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A blood cell lysate and method for extracting nucleic acid in blood using the lysate

A blood cell and lysate technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA preparation, etc., can solve problems such as low efficiency, affecting follow-up tests, and easy contamination, achieving high yield and saving detection time , the effect of not easy to pollute

Active Publication Date: 2021-07-23
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 disadvantages of low efficiency, influence on follow-up tests and easy contamination of the blood cell nucleic acid extraction method in the prior art, and provides a blood cell lysate and a method for extracting nucleic acid in blood using the lyse

Method used

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  • A blood cell lysate and method for extracting nucleic acid in blood using the lysate
  • A blood cell lysate and method for extracting nucleic acid in blood using the lysate
  • A blood cell lysate and method for extracting nucleic acid in blood using the lysate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: Real-time fluorescent quantitative PCR detection of EBV DNA

[0059] The reagents prepared according to the above-mentioned lysate solution mixed with heparin magnetic beads scheme 1) were used to confirm the clinical diagnosis and the quantitative value of EBV DNA after detection was 2×10 2 IU / ml patient blood is operated as follows:

[0060] Step 1) Put the magnetic bead chromatography column in a nuclease-free 1.5mL centrifuge tube,

[0061] Step 2) Add 20 μl of the lysate mixed with heparin magnetic beads to the bottom of the magnetic bead chromatography column tube; take 120 μl of the sample to be tested and add it to the lysate mixed with heparin magnetic beads, mix well, and stand at 95°C for 10 minute;

[0062] Step 3) Move the sleeve to a centrifuge for 5 minutes at 15,000 rpm, and the precipitated liquid is the extraction of nucleic acid.

[0063] Step 4) Taq DNA polymerase of 4.0 μl (0.5U / μl) unit is mixed with 36.0 μl PCR reaction solution (...

Embodiment 2

[0068] Embodiment 2: Real-time fluorescent quantitative PCR detection of EBV DNA

[0069] The reagents prepared by scheme 2) respectively have a clear clinical diagnosis and a quantitative value of 2×10 EBV DNA after testing. 2 IU / ml hepatitis B patient's serum carries out the operation of following steps:

[0070] Step 1) Put the magnetic bead chromatography column in a nuclease-free 1.5mL centrifuge tube,

[0071] Step 2) Add 20 μl of the lysate mixed with heparin magnetic beads to the bottom of the magnetic bead chromatography column tube; take 120 μl of the sample to be tested and add it to the lysate mixed with heparin magnetic beads, mix well, and stand at 85°C for 5 minute;

[0072] Step 3) Move the cannula to a centrifuge for 10 minutes at 10,000 rpm, and the precipitated liquid is the extraction of nucleic acid.

[0073] Step 4) Taq DNA polymerase of 4.0 μl (0.5U / μl) unit is mixed with 36.0 μl PCR reaction solution (the reaction solution is to include concentration...

Embodiment 3

[0078] Embodiment 3: Real-time fluorescent quantitative PCR detection of EBV DNA

[0079] The reagents prepared according to the above-mentioned lysate solution mixed with heparin magnetic beads scheme 1) were used to confirm the clinical diagnosis and the quantitative value of EBV DNA after detection was 2×10 2 IU / ml patient blood is operated as follows:

[0080] Step 1) Put the magnetic bead chromatography column in a nuclease-free 1.5mL centrifuge tube,

[0081] Step 2) Add 20 μl of the lysate mixed with heparin magnetic beads to the bottom of the magnetic bead chromatography column; take 120 μl of the sample to be tested and add it to the lysate mixed with heparin magnetic beads, mix well, and stand at 90°C 8 minutes;

[0082] Step 3) Move the cannula to a centrifuge for 5 minutes at 12000 rpm, and the precipitated liquid is the extraction of nucleic acid.

[0083] Step 4) Taq DNA polymerase of 4.0 μl (0.5U / μl) unit is mixed with 36.0 μl PCR reaction solution (the react...

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Abstract

The invention discloses a blood cell lysate and a method for extracting nucleic acid in blood by using the lysate. The blood cell lyse contains heparin magnetic beads with a mass fraction of 3% to 10%, and Triton X-100 with a mass fraction of 0.5 to 1.5%. , urea with a final concentration of 0.5-1.0M, mass fraction of 0.02-0.08% sodium lauroyl sarcosinate, and calcium chloride and / or magnesium chloride with a final concentration of 0.1-0.3M. The compound detergent in the lysate of the present invention can effectively lyse cells, and the heparin magnetic beads can effectively adsorb proteins, and under the action of calcium (magnesium) ions, PCR interfering substances such as proteins can be solidified rapidly and nucleic acids can be separated out by warm treatment. Nucleic acids of high purity are obtained by centrifugation. The lysate of the present invention is stable in nature and temperature, and is not affected by seasons, temperatures, salt ion concentrations, etc., and the method is simple and fast in operation, is not easy to be polluted, has a high yield of nucleic acid extraction, and is suitable for clinical and scientific research.

Description

technical field [0001] The invention relates to the field of molecular biology detection, in particular to a blood cell lysate and a method for extracting nucleic acid in blood using the lyse. Background technique [0002] Nucleic acid is the carrier of genetic information, the most important biological information molecule, and the main object of molecular biology research. Therefore, nucleic acid extraction is the most important and basic operation in molecular biology experimental techniques. Nucleic acid extraction refers to the process of separating nucleic acid from the carrier by physical and chemical methods. At present, most medical institutions and scientific research fields in China need to extract nucleic acid from a large number of blood samples. Therefore, in order to ensure that high-concentration nucleic acids are extracted, new technologies are constantly being developed. [0003] There are many methods for nucleic acid extraction, including the traditiona...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/6806
Inventor 王海滨窦瑞燕周其玲王维王奇高智强
Owner BEIJING NAGENE DIAGNOSTIC REAGENT CO LTD
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