About Bovine viral diarrhea
Bovine viral diarrhea (mucosal disease) is an infectious disease caused by bovine viral diarrhea virus. Cattle of all ages are susceptible to infection, with young cattle being most susceptible.
Bovine viral diarrhea-mucosal disease can pass through the placenta, especially in early pregnancy. Bovine viral diarrhea-Once infected cows with mucosal virus serum antibodies are negative, often through the placenta, the fetus is immunosuppressed, causing persistent viremia. If the calf is produced normally, viremia can continue to reach adulthood. Breeding cattle are clinically healthy and there are no protective antibodies in the serum, but the body is always poisonous and is the most dangerous source of infection in the herd.
Rapid Test Kit principle
This kit uses double antibody sandwich enzyme-linked immunoassay (ELISA) to determine the bovine diarrhea virus (BVDV) in the specimen. The microbore plate is coated with purified bovine diarrhea virus (BVDV) antibody to make a solid-phase antibody, which can be combined with bovine diarrhea virus (BVDV) in the sample. The HRP-labeled bovine diarrhea virus (BVDV) antibody binds to form an antigen-antibody-enzyme-labeled antigen complex. 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 absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and compared with the CUTOFF value to determine the presence or absence of bovine diarrhea virus (BVDV) in the specimen.
Rapid Test Kit sample processing
1. Serum and plasma samples can be directly detected;
2.2. Perform the experiment as soon as possible after the specimen is prepared as required. If it cannot be detected in time, the specimen can be stored at -20 ℃ for one month, but repeated freezing and thawing should be avoided;
3. Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
Operation steps of Rapid Test Kit
1. Numbering: Samples corresponding to the microwells are numbered in sequence, and each plate should be set with 2 wells for negative control, 2 wells for positive control, and 1 well for blank control (the blank control well does not add sample and enzyme labeling reagents, the remaining steps are the same)
2. Add samples: add negative control and positive control 50μl to the negative and positive control wells respectively. Then add 40μl of sample diluent to the sample well to be tested, and then add 10μl of the sample to be tested. 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, gently shake to mix;
3. Incubation: seal the plate with a sealing film and incubate at 37 °C for 30 minutes;
4. Mixing solution: add 20 times concentrated washing liquid to distilled water to 600ml and reserve;
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let it stand for 30 seconds and then discard, repeat 5 times, pat dry;
6. Add enzyme: add 50μl of enzyme label reagent to each well, except the 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, and then add 50μl of developer B, mix gently, and develop at 37 °C in the dark; wait 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).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be performed within 15 minutes after adding the stop solution.
Rapid Test Kit result judgment
Test effectiveness: the average value of positive control wells ≥1.00; the average value of negative control ≤0.10 critical value (CUT OFF) calculation: critical value = average value of negative control wells +0.15;
Negative judgment: the sample with OD value
Notes for Rapid Test Kit
1. The operation is carried out in strict accordance with the instructions. The components of different batches of this reagent must not be mixed.
2. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme-coated plate is unsealed after opening, the strip should be stored in a sealed bag.
3. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in the water bath during dilution, and the result will not be affected during washing.
4. The sealing film is limited to one-time use to avoid cross-contamination.
5. Please keep the substrate away from light.
6. The test result must be determined by the reading of the microplate reader. When using dual-wavelength detection, the reference wavelength is 630nm
7. All samples, washing liquids and various wastes should be treated as infectious agents. The stop solution is 2M sulfuric acid, and you must pay attention to safety when using it.
Generally speaking, professional BVD testing cost is relatively expensive, and the average small and medium-sized farms cannot afford it. Therefore, the appearance of BVD test kits can greatly reduce the farm's testing costs and ensure the health of the tested cattle.