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Pullorum antibody latex agglutination negative screening kit, preparation method and application

A detection kit and latex agglutination technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of non-specific reactions, false positives, false negatives, etc., and achieve the goal of getting rid of variation and instability, strong specificity, The effect of high sensitivity

Active Publication Date: 2018-06-29
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 antibody latex agglutination negative screening kit, preparation method and application
  • Pullorum antibody latex agglutination negative screening kit, preparation method and application
  • Pullorum antibody latex agglutination negative screening kit, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0041] One, the preparation method of Salmonella surface protein InvJ recombinant protein, its step is:

[0042] 1. Cloning of InvJ gene

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

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

[0045] Genomic DNA of Salmonella Enteritidis (CVCC ...

Embodiment 2

[0068] Optimization of the preparation method of latex reagent for sensitization of recombinant protein of Salmonella surface protein InvJ and secreted protein SopA C-terminus

[0069] Salmonella surface protein InvJ and secretory protein SopA C-terminal recombinant protein were used as antigens to prepare sensitized latex reagents, and latex agglutination test negative screening was used to detect pullorum antibody.

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

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

Embodiment 3

[0132] Application of Latex Agglutination Negative Screening Kit for Pullorum Antibody

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

[0134] (1) Sampling and testing

[0135] Collect the venous blood of the chickens to be tested and separate the serum; draw two 10 μL samples of the serum to be tested and drop them on the glass slide, add an equal volume of InvJ recombinant protein sensitized latex reagent and SopA C-terminal recombinant protein sensitized latex reagent respectively, and stir evenly After shaking for 30 seconds, observe the test results. Positive and negative control sera were processed in the same manner as the samples.

[0136] (2) Result judgment

[0137] The InvJ protein latex agglutination test and the SopA C-terminal protein latex agglutination test were judged respectively; when the agglutination degree of the positive control serum was 100% agglutination (++++), and the negative control serum had no a...

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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 technical field of bioengineering and detection, and in particular relates to a latex agglutination negative screening detection kit for Salmonella pullorum antibody, and at the same time relates to the latex reagent for sensitization of InvJ recombinant protein and the latex reagent for recombinant protein sensitization of SopA C-terminal in the detection kit The preparation method also relates to the application of the kit in detecting antibodies against Salmonella pullorum pullorum infection. Background technique [0002] Salmonella has a variety of serotypes, and the main ones that can infect chickens are Salmonella pullorum, Salmonella enteritidis and other serotypes. Among them, Pullorum Disease (PD) is a kind of infectious disease caused by Salmonella Pullorum (SG), and it is one of the main infectious diseases that endanger the chicken industry at present. Salmonella pullorum can be transmitted vertically and horiz...

Claims

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Application Information

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Patent Type & Authority Patents(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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