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, reduced sensitivity, complicated process, etc., and achieves the effects of improving specificity, improving sensitivity, and reducing pollution
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specific Embodiment 1
[0035] A method for detecting Salmonella typhimurium based on BCA-RPA and CRISPR-Cas12a system, such as figure 1 shown, including the following steps:
[0036] (1) Synthesis of AuNPs probes
[0037] A. Add 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 turned from light yellow to wine red, and the magnetic stirring was continued for 15 minutes to obtain the AuNPs solution, which was stored at 4 °C in the dark for later use ;
[0038] B. Dilute the AuNPs solution with Na 2 CO 3 After the pH of the solution was adjusted to 7.4, 10 μL of 1 mg / mL monoclonal antibody solution against typhoid fever was added, and the ligated DNA was added to a final concentration of 3 μM, reacted at -80 °C for 15 minutes, at 9000 rpm, 4 Centrifuge at ℃ for 15 minutes to remove the supernatant, disperse the precipitate in 1 mL of 0.01 M ph...
specific Embodiment 2
[0049] In order to verify the value of specific embodiment one detection method of the present invention in practical application, in milk, add Salmonella typhimurium standard solution as actual sample, the Salmonella typhimurium of different concentrations in milk is detected, and the result is as follows: Figure 5 , 6 shown;
[0050] Depend on Figure 5 It can be seen that different concentrations (10 0 -10 6 CFU / mL) in the presence of Salmonella typhimurium, the results can be read with naked eyes, and can be used for the detection of Salmonella typhimurium in actual samples.
[0051] 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 has a linear relationship with the concentration of Salmonella typhimurium. The correlation coefficient R=0.990, and the linear relationship is good.
specific Embodiment 3
[0053] Figure 7 For specific embodiment one detection method respectively to 10 5 CFU / mL Salmonella typhimurium, 10 5 CFU / mL Escherichia coli, 10 5 CFU / mL Listeria monocytogenes, 3×10 5 CFU / mL Salmonella typhimurium: 3×10 5 CFU / mL Escherichia coli: 3×10 5 CFU / mL Listeria monocytogenes=1:1:1 detects the fluorescence intensity value; Figure 7 It can be seen that when Salmonella typhimurium exists, the detected fluorescence intensity value is far greater than the fluorescence intensity value of interfering foodborne pathogens, indicating that the sensor has specific detection for Salmonella typhimurium.
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