Nucleic acid extraction method and nucleic acid extraction reagent for biological samples

A biological sample, nucleic acid technology, applied in the field of nucleic acid extraction, can solve the problems of long time and complicated operation steps, and achieve the effect of weakening the agglutination effect, increasing the pH value, and enhancing the hydrolysis ability and

Active Publication Date: 2015-12-23
USTAR BIOTECHNOLOGIES (HANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the above methods, this method needs to digest the sample with protease, although the dissolution effect of the sample is significantly improved, but the operation steps are too complicated and time-consuming

Method used

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  • Nucleic acid extraction method and nucleic acid extraction reagent for biological samples
  • Nucleic acid extraction method and nucleic acid extraction reagent for biological samples
  • Nucleic acid extraction method and nucleic acid extraction reagent for biological samples

Examples

Experimental program
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Effect test

Embodiment 1

[0050] 1. Sample pretreatment: add 2 times the volume of 4% NaOH to <5ml of sputum, shake and mix well, and leave it at room temperature for 20-30 minutes to fully liquefy, that is, there is no obvious solid and no dragging when sucking out Phenomenon;

[0051] 2. Cleavage sample: take 0.25ml of pretreatment sample, add 0.15ml of lysate, pipette and mix, and bathe in 100°C water for 10 minutes; the pH value of the system is 13; the lysate contains guanidine hydrochloride 6mol / L, ethylenediaminetetraacetic acid Disodium 20mmol / L, glycogen 0.15mmol / L, trishydroxymethylaminomethane 60mmol / L and polyethylene glycol octylphenyl ether 1.2v / v%;

[0052] 3. Adsorption: After the sample is cooled to room temperature, add 0.4ml ≥ 95% ethanol and mix well, use the instrument-free nucleic acid extraction device in ZL200920312749.4 to suck the sample to be tested, and absorb the nucleic acid molecules in the sample to be tested on the filter membrane , discard the filtrate;

[0053] 4. C...

Embodiment 2

[0056] 1. Sample pretreatment: After sampling the oral mucosa, pharynx, vagina, and prostate secretions with cotton swabs, elute the cotton swabs in 1ml of normal saline, stir several times, and discard the cotton swabs; take 0.15ml of eluent Add 0.1ml 2% NaOH, mix well and set aside;

[0057] 2. Cleavage sample: take 0.25ml of pretreatment sample, add 0.15ml of lysate, pipette and mix, and treat at room temperature for 10 minutes; the pH value of the system is 11; the lysate contains guanidine thiocyanate 6mol / L, ethylenediaminetetra Disodium acetate 25mmol / L, glycogen 0.08mmol / L, trishydroxymethylaminomethane 50mmol / L and polyethylene glycol octylphenyl ether 0.8v / v%;

[0058] 3. Adsorption: After the sample is cooled to room temperature, add 0.4ml ≥ 98% ethanol and mix well, use the instrument-free nucleic acid extraction device in ZL200920312749.4 to suck the sample to be tested, and absorb the nucleic acid molecules in the sample to be tested on the filter membrane , dis...

Embodiment 3

[0062] 1. Bacterial cloning: Take a small amount of bacteria with a toothpick and dissolve in 0.15ml of normal saline, add 0.1ml of 3% NaOH, mix well and set aside;

[0063] 2. Cleavage sample: take 0.25ml of pretreatment sample, add 0.15ml of lysate, pipette and mix well, treat at room temperature or 95-100°C water bath for 10 minutes; the pH value of the system is 12; the lysate contains guanidine thiocyanate 6mol / L, disodium edetate 25mmol / L, glycogen 0.08mmol / L, trishydroxymethylaminomethane 50mmol / L and polyethylene glycol octylphenyl ether 0.8v / v%;

[0064] 3. Adsorption: After the sample is cooled to room temperature, add 0.5ml ≥ 95% ethanol and mix well, use the instrument-free nucleic acid extraction device in ZL200920312749.4 to suck the sample to be tested, and absorb the nucleic acid components in the sample to be tested on the filter membrane , discard the filtrate;

[0065] 4. Cleaning: Use a trace nucleic acid extraction device to suck 0.5ml of cleaning soluti...

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Abstract

The present invention relates to the field of nucleic acid extraction, particularly to a nucleic acid extraction method and a nucleic acid extraction reagent for biological samples. The specific method comprises: adding a sodium hydroxide solution to a biological sample, adding a lysate, and carrying out lysis to obtain a lysis reaction liquid, wherein the lysate contains a guanidine salt, disodium edetate, a surfactant and a buffer agent; and adding an ethanol solution to the lysis reaction liquid, and achieving nucleic acid adsorption, cleaning and elution by using an instrument-free nucleic acid extraction apparatus having a silica gel membrane or a centrifugal column. According to the present invention, the sodium hydroxide is added to pre-treat and then the lysate is added to carry out lysis, such that the protein in the sample can be well dissolved, and the binding of the nucleic acid and the adsorption film is prompted; and the lysate component is optimized, such that the binding capacity of the nucleic acid molecule and the silica gel membrane under the high salt and high pH value conditions is significantly enhanced, and the good nucleic acid extraction efficiency is ensured.

Description

technical field [0001] The invention relates to the field of nucleic acid extraction, in particular to a nucleic acid extraction method and reagents for biological samples. Background technique [0002] Nucleic acid extraction is the first step in many clinical tests. The concentration and purity of the nucleic acid templates obtained in this link directly affect the accuracy of subsequent test results. The spin column method is a common nucleic acid extraction method in clinical testing. This method utilizes the characteristic that nucleic acid molecules bind to silica gel membrane in high-salt and low-pH environment, and dissociate from silica gel membrane in low-salt and high-pH environment. Enrich and purify nucleic acid templates. Combined with a high-speed centrifuge or an automated nucleic acid extractor, the spin column method has the advantages of large throughput, fast speed, high extraction efficiency, and good template purity, which can meet the inspection req...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 范倻王岱桑金成芳朱勤锋孙刚胡林尤其敏
Owner USTAR BIOTECHNOLOGIES (HANGZHOU) CO LTD
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