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A lysate for extracting nucleic acid by magnetic bead method and method for extracting nucleic acid using the lysate

A technology for extracting nucleic acid and lysate, applied in the field of molecular biology, can solve the problems of nucleic acid loss, nucleic acid leakage pollution, low extraction efficiency, etc., and achieve the effects of avoiding pollution, good repeatability, and reducing nucleic acid loss.

Active Publication Date: 2021-08-13
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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  • A lysate for extracting nucleic acid by magnetic bead method and method for extracting nucleic acid using the lysate
  • A lysate for extracting nucleic acid by magnetic bead method and method for extracting nucleic acid using the lysate
  • A lysate for extracting nucleic acid by magnetic bead method and method for extracting nucleic acid using the lysate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0065] Using the above lysate and rinse solution, the clinical diagnosis is clear and the quantitative value of HBV DNA after testing is 1.5×10 6 The hepatitis B patient serum of IU / mL and 30IU / mL carries out the operation of following steps:

[0066] (1) Dispensing of PCR lysate: Dispense the prepared lysate containing magnetic beads into dedicated PCR tubes at a rate of 100 μL per tube.

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

[0068] (3) Absorb and discard the liquid: Place the above PCR tubes on the eight-row magnetic stand, let it stand for 2 minutes, and use a negative pressure pump to suck up the liquid on the opposite side of the magnetic beads, and be careful not to suck off the magneti...

Embodiment 2

[0080] Example 2: Comparison of whether the sample and the lysate are mixed evenly to detect the repeatability of 80IU / mL HBV DNA

[0081] In order to verify the impact of whether the sample and lysate are mixed uniformly on the repeatability of low-value samples, the non-mixed method of the present invention is compared with the mixed method.

[0082] Using the same method as in Example 1, the serum of hepatitis B patients with a clear clinical diagnosis and a quantitative HBV DNA value of 80 IU / mL was tested repeatedly in 10 wells.

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

Embodiment 3

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

[0085] In order to verify the effect of blowing off the magnetic beads during rinsing on the efficiency of nucleic acid extraction, use the above lysate and rinsing solution to determine the clinical diagnosis and the tested HBV DNA quantitative value is 1.5×10 8 IU / mL and 1.5×10 4IU / mL serum from patients with hepatitis B was tested. Scheme 1) blowing off the magnetic beads during rinsing, scheme 2) not blowing off the magnetic beads during rinsing, and other steps are the same as in Example 1.

[0086] Experimental results such as Figure 4 As shown, the results show that according to the example scheme 2) the inventive method of not blowing off the magnetic beads during rinsing, for 1.5 × 10 8 IU / mL and 1.5×10 4 When the IU / mL hepatitis B patient serum sample is tested, not only can it be quantified effectively, but also its PCR efficiency is higher than the PCR effi...

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Abstract

The invention discloses a lysate for extracting nucleic acid by a magnetic bead method. The lysate contains potassium hydroxide with a final concentration of 0.3-0.5N, sodium acetate with a final concentration of 0.2-0.4M, and a mass fraction of 0.03-0.07%. N-lauroyl sarcosinate sodium, EDTA with a final concentration of 3-7mM, Tween 20 with a volume fraction of 0.5-1.5%, and trehalose with a final concentration of 0.1-0.3M. When using the lysate or kit of the present invention to conduct experiments, it is possible to add samples without mixing or heating, to lyse nucleic acids without shaking, and to rinse magnetic beads without blowing them away, and only need to soak once statically, thus reducing the need for laboratory testing. Contamination and loss of nucleic acid, no need to mix the reaction solution and magnetic beads, no elution for nucleic acid amplification, all the obtained magnetic bead nucleic acid participates in the PCR amplification reaction, avoiding the possibility of contamination caused by pipetting, greatly shortening the detection time, And low-value samples have good repeatability.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a lysate for extracting nucleic acid by a magnetic bead method and a method for extracting nucleic acid using the lyse. Background technique [0002] Nucleic acid extraction technology is the basis of molecular biology research, and it is also a common research method for pathogenic microorganism detection, species identification, paternity identification, forensic evidence collection, etc. However, a method with simple operation, few operation steps, high yield of nucleic acid, and no aerosol pollution in the laboratory is the focus and difficulty of the quality construction of clinical molecular biology laboratories. [0003] At present, the magnetic bead method has become an emerging nucleic acid extraction technology. Its main principle is to adsorb nucleic acid based on magnetic nanomaterials. After magnetic separation and impurity rinsing, the nucleic acid is eluted from th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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