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Staphylococcus aureus immune PCR (polymerase chain reaction) detection kit

A staphylococcus and kit technology, applied in the fields of biotechnology and immunology, can solve the problems of long detection cycle, cumbersome operation, and dependence on imports of detection products.

Active Publication Date: 2015-07-15
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 Staphylococcus aureus mainly relies on the biochemical identification stipulated by 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 Staphylococcus aureus is the detection target, and the claimed technology involves rapid detection products of Staphylococcus aureus

Method used

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  • Staphylococcus aureus immune PCR (polymerase chain reaction) detection kit
  • Staphylococcus aureus immune PCR (polymerase chain reaction) detection kit
  • Staphylococcus aureus immune PCR (polymerase chain reaction) detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Preparation of anti-Staphylococcus aureus monoclonal antibodies 5D2G2D9C1 and 5D6D9G3B4

[0073] 1. Preparation of immunogen and positive standard

[0074] Staphylococcus aureus (ATCC No.27660) was inoculated in Brain Heart Infusion Broth (BHI), 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 Staphylococcus aureus (ATCC No.27660) 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 Staphylococcus aureus liquid was used as the positive control standard, and the improved brain heart infusion broth (BHI) was used as the negative control standard.

[0075] 2. Preparation of monoclonal antibodies

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

[0077] 2) Immunization method: each mou...

Embodiment 2

[0098] Example 2. Characterization of monoclonal antibodies 5D2G2D9C1 and 5D6D9G3B4

[0099] 1. Monoclonal Antibody Subclass Identification

[0100] 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.

[0101] 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.

[0102] 3. Add monoclonal antibody hybridoma cell supernatant, 8 microwells for each sample, 50 μl per well. Incubate for 1 hour at 37°C.

[0103] 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.

[0104] 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.

[0105] 6. After washing t...

Embodiment 3

[0118] The preparation of embodiment 3PCR reaction tube

[0119] The specific coating method of the PCR reaction tube for detecting Staphylococcus aureus of the present invention is as follows:

[0120] 1. Dilute the anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 or 5D6D9G3B4 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 into the immuno-PCR tube , overnight at 4°C;

[0121] 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 (polymerase chain reaction) tube coated by a specific antibody and an immune PCR (polymerase chain reaction) detection kit, which are used for detecting staphylococcus aureu. A monoclonal antibody capable of specifically enriching staphylococcus aureu in a sample is pre-coated on the PCR tube, the detection kit disclosed by the invention is capable of fast, accurately and sensitively detecting staphylococcus aureu from samples such as food, the sensitivity can achieve 10<3>-10<4>cfu / ml, and is improved by 10-100 times in comparison with that of the conventional PCR. The immunology and a molecular biology method are organically combined to enrich and detect the staphylococcus aureu in the sample in the PCR tube, the operation is simple, the cost is low, the detection is fast and the result is 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 Staphylococcus aureus. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is the most common food-borne pathogen, widely present in the natural environment. Staphylococcus aureus mainly contaminates animal products such as milk, meat, eggs, fish and their products, including beverages made of milk, cold drinks, cakes, cooked meat, canned meat products, leftovers, fried eggs, glutinous rice cakes, jelly, Rice and rice wine are left, commonly known as "flesh-loving bacteria". After Staphylococcus aureus infects the human body, it will mainly cause the following human diseases. The first is invasive diseases such as causing suppurative infections, such as wound suppuration, cellulitis, pneumonia, meningitis, pericarditis, sepsis, etc.; The toxin disease produced by Staphylococcus aureus can ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/14
CPCC12Q1/14C12Q1/6804C12Q2531/113
Inventor 刘箐吴嫚
Owner UNIV OF SHANGHAI FOR SCI & TECH
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