Sample releasing agent

Abstract

The invention discloses a sample releasing agent, which comprises 0.01-0.1 M of a basic solution and 0.02-0.05% of a surfactant, the pH value of the sample releasing agent is 7.4-8.0, and the basic solution comprises Na2HPO4, KH2PO4, NaCl and KCl; the surfactant is prepared from TritonX-100 or Tween20, and the surfactant is prepared from the following raw materials: TriconX-100 The method has the beneficial effects that compared with a traditional method, the sample releasing agent (fungus-nucleic acid) is subjected to the steps of optimized splitting decomposition, washing, elution and the like, so that the whole extraction process is simpler, the loss of nucleic acid in the extraction process is reduced by the optimized extraction method, and more and more complete nucleic acid samples are obtained; meanwhile, the sample releasing agent does not involve harmful organic solvents, potential hazards to operators are reduced, fungal nucleic acid is rapidly released, time and labor are saved, the sample releasing agent is compatible with laboratories and any site, and the sample releasing agent has wide application prospects in the field of microbiological inspection.

Description

Technical Field

[0001] The present invention relates to the technical field of fungal nucleic acid extraction, and particularly to a sample releasing agent. Background Art

[0002] Fungi belong to filamentous fungi and are also important plant pathogens. In recent years, fungi have mutated rapidly and there are many varieties. It is difficult to completely identify the categories using ordinary cultivation. Therefore, nucleic acid detection has become an important means for accurate identification at present. The prerequisite for nucleic acid detection is to extract sufficient DNA or RNA with acceptable quality (nucleic acid concentration / purity). However, the fungal cell wall has a strong structure and is difficult to be destroyed. The main component of the fungal cell wall is polysaccharide, which is easily precipitated together with nucleic acid during the release process, resulting in a decrease in nucleic acid purity.

[0003] Currently, the extraction methods include CTAB method, microwave method, SDS method, urea method, ammonium acetate method, and kit method. Their steps are cumbersome, time-consuming, and improper handling will lead to low extraction efficiency. At the same time, there are toxic and harmful substances such as phenol and chloroform in the extraction reagents, which are likely to cause harm to operators. Summary of the Invention

[0004] The purpose of the present invention is to provide a sample releasing agent to solve the above problems.

[0005] The present invention realizes the above purpose through the following technical solutions:

[0006] A sample releasing agent, which includes a base solution and a surfactant. The base solution includes Na 2 HPO 4 、KH 2 PO 4 、NaCl and KCl; and the surfactant includes TritonX-100 or Tween20.

[0007] Furthermore, the concentration of the base solution is 0.01 - 0.1M, and the concentration of the surfactant is 0.02 - 0.05%.

[0008] Furthermore, preferably, the concentration of the base solution is 0.01M, and the concentration of the surfactant is 0.05%. Research shows that when the concentration of the base solution is 0.01M and the concentration of the surfactant is 0.05%, it is more conducive to the extraction of fungal nucleic acid.

[0009] Furthermore, the pH of the surfactant is 7.4 - 8.

[0010] Furthermore, preferably, the pH of the surfactant is 7.5 - 7.6.

[0011] Furthermore, the nucleic acid extraction process using this sample releasing agent is as follows: Take about 100 mg of the fungal mycelium to be tested and put it into a 1.5 ml centrifuge tube. Add 100 μl of the sample releasing agent and manually shake well so that the mycelium is completely immersed in the solution. Place the test tube in a constant temperature metal bath at 96 °C and heat for 3 minutes. After heating, cool and let it stand still. Centrifuge at 12,000 rpm for 2 minutes and take the supernatant for the upper machine experiment.

[0012] Furthermore, the experimental results of the nucleic acid extraction process using this sample releasing agent are as follows in the table:

[0013] Serial number Sample number Nucleic acid concentration (ng / ul) Nucleic acid purity (A260 / A280) 1 LDJ3 63.374 1.636 2 LDJ5 200.876 1.090 3 LDJ7 167.113 1.679 4 LDJ8 117.548 1.637 5 LDJ10 165.437 1.349

[0014] The beneficial effects of the present invention are as follows:

[0015] Compared with the traditional method, the sample releasing agent (fungal-nucleic acid) of the present invention optimizes steps such as lysis, washing, and elution, making the whole extraction process more concise. The optimized extraction method reduces the loss of nucleic acid during the extraction process, ensuring more and more complete nucleic acid samples are obtained. At the same time, the sample releasing agent does not involve harmful organic solvents, reducing potential harm to operators. In addition, it can quickly release fungal nucleic acid, saving time and effort, and is compatible with laboratories and any on-site, having a wide application prospect in the field of microbial inspection. Description of the Drawings

[0016] Figure 1 It is the amplification curve diagram of isothermal amplification fluorescence method detection after nucleic acid extraction by a sample releasing agent described in the present invention;

[0017] Figure 2 It is the melting curve diagram of isothermal amplification fluorescence method detection after nucleic acid extraction by a sample releasing agent described in the present invention. Detailed Embodiments

[0018] A sample releasing agent, which includes a base solution and a surfactant. The base solution includes Na 2 HPO 4 、KH 2 PO 4 、NaCl and KCl; and the surfactant includes Triton X-100 or Tween 20.

[0019] In this embodiment, the concentration of the base solution is 0.01 - 0.1 M, and the concentration of the surfactant is 0.02 - 0.05%.

[0020] In this embodiment, preferably, the concentration of the base solution is 0.01 M, and the concentration of the surfactant is 0.05%.

[0021] In this embodiment, the pH of the surfactant is 7.4 - 8.

[0022] In this embodiment, preferably, the pH of the surfactant is 7.5 - 7.6.

[0023] In this embodiment, the nucleic acid extraction process using this sample releasing agent is as follows: Take about 100 mg of the fungal hyphae to be tested and put it into a 1.5 ml centrifuge tube. Add 100 μl of the sample releasing agent and manually shake well so that the hyphae are completely immersed in the solution; Put the test tube into a constant temperature metal bath at 96 °C and heat for 3 minutes. After heating, cool and let it stand still, centrifuge at 12,000 rpm for 2 minutes, and take the supernatant for the experiment on the machine.

[0024] In this embodiment, the experimental results of the nucleic acid extraction process using this sample releasing agent are as shown in the following table:

[0025] Serial number Sample number Nucleic acid concentration (ng / ul) Nucleic acid purity (A260 / A280) 1 LDJ3 63.374 1.636 2 LDJ5 200.876 1.090 3 LDJ7 167.113 1.679 4 LDJ8 117.548 1.637 5 LDJ10 165.437 1.349

[0026] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A sample release agent, characterized in that: The sample release agent includes a base solution and a surfactant, wherein the base solution includes Na2HPO4, KH2PO4, NaCl and KCl; and the surfactant includes TritonX-100 or Tween20.

2. A sample release agent according to claim 1, characterized in that: The concentration of the base liquid is 0.01-0.1 M, and the concentration of the surfactant is 0.02-0.05%.

3. A sample release agent according to claim 2, characterized in that: Preferably, the concentration of the base liquid is 0.01 M, and the concentration of the surfactant is 0.05%.

4. A sample release agent according to claim 2, characterized in that: The pH of the surfactant is 7.4-8.

5. A sample release agent according to claim 4, characterized in that: Preferably, the pH of the surfactant is 7.5-7.

6.

6. A sample release agent according to claim 1, characterized in that: The nucleic acid extraction process using the sample releaser is as follows: take about 100 mg of the fungal hyphae to be tested, put it into a 1.5 ml centrifuge tube, add 100 ul of the sample releaser and shake it manually so that the hyphae are completely immersed in the solution; put the test tube into a 96°C constant temperature metal bath and heat it for 3 minutes. After the heating is completed, cool it down and let it stand, centrifuge it at 12000 rpm for 2 minutes, take the supernatant and put it on the machine for experiment.

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