Real-time fluorescence quantification PCR method with magnetic bead nucleic acid extraction and amplification conducted in one tube

A real-time fluorescence quantitative and nucleic acid extraction technology, applied in the field of molecular biology, can solve the problems of unstable nucleic acid cleavage efficiency, nucleic acid loss, nucleic acid pollution, etc., and achieve the effect of optimized nucleic acid extraction effect, low cost, and complete protein removal

Active Publication Date: 2016-03-23
BEIJING NAGENE DIAGNOSTIC REAGENT CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The technical problem to be solved in the present invention is to extract nucleic acid by magnetic bead method and real-time fluorescent quantitative PCR amplification in the prior art separately and need multiple pipetting, nucleic acid cracking efficiency is unstable, magnetic bead nucleic acid elution causes nucleic acid loss, nucleic acid pollution etc., a real-time fluorescent quantitative PCR method for nucleic acid extraction and amplification in one tube is provided

Method used

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  • Real-time fluorescence quantification PCR method with magnetic bead nucleic acid extraction and amplification conducted in one tube
  • Real-time fluorescence quantification PCR method with magnetic bead nucleic acid extraction and amplification conducted in one tube
  • Real-time fluorescence quantification PCR method with magnetic bead nucleic acid extraction and amplification conducted in one tube

Examples

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Effect test

Embodiment 1

[0075] Embodiment 1: The real-time fluorescent quantitative PCR detection of HBVDNA

[0076] The reagents prepared according to the above-mentioned lysate scheme 1) and washing solution scheme 1) mixed with magnetic beads were used respectively for the clinical diagnosis and the tested HBVDNA quantitative value of 2×10 1 IU / ml hepatitis B patient's serum carries out the operation of following steps:

[0077] (1) Dispensing of PCR lysate: Dispense the prepared lysate mixed with magnetic beads into dedicated PCR tubes at a rate of 100 μl per tube.

[0078] (2) Adding samples: Take 100 μl of serum and add it to the above-mentioned PCR tube containing the lysate mixed with magnetic beads, gently blow and mix 5 times with a tip, and let stand at room temperature for 10 minutes.

[0079] (3) Aspirate and discard the liquid: Place the above PCR tubes on the eight-row magnetic stand, let it stand for 2 minutes, and use a pipette to absorb the liquid on the opposite side of the magnet...

Embodiment 2

[0087] Embodiment 2: The real-time fluorescent quantitative PCR detection of HBVDNA

[0088] The reagents prepared according to the above-mentioned lysate scheme 2) and washing solution scheme 2) mixed with magnetic beads were used respectively for the clinical diagnosis and the tested HBVDNA quantitative value of 2×10 1 IU / ml hepatitis B patient's serum carries out the operation of following steps:

[0089] (1) Dispensing of PCR lysate: Dispense the prepared lysate mixed with magnetic beads into dedicated PCR tubes at a rate of 100 μl per tube.

[0090] (2) Adding samples: Take 100 μl of serum and add it to the above-mentioned PCR tube containing the lysate mixed with magnetic beads, gently blow and mix 5 times with a pipette tip, and let stand at room temperature for 5 minutes.

[0091] (3) Aspirate and discard the liquid: place the above-mentioned PCR tubes on the eight-row magnetic stand, let it stand for 2 minutes, and use a pipette to absorb the liquid on the opposite s...

Embodiment 3

[0099] Embodiment 3: Sensitivity test of real-time fluorescent quantitative PCR method of the present invention

[0100] Serum samples negative for HBVDNA were used as diluent, and the clinical diagnosis was confirmed and the quantitative value of HBVDNA after testing was 2×10 9 The IU / ml serum of hepatitis B patients was diluted sequentially to obtain HBVDNA concentrations of 5IU / ml, 10IU / ml, 20IU / ml, and 2×10 2 IU / ml, 2×10 3 IU / ml, 2×10 4 IU / ml, 2×10 5 IU / ml, 2×10 6 IU / ml, 2×10 7 IU / ml, 2×10 8 IU / ml, 2×10 9 IU / ml of sample. The same method as in Example 1 was used to perform real-time fluorescent quantitative PCR detection on the above diluted samples.

[0101] Experimental results such as image 3 Shown, where the curve from right to left represents the concentration of 5IU / ml, 10IU / ml, 20IU / ml, 2×10 2 IU / ml, 2×10 3 IU / ml, 2×10 4 IU / ml, 2×10 5 IU / ml, 2×10 6 IU / ml, 2×10 7 IU / ml, 2×10 8 IU / ml, 2×10 9 IU / ml of sample. The results show that the present inventi...

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Abstract

The invention provides a real-time fluorescence quantification PCR method with magnetic bead nucleic acid extraction and amplification conducted in one tube. A lysis solution mixed with magnetic beads and a sample to be detected are added to a PCR amplification tube, even mixing and standing are conducted, mixed liquor is extracted after magnetic extraction, and obtained magnetic beads are washed once; a well prepared PCR liquid is added into the PCR amplification tube for real-time fluorescence quantification PCR of target nucleic acid; the lysis solution is prepared from 0.2-0.4N sodium hydroxide, 0.3-0.6M potassium chloride, 0.01-0.05%N-sodium lauroyl sarcosine, 5mM EDTA, 0.3-0.6M Tris-HCL, and 1-2% Triton X-100. According to the method, heating is not needed, indoor-temperature pyrolysis is conducted for about 5-10 min only, static washing is needed only once, and then laboratory pollution and magnetic bead nucleic acid loss are reduced, pollution possibility brought by step-by-step operation is avoided and detection time is shortened. By taking HBV DNA quantification as an example, sensitivity can be as low as 5 IU / ML, and repeatability is high.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a real-time fluorescent quantitative PCR method for extracting nucleic acid and amplifying magnetic beads in one tube. Background technique [0002] Polymerase Chain Reaction (Polymerase Chain Reaction, PCR) is a technology designed according to the nature of DNA replication in vivo to rapidly amplify specific DNA sequences in vitro. The PCR reaction system is mainly composed of nucleic acid primers, 4 kinds of dNTPs, DNA polymerase, template DNA and PCR reaction buffer system. Since the invention of polymerase chain reaction (PCR) in 1985 by Kary Mullis and his colleagues in the human genetic laboratory of Cetus Corporation in the United States, PCR technology and its derivative technologies have been rapidly developed and widely used in various nucleic acid detection. Especially for the detection of viruses or other pathogens, when a specific gene segment of the pathogen to be...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q1/6851C12Q2531/113C12Q2563/107C12Q2523/308
Inventor 王海滨王棽周其玲冯小霞
Owner BEIJING NAGENE DIAGNOSTIC REAGENT CO LTD
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