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Salmonella choleraesuis immune PCR detection kit

A Salmonella, kit technology for use in biotechnology and immunology

Active Publication Date: 2014-05-28
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[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. This patent uses Salmonella choleraesuis as the detection target, and the technology claimed Involving rapid testing products for Salmonella choleraesuis

Method used

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  • Salmonella choleraesuis immune PCR detection kit
  • Salmonella choleraesuis immune PCR detection kit
  • Salmonella choleraesuis immune PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1. Preparation of anti-Salmonella choleraesuis monoclonal antibodies MAB05-D10 and Mab05-F10

[0074] 1. Preparation of immunogen and positive standard

[0075] Salmonella choleraesuis (CICC21493) was inoculated in peptone water (BPW), cultured with shaking at 37°C and 150r / min for 17h, counted, and inactivated by adding 0.3% formaldehyde solution at room temperature for 1 day. Adjust the concentration of Salmonella choleraesuis (CICC21493) to 5×10 with normal saline 9 CFU / ml was used as immunogen; the concentration was adjusted to 10 with normal saline 8 The cfu / ml Salmonella choleraesuis liquid was used as a positive control standard, and buffered peptone water (BPW) was used as a negative control standard.

[0076] 2. Preparation of monoclonal antibodies

[0077] 1) Experimental animals: Three 8-week-old, weighing about 20g female Balb / c mice were selected as experimental animals.

[0078] 2) Immunization method: each mouse was intraperitoneally injected ...

Embodiment 2

[0102] Example 2. Characterization of monoclonal antibodies MAB05-D10 and Mab05-F10

[0103] 1. Monoclonal Antibody Subclass Identification

[0104] 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.

[0105] 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.

[0106] 3. Add monoclonal antibody hybridoma cell supernatant, 8 microwells for each sample, 50 μl per well. Incubate for 1 hour at 37°C.

[0107] 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.

[0108] 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.

[0109] 6. After washing t...

Embodiment 3

[0124] The preparation of embodiment 3PCR reaction tube

[0125] The concrete coating method of the PCR reaction tube that is used to detect Salmonella choleraesuis of the present invention is as follows:

[0126] 1. Dilute the anti-Salmonella choleraesuis monoclonal antibody MAB05-D10 or Mab05-F10 of the present invention to 1:300 times with coating buffer, and the antibody concentration is 10 μg / mL, and add 50 uL of the diluted monoclonal antibody to each tube for immunization In a PCR tube, coat overnight at 4°C;

[0127] 2. The formula of the coating buffer (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 PCR reaction tube coated with a specific antibody and used for detecting salmonella choleraesuis and an immune PCR detection kit. The PCR reaction tube is pre-coated with the monoclonal antibody which can specifically enrich salmonella choleraesuis in samples. The detection kit can fast, accurately and sensitively detect salmonella choleraesuis from food and other samples, and the sensitivity can reach 103-104 cfu / ml and is increased by 10-100 times than the sensitivity of a conventional PCR. An immunology method and a molecular biology method are organically combined into a whole, enrichment and detection of salmonella choleraesuis in the samples can be achieved in one PCR tube, the operation is simple, the cost is low, the detection is fast, and results are accurate.

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 Salmonella choleraesuis. Background technique [0002] Salmonella choleraesuis (Salmonella choleraesuis) is a kind of non-typhoidal Salmonella, which is the main pathogen causing paratyphoid fever in piglets. It is also an important food-borne pathogen, which can enter the human body along with pork products, causing food safety problems such as typhoid, paratyphoid, and infectious diarrhea. At present, the detection of Salmonella choleraesuis 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/10C12Q1/6804C12Q2531/113Y02A50/30
Inventor 刘箐陈国薇
Owner UNIV OF SHANGHAI FOR SCI & TECH
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