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Kit for magnetic-bead-method rapid nucleic acid extraction and application of kit

A magnetic bead method and a kit technology, applied in the field of rapid nucleic acid extraction lysate by magnetic bead method, can solve the problems of nucleic acid damage, complicated operation, long extraction time, etc., and achieve the effects of preventing degradation, quick and easy operation, and simple operation.

Active Publication Date: 2021-01-15
北京健为医学检验实验室有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the extraction process of commercial extraction kits based on the magnetic bead method on the market generally requires heating steps for lysis incubation and nucleic acid elution, and heating will cause a certain degree of damage to nucleic acids, especially for RNA in samples. If heating at 50-60°C is used during the process, the RNase contained in the sample, reagent consumables or introduced in the environment will reach the optimum temperature condition, which will lead to the degradation and loss of RNA samples
This kind of loss is even more unacceptable when the sample size is not high and the virus content in the sample is small, and it is easy to cause serious misjudgment of results.
In addition, whether it is adsorption column extraction or magnetic bead nucleic acid extraction, there are problems such as complicated operation and long extraction time. The whole extraction process usually takes 30 minutes or even longer, which cannot meet the needs of simpler and faster extraction in the case of an outbreak. nucleic acid extraction needs

Method used

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  • Kit for magnetic-bead-method rapid nucleic acid extraction and application of kit
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  • Kit for magnetic-bead-method rapid nucleic acid extraction and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The magnetic bead method rapid nucleic acid (DNA / RNA) extraction kit used includes flying magnetic lysis solution, magnetic bead suspension, flying magnetic washing solution and flying magnetic eluent; among them: (1) The composition of flying magnetic lysis solution is: 50mM Tris-HCl, 3 M guanidine hydrochloride, 100 mM sodium chloride, 20 mM EDTA buffer, 0.5% Triton X-100, 50% isopropanol, 8 mg / mL nucleic acid protection peptide, the pH of the lysate is 7.0; (2 ) The magnetic bead suspension is composed of magnetic balls with a volume fraction of 70%, the particle size of the magnetic beads is between 10-50nm, 30% of nuclease-free water, and the pH of the magnetic bead suspension is 5.0; (3) The flying magnetic rinse solution is composed of The following components are 20mM Tris-HCl, 10mM NaCl, 2mM EDTA, 80% ethanol; (4) The composition of the flying magnetic eluent is: 5mM Tris-HCl (pH8.0).

[0032] We use porcine circovirus (PCV, DNA virus, non-infectious to humans)...

Embodiment 2

[0040] Using the same kit as in Example 1, used in conjunction with the automatic nucleic acid extractor CWE2100, the nucleic acid extraction test was performed on the 4 porcine PCV virus swab samples and 4 poultry IBV virus swab samples in Example 1, specifically including the following steps :

[0041] (1) Sample and reagent preparation

[0042] Take the 96-well deep-well plate (CWE2100 matching consumables), and add the corresponding reagents to the 96-well deep-well plate according to the following table:

[0043]

[0044] (2) Nucleic acid extraction

[0045] Put the 96-well deep-well sample plate with samples and reagents added in the previous step into the CWE2100 instrument, put it into the magnetic bar cover, and run the program

[0046]

[0047] After about 7 minutes, take out the 96-well deep-well plate, transfer the nucleic acid samples in columns 6 and 12 to a clean centrifuge tube (nuclease-free, self-prepared), and store at -80°C.

[0048] Similarly, acc...

Embodiment 3

[0050] (1) Detection of nucleic acid concentration and purity:

[0051] For swab samples of porcine PCV and poultry IBV virus extracted in Example 1 and Example 2, the concentration of the extracted total nucleic acid was measured using Qubit, and the DNA purity was measured by Nanodrop. The results are shown in Table 1. Manual extraction and CWE series The nucleic acid extracted by the automatic extractor, the total amount (concentration) and purity (OD 260 / 280 ) are basically the same, there is no significant difference, the nucleic acid purity is better, and the protein contamination is less; the effect is obviously better than that of the control group using proteinase K.

[0052] Table 1. Quality detection of total nucleic acid extracted by manual extraction and CWE2100 automatic extraction instrument

[0053]

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Abstract

The invention relates to a lysate for magnetic-bead-method rapid nucleic acid extraction and application of the lysate in respiratory tract virus detection. A kit for magnetic-bead-method rapid nucleic acid extraction with excellent preservation capability is prepared through designing a fly-magnetic lysate containing novel nucleic acid protecting peptides. The kit disclosed by the invention can be used for rapidly completing nucleic acid extraction on a swab sample, the operation is rapid, simple and convenient, and one extraction process can be completed by manual operations by only 5 minutes; and the protecting peptides can provide an excellent protective action on nucleic acid extraction during extraction.

Description

technical field [0001] The present invention relates to the technical field of nucleic acid extraction, in particular to the technical field of nucleic acid extraction lysate, and specifically refers to a rapid nucleic acid extraction lysate by magnetic bead method, its preparation method and its application in respiratory virus detection. Background technique [0002] Respiratory tract infections, especially acute respiratory infectious diseases, have always been an important cause of death and disability worldwide. Viruses are the main cause of respiratory tract infections, and more than 50% of respiratory infections are caused by viruses. Common respiratory infection viruses include influenza A virus, influenza B virus, parainfluenza virus, respiratory syncytial virus, coronavirus, etc. According to statistics, more than 100 million people were killed by influenza virus in the last century. Among them, the "Spanish flu" that broke out in 1918 caused about 50 million deat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 刘淑君庄志华殷剑峰王春香
Owner 北京健为医学检验实验室有限公司