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Pullorum disease antibody latex agglutination negative selection detection kit as well as preparation method and application thereof

A detection kit, latex agglutination technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of non-specific reaction, false negative, detection false positive, etc., to get rid of variation and instability, high sensitivity, Antigen stabilization effect

Active Publication Date: 2017-05-24
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the whole blood or serum glass plate agglutination test is mainly used, and the antigenic component is inactivated whole bacteria. There are plate staining antigen products used to detect pullorum in my country, but the inactivated whole bacteria are used as antigens in practical applications. Bacteria batch, growth state and production process, etc., can cause non-specific reactions caused by unstable antigen components, resulting in false positives and false negatives in the detection, resulting in inaccurate elimination of pullorum positive chickens, causing farmers Serious economic loss, so there is an urgent need for a detection method that can more accurately and efficiently identify Salmonella pullorum infection

Method used

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  • Pullorum disease antibody latex agglutination negative selection detection kit as well as preparation method and application thereof
  • Pullorum disease antibody latex agglutination negative selection detection kit as well as preparation method and application thereof
  • Pullorum disease antibody latex agglutination negative selection detection kit as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Preparation method of C-terminal recombinant protein of Salmonella surface protein InvJ and secreted protein SopA

[0041] 1. The preparation method of Salmonella surface protein InvJ recombinant protein, the steps are:

[0042] 1. Cloning of InvJ gene

[0043] The amino acid sequence of the InvJ recombinant protein of Salmonella enteritidis is shown in SEQ ID NO:1 (namely, the accession number is: the amino acid sequence from position 1 to position 336 of InvJ of KOX81849.1), and the nucleotide sequence of the coding gene is shown in SEQ ID NO : Shown in 3 (namely, the nucleotide sequence from position 1 to position 1008 of the InvJ gene with accession number LIHI01000011.1).

[0044] Design primers based on the InvJ gene sequence: upstream primer InvJ F: 5’-CGC GGATCC ATGGGCGATGTGTCAGCTGT-3’, underlined part is BamHI site; downstream primer InvJ R: 5’-CCC AAGCTT GGCGTCATCCTCCTCGCA-3', the underlined part is Hind III site.

[0045] The genomic DNA of Salmonella enteritidis (...

Embodiment 2

[0068] Optimization of preparation method of C-terminal recombinant protein sensitized latex reagent of Salmonella surface protein InvJ and secreted protein SopA

[0069] Salmonella surface protein InvJ and secreted protein SopA C-terminal recombinant protein were used as antigens to prepare sensitized latex reagent, and then the pullorum antibody was detected by negative screening of latex agglutination test.

[0070] 1. Latex agglutination test method and result judgment

[0071] Drop about 10μL of the sample solution to be tested on a clean glass slide, then add about 10μL of sensitizing latex reagent, stir and mix well, shake the slide, and observe against a black background within 3 minutes, and set a positive control and a negative control at the same time. ++++ is 100% latex agglomerated, all agglomerated into flocculent masses, and the liquid is clear; +++ is 75% latex coagulated into small particles, the liquid is slightly turbid; ++ is 50% latex coagulated into finer Parti...

Embodiment 3

[0132] Application of latex agglutination negative screening test kit for pullorum antibody

[0133] 1. Application method of latex agglutination negative screening test kit for pullorum antibody

[0134] (1) Sampling and testing

[0135] Collect the venous blood of the chickens to be tested and separate the serum; draw 2 10μL samples of the serum to be tested and drop them on a glass slide, add equal volumes of InvJ recombinant protein sensitized latex reagent and SopA C-terminal recombinant protein sensitized latex reagent, and stir well After shaking for 30 seconds, observe the test results. The processing method of the positive and negative control serum is the same as the sample.

[0136] (2) Results judgment

[0137] Judge the InvJ protein latex agglutination test and SopA C-terminal protein latex agglutination test respectively; when the positive control serum agglutination degree is 100% agglutination (++++), and the negative control serum has no agglutination, the test sample...

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Abstract

The invention discloses a pullorum disease antibody latex agglutination negative selection detection kit, comprising a salmonella surface protein InvJ recombinant protein sensitized latex reagent and a secreted protein SopA C-terminal recombinant protein sensitized latex reagent, wherein the InvJ recombinant protein sensitized latex reagent can rapidly detect a salmonella antibody by virtue of a latex agglutination test, the SopA C-terminal recombinant protein sensitized latex reagent can detect serotype salmonella antibodies except for the pullorum disease by virtue of the latex agglutination test, and the combination of the two reagents is used for negative selection of a pullorum disease antibody positive blood or serum sample. The invention also discloses a preparation method of salmonella surface protein InvJ recombinant protein and secreted protein SopA C-terminal recombinant protein sensitized latex reagents. The pullorum disease antibody latex agglutination negative selection detection kit disclosed by the invention has the advantages of stable antigen, strong specificity and high sensitivity, and solves the problem of antigen instability caused by potential variability and changed culture conditions of an existing pullorum disease agglutination antigen production strain.

Description

Technical field [0001] The invention belongs to the field of bioengineering and detection technology, and specifically relates to a negative screening detection kit for Salmonella pullorum antibody latex agglutination, and also relates to the InvJ recombinant protein sensitized latex reagent and SopA C-terminal recombinant protein sensitized latex reagent in the detection kit The preparation method also relates to the application of the kit in detecting antibodies against Salmonella pullorum infection. Background technique [0002] Salmonella has a variety of serotypes. The main serotypes that can infect chickens are Salmonella pullorum and Salmonella enteritidis. Among them, pullorum disease (Pullorum Disease, PD) is a type of infectious disease caused by Salmonella pullorum (SG), and it is currently one of the main infectious diseases that harm the chicken industry. Salmonella pullorum can spread vertically and horizontally, and it is very harmful to the chicks. Infected chick...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/559
CPCG01N33/559G01N33/56916
Inventor 张腾飞李丽罗青平王红琳邵华斌温国元张蓉蓉罗玲卢琴汪宏才艾地云毛光明
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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