A method for detecting Salmonella typhimurium based on bca-rpa and CRISPR-cas12a system
A technology for Salmonella and Typhimurium, applied in the field of detection of Salmonella Typhimurium, can solve the problems of barcode DNA loss, complicated process, and reduced sensitivity, and achieve the effects of improving specificity, reducing pollution and improving sensitivity
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specific Embodiment 1
[0034] A method for detecting Salmonella typhimurium based on BCA-RPA and CRISPR-Cas12a system, such as figure 1 shown, including the following steps:
[0035] (1) Synthesis of AuNPs probes
[0036] A. Put 100 mL of 1 mM HAuCl 4 After the aqueous solution was heated to boiling with magnetic stirring, 10 mL of 38.8 mM trisodium citrate aqueous solution was added, and the heating was stopped when the solution changed from light yellow to wine red, and the magnetic stirring was continued for 15 minutes to obtain AuNPs solution, which was stored at 4 °C in the dark for later use. ;
[0037] B. AuNPs solution with Na 2 CO 3 After the solution was adjusted to pH 7.4, 10 μL of 1 mg / mL anti-typhoid fever monoclonal antibody solution was added, and ligated DNA was added to a final concentration of 3 μM, and the reaction was carried out at -80 °C for 15 minutes, at 9000 rpm, 4 Centrifuge at ℃ for 15 minutes to remove the supernatant, disperse the pellet in 1 mL of 0.01 M phosphate...
specific Embodiment 2
[0047] In order to verify the value of the detection method of the specific embodiment of the present invention in practical application, a standard solution of Salmonella typhimurium was added in milk as an actual sample, and different concentrations of Salmonella typhimurium in the milk were detected, and the results were as follows: Figure 5 , 6 shown;
[0048] Depend on Figure 5 It can be seen that different concentrations (10 0 -10 6 CFU / mL) fluorescence intensity in the presence of Salmonella typhimurium, the result can be read by naked eyes, and can be used for the detection of Salmonella typhimurium in actual samples.
[0049] Depend on Image 6 It can be seen that the method of the present invention detects Salmonella typhimurium in milk, and the fluorescence intensity of different concentrations of Salmonella typhimurium is linearly related to the concentration of Salmonella typhimurium, the correlation coefficient R=0.990, and the linear relationship is good....
specific Embodiment 3
[0050] Figure 7 For a specific embodiment, a detection method is respectively paired with 10 5 CFU / mL Salmonella typhimurium, 10 5 CFU / mL E. coli, 10 5 CFU / mL Listeria monocytogenes, 3×10 5 CFU / mL Salmonella typhimurium: 3×10 5 CFU / mL E. coli: 3×10 5 CFU / mL Listeria monocytogenes = 1:1:1 fluorescence intensity value for detection; Figure 7It can be seen that when Salmonella typhimurium exists, the detected fluorescence intensity value is far greater than the fluorescence intensity value of interfering food-borne pathogens, indicating that the sensor has specific detection of Salmonella typhimurium.
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