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Rapid DNA (deoxyribonucleic acid) extraction kit with magnetic beads for fluorescent quantitative PCR (polymerase chain reaction) detection and extraction method of rapid DNA extraction kit

A fluorescence quantification and extraction method technology, applied in the field of bioengineering, can solve problems such as affecting the stability of downstream detection, interfering with downstream nucleic acid detection, affecting the accuracy of nucleic acid, etc., to achieve automation and high-throughput quantification, storage and transportation are more stable and safer. The effect of improving quality and extraction efficiency

Pending Publication Date: 2022-06-24
珠海宝锐生物科技有限公司
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, there are still some defects in the existing methods of nucleic acid extraction using magnetic beads, including: 1) In addition to adsorbing specific DNA on the surface of magnetic beads, some impurities such as foreign proteins are often adhered to the surface during the nucleic acid extraction process. It is easy to interfere with downstream nucleic acid detection; 2) Most of the existing nucleic acid extraction or purification reagents often introduce ethanol in the cleaning step, and the magnetic beads need to be fully dried after being washed with a solution containing ethanol, otherwise it will affect the accuracy of subsequent nucleic acid detection ; 3) In the process of nucleic acid extraction for most of the existing similar products, the magnetic beads are sometimes easy to remain in the nucleic acid eluent, which has an inhibitory effect on the downstream fluorescent quantitative PCR detection to a certain extent; 4) For samples with lower concentrations, The effect of nucleic acid extraction or purification is not good, which affects the stability of downstream detection

Method used

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  • Rapid DNA (deoxyribonucleic acid) extraction kit with magnetic beads for fluorescent quantitative PCR (polymerase chain reaction) detection and extraction method of rapid DNA extraction kit
  • Rapid DNA (deoxyribonucleic acid) extraction kit with magnetic beads for fluorescent quantitative PCR (polymerase chain reaction) detection and extraction method of rapid DNA extraction kit
  • Rapid DNA (deoxyribonucleic acid) extraction kit with magnetic beads for fluorescent quantitative PCR (polymerase chain reaction) detection and extraction method of rapid DNA extraction kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] In this experiment, HBV positive samples were used as test samples, and the concentration gradients of HBV samples diluted with human negative plasma were respectively 10^4IU / mL, 50IU / mL, and 10IU / mL as samples to be tested. extraction by manual extraction. After the extraction, take 23.5 μl of nucleic acid elution reaction solution EB containing magnetic beads into a 200 μl PCR reaction tube, and configure the fluorescence quantitative PCR reaction system according to the following table:

[0114] Table 1 Fluorescence quantitative PCR reaction system

[0115] Reagent components Dosage (μl / T) Nucleic acid elution reaction solution EB (containing magnetic beads) 23.5 HBV pbmix 1 50×Fast Direct Enzyme / UNG mix 0.5 total capacity 25.0

[0116] Among them, HBV pbmix is ​​the fluorescent quantitative PCR primer and probe premix reagent independently configured by Zhuhai Baorui Biotechnology Co., Ltd., and 50×Fast Direct Enzyme / UNG mix ...

Embodiment 2

[0133] In this experiment, HBV positive samples were used as test samples, and whole blood samples were used to dilute HBV samples with concentration gradients of 10^4IU / mL, 50IU / mL, and 10IU / mL as samples to be tested. The extraction of HBV-DNA adopts the method of manual extraction. After the extraction, take 23.5 μl of the nucleic acid elution reaction solution EB containing magnetic beads into a 200 μl PCR reaction tube, and configure the fluorescence quantitative PCR reaction system according to the following table, as shown in Table 5:

[0134] Table 5 Fluorescence quantitative PCR reaction system

[0135]

[0136]

[0137] Among them, HBV pbmix is ​​the fluorescent quantitative PCR primer and probe premix reagent independently configured by Zhuhai Baorui Biotechnology Co., Ltd., and 50×Fast Direct Enzyme / UNG mix is ​​the DNA polymerase reaction premix independently configured by Zhuhai Baorui Biotechnology Co., Ltd. .

[0138] Among them, the HBV pbmix primer pr...

Embodiment 3

[0154] In this experiment, the oral cell swab lotion of normal people was used as the test sample, and the human genomic DNA in the swab sample was extracted by using the reagent provided by the present invention, and the method of manual extraction was adopted. After the extraction, take 23.5 μl of the nucleic acid elution reaction solution EB containing magnetic beads into a 200 μl PCR reaction tube, and configure the fluorescence quantitative PCR reaction system according to the following table to detect EF-1-α and UbcH5B fragments in human genomic DNA, such as As shown in Table 9:

[0155] Table 9 Fluorescence quantitative PCR reaction system

[0156] Reagent components Dosage (μl / T) Nucleic acid elution reaction solution EB (containing magnetic beads) 23.5 EF-1-α / UbcH5B pbmix 1 50×Fast Direct Enzyme / UNG mix 0.5 total capacity 25.0

[0157] Among them, EF-1-α / UbcH5B pbmix is ​​a fluorescent quantitative PCR primer and probe premix r...

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Abstract

The invention discloses a rapid DNA (deoxyribonucleic acid) extraction kit with magnetic beads for fluorescent quantitative PCR (polymerase chain reaction) detection and an extraction method thereof, and belongs to the technical field of bioengineering. The rapid DNA extraction kit comprises a rapid DNA extraction kit body; the rapid DNA extraction kit comprises a lysis binding solution LB, a washing solution W1, a washing solution W2, a nucleic acid elution reaction solution EB, magnetic beads MB and protease K. The extraction method comprises a manual nucleic acid extraction method or a method for realizing high-flux automatic DNA extraction by matching with an automatic instrument. The rapid DNA extraction reagent with the magnetic bead amplification function can meet DNA extraction of various sample types such as whole blood, plasma and swabs, an extracted product can be directly applied to fluorescent quantitative PCR amplification detection reaction, separation of magnetic beads and nucleic acid is not needed, downstream detection is not affected, and nucleic acid extraction or purification can be achieved more simply, more sensitively and more rapidly.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a rapid DNA extraction kit with magnetic beads for fluorescence quantitative PCR detection and an extraction method thereof. Background technique [0002] Nucleic acid is a kind of biopolymer, which is an indispensable constituent of all known life, widely exists in animal and plant cells, microorganisms, and is divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acid detection is currently inseparable from various research fields such as medicine and molecular biology. One of the core steps of nucleic acid detection is the extraction and purification of nucleic acid, which is also the basis for molecular biology research and clinical testing. Existing nucleic acid extraction methods mainly include alkaline lysis method, phenol-chloroform extraction method, chelating resin method, spin column membrane adsorption method, magnetic bead m...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6851C12Q1/70C12N15/11C12R1/93
CPCC12Q1/6806C12Q1/6851C12Q1/706C12Q2523/308C12Q2521/537C12Q2527/125C12Q2531/113C12Q2563/107
Inventor 林楚江李倩茜时彦祎王进
Owner 珠海宝锐生物科技有限公司
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