Instruction Manual of Rice Bacterial Stripe Kit

Detection range: 96T

1.5ng / L-45ng / L

purpose of usage:

Rice bacterial stripe disease (RBS) kit is used to determine the content of rice bacterial stripe disease (RBS) in plant tissue, cells and other related samples.

Experimental principle

This kit uses the double-antibody sandwich method to determine the level of bacterial stripe disease (RBS) in rice. Microporous plates were coated with purified rice bacterial stripe disease (RBS) antibody to make solid-phase antibodies. Rice bacterial stripe disease (RBS) was added to the microwells coated with monoclonal antibody in turn, and then labeled with HRP Rice bacterial stripe disease (RBS) antibody binds to form an antibody-antigen-enzyme-labeled antibody complex, and after thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with rice bacterial stripe disease (RBS) in the samples. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of rice bacterial stripe disease (RBS) in the sample was calculated by a standard 45 curve.

Kit composition

1

30 times concentrated washing solution

20ml × 1 bottle

7

Stop solution

6ml × 1 bottle

2

Enzyme reagent

6ml × 1 bottle

8

Standard product (80ng / L)

0.5ml × 1 bottle

3

Enzyme coated plate

12 holes × 8

9

Standard dilution

1.5ml × 1 bottle

4

Sample diluent

6ml × 1 bottle

10

Instructions

1 serving

5

Developer A liquid

6ml × 1 bottle

11

Sealing film

2 sheets

6

Developer B liquid

6ml × 1 / bottle

12

sealed bag

1

Specimen requirements

1. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided

2. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.

Steps

1. Dilution of standard products: This kit provides one original standard product. The user can perform dilution in a small test tube according to the following chart.

40ng / L

Standard No. 5

150μl of original standard is added to 150μl standard dilution

20ng / L

Standard 4

150μl Standard No. 5 is added to 150μl standard dilution

10ng / L

Standard 3

150μl Standard No. 4 is added to 150μl Standard Diluent

5ng / L

Standard No. 2

150μl Standard No. 3 is added to 150μl Standard Diluent

2.5ng / L

Standard 1

150μl Standard No. 2 is added to 150μl Standard Diluent

2. Sample addition: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the operation is the same), standard wells, sample wells to be tested. Accurately add 50μl of the standard on the enzyme-coated plate, add 40μl of sample diluent to the sample well, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix.

3. Incubation: Seal the plate with a sealing plate and incubate at 37 ° C for 30 minutes.

4. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 times and reserve

5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat 5 times and pat dry.

6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.

7. Incubation: The operation is the same as 3.

8. Washing: The operation is the same as 5.

9. Color development: add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes.

10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).