Method for detecting Salmonella based on nucleic acid chromatography biosensing technology
A Salmonella and nucleic acid technology, applied in the direction of measuring devices, analysis materials, instruments, etc., can solve the problems of long detection period, cumbersome operation, and difficult analysis results of molecular biology methods
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Embodiment 1
[0048] Embodiment 1 Method for detecting Salmonella based on nucleic acid chromatography biosensing technology
[0049] 1. Experimental materials
[0050] The information on Salmonella and non-Salmonella strains used in this example is shown in Table 1.
[0051] Table 1 Information on Salmonella and non-Salmonella used
[0052]
[0053] The specificity of the nanoenzyme sensor was determined using Salmonella and other bacterial strains. All strains were stored in 20% (v / v) glycerol solution at -80°C until use. It was then activated overnight in LB medium. Salmonella concentrations were determined spectrophotometrically.
[0054] 2. Salmonella genome extraction
[0055] Using the bacterial genomic DNA extraction kit from New Industry, the specific steps are as follows:
[0056] Using the bacterial genomic DNA extraction kit from New Industry, the specific steps are as follows:
[0057] Using the bacterial genomic DNA extraction kit from New Industry, the specific step...
Embodiment 2
[0095] Example 2 Optimization of Nanozyme Nucleic Acid Test Strips
[0096] Synthesis of Fe by hydrothermal method 3 O 4 Magnetic particles were then incubated with biotin secondary antibody (goat anti-mouse IgG) to prepare nanozyme probes. The T-line and C-line positions on the NC membrane were marked with FITC antibody and biotin antibody, respectively, and then assembled into nanozyme nucleic acid test strips after drying. In order to improve the sensitivity of the nanoenzyme sensor, a systematic analysis was carried out by comparing the properties of membrane materials, the concentration of FITC antibody in the detection area, the amount of nanoenzyme probes, and the reaction time. The results show that the nanoenzyme sensor using Millipore135S nitrocellulose membrane has better performance ( figure 2 A). The highest signal peak area was obtained with 1 mg / mL FITC antibody and 1 mg / mL goat anti-mouse IgG ( figure 2 B). In addition, the amount of nanozyme probes aff...
Embodiment 3
[0097] Example 3 Performance detection of nano-enzyme sensor
[0098] The principle of the nanoenzyme sensor is as follows. First, the sample is treated with PMA (step 1). PMA can selectively penetrate the damaged cell membrane of dead cells, bind to intracellular DNA, and make it unavailable for subsequent LAMP amplification, but if it is an intact cell membrane of living cells, PMA cannot enter the cell. Then, many BIO- and FITC-linked duplex DNAs were generated in a short time using LAMP (step 2). In the presence of the target substance invA-specific sequence, it is recognized and amplified by the four primers. The third is the visual interpretation of the nanozyme nucleic acid test strip (step 3). FITC antibody and goat anti-mouse IgG were immobilized on nitrocellulose membrane by physical adsorption to form detection zone (TL) and quality control zone (CL), respectively. If the sample is positive, after LAMP amplification, the 5' end of the target substance is labeled...
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