Bacterial nucleic acid extraction lysate as well as preparation method and application thereof

A bacterial nucleic acid and lysate technology, applied in the biological field, can solve the problems of unfavorable detection accuracy, increased nucleic acid contamination, and difficulty in obtaining high-purity and high-yield nucleic acid.

Pending Publication Date: 2020-10-23
广州源古纪科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Secondly, in the nucleic acid extraction operation of the prior art, the lysis of the sample and the binding of magnetic beads and DNA are often carried out step by step, and the corresponding kits often include separately packaged lysate and binding liquid, which not only makes the nucleic acid extraction process Complicated, and greatly increases the probability of nucleic acid contamination, which is not conducive to subsequent detection and detection accuracy
[0004] However, the extraction rate of traditional nucleic acid extraction lysate is low, especially for samples with more impurities such as feces and saliva, it is difficult to obtain high-purity and high-yield nucleic acids, and it is difficult to meet the amount required for subsequent related tumor detection

Method used

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  • Bacterial nucleic acid extraction lysate as well as preparation method and application thereof
  • Bacterial nucleic acid extraction lysate as well as preparation method and application thereof
  • Bacterial nucleic acid extraction lysate as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] In this example, human feces were used as biological samples for nucleic acid extraction and detection.

[0021] 1. Preparation of lysate: Prepare 7 parts of lysate according to the ratio of raw material components, as shown in the table below:

[0022] Table 1 lysate 1-7 component list

[0023]

[0024]

[0025] 2. Stool sample processing: For 7 stool samples of patients with diarrhea, draw 1.5mL of stool samples into 2mLEP tubes, centrifuge at room temperature, 2500rpm, 90s, draw 800μL of supernatant and mix well, then quickly dispense 200μL of supernatant to 3 new 2mL EP tubes and place on ice.

[0026] 3. Nucleic acid extraction: Centrifuge the above-mentioned 2mL EP tube containing 200 μL supernatant at 12000 rpm for 2 minutes to discard the supernatant, add 100 μL of the lysate of the present invention to the collected precipitate, mix well, and place in a metal bath at 37 °C Heated at medium temperature for 20 minutes, and vortexed 3-4 times during the he...

Embodiment 2

[0031] Use saliva as a biological sample for nucleic acid extraction and detection.

[0032] 1. Sample processing and nucleic acid extraction: Take 7 samples of saliva without pretreatment. After mixing them upside down, add 7 parts of the lysate prepared in Example 1, mix well, and heat in a metal bath at 37°C for 20 minutes. , and vortex 3-4 times during the heating process. After the heating is completed, vortex again to mix and boil in a metal bath at 100°C for 10 minutes; after cooling slightly, centrifuge at 12000 rpm for 2 minutes, and the supernatant obtained can be used for nucleic acid amplification. Increased DNA solution.

[0033] 2. Nucleic acid detection: 7 parts of the extracted saliva samples in Example 1 were respectively tested for nucleic acid concentration, and the test results are shown in Table 3 below; it shows that the lysate of the present invention is used to extract nucleic acid with high efficiency and high purity.

[0034] Table 3 Detection of Nuc...

Embodiment 3

[0037] Nasal swabs were used as biological samples for nucleic acid extraction and detection.

[0038] 1. Sample processing and nucleic acid extraction: Take 7 nasal swab samples without pretreatment. After mixing them upside down, add 7 lysates prepared in Example 1, mix well, and place them in a metal bath at 37°C Heat for 20 minutes, and vortex 3-4 times during the heating process. After the heating is completed, vortex and mix again, then place in a 100°C metal bath and boil for 10 minutes; after a little cooling, centrifuge at 12000rpm for 2 minutes, and the supernatant obtained can be used DNA solution for nucleic acid amplification.

[0039] 2. Nucleic acid detection: 7 parts of the extracted nasal cavity swab samples in Example 1 were respectively tested for nucleic acid concentration, and the test results are shown in Table 4 below; it shows that the efficiency and purity of nucleic acid extraction using the lysate of the present invention are high.

[0040] Table 4 ...

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Abstract

The invention provides a bacterial nucleic acid extraction lysate, which comprises the following components: 1-15 mM of a metal ion chelating agent, 0.05-5% of an anionic surfactant, 0.5-5% of a nonionic surfactant, 5-30 mM of a buffer solution, 0.05-0.2 mg/mL of lysozyme, 5-8 M of a high ionic liquid salt, a pH regulator and a solvent. And the pH regulator is used for regulating the pH of the lysis solution to 7-8. The method is simple, convenient and rapid to operate and low in cost, can provide a basis for nucleic acid amplification detection, provides support for rapid diagnosis of gastrointestinal tract related diseases, also reduces the time cost and economic cost in the definite diagnosis process, has broad market prospects and great economic and social benefits, and is suitable forlarge-scale popularization and application.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a bacterial nucleic acid extraction lysate, a preparation method and an application. Background technique [0002] Nucleic acid extraction methods include traditional phenol-chloroform method, salting out method, membrane spin column method, etc. Traditional nucleic acid extraction methods have their own advantages and disadvantages, but the extraction effect is not good. The current mainstream nucleic acid extraction methods include silica gel membrane adsorption column method, immunoaffinity method of anti-DNA monoclonal antibody, magnetic bead nucleic acid extraction method widely used in automation platforms, etc. Nucleic acid is a biological macromolecular compound synthesized by many nucleotides, and is one of the most basic substances of life. Nucleic acid extraction refers to the process of separating nucleic acid from samples by physical and chemical methods. Isolating hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 段志峰
Owner 广州源古纪科技有限公司
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