Rapid no-heating nucleic acid extraction kit based on magnetic bead method

A technology of nucleic acid extraction reagent and magnetic bead method, which is applied in the field of kits and nucleic acid extraction kits, can solve problems such as high experimental requirements, false positives, and complicated nucleic acid extraction process, and achieve simple and fast operation, simple steps, and technical Significant progress

CN109750030APending Publication Date: 2019-05-14SHANGHAI KEHUA BIO ENG
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2019-05-14

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Abstract

The invention provides a rapid no-heating nucleic acid extraction kit based on a magnetic bead method. The kit comprises a solution containing magnetic beads, protease K, a polyA solution, deproteinizing liquid, washing liquid, eluant and lysate. The invention also provides a method for extracting nucleic acid by using the kit. The method includes the steps that the lysate and the magnetic beads are added to a sample and fully shaken for uniform mixing, and when lytic cells release nucleic acid, the nucleic acid is captured with the magnetic beads; then, the specific deproteinizing liquid andwashing liquid are adopted for washing respectively to remove protein, salt ions and other impurities on the surfaces of the magnetic beads; finally, the specific eluant is used for rapidly eluting the nucleic acid at room temperature. Compared with a conventional nucleic acid extraction kit based on a magnetic bead method, the kit has the advantages that the steps are simple and rapid, and the whole process only takes about 15 min; besides, heating is not needed, so that the probability of sample room contamination possibly caused by aerosol is reduced; no chloroform phenol or other toxic reagents are used, so that the kit is safe and free of pollution.
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Description

technical field

[0001] The invention belongs to the field of bioengineering and relates to a kit, in particular to a rapid and non-heating nucleic acid extraction kit based on a magnetic bead method. Background technique

[0002] With the development of molecular biology, the detection and typing of disease-associated viruses is an important application field of molecular detection, and the efficient and simple extraction and purification of target nucleic acid from biological materials is the key technology of molecular detection. At present, there are mainly three types of viral nucleic acid purification, namely boiling method, alcohol precipitation method, column membrane method and magnetic bead method.

[0003] The boiling method is to add a lysate to the sample, and the cells are lysed to release nucleic acid by heating. This method is simple to operate, but the purity of the extracted nucleic acid is low, and the throughput is low, which is not conducive to subsequent...

Examples

Embodiment 1

[0027] Detection of HBV virus in plasma (comparative study with a domestic manufacturer's magnetic bead method kit)

[0028] (1) Extraction of viral nucleic acid

[0029] 1. Add 300ul of plasma to a 2ml centrifuge tube (WHO HBV standard with a final concentration of 10IU is added to the negative plasma), add 600μL of lysate, 15μL of magnetic bead solution, 20μL of proteinase K solution and 5μL of polyA solution, and vortex for 1min to Mix the sample well, then shake and mix for 3-5min.

[0030] 2. After instantaneous centrifugation, place the centrifuge tube on the magnetic stand and let it stand for 1 min. After the magnetic beads are completely absorbed, the liquid is sucked off.

[0031] 3. Add 500 μL buffer deproteinized solution, mix well, and vortex for 1 min.

[0032] 4. After instantaneous centrifugation, place the centrifuge tube on the magnetic stand and let it stand for 1-2 minutes. After the magnetic beads are completely absorbed, absorb the liquid.

[0033] 5. ...

Embodiment 2

[0050] Embodiment 2: Detection of HCV virus in plasma (comparative research with a certain domestic manufacturer's magnetic bead method kit)

[0051] (1) Extraction of viral nucleic acid

[0052] 1. Add 300 μL of plasma to a 2ml centrifuge tube (WHO HCV standard with a final concentration of 20 IU is added to the negative plasma), add 600 μL of lysate, 15 μL of magnetic bead solution, 20 μL of proteinase K solution and 5 μL of polyA solution, and vortex for 1 min to Mix the sample well, then shake and mix for 3-5min.

[0053] 2. After instantaneous centrifugation, place the centrifuge tube on the magnetic stand and let it stand for 1 min. After the magnetic beads are completely absorbed, the liquid is sucked off.

[0054] 3. Add 500 μL buffer deproteinized solution, mix well, and vortex for 1 min.

[0055] 4. After instantaneous centrifugation, place the centrifuge tube on the magnetic stand and let it stand for 1-2 minutes. After the magnetic beads are completely absorbed, ...