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Kit for extracting viral genome nucleic acid and use method thereof

A virus genome and kit technology, applied in the field of viral genome nucleic acid extraction, can solve the problems of insufficient adsorption capacity, low concentration of DNA and RNA, etc., and achieve the effect of convenient and quick extraction and large adsorption capacity of nucleic acid.

Inactive Publication Date: 2015-07-29
EBIOS BEIJING BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Viral genomic nucleic acid uses magnetic beads or silicon-based magnetic beads to utilize the ability to specifically adsorb DNA and RNA, remove proteins and other impurities by rinsing, and then elute with low-salt solution to obtain genomic DNA. No organic substances such as phenol and chloroform are used in the operation. Solvent and strong base, but the DNA and RNA adsorption capacity of magnetic beads or silicon-based magnetic beads is insufficient, and the concentration of DNA and RNA obtained is not high

Method used

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  • Kit for extracting viral genome nucleic acid and use method thereof
  • Kit for extracting viral genome nucleic acid and use method thereof
  • Kit for extracting viral genome nucleic acid and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Virus Genome Nucleic Acid Extraction Kit

[0078] The components of the kit are as follows:

[0079] 1) Proteinase K, take 100mg proteinase K dry powder, add 5ml sterilized ddH 2 O, mix upside down, prepare to a concentration of 20mg / ml, dispense into 1ml sterile plastic tubes, 1ml per tube, and store in a -20°C refrigerator for later use;

[0080] 2) Silicon-based modified microsphere G, take 1ml of 100mg / ml silicon-based modified microsphere G stock solution, add 9ml 1×PBS (pH=7.4) to dilute, mix upside down, prepare to a concentration of 10mg / ml, and dispense into 1ml without Bacteria in plastic tubes, 1ml per tube, stored in a 4°C refrigerator for later use;

[0081] 3) Extraction buffer 1: Dissolve 60.55g Tris base in 400ml ddH2O, adjust the pH value to 5.5 with HCl (concentrated HCl adds about 3.5ml), and use ddH 2 O was fixed to 500ml, and sterilized at 121°C for 20 minutes under a pressure of 100 kPa to prepare 1mol / L Tris-HCl (pH 8.0);

[0082] 18...

Embodiment 2

[0089] Embodiment two uses kit of the present invention to extract hepatitis B virus genome nucleic acid in plasma

[0090] Collect 5ml EDTA anticoagulant blood from 10 clinical hepatitis B virus carriers and 5 normal people (a scientific research cooperation project with a cooperative unit, approved by the hospital ethics committee, and signed an informed consent form with the volunteers).

[0091] 1) First take a 1.5ml nuclease-free centrifuge tube, add 20μl proteinase K and 15μl magnetic bead suspension G (use a pipette or vortex to mix the magnetic beads before use), then add 300μl extraction buffer 1.

[0092] 2) Take 200 μl of plasma sample taken out from the refrigerator, equilibrate at room temperature for 30 minutes, add to the above 1.5ml centrifuge tube, blow or vortex with a pipette to mix; incubate at 56°C for 10 minutes, and mix up and down every 3 minutes for 10 seconds during this period , so that the magnetic beads and nucleic acid are fully combined.

[009...

Embodiment 3

[0098] Example 3 Use the kit of the present invention to extract the virus genome in the vaginal exfoliated cells of HPV patients

[0099] nucleic acid

[0100] The vaginal exfoliated cells of 10 HPV clinical patients were collected with sterile cotton swabs (approved by the hospital ethics committee, and informed consent was signed with the volunteers), and stored in a 4°C refrigerator. The step of extracting HPV genomic nucleic acid is completed according to steps 1-6 in Example 2, and the extracted genomic nucleic acid is detected by 0.8% agarose gel electrophoresis. figure 2 It shows the extracted HPV genomic DNA detected by agarose gel electrophoresis, and the DNA electrophoresis pattern is all a single band, without tailing and miscellaneous bands.

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Abstract

The invention relates to a kit for extracting viral genome nucleic acid and a use method thereof. The kit is characterized by comprising protease K, silica-based modified magnetic beads, an extraction buffer solution, a rinsing liquid I, a rinsing liquid II, RNase-free water and the like, and further comprising a magnetic frame in a matched manner. The use method is characterized by comprising the following steps: adding protease K and the silica-based modified magnetic beads into the extraction buffer solution for one-time digestion pyrolysis of cells and adsorption pyrolysis of released virus nucleic acid; extracting guanidinium thiocyanate in the extraction buffer solution with the pH value of 5.5, so that the silica-based modified magnetic beads can adsorb more nucleic acid, that is, each gram of the silica-based modified magnetic beads can adsorb 10 mg or more of nucleic acid; utilizing hexadecyl trimethyl ammonium bromide contained in the extraction buffer solution for removing protein, polyose, phenols and other impurities, and utilizing polyvinylpyrrolidone K25 for removing pigments in the extraction buffer solution; conducting further purification on the silica-based modified magnetic beads adsorbed on the magnetic frame to obtain high-concentration genome nucleic acid. When the kit is utilized for extracting genome nucleic acid, independent digestion pyrolysis, washing and re-extraction on a source sample are not required in advance, so that the clinical test time is shortened, and the cost is greatly reduced.

Description

technical field [0001] The invention relates to a kit for extracting viral genome nucleic acid from clinical specimens and a method for extracting viral genome nucleic acid from clinical specimens using the kit of the invention. The kit of the present invention can be used for plasma, serum, lymph fluid, exfoliated cells, saliva, urine, feces, and supernatant of cultured cells, etc., to obtain virus genome nucleic acid with high efficiency, and can be used for PCR amplification, mutation screening, gene analysis, etc. Scientific research or clinical diagnostic analysis such as typing, gene sequencing and sample storage. Background technique [0002] Complete virus particles include coat protein and internal genomic DNA or RNA, and each virus particle contains only one nucleic acid, either DNA or RNA. Viruses must rely on host cells, use the host's replication system, and reproduce new viruses in accordance with the instructions of viral genes to reproduce. [0003] Studies...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 曾沃坦黄启明梁辰严兰兰徐晓洁廖文敏
Owner EBIOS BEIJING BIOTECH
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