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Campylobacter jejuni immune PCR detection kit

A technology of Campylobacter jejuni and a kit, which is applied in the fields of biotechnology and immunology, and can solve problems such as long detection cycle, cumbersome operation, and inability to adapt to sample screening

Active Publication Date: 2017-06-16
上海慧耘生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of Campylobacter jejuni mainly relies on the biochemical identification stipulated in the national standard. The disadvantage is that the operation is cumbersome, the detection cycle is long, and it cannot adapt to the screening of a large number of samples.
[0003] Immunological detection is the fastest, accurate and stable rapid detection method that has appeared in recent years, but there is no rapid detection product that can be used in detection practice at home and abroad. The patent uses Campylobacter jejuni as the detection target, and the technology claimed Involving Campylobacter jejuni rapid detection products

Method used

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  • Campylobacter jejuni immune PCR detection kit
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  • Campylobacter jejuni immune PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Preparation of anti-Campylobacter jejuni monoclonal antibodies 4C9E1G7H3 and 3G2D8H3G9

[0057] 1. Preparation of immunogen and positive standard

[0058] Campylobacter jejuni (CICC 22937) was inoculated on Brunner's broth, cultured under anaerobic conditions at 37°C and 150r / min shaking for 17h, counted, and inactivated by adding 0.3% formaldehyde solution at room temperature for 1 day. Adjust the concentration of Campylobacter jejuni (CICC No.22937) to 5×10 with normal saline 9 CFU / ml was used as immunogen; the concentration was adjusted to 10 with normal saline 8 cfu / ml was used as a positive control standard, and Brooke's broth as a negative control standard.

[0059] 2. Preparation of monoclonal antibodies

[0060] 1. Experimental animals: Three 8-week-old, weighing about 20 g female Balb / c mice were selected as experimental animals.

[0061] 2. Immunization method: each mouse was intraperitoneally injected with 0.2ml of immunogen, and injected with ...

Embodiment 2

[0070] Example 2. Characterization of Monoclonal Antibodies 4C9E1G7H3 and 3G2D8H3G9

[0071] 1. Monoclonal Antibody Subclass Identification

[0072] 1. Antigen coating: Coat goat anti-mouse secondary antibody IgG+A+M with 0.01M PBS, 50 μl per well, coat overnight at 4°C, discard the liquid in the well the next day, and wash the plate 3 times.

[0073] 2. Blocking: add 200 μl of 1% BSA to each well, and block overnight at 4°C. Pat the board dry the next day without washing it.

[0074] 3. Add monoclonal antibody hybridoma cell supernatant, 8 microwells for each sample, 50 μl per well. Incubate for 1 hour at 37°C.

[0075] 4. After washing the plate 4 times, add specific binding rabbit anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, κ, λ, and incubate at 37°C for 1 hour.

[0076] 5. After washing the plate 4 times, add diluted horseradish peroxidase-labeled anti-rabbit secondary antibody IgG (H+L) to each well, and incubate at 37°C for 30 minutes.

[0077] 6. After washing t...

Embodiment 3

[0092] Embodiment 3 Preparation of PCR reaction tube

[0093] The specific coating method of the PCR reaction tube for detecting Campylobacter jejuni of the present invention is as follows:

[0094] 1. Dilute the anti-Campylobacter jejuni monoclonal antibody 4C9E1G7H3 or 3G2D8H3G9 of the present invention to 1:300 times with coating buffer solution, and the antibody concentration is 10 μg / mL, and add 50 uL of the diluted monoclonal antibody into the immuno-PCR tube, Coating overnight at 4°C;

[0095] 2. The formula of Carbonate Coating buffer (1×) is: anhydrous sodium carbonate Na 2 CO 3 1.59g, sodium bicarbonate NaHCO 3 2.93g, sodium azide NaN 3 0.2g, dissolve in 1000ml of distilled water, adjust the pH to 9.6, and store at 4°C.

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Abstract

The invention discloses a specific antibody-coated PCR reaction tube and immune PCR detection kit for detecting campylobacter jejuni. A monoclonal antibody which can specifically enrich the campylobacter jejuni in samples pre-coats the PCR reaction tube. The detection kit has the advantages that the detection kit can fast, accurately and sensitively detect the campylobacter jejuni from the samples such as food, the sensitivity of the detection kit can reach 103-104cfu / ml, and the sensitivity of the detection kit is 10-100 times higher than that of conventional PCR; the detection kit organically and integrally combines immunology and molecular biology methods, enrichment and detection of the campylobacter jejuni in the samples can be realized in one PCR tube, and the detection kit is simple to operate, low in cost, fast in detection and accurate in result.

Description

technical field [0001] The invention belongs to the fields of biotechnology and immunology, and in particular relates to an immuno-PCR detection kit for detecting campylobacter jejuni. Background technique [0002] Campylobacter jejuni enteritis was isolated from the feces of patients with diarrhea by Butzler in 1973, and it has been recognized as one of the main pathogenic bacteria of human diarrhea. The incidence of Campylobacter jejuni enteritis exceeds bacillary dysentery in developed countries and is almost equal to bacillary dysentery in developing countries. As an important food-borne pathogen, this bacterium rapidly multiplies in an environment containing trace oxygen after food enters the intestinal tract, mainly invades the jejunum, ileum and colon, invades the intestinal mucosa, and causes congestion and hemorrhagic injury. To some strains can produce cholera-like enterotoxin, causing increased fluid secretion in the intestinal lumen. At present, the detection o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12R1/01
CPCC12Q1/6804C12Q1/689C12Q2545/101
Inventor 万绍业方水琴杨标郭慧琴胡永辉刘箐
Owner 上海慧耘生物科技有限公司
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