Glass fiber filter paper-based viral nucleic acid extraction method

An extraction method and filter paper technology, applied in biochemical equipment and methods, resistance to vector-borne diseases, measurement/inspection of microorganisms, etc., can solve the problems of large volume, cumbersome enrichment and elution processes, etc., and achieve small sample size , The effect of quick and convenient extraction operation

Active Publication Date: 2020-05-29
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the spin column extraction method and nano-magnetic bead extraction method have been widely used, but the disadvantage is that the spin-column method needs repeated centrifugation to extract nucleic acid, and equipment such as a centrifuge is required to meet the demand, while the nano-magnetic bead extraction method requires an external magnetic field , and the required sample is usually 200uL and above, the volume is relatively large, and the enrichment and elution process is relatively cumbersome

Method used

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  • Glass fiber filter paper-based viral nucleic acid extraction method
  • Glass fiber filter paper-based viral nucleic acid extraction method
  • Glass fiber filter paper-based viral nucleic acid extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A method for extracting nucleic acid based on glass fiber filter paper, comprising the following steps:

[0035] Step 1: Take two 50uL serums containing hepatitis B virus (HBV virus) and place them in 1.5mL centrifuge tubes as experimental group 1 (and marked as HBV11, HBV16) as the biological samples to be extracted nucleic acid, add 5uL proteinase K After mixing quickly, a mixed solution was obtained;

[0036] Step 2: Add 80uL binding buffer and 0.7uL Carrier RNA to the mixed solution obtained in step 1, mix well and incubate at room temperature for 10 minutes;

[0037] Step 3, add the mixed solution obtained in step 2 dropwise on the glass fiber filter paper, pad the absorbent paper under the filter paper, and absorb the filtered waste liquid with the absorbent paper;

[0038] Step 4, slowly drop 125uL of washing buffer solution on the above-mentioned glass fiber filter paper, put absorbent paper under the filter paper, absorb the filtered waste liquid with absorben...

Embodiment 2

[0049] A method for extracting nucleic acid based on glass fiber filter paper, comprising the following steps:

[0050]Step 1: Take two 50uL serums containing hepatitis B virus (HBV virus) and place them in 1.5mL centrifuge tubes as experimental group 2 (and marked as HBV1, HBV5) as biological samples to be extracted nucleic acid, add 5uL proteinase K After mixing quickly, a mixed solution was obtained;

[0051] Step 2: Add 80uL binding buffer and 0.7uL Carrier RNA to the mixed solution obtained in step 1, mix well and incubate at room temperature for 10 minutes;

[0052] Step 3, drop the mixed solution obtained in Step 2 on the glass fiber filter paper, the glass fiber filter paper is fixed with a double-circle filter paper as a support, and absorbent paper is placed under the filter paper, and the filtered waste liquid is absorbed by the absorbent paper;

[0053] Step 4, slowly drop 125uL of washing buffer solution on the glass fiber filter paper in step 3, put absorbent pa...

Embodiment 3

[0066] A method for extracting nucleic acid based on glass fiber filter paper, comprising the following steps:

[0067] Step 1: Take a portion of 150uL serum containing hepatitis B virus (HBV virus) and divide it evenly into three portions and place them in 1.5mL centrifuge tubes (marked as LZ, BX, F5 respectively) as biological samples to be extracted nucleic acid, add Mix 5uL proteinase K quickly to obtain a mixed solution;

[0068] Step 2: Add 80uL binding buffer and 0.7uL Carrier RNA to the mixed solution obtained in step 1, mix well and incubate at room temperature for 10 minutes;

[0069] Step 3: Drop the mixed solution obtained in Step 2 marked as LZ on the double-circle filter paper, drop the liquid marked as BX on the glass fiber filter paper, and add the mixed solution marked as F5 on the Fusion 5 filter membrane. Absorbent paper is placed on the lower pad, and the filtered waste liquid is absorbed by absorbent paper;

[0070] Step 4: Slowly add 125uL of washing bu...

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Abstract

The invention discloses a glass fiber filter paper-based viral nucleic acid extraction method. The method comprises the following steps of 1, adding protease K in a biological sample and carrying outrapid mixing to obtain mixed solution; 2, adding binding buffer and Carrier RNA and turning upside down to sufficiently mixing the materials; 3, dropwise adding the mixed solution on glass fiber filter paper, fixing the glass fiber filter paper by taking double-loop filter paper as a support, and absorbing the filtered waste liquid by using absorbent paper; 4, carrying out cleaning by using washing buffer and absorbing the waste liquid by using absorbent paper; and 5, dropwise adding eluting buffer on the glass fiber filter paper, and collecting eluent to obtain required nucleic acid. According to the method, the glass fiber filter paper is imported into the nucleic acid extraction method and can be used for the enrichment and extraction of nucleic acid in biological samples. The method iscapable of simplifying the nucleic acid extraction step and the required instrument equipment, thereby saving the operation time, reducing the extraction cost and ensuring the high-efficiency nucleicacid recovery rate at the same time.

Description

technical field [0001] The invention belongs to the technical field of sample nucleic acid extraction for medical testing, in particular relates to a method for rapidly extracting nucleic acid from biological samples, and more particularly relates to a method for extracting viral nucleic acid based on glass fiber filter paper. Background technique [0002] Body fluids such as plasma, serum, and interstitial fluid can be used as primary assay samples for a variety of blood-borne infectious diseases. Common blood-borne infectious diseases include malaria, chronic hepatitis B, acquired immunodeficiency syndrome, etc. These diseases are caused by pathogens parasitic in human blood and lymph, mainly through contact and exchange of blood and wounds Pass the disease to another body. Therefore, by extracting and detecting the nucleic acid content of pathogens in the blood, it can help to determine whether or not to be infected with a disease, the progress of the infection, and the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1006C12Q2521/537Y02A50/30
Inventor 刘斐李嘉豪单衍可李悦
Owner NANJING AGRICULTURAL UNIVERSITY
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