Quick extraction kit for common pathogenic bacterium genomes in food and method

A pathogenic bacteria and genome technology, which is applied in the rapid extraction kit and field of common pathogenic bacteria in food, can solve the problems of few applications and high price, and achieve the effect of wide application range, reduction of health damage and rapid extraction method.

Inactive Publication Date: 2017-04-26
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the magnetic bead kits are launched by foreign biotechnology companies, which are expensive and rarely used in the extraction of genomic DNA of food pa

Method used

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  • Quick extraction kit for common pathogenic bacterium genomes in food and method
  • Quick extraction kit for common pathogenic bacterium genomes in food and method
  • Quick extraction kit for common pathogenic bacterium genomes in food and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] This example is to use the extraction method and kit of the present invention to test the extraction effect of 6 kinds of pathogen DNA simulated in different food samples.

[0044] 1. Make DNA extraction kits for 6 common pathogenic bacteria in food according to the following formula:

[0045] (a) Reagent A: PBS solution with pH 8.0;

[0046] (b) Reagent B: containing 70 mM SDS, 50 mM EDTA, 0.1 M Tris-Hcl, pH8.0;

[0047] (c) Reagent C: proteinase K at a concentration of 20 mg / mL;

[0048] (d) Reagent D: isopropanol;

[0049] (e) Reagent E: carboxylated magnetic bead solution;

[0050] (f) Reagent F: absolute ethanol;

[0051] (g) Reagent G: 70% ethanol;

[0052] (h) Reagent H: pH 8.0 TE solution containing 100 mM Tris-HCl (pH 8.0) and 10 mM EDTA.

[0053] 2. Listeria monocytogenes IQCC42222, IQCC42223; Staphylococcus aureus ATCC25923, ATCC27217; Salmonella IQCC10546, IQCC10547; Escherichia coli O157:H7 EDL933, IQCC10102; Cronobacter ATCC29544, ATCC51329; Shigella...

Embodiment 2

[0067] This example is to use the extraction method and kit of the present invention to extract 6 kinds of pathogenic bacteria DNA simulated in different food samples and compare them with the extraction effects of 2 commonly used commercially available kits, Wizard Genomic DNA Purification Kit and TIANampBacteria DNA Kit.

[0068] 1. Make DNA extraction kits for 6 common pathogenic bacteria in food according to the following formula:

[0069] (a) Reagent A: PBS solution with pH 8.0;

[0070] (b) Reagent B: containing 70 mM SDS, 50 mM EDTA, 0.1 M Tris-Hcl, pH8.0;

[0071] (c) Reagent C: proteinase K at a concentration of 20 mg / mL;

[0072] (d) Reagent D: isopropanol;

[0073] (e) Reagent E: carboxylated magnetic bead solution;

[0074] (f) Reagent F: absolute ethanol;

[0075] (g) Reagent G: 70% ethanol;

[0076] (h) Reagent H: pH 8.0 TE solution containing 100 mM Tris-HCl (pH 8.0) and 10 mM EDTA.

[0077] 2. Listeria monocytogenes IQCC42222; Staphylococcus aureus ATCC25...

Embodiment 3

[0092] This example is the detection result of real-time fluorescent PCR after using the extraction method and kit of the present invention to extract the DNA of 6 pathogenic bacteria in different food samples.

[0093] 1. Make DNA extraction kits for 6 common pathogenic bacteria in food according to the following formula:

[0094] (a) Reagent A: PBS solution with pH 8.0;

[0095] (b) Reagent B: containing 70 mM SDS, 50 mM EDTA, 0.1 M Tris-Hcl, pH8.0;

[0096] (c) Reagent C: proteinase K at a concentration of 20 mg / mL;

[0097] (d) Reagent D: isopropanol;

[0098] (e) Reagent E: carboxylated magnetic bead solution;

[0099] (f) Reagent F: absolute ethanol;

[0100] (g) Reagent G: 70% ethanol;

[0101] (h) Reagent H: pH 8.0 TE solution containing 100 mM Tris-HCl (pH 8.0) and 10 mM EDTA.

[0102] 2. Listeria monocytogenes IQCC42222, IQCC42223; Staphylococcus aureus ATCC25923, ATCC27217; Salmonella IQCC10546, IQCC10547; Escherichia coli O157:H7 EDL933, IQCC10102; Cronobacte...

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Abstract

The invention relates to a quick extraction kit for common pathogenic bacterium genomes in food and a use method. The kit comprises carboxyl-modified silica-coated magnetic beads, a pretreatment buffer solution, a pyrolysis buffer solution, a rinsing solution, proteinase K, nucleic acid dissolution solution and the like. The use method of the quick extraction kit for common pathogenic bacterium genomes in food comprises steps as follows: a to-be-extracted sample is washed with the pretreatment buffer solution for removal of food substrate, enrichment broth and other impurities, the pyrolysis solution, proteinase K and the carboxyl-modified silica-coated magnetic beads are added to pyrolyse cells and release nucleic acid, the carboxyl-modified silica-coated magnetic beads are adsorbed on a magnetic grate after isopropyl alcohol is added, and high-purity nucleic acid is obtained through further purification. When the kit is used for extracting the common pathogenic bacterium genomes in food, the sample is not required to be pyrolyzed in advance, the effect of food substrate is smaller, extraction is quick, and the time and the cost are saved for laboratory detection.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the invention relates to a kit for extracting genomes of common pathogenic bacteria in food. The kit contains carboxyl-modified silicon-coated magnetic beads, a washing buffer, an extraction buffer, and a rinsing solution. Proteinase K and nucleic acid solution, etc., are used for the rapid extraction of genomic nucleic acids of common pathogenic bacteria in food, and their application in food sample detection. Background technique [0002] Food safety is closely related to human health. In recent years, food safety incidents have occurred frequently in countries all over the world. Foodborne diseases caused by contamination by pathogenic microorganisms are the main factors affecting food safety. The detection and regulation of pathogenic microorganisms in the process of food production and consumption is crucial to ensure food safety. At present, the detection of foodborne pathogenic ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 陈颖王娉赵晓美
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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