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Lysis solution for extracting nucleic acid by paramagnetic particle method and method for extracting nucleic acid by using lysis solution

A technology for extracting nucleic acid and lysate, applied in the field of molecular biology, can solve problems such as low extraction efficiency, nucleic acid loss, nucleic acid leakage pollution, etc., and achieve the effect of ensuring accuracy and effectiveness, protecting nucleic acid, and simplifying operation steps.

Active Publication Date: 2019-12-24
BEIJING NAGENE DIAGNOSTIC REAGENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] One aspect of the present invention is aimed at the existence of power mixing in the dynamic nucleic acid preparation method in the prior art, which leads to nucleic acid leakage pollution, magnetic bead rinsing and PCR reaction solution and nucleic acid magnetic beads need to be blown and mixed, and a large amount of nucleic acid is adhered to the tip. To solve the problem of loss of nucleic acid and low extraction efficiency, a lysate for extracting nucleic acid by magnetic bead method and a method for extracting nucleic acid using the lysate are provided

Method used

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  • Lysis solution for extracting nucleic acid by paramagnetic particle method and method for extracting nucleic acid by using lysis solution
  • Lysis solution for extracting nucleic acid by paramagnetic particle method and method for extracting nucleic acid by using lysis solution
  • Lysis solution for extracting nucleic acid by paramagnetic particle method and method for extracting nucleic acid by using lysis solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Comparison of whether the sample and the lysate are mixed to detect HBV DNA

[0065] Use the above lysis solution and rinsing solution to make a clear clinical diagnosis and the quantitative value of HBV DNA after detection is 1.5×10 6 The IU / mL and 30IU / mL hepatitis B patient serum is operated as follows:

[0066] (1) Aliquoting of PCR lysis solution: Aliquot the prepared lysis solution containing magnetic beads into special PCR tubes according to 100μL per tube.

[0067] (2) Adding samples: Take 100 μL of serum and add it to the above PCR tube containing the lysing solution containing magnetic beads. The sample and the lysing solution are not mixed, and let stand at room temperature for 5 minutes.

[0068] (3) Aspirate and discard liquid: Place the above PCR tube on the eight-row magnetic rack and let it stand for 2 minutes. Use a negative pressure pump to suck off the liquid on the opposite side of the magnetic beads. Be careful not to suck off the magnetic beads. ...

Embodiment 2

[0080] Example 2: Comparison of the reproducibility of 80IU / mL HBV DNA detection if the sample and lysate are mixed

[0081] In order to verify the influence of whether the sample and the lysis solution are evenly mixed on the reproducibility of the low-value sample, the unmixing method of the present invention is compared with the mixing method.

[0082] The same method as in Example 1 was used to carry out 10 repetitive tests on the serum of hepatitis B patients whose clinical diagnosis was clear and the HBV DNA quantitative value was 80 IU / mL.

[0083] The result is figure 2 , image 3 As shown, the experimental results show that when the sample to be tested is added to the lysis solution, the reproducibility of 80IU / mL amplified by the unmixing method is significantly higher than that of the 80IU / mL amplified by the mixing method.

Embodiment 3

[0084] Example 3: Comparison of whether to blow off the magnetic beads during rinsing to detect HBV DNA

[0085] In order to verify whether the magnetic beads are blown away during rinsing, the effect on nucleic acid extraction efficiency, the above lysis solution and rinsing solution are used to make a clear clinical diagnosis and the quantitative value of HBV DNA after detection is 1.5×10 8 IU / mL and 1.5×10 4 IU / mL serum of hepatitis B patients was tested. Scheme 1) Blow off the magnetic beads during rinsing, Scheme 2) Do not blow off the magnetic beads during rinsing, and the other steps are the same as in Example 1.

[0086] The experimental results are as Figure 4 As shown, the results show that, according to the inventive method of scheme 2) of the present example that the magnetic beads are not blown off during rinsing, the value of 1.5×10 8 IU / mL and 1.5×10 4 When detecting IU / mL serum samples of hepatitis B patients, it can not only quantify it effectively, but also its PC...

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Abstract

The invention discloses a lysis solution for extracting nucleic acid by a paramagnetic particle method. The lysis solution contains: potassium hydroxide with a final concentration of 0.3-0.5, sodium acetate with a final concentration of 0.2-0.4, N-sodium lauroyl sarcosine with a mass fraction of 0.03-0.07%, EDTA with a final concentration of 3-7mM, Tween 20 with a volume fraction of 0.5-1.5%, andtrehalose with a final concentration of 0.1-0.3%. When used for experiments, the lysis solution or a kit provided by the invention can realizes the effects that: samples can be added without uniform mixing and heating; nucleic acid lysis requires no oscillation; magnetic beads do not need to be blown away during rinsing; only one static soaking is required to reduce laboratory contamination and nucleic acid loss; a reaction liquid and magnetic beads do not need to be uniformly mixed; the nucleic acid amplification does not need elution; and the obtained magnetic bead nucleic acid completely participates in the PCR amplification reaction, so that the possibility of pollution caused by tube transfer is avoided; the detection time is greatly shortened; and the repeatability of a low-value sample is good.

Description

Technical field [0001] The present invention relates to the field of molecular biology, in particular to a lysis solution for extracting nucleic acid by a magnetic bead method and a method for extracting nucleic acid using the lysis solution. Background technique [0002] Nucleic acid extraction technology is the basis of molecular biology research, and it is also a commonly used research method for pathogenic microorganism detection, species identification, paternity identification, forensic evidence collection, and so on. However, a method that has simple operation, few operation steps, high nucleic acid yield, and does not cause laboratory aerosol pollution is the focus and difficulty of the current clinical molecular biology laboratory quality construction. [0003] At present, the magnetic bead method has become an emerging nucleic acid extraction technology. Its main principle is to absorb nucleic acid based on magnetic nanomaterials. After magnetic separation and impurity ri...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1013C12Q1/6806C12Q2527/125C12Q2531/113
Inventor 王海滨
Owner BEIJING NAGENE DIAGNOSTIC REAGENT CO LTD
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