African swine fever virus detection kit and application thereof
A technology of African swine fever virus and detection kit, which is applied in the biological field, can solve the problems of unfavorable rapid detection, long detection time, and low sensitivity of paper strips, so as to facilitate rapid on-site detection and diagnosis, shorten detection time, and reduce false positives Effect
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Embodiment 1
[0050] Embodiment 1 Preparation, purification and identification of African swine fever virus P30 protein monoclonal antibody
[0051] 1.1 Preparation and purification of African swine fever virus P30 protein
[0052] With reference to the gene sequence published by GenBank (GenBank accession number: MH766894.1), the ASFV P30 protein gene fragment was synthesized, induced and expressed using the Escherichia coli expression system, and the expressed protein was purified by protein affinity chromatography. After purification, the measured content was 2.4 mg / mL.
[0053] 1.2 Preparation and purification of African swine fever virus P30 protein monoclonal antibody
[0054] The ASFV P30 protein was mixed with an equal volume of complete Freund's adjuvant, and 10 μL of Magic adjuvant from Ambec Biotechnology Co., Ltd. was added, mixed and emulsified, and mice were immunized once. Then, the ASFV P30 protein was mixed with an equal volume of Freund's incomplete adjuvant and 10 μL of...
Embodiment 2
[0065] Preparation and detection method of embodiment 2 African swine fever virus detection kit
[0066] 2.1 Preparation and identification of enzyme-labeled monoclonal antibodies
[0067] The purified monoclonal antibody was labeled with horseradish peroxidase (HRP) by the modified sodium periodate method. Three batches of HRP-labeled enzyme-labeled monoclonal antibodies were prepared from two monoclonal antibodies 3H7 and 5B4, all of which were brownish-red clear liquids, with a molar ratio of 1.679-1.790, not less than 1.5; the labeling rate was 0.544-0.574, and the average Not less than 0.5; direct ELISA titer for activity detection is 1:6400-1:25600.
[0068] 2.2 Study on pairing mode of monoclonal antibody
[0069]Monoclonal antibodies 3H7 and 5B4 were coated and labeled with HRP enzyme respectively, and three African swine fever virus negative blood samples (N1-N3) and African swine fever virus positive blood samples ( P1-P3), the results showed that when 3H7 was use...
Embodiment 3
[0113] The evaluation of embodiment 3 African swine fever virus detection kit
[0114] Sensitivity: The ASFV P30 protein prepared in Example 1 was serially diluted, and detected according to the detection method. As a result, the lower limit of detection of the African swine fever virus detection kit was 20 ng / mL.
[0115] Sensitivity: get 50 samples with clear background and PCR detection of ASFV nucleic acid negative (containing 35 parts of organ tissue grinding liquid supernatant, 12 parts of serum plasma or whole blood, 3 parts of saliva), the ASFV P30 prepared in Example 1 The protein was diluted and added to the 50 samples, so that the final concentration of P30 protein in the sample was 5-80ng / mL, and the kit was used for detection. As a result, the kit detected 47 positives (the final concentration of P30 protein was 20-80ng / mL). 80ng / mL), 3 copies were negative (the final concentration of P30 protein was 5-10ng / mL), and the sensitivity was 94.0% (47 / 50).
[0116] Spe...
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