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ELISA kit for detecting Salmonella pullorum antibody

A technology for Salmonella and pullorum, applied in the field of ELISA kits for detecting Salmonella pullorum antibodies, can solve the problems of high stress in chickens, agglutination of negative serum, decreased production performance of chickens, etc., achieves good specificity and sensitivity, and improves economical efficiency. Benefit, the effect of reducing the stress of chickens

Active Publication Date: 2014-08-20
WENS FOOD GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the great stress on the chickens through blood collection by acupuncture, it is easy to cause the decline in the production performance of the chickens, and even the rupture of the liver and spleen, especially in laying hens. Agglutination can also occur, which can easily cause false positives

Method used

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  • ELISA kit for detecting Salmonella pullorum antibody
  • ELISA kit for detecting Salmonella pullorum antibody
  • ELISA kit for detecting Salmonella pullorum antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Screening of the dominant antigen of Salmonella pullorum by immunoprecipitation (pulldown) technology

[0046] 1 Plate agglutination experiment

[0047] Take 10μL of pullorum and typhoid multivalent staining plate agglutination test antigen and drop it on a clean glass slide, then take 10μL of serum collected from chicken farm and drop it on the slide, turn it upside down to mix thoroughly, agglutinated particles appear within 2 minutes, and the liquid is clear It was judged to be positive, and no agglutinated particles were judged to be negative. SPF chicken was used as a negative control. The yin and yang serum of pullorum were identified for immunoprecipitation.

[0048] 2 Purified IgG from chicken pullorum negative and positive serum

[0049] 2.1 Dialysis bag treatment: Take 200ml of solution I (prepared with distilled water, containing 2% (W / V) sodium bicarbonate, 1mmol EDTA, and sodium hydroxide adjusted to pH 8.0) in a beaker, and place the beaker in a water b...

Embodiment 2

[0066] Example 2 Construction of Prokaryotic Expression Vector of Salmonella Pullorum GroEL

[0067] 1 Extraction of the whole genome of Salmonella pullorum

[0068] Take 10 μL of Salmonella pullorum standard strain 533 Glycerin bacteria inoculate 5ml LB liquid medium, shake overnight at 37°C.

[0069] Follow the instructions of the bacterial genomic DNA rapid extraction kit: take 2ml of culture broth, centrifuge at 10000rpm for 30s, aspirate and discard the supernatant as much as possible, and collect the bacteria. Add 200μL of buffer RB to resuspend, centrifuge at 10000rpm for 30s, and discard the supernatant. Resuspend the cells in 180 μL buffer RB with shaking. Add 20μL of proteinase K (20mg / ml), mix thoroughly, then add 200μL of binding solution CB, vortex and shake to mix thoroughly, and place at 70°C for 10 minutes. After cooling, add 100μL of isopropanol, vortex and shake to mix thoroughly. At this time, flocculent precipitation may appear. Add the mixture from the previo...

Embodiment 3

[0101] Example 3 Expression and purification of recombinant protein

[0102] 1 Transformation and induced expression of recombinant prokaryotic expression vector

[0103] The constructed recombinant expression plasmid pET-28a-GroEL prokaryotic expression vector was transformed into Ecoli.BL21 competent cells respectively, spread on LBK plates, cultured overnight at 37°C, and the recombinant transformants were screened by kanamycin resistance. Randomly pick 3 single colonies and pET-28aEcoli.BL21 empty vector to 5ml LBK liquid medium, shake culture at 37°C for about 4h to OD600=0.6-0.8, sample 1ml respectively, and then add IPTG at a final concentration of 1mmol / L. Shake at 37°C for about 6 hours, and sample 1 ml respectively. For the samples before and after induction, the cells were collected by centrifugation at 5000 rpm for 10 min. Add a certain amount of distilled water and 6×SDS loading buffer to boil for 10 minutes, and then perform SDS-PAGE electrophoresis to analyze the p...

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Abstract

An ELISA kit for detecting a Salmonella pullorum antibody is established by screening Salmonella pullorum dominant antigen GroEL through an immunoprecipitation technology, expressing GroEL recombinant protein through utilizing a prokaryotic expression vector, and utilizing an antigen protein. The kit can reduce the response of chicken in the detection process of the Salmonella pullorum antibody, and can improve the detection specificity and the sensitivity.

Description

Technical field [0001] The invention relates to an antigen preparation and an ELISA kit, in particular to an ELISA kit for detecting Salmonella pullorum antibody. Background technique [0002] Pullorum pullorum is a septic infectious disease caused by Salmonella pullorum. Its excrement is an important transmission vector, and it can also be transmitted vertically through eggs. It is one of the most serious diseases that endanger the chicken industry. [0003] Salmonella pullorum mostly invades young chicks less than 20 days old, causing white diarrhea, with a very high mortality rate. Adult chickens can carry the bacteria without clinical symptoms. The disease can spread both vertically and severely horizontally, so the harm and economic losses caused are huge. At present, Western countries have completely eliminated pullorum, but the outbreak is still serious in my country. The conventional method for detection of pullorum is plate agglutination. Blood is collected by acupunctur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12N15/70C12N15/66
CPCC12N15/70G01N33/56916G01N2333/255
Inventor 郑世军徐志超李晓齐王永强曹红
Owner WENS FOOD GRP CO LTD
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