Nucleic acid releasing agent and nucleic acid on-site releasing method

A technology of nucleic acid release agent and liquid preparation, which is applied in the fields of biochemical equipment and methods, DNA preparation, microbial measurement/inspection, etc. It can solve the problems of long sample processing time, long time, and not widely used, so as to solve the loss of nucleic acid and pollution, low manufacturing cost and safe use

Inactive Publication Date: 2019-02-05
宁波奇天基因科技有限公司
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The boiling extraction method requires multiple steps such as changing tubes and centrifugation, the sample processing time is long, and there are still a small amount of substances that inhibit amplification, which will have an inhibitory effect on subsequent amplification; the extraction effect of the spin column method is better, but there are many steps. Moreover, there is the disadvantage that nucleic acid is easily lost or polluted in this process; the magnetic bead method has simple extraction steps, high recovery rate, and easy automatic extraction, but the product is very expensive and is not widely used at present
[0003] In summary, the current nucleic acid extraction methods have the problems of large sample demand, long time, and poor extraction effect

Method used

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  • Nucleic acid releasing agent and nucleic acid on-site releasing method
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  • Nucleic acid releasing agent and nucleic acid on-site releasing method

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preparation example Construction

[0036] The present invention also provides a preparation method of a nucleic acid rapid release agent, comprising the following steps: mixing nonionic surfactant, potassium chloride, guanidine isothiocyanate, bovine serum albumin, ethylenediaminetetraacetic acid, trehalose, Tris base, proteinase K and water are mixed to prepare a solution, the pH value is adjusted to 7.2-7.7 with HCl, and the solution is sterilized by filtration to obtain the nucleic acid releasing agent. The preparation method of the nucleic acid releasing agent in the present invention is simple and meets the requirement of convenient detection.

[0037] The present invention also provides a nucleic acid releasing agent on-site nucleic acid release method, comprising the following steps: 1) mixing the nucleic acid releasing agent with a sample to obtain a mixed solution; 2) leaving the mixed solution in step 1) for 3- After 10 minutes, the solid-liquid separation was performed, and the solid-phase components...

Embodiment 1

[0043] The method of the present invention, the boiling method, the centrifugal column extraction method, and the magnetic bead extraction method are respectively used to extract DNA from a Salmonella culture, measure the OD value of nucleic acid, and perform electrophoresis detection at the same time.

[0044] The proportion of each component of the nucleic acid releasing agent described in the present embodiment is: the nonionic surfactant of 0.6% by volume, the potassium chloride of 100mM, the guanidine isothiocyanate of 20mM, the bovine serum albumin of 1.5mM, 3mM EDTA, 0.25% by mass volume trehalose, 3mg / ml proteinase K, 1.5mM Tris.HCl (PH7.5), and sterile water as the balance. Extraction steps: draw 10 μL of nucleic acid release agent and 20 μL of Salmonella culture sample respectively, put them into a 0.5mL Eppendorf centrifuge tube, repeatedly blow and beat several times with a pipette, mix well, then let the mixed solution stand for 5min, and centrifuge at 2000rpm to o...

Embodiment 2

[0051] The proportion of each component of the nucleic acid release agent described in the present embodiment is: 0.3% by volume of non-ionic surfactant, 80mM potassium chloride, 20mM guanidine isothiocyanate, 2mM bovine serum albumin, 5mM EDTA, 0.5% trehalose by mass volume, 0.5 mg / ml proteinase K, 1 mM Tris.HCl (pH 7.5), and sterile water as the balance. Extraction steps: draw 5 μL of nucleic acid release agent and 5 μL of hepatitis B virus sample respectively, put them into a 0.5mL Eppendorf centrifuge tube, repeatedly blow and beat several times with a pipette, mix well, then let the mixed solution stand for 5min, and centrifuge at 2000rpm to obtain the target DNA nucleic acid.

[0052] In this embodiment, nucleic acid release agents were used to extract 4 cases of hepatitis B virus positive serum samples. After nucleic acid extraction, 2 μL of nucleic acid extraction solution was respectively drawn, and added to the hepatitis B virus fluorescent PCR commercial detection k...

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Abstract

The invention provides a nucleic acid releasing agent, and belongs to the technical field of nucleic acid extraction. The nucleic acid releasing agent is a liquid preparation. In the nucleic acid releasing agent, volume percentage content of a nonionic surfactant is 0.2-2%, mass volume percentage content of trehalose is 0.01-2%, mass volume concentration of protease K is 0.1-5 mg / ml, concentrationof potassium chloride is 50-200 mmol / L, the concentration of guanidinium isothiocyanate is 20-100 mmol / L, the concentration of bovine serum albumin is 0.5-10 mmol / L, the concentration of ethylene diamine tetraacetic acid is 1-10 mmol / L, and the concentration of Tris-HCl is 0.2-5 mmol / L. The nucleic acid releasing agent further comprises the balance of water. The nucleic acid releasing agent is capable of eliminating a recovering step of nucleic acid, and avoiding nucleic acid losing or polluting. The nucleic acid releasing agent can be applied for realizing on-site releasing and detecting ofthe nucleic acid.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid extraction, and in particular relates to a nucleic acid release agent and a nucleic acid release method on site. Background technique [0002] At present, nucleic acid detection has been widely used in many fields such as clinical medicine, inspection and quarantine due to its high sensitivity, strong specificity, easy operation, and short time required, especially nucleic acid amplification technologies such as PCR and isothermal amplification. However, nucleic acid amplification technology is more sensitive to interfering substances in clinical or directly obtained specimens, which may cause false negative test results. Therefore, sample nucleic acid extraction and purification are required before nucleic acid amplification. However, the extraction and purification of nucleic acids in samples has always been the most time-consuming and cumbersome process in the entire experiment, which seri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1003C12Q1/6806
Inventor 周冬根应清界郭利川汤赛君王智宏
Owner 宁波奇天基因科技有限公司
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