Sorghum mosaic virus indirect ELISA detection method and its kit and application
A technology of sorghum mosaic virus and detection kit, which is applied in the directions of measuring devices, instruments, scientific instruments, etc., can solve the problems of immature, difficult field disease, healthy sugarcane seedling breeding, etc., and achieves low cost and is suitable for large-scale samples. Detection, the effect of high detection sensitivity
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Embodiment 1
[0029] Example 1 Application of Sorghum Mosaic Virus ELISA Detection Method in Field Detection
[0030] A. Sample processing: Take about 0.5g of the sugarcane sample to be tested and put it into a mortar, add an appropriate amount of quartz sand, and then add 1-5ml of PBS buffer; grind until the sample is homogenous, let it settle for 15 minutes, and take the supernatant The solution was diluted 2-8 times with coating buffer for later use.
[0031] B. Sample coating: Take 100 μL of appropriately diluted test sample solution and add it to a 96-well microtiter plate, use SrMV antigen standard solution as a positive control, and healthy sugarcane sample solution as a negative control, and coat 1- 2h;
[0032] C. Washing: Remove the coating solution in the microtiter plate, add 100 μL PBST buffer to each well and rinse 3 times, each time for 5 minutes;
[0033] D. Blocking: remove the PBST buffer in the ELISA plate, add 100 μL of blocking solution (PBST solution containing 5% sk...
Embodiment 2
[0038] Example 2 Application of Sorghum mosaic virus ELISA detection method in the detection of a large number of samples
[0039] A. Sample treatment: Weigh about 0.2g of the sugarcane sample to be tested and put it into a 2mL EP tube, add 2 small steel balls and add 0.5-1.5mL of lysate. After being processed by the sample processing system (FastPrep-24, MP Biomedicals, USA) for 1-3 minutes, the sample can be homogenized, centrifuged at 8000-12000r / min for 5 minutes, and the supernatant is diluted 2-8 times with coating buffer for later use. .
[0040] B. Sample coating: Take 100 μL of appropriately diluted test sample solution and add it to a 96-well microtiter plate, use SrMV antigen standard solution as a positive control, and healthy sugarcane sample solution as a negative control, and coat 1- 2h;
[0041] C. Washing: Remove the coating solution in the ELISA plate, add 100 μL PBST buffer to each well and rinse for 3 times, each time for 5 minutes;
[0042] D. Blocking:...
Embodiment 3
[0049] Example 3 Assembly and Application of Sorghum Mosaic Virus ELISA Detection Kit
[0050] For ease of use, the assembly detection kit (100 times) is as follows with reference to the aforementioned content of the invention and the embodiments:
[0051] Kit components
[0052]
[0053] The above reagents are stored at -20°C
[0054]
[0055] The above reagents were stored at 4°C
[0056] 2ml EP tube 100pcs
[0057] 50 small steel balls with a diameter of 0.2-0.7cm
[0058] The above materials were stored at room temperature.
[0059] Among them, the SrMV-P3N polyclonal antibody is prepared as follows: obtain the P3 gene sequence of SrMV from the NCBI website, predict the amino acid sequence of the P3 protein, select the polypeptide whose N-terminus is exposed to the appearance of the natural protein as an antigen, and synthesize the polypeptide MFKPEGMKKIIEEEC (SEQ.ID. No.1), the polypeptide was mixed with Freund's adjuvant to immunize rabbits, the blood of the ...
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