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Viral nucleic acid extraction reagent

A virus nucleic acid and reagent technology, applied in the field of biotin-labeled specific probes, can solve the problems of reducing the ability of magnetic beads to adsorb viral nucleic acids, coating magnetic beads, and reducing the sensitivity of magnetic bead methods, etc.

Active Publication Date: 2014-04-02
SANSURE BIOTECH INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In China, methods for extracting nucleic acids based on magnetic beads have been used in clinical testing and nucleic acid blood screening. These magnetic bead reagents have the following disadvantages: 1) The nucleic acid extraction process has many steps, sample processing takes a long time, and RNA is easily detected by RNA after long-term exposure. Enzymatic hydrolysis, reducing the sensitivity of the reagent
2) Conventional magnetic bead extraction requires heating, and the solution causes aerosol pollution, resulting in the indistinguishability of negative and positive samples for testing
3) The conventional magnetic bead method does not label specific oligonucleotide chains, and cannot specifically enrich viral nucleic acids. Magnetic beads are easily coated with human genomic nucleic acid, which reduces the ability of magnetic beads to adsorb viral nucleic acids and reduces the ability of magnetic beads to absorb viral nucleic acids. Sensitivity

Method used

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Examples

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Embodiment 1

[0027] A viral nucleic acid extraction kit is provided in this embodiment, which contains a viral nucleic acid extraction reagent, and the reagent contains three biotin-labeled specific probes, namely biotin-labeled hepatitis B virus probe, biotinylated hepatitis C virus probe and biotinylated human immunodeficiency virus probe. Specifically, the kit includes the following components:

[0028] ① Magnetic beads containing specific probes: The magnetic beads containing specific probes include the following three types:

[0029] Magnetic beads containing HBV capture probe: magnetic beads-streptavidin-biotin-TTTTTTTTTTTTTT-TTGGGTGGCTTTGGGGCATGG;

[0030] Magnetic beads containing HCV capture probe: magnetic beads-streptavidin-biotin-TTTTTTTTTTTTTT-TGGTACTGCCTGATAGGGTGCTTGCG;

[0031] Magnetic beads containing HIV-I capture probe: magnetic beads-streptavidin-biotin-TTTTTTTTTTTTTT-CCAGGCCAGATGAGAGAACCAAGGG.

[0032] The streptavidin magnetic beads described in the present inventi...

Embodiment 2

[0039] This example provides a method for extracting the HBV / HCV / HIV-I virus (HBV DNA in this example) in the sample to be tested using the kit described in Example 1, as well as the detection of the obtained viral nucleic acid by fluorescent quantitative PCR and its result.

[0040] First of all, the sample to be tested in the present invention is: the concentrations of the quantitative standards of hepatitis B virus (HBV) nucleic acid of the China Institute for the Control of Biological Products (National Institute for Inspection and Control) are 5.0×10 6 IU / ml, 5.0×10 4 IU / ml and 5.0×10 2 Three concentrations of HBV DNA positive serum in IU / ml.

[0041] The steps of nucleic acid extraction and detection are as follows:

[0042] 1) Cell lysis: add the sample to be tested into a centrifuge tube, add cell lysate to lyse the cells to release nucleic acids;

[0043] 2) Nucleic acid adsorption: Add magnetic beads containing specific probes to the solution, and the HBV / HCV / HIV...

Embodiment 3

[0053] This example provides a method for extracting the HBV / HCV / HIV-I virus (HCV RNA in this example) in the sample to be tested using the kit described in Example 1, as well as the detection of the obtained viral nucleic acid by fluorescent quantitative PCR and its result.

[0054] The samples to be tested in the present invention are: srea care HCV typing reference products diluted 10 times (20 kinds in total). In the present invention, two control systems (Abbott RealTime HCV m2000 detection system, Roche AmpliPrep / COBAS TaqMan Test detection system) and the kit of the present invention are used to detect various subtypes of HCV in the serum of the test sample.

[0055] The extraction and detection method of the present invention: add 200 μl of the sample to be tested into a 1.5ml centrifuge tube, and then add 600 μl of RNA extraction solution 1 (a mixture of cell lysate and magnetic beads containing specific probes): the RNA extraction Solution 1 was composed of 0.5% (W / ...

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Abstract

The invention firstly provides a biotin-labeled specific probe which is a biotin-labeled hepatitis c virus probe and has a sequence of biotin-(T)n-TGGTACTGCCTGATAGGGTGCTTGCG, wherein n is the number selected from 3 to 30, preferably 5 to 20. Correspondingly, the invention further provides a viral nucleic acid extraction reagent which comprises the biotin-labeled specific probe. The invention further provides the biotin-labeled hepatitis c virus probe and a biotin-labeled human immunodeficiency virus probe. The viral nucleic acid extraction reagent comprising the three biotin-labeled specific probes can be used for blood screening for three viruses.

Description

technical field [0001] The invention relates to a virus nucleic acid extraction reagent, a corresponding virus nucleic acid extraction kit, and a biotin-labeled specific probe in the virus nucleic acid extraction reagent. Background technique [0002] Nucleic acid extraction and purification technology is a basic technology of biochemistry and molecular biology. As molecular biology techniques are widely used in biology, medicine and related fields, nucleic acid extraction and purification techniques have also been further developed. [0003] Nucleic acid is always combined with various proteins in cells. Nucleic acid extraction mainly refers to the separation of nucleic acid from biological macromolecules such as proteins, polysaccharides, and fats. Most nucleic acid extraction methods generally include cell lysis, removal of biological macromolecules such as proteins, polysaccharides, and lipids bound to nucleic acids, removal of other unnecessary nucleic acid molecules,...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12Q1/70C12Q1/68
Inventor 戴立忠龚小鹏
Owner SANSURE BIOTECH INC
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