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Direct real-time quantitative fluorescent PCR method of layered packaging tape indicator

A real-time quantitative fluorescence and indicator technology, applied in the field of molecular biology, can solve the problems of easy pollution, low PCR sensitivity, and complicated nucleic acid extraction operations, and achieve the effect of simple operation and high detection sensitivity.

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

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a direct real-time quantitative fluorescent method with good reagent stability, easy operation and accurate quantification, aiming at the disadvantages of complex nucleic acid extraction operation, easy pollution, and low PCR sensitivity in the real-time quantitative fluorescent PCR process. PCR method

Method used

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  • Direct real-time quantitative fluorescent PCR method of layered packaging tape indicator
  • Direct real-time quantitative fluorescent PCR method of layered packaging tape indicator
  • Direct real-time quantitative fluorescent PCR method of layered packaging tape indicator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: Real-time quantitative fluorescent PCR detection to HBVDNA

[0048] 1. Coating of hot start DNA polymerase:

[0049] Take 1 μL (2.5-3.5U / μL) of hot-start DNA polymerase, and coat the bottom of the PCR amplification tube with 10-15 μL of paraffin oil mixture with a melting point of 50°C-55°C through melting and condensation.

[0050] 2. Preparation of fluorescent PCR amplification reaction solution:

[0051] The concentration is 40nmol / μL upstream and downstream primers and 30nmol / μL probe; 20nmol / μL Tris base; 20mmol / μL magnesium chloride; 150mmol / μL potassium chloride, 200umol / μL dNTP and no more than 0.001wt% stone green indicator reagents to prepare fluorescent PCR amplification reaction solution.

[0052] 3. Coating of fluorescent PCR amplification reaction solution:

[0053] Dispense 27 μL of the fluorescent PCR amplification reagent to the bottom of the PCR amplification tube in step 1, and add 25 μl of solid paraffin oil with a melting point of 4...

Embodiment 2

[0062] Embodiment 2: Real-time quantitative fluorescent PCR detection to HBVDNA

[0063] 1. Coating of hot start DNA polymerase:

[0064] Take 1 μL of 1.5U / μL hot-start Taq DNA polymerase, and coat the bottom of the PCR amplification tube with 10-15 μL of paraffin oil mixture with a melting point of 50°C-55°C through melting and condensation.

[0065] 2. Preparation of fluorescent PCR amplification reaction solution:

[0066] Each 1 μL concentration is 20 μmol / L upstream and downstream primers and 1 μL 10 μmol / L probe; 20 nmol / μL Tris base; 4 μl 25 mmol / L magnesium chloride; 2 μL 500 mmol / L potassium chloride, 1 μL each 10 mmol / L dNTP and no more than 0.001 wt% The stone green indicator was used to prepare the fluorescent PCR amplification reaction solution.

[0067] 3. Coating of fluorescent PCR amplification reaction solution:

[0068] Dispense 20 μL of the fluorescent PCR amplification reagent to the bottom of the PCR amplification tube in step 1, and add 30 μL of solid ...

Embodiment 3

[0077] Embodiment 3: to the real-time quantitative fluorescent PCR detection repeatability test of HBVDNA

[0078] In addition to using the clinical HBVDNA quantitative detection value of 2.0×10 3 IU / ml, 2.0×10 5 IU / ml and 2.0×10 8 Except the hepatitis B patient's serum of IU / ml, utilize the method identical with embodiment 1 to carry out real-time quantitative fluorescent PCR detection. At the same time, a comparative test was carried out with a commercial kit, and the commercial reagent was the Roche COBAS automatic nucleic acid detection system. Experimental results such as image 3 As shown in Table 1, the results show that the detection repeatability CV% value of the method of the present invention is basically the same as that of commercial Roche COBAS reagents.

[0079] The repeatability contrast of table 1 inventive method and Roche COBAS reagent

[0080]

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Abstract

The invention provides a direct real-time quantitative fluorescent PCR method of a layered packaging tape indicator. Hot-start DNA polymerase and PCR amplification reaction liquid are both packaged in a PCR amplification tube in layers. Nucleic acid lysis liquid and a sample to be detected are added when the method is used so that real-time quantitative fluorescent PCR can be carried out. By means of the method, the virus quantity lower to 20IU / ml can be detected, and the preferential detection range is 30-2*10<9>IU / ml. Detection results are not limited by the professional level of operators and experiment conditions in a laboratory, and the method has the advantages of being easy, convenient and quick to operate.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a direct real-time quantitative fluorescent PCR method with layered packaging and indicators. 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 detected is known, speci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/70C12Q2521/101C12Q2547/107C12Q2561/113C12Q2563/107C12Q2531/113
Inventor 王海滨王棽周其玲冯小霞
Owner BEIJING NAGENE DIAGNOSTIC REAGENT CO LTD
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