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Immune PCR (polymerase chain reaction) detection kit for Listeria monocytogenes

A technology for mononucleosis and Listeria, applied in the fields of biotechnology and immunology, can solve the problems of detection products relying on imports, inability to adapt to sample screening, long detection cycle, etc.

Active Publication Date: 2014-07-23
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of Listeria monocytogenes 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 emerged in recent years, but all detection products rely on imports. At present, there is no rapid detection product with completely independent intellectual property rights in my country that can be used in detection practice. The patent is based on Listeria monocytogenes is the detection target, and the claimed technology involves rapid detection products for Listeria monocytogenes

Method used

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  • Immune PCR (polymerase chain reaction) detection kit for Listeria monocytogenes
  • Immune PCR (polymerase chain reaction) detection kit for Listeria monocytogenes
  • Immune PCR (polymerase chain reaction) detection kit for Listeria monocytogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Preparation of anti-Listeria monocytogenes monoclonal antibodies 6D4H7C8B6 and 1B4E7A6C9

[0073] 1. Preparation of immunogen and positive standard

[0074] Listeria monocytogenes (ATCC No.43251) was inoculated in Listeria broth at 37°C and shaken at 150r / min for 17h, counted, and inactivated by adding 0.3% formaldehyde solution at room temperature for 1 day. Adjust the concentration of Listeria monocytogenes (ATCC No.43251) 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 Listeria monocytogenes liquid as a positive control standard, Listeria broth as a negative control standard.

[0075] 2. Preparation of monoclonal antibodies

[0076] 1) Experimental animals: Three 8-week-old, weighing about 20 g female Balb / c mice were selected as experimental animals.

[0077] 2) Immunization method: each mouse was intraperitoneally injected with 0.2ml of immunogen, and the same dose was boosted ...

Embodiment 2

[0097] Example 2. Characterization of monoclonal antibodies 6D4H7C8B6 and 1B4E7A6C9

[0098] 1. Monoclonal Antibody Subclass Identification

[0099] 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.

[0100] 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.

[0101] 3. Add monoclonal antibody hybridoma cell supernatant, 8 microwells for each sample, 50 μl per well. Incubate for 1 hour at 37°C.

[0102] 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.

[0103] 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.

[0104] 6. After washing t...

Embodiment 3

[0117] The preparation of embodiment 3PCR reaction tube

[0118] The specific coating method of the PCR reaction tube for detecting Listeria monocytogenes of the present invention is as follows:

[0119] 1. Dilute the anti-Listeria monocytogenes monoclonal antibody 6D4H7C8B6 or 1B4E7A6C9 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 Immuno-PCR tubes were coated overnight at 4°C;

[0120] 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 (polymerase chain reaction) tube and an immune PCR detection kit which are used for detecting Listeria monocytogenes. The PCR reaction tube is precoated with monoclonal antibodies which can specifically enrich Listeria monocytogenes in samples. The detection kit can quickly, accurately and sensitively detect the Listeria monocytogenes from the samples like food, and the sensitivity can be 10<3>-10<4>cfu / ml and is increased by 10-100 times compared with the sensitivity of the conventional PCR. The PCR reaction tube and the immune PCR detection kit have the advantages that immunology and a molecular biological method are organically combined into a whole, enrichment and detection of Listeria monocytogenes in the samples can be realized in one PCR tube, operation is easy, cost is low, detection is quick 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 Listeria monocytogenes. Background technique [0002] Listeria monocytogenes (Listeria monocytogenes) is a short Gram-positive non-spore facultative anaerobic bacilli, the most pathogenic bacteria in the Listeria genus, and the only one that is pathogenic to humans. Typical intracellular parasites that can cause severe zoonotic diseases such as meningitis, sepsis, miscarriage, and mononucleosis. In 1988, the World Health Organization (WHO) published the "Foodborne Listeriosis Advice" to guide countries around the world on how to prevent Listeria contamination and poisoning. Since then, LM has become a new important food-borne disease pathogen, and WHO listed it as the four major food-borne pathogens in the 1990s along with E.coli O157, Salmonella and Staphylococcus aureus. The bacteria mainly contaminate milk and...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6804C12Q2545/101
Inventor 刘箐宋春美吴嫚
Owner UNIV OF SHANGHAI FOR SCI & TECH
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