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Method for detecting Haemophilus parasuis antibody and kit thereof

A technology of Haemophilus parasuis and a detection kit, which is applied in the field of ELISA detection method and detection kit for rapid detection of Haemophilus parasuis antibody, can solve the problem of the absence of Haemophilus parasuis antibody detection kit, Haemophilus parasuis antibody Problems such as unsatisfactory specificity of bacillus antibody detection method

Active Publication Date: 2019-10-25
NANJING AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide an indirect ELISA detection method for detecting Haemophilus parasuis antibody and its kit, which can solve the current situation that there is no commercialized Haemophilus parasuis antibody detection kit in China. The unsatisfactory specificity of the detection method of Haemophilus parasuis antibody

Method used

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  • Method for detecting Haemophilus parasuis antibody and kit thereof
  • Method for detecting Haemophilus parasuis antibody and kit thereof
  • Method for detecting Haemophilus parasuis antibody and kit thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Example 1: Prokaryotic expression of Haemophilus parasuis truncated P2 protein

[0069] 1.1 Amplification of the target gene P2

[0070] According to the P2 gene sequence of Haemophilus parasuis Nagasaki strain (GenBank: CP018034.1), a pair of cloning primers P2F / P2R were designed for the P2 gene, and the 5' ends of the primers contained restriction enzymes BamH I and Sal I respectively The enzyme cleavage sites and protective bases, the primers were designed with Primer Premier 5.0 software and synthesized by Nanjing GenScript Biotechnology Co., Ltd. The primer sequences are as follows:

[0071] P2F: 5'-CGGGATCCACAGTTTATGAAAATGAAG-3' (SEQ ID NO.3);

[0072] P2R: 5'-GCGTCGACTCTTGCGCCAGTTCTTACG-3' (SEQ ID NO.4);

[0073] Using the genomic DNA of the Haemophilus parasuis serotype 5 strain as a template, the Haemophilus parasuis serotype 5 strain can be purchased from commercial channels such as the "China Veterinary Microbiological Culture Collection and Management Cent...

Embodiment 2

[0081] Example 2: Optimization of the reaction conditions of the indirect ELISA detection method for Haemophilus parasuis antibody

[0082] 2.1 Determination of the optimal coating concentration of antigen and the optimal dilution of serum

[0083]Using the square array titration method, the purified P2 protein was diluted 2-fold with the coating solution (0.05mol / L carbonate buffer at pH 9.6), and the final concentrations were 1, 2, 4, 8, 16 μg / mL, add 100 μL to each well, repeat two rows for each dilution, and add a control without antigen coating, that is, the final antigen concentration is 0, and coat at 4°C overnight; then use washing solution (PBST solution with pH 7.4 ) washed 4 times, 300 μL per well each time, 3 min, and dried; add 250 μL blocking solution (2% gelatin) to each well and seal at 37 ° C for 2 h; wash as above; use Haemophilus parasuis negative serum and positive serum with PBST was diluted 1:50, 1:100, 1:200, and 1:400, respectively, and 100 μL was add...

Embodiment 3

[0130] Embodiment 3: Sensitivity test of Haemophilus parasuis antibody indirect ELISA detection kit

[0131] Immunize 65 weaned piglets at the age of 3 weeks with the commercialized Haemophilus parasuis inactivated vaccine, carry out the second immunization 21 days after the first immunization, and 14 days after the second immunization, collect blood from the anterior vena cava of all immunized piglets, separate serum, and use the present invention to establish ELISA method for the detection of Haemophilus parasuis antibody. Simultaneously, the isolated serum is diluted 20, 40, 80, 160, 320, 640 times, and the ELISA method established by the present invention is used to detect the positive serum of different dilutions of Haemophilus parasuis to confirm the sensitivity of the detection kit. sex.

[0132] The results showed that 63 of the 65 Haemophilus parasuis positive sera were positive, with a positive rate of 96.9%. The results were consistent with those of the commercial ...

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Abstract

The invention discloses a novel indirect ELISA detection method for Haemophilus parasuis antibody, relates to the field of antibody detection, and especially relates to an indirect ELISA detection method for Haemophilus parasuis antibody with the use of recombinant truncated proteins P2 and a kit thereof. According to the method, by truncating recombinant Haemophilus parasuis outer membrane proteins P2 and using the proteins as a coating antigen, the accurate detection of the Haemophilus parasuis antibody can be achieved by optimizing an ELISA reaction condition, a detection mode and a critical value determining mode, the method has good specificity and sensitivity, the problem of an over-high background in the process of antibody detection is effectively solved, and the cost of detectionis greatly reduced.

Description

technical field [0001] The present invention relates to the field of veterinary medicine. Specifically, the present invention relates to an ELISA detection method for rapid detection of Haemophilus parasuis (HPS) antibody and a detection kit thereof. Background technique [0002] Haemophilus parasuis (HPS) can cause Haemophilus parasuis disease in pigs. The clinical symptoms of the disease are polyarthritis, serositis, and meningitis. It often occurs in the nursery stage of piglets, and the mortality rate can reach 50%. With the rapid expansion of the scale of the pig industry and the change of the pig farming model, the epidemic trend of this disease has become increasingly serious in recent years, which has brought huge economic losses to the pig industry in my country. The detection of serum antibodies to Haemophilus parasuis can accurately diagnose whether pigs have ever been infected by this bacteria, and evaluate the immune protection effect of related vaccines. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569G01N33/535
CPCG01N33/535G01N33/56911G01N33/6854G01N2333/285
Inventor 范红结张鹏云周红蔺辉星
Owner NANJING AGRICULTURAL UNIVERSITY
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