High-efficiency extracting method of food-borne pathogen nucleic acid

A technology of food-borne pathogenic bacteria and extraction method, which is applied in the field of efficient nucleic acid extraction of food-borne pathogenic bacteria, can solve the problems of time-consuming nucleic acid extraction and purification, slowing down the detection speed, and cumbersomeness, and achieves the elimination of food and culture. Interference of base components, high purity, high yield effect

Inactive Publication Date: 2010-06-23
INSPECTION & QUARANTINE TECH CENT OF HEILONGJIANG ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

The extraction and purification of nucleic acids from samples has long be

Method used

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  • High-efficiency extracting method of food-borne pathogen nucleic acid
  • High-efficiency extracting method of food-borne pathogen nucleic acid

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Embodiment 1

[0024] A method for efficiently extracting nucleic acids from food-borne pathogenic bacteria, the composition of which includes: centrifuging the bacterial culture solution, adding buffer A to the centrifuged sediment, shaking and adding lysozyme, adding proteinase K after incubation, adding buffer B, shaking vigorously Afterwards, incubate at a temperature of 65°C for 10 minutes to make a lysis solution, pass through the silicon matrix SiO 2 The adsorption membrane of the material collects high-quality nucleic acid extraction nucleic acid solution.

Embodiment 2

[0026] In the method for efficiently extracting nucleic acid from food-borne pathogenic bacteria, the centrifugation of the bacterial culture solution is to centrifuge 1-5 mL of the bacterial culture solution at a speed of 8,000 rpm for 5 minutes, and suck up the supernatant.

Embodiment 3

[0028] The method for efficiently extracting nucleic acids from food-borne pathogenic bacteria includes adding buffer A to the centrifuge, shaking and adding lysozyme, adding proteinase K after incubation, adding buffer B, and shaking vigorously at temperature Incubate at 65°C for 10 minutes to prepare a lysis solution: add 250 μL of buffer A to the bacterial pellet: 20 mol / L TrisHCl, 2 mol / L EDTA, 1.2% polyethylene Diol octylphenyl ether Triton, shake until the cells are completely suspended, add lysozyme at a final concentration of 20mg / mL, incubate at 37°C for 30 minutes, add 20μL of proteinase K at a concentration of 20mg / mL to the tube Solution, mix well, then add 200 μL buffer B: 0.05mol / L TrisHCl, 0.1mol / L sodium chloride NaCI with pH 7.6, 0.05mol / L EDTA, 2% Sodium dodecyl sulfate SDS, after vigorous shaking, incubated at a temperature of 65°C for 10 minutes.

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Abstract

The present invention provides a high-efficiency extracting method of food-borne pathogen nucleic acid. A PCR technology has been widely used for the detection of pathogenic microorganisms currently. A first step of the PCR detecting technology is the preparation of template nucleic acid, that is, the extraction of nucleic acid in a sample, which directly affects the result of PCR reaction. For a long time, the processes of extraction and purification of the nucleic acid in a sample consume time, and are fussy all the time, so detection speed is seriously slowed down. Thus, a plurality of scholars searches after the extracting method of various nucleic acids. The high-efficiency extracting method of food-borne pathogen nucleic acid comprises the following steps: bacterial culture solution is centrifuged; buffer solution A is added into centrifugal sediment to be vibrated, and muramidase is added; prolease K is added after incubation; buffer solution B is added to be incubated at the temperature of 65 DEG C for 10 minutes after being vibrated violently to be prepared into cracking solution; and high-quality nucleic acid is collected by an adsorption film of silicon matrix SiO2 material is used for extracting nucleic acid solution. The present invention is applied to the detection of the pathogenic microorganisms.

Description

Technical field: [0001] The invention relates to a method for efficiently extracting nucleic acid of food-borne pathogenic bacteria. Background technique: [0002] PCR technology has been widely used in the detection of pathogenic microorganisms. The first step of PCR detection technology is the preparation of template nucleic acid, that is, the extraction of nucleic acid in the sample, which directly affects the result of PCR reaction. The extraction and purification of nucleic acids from samples has long been a time-consuming, cumbersome process that severely slows down detection. Therefore, many scholars have been exploring various nucleic acid extraction methods. [0003] The extraction of nucleic acids in samples is divided into two steps: lysing cells and extracting nucleic acids. To extract nucleic acid from a sample, cells must first be lysed by physical, chemical or enzymatic action to release the nucleic acid. Commonly used methods include physical methods (boi...

Claims

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Application Information

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IPC IPC(8): C12P19/34
Inventor 李苏龙
Owner INSPECTION & QUARANTINE TECH CENT OF HEILONGJIANG ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C
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