DNA were 34.7 pg and 11.7 fg,respectively.The specificity was good and could be used for the detection of Salmonella and Staphylococcus aureus.（2）On the basis of the optimized PCR,PCR-lateral flow dipstick（LFD）for Salmonella and Staphylococcus aureus were prepared respectively.The 5’end of the upstream primer was modified with Digoxin（DIG）,and the 5’end of the downstream primer was modified with biotin.Colloidal gold-anti-digoxin antibody was dropped on the binding pad,0.8 mg/m L streptavidin was sprayed on the detection line,and 1.0 mg/m L goat anti-mouse Ig G was sprayed on the quality control line.When the sample to be tested contains the target DNA,the streptavidin on the detection line will have an affinity with biotin,showing red.There was no cross-reaction with Escherichia coli O157,Streptococcus agalactiae and Bifidobacterium detected by this test strip,indicating that the PCR-LFD was highly specific for Salmonella and Staphylococcus aureus.The detection sensitivity of PCR-LFD for Salmonella and Staphylococcus aureus was determined.The results showed that the sensitivity of Salmonella and Staphylococcus aureus DNA reached 347 pg and 117 fg,respectively.The sensitivity of Salmonella and Staphylococcus aureus was 1.85×10³CFU/m L and 1.35×10³CFU/m L,which was compared with the conventional PCR-electrophoresis detection method.The sensitivity of Salmonella and Staphylococcus aureus can be increased by 10 times.The detection effect of PCR-LFD was evaluated by artificial simulated milk samples.The results showed that the detection limit of Salmonella in artificial simulated milk samples was 1.04×10⁴CFU/m L,and the detection limit of Salmonella DNA in artificial simulated milk was 1.44 ng.The detection limit of Staphylococcus aureus in artificial simulated milk samples was 0.5×10⁰CFU/m L,and the detection limit of Staphylococcus aureus DNA in artificial simulated milk samples was 0.523 pg.Therefore,the PCR-LFD developed in this study can be used for visual detection of Salmonella and Staphylococcus aureus in milk.