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One-step real-time fluorescent RT-PCR reaction buffer and reaction system and PCR method thereof

A technology of RT-PCR and reaction buffer, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., to achieve the effect of ensuring uniformity, high sensitivity, and strong temperature compatibility

Active Publication Date: 2018-12-14
SHENZHEN ZIJIAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a one-step real-time fluorescent RT-PCR reaction buffer solution and its reaction system and real-time fluorescent PCR method that can solve the low efficiency of PCR reaction

Method used

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  • One-step real-time fluorescent RT-PCR reaction buffer and reaction system and PCR method thereof
  • One-step real-time fluorescent RT-PCR reaction buffer and reaction system and PCR method thereof
  • One-step real-time fluorescent RT-PCR reaction buffer and reaction system and PCR method thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0064] Preparation of template: Take 200 μL of H5N6 inactivated HI antigen purchased from Harbin Veterinary Research Institute, and extract viral RNA in an automatic nucleic acid extraction workstation according to the operation manual of the MagMAX nucleic acid extraction kit. Extract the template from 10 0 Serial 10-fold serial dilutions to 10 -10 , as a reaction template.

[0065] Hydrolysis probe H5-P sequence:

[0066] 5'-FAM-GGGTGATTATTTATTATTGTTGTTGTTGGGYTGGT-3'-BHQ1 (SEQ ID NO: 1), with a fluorescent group 5'-FAM (5'-carboxyfluorescein) at its 5' end and a quencher group at its 3' end BHQ1.

[0067]Upstream primer H5-F sequence: 5'-AAATTATTCTGAGGGTGATTATTTATTATTGT-3' (SEQ IDNO: 2)

[0068] Downstream primer H5-R sequence: 5'-ATCGGTAGTAAGCCTGCTAATAGGA-3'(SEQ IDNO:3)

[0069] 2) Reagent

[0070] Tris-HCl buffer (Tris-HCl) at a concentration of 1M, potassium chloride (KCl) at a concentration of 1M, magnesium chloride (MgCl) at a concentration of 1M 2 ), a concentra...

Embodiment 1

[0072] 1) Configure one-step real-time fluorescent RT-PCR reaction buffer; mix Tris-HCl, KCl solution, MgCl 2 solution, (NH 4 ) 2 SO 4 Solution, DMSO, dATP, dGTP, dCTP, dTTP, Glycerol, BSA, Tween 20, NaN 3 Add the solution, Roxreference dye, and deionized water into the reaction container according to the concentration and ratio in the following table, and configure 100mL of one-step real-time fluorescent RT-PCR reaction buffer.

[0073] Table 1: Raw material concentration and proportioning in embodiment 1

[0074] Element

Mother liquor concentration

The amount used to prepare 100ml

2× final concentration of reaction solution

Tris-HCl

1M

2mL

20mM

KCl

1M

1mL

100mM

MgCl 2

1M

300μL

3mM

(NH 4 ) 2 SO 4

3.2M

625μL

20mM

Dimethylsulfoxide (DMSO)

stock solution

5mL

0.07mM

dATP

100mM

2mL

2mM

dGTP

100mM

2mL

2mM

dCTP

10...

Embodiment 2

[0082] 1) Configure one-step real-time fluorescent RT-PCR reaction buffer; mix Tris-HCl, KCl solution, MgCl 2 solution, (NH 4 ) 2 SO 4 Solution, DMSO, dATP, dGTP, dCTP, dTTP, Glycerol, BSA, Tween 20, NaN 3 Add the solution, Roxreference dye, and deionized water into the reaction container according to the concentration and ratio in the following table, and configure 100mL of one-step real-time fluorescent RT-PCR reaction buffer.

[0083] Table 2: Raw material concentration and proportioning in embodiment 2

[0084] Element

[0085] Where (2×) means that the concentration is doubled

[0086] 2) The reaction system formed by the one-step real-time fluorescent RT-PCR reaction buffer prepared by the present invention is as follows:

[0087] 25μL fluorescent RT-PCR reaction system:

[0088] 2×RT-PCR Buffer

[0089] 3) The fluorescent RT-PCR reaction system was placed in a fluorescent quantitative PCR instrument for reaction, and the denaturation reaction w...

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Abstract

The invention relates to a one-step real-time fluorescent RT-PCR reaction buffer and a reaction system and a PCR method thereof. The one-step real-time fluorescent RT-PCR reaction buffer comprises thefollowing raw materials: a trishydroxymethyl aminomethane-hydrochloric acid buffer, potassium chloride, magnesium chloride, ammonium sulfate, dimethyl sulfoxide, deoxyribonucleoside triphosphate, glycerin, bovine serum albumin, Tween 20, sodium trinitride, a Rox reference dye and deionized water. The one-step real-time fluorescent RT-PCR reaction buffer increases NH4<+> and N3<->, improves the PCR reaction efficiency, is applicable to high-efficiency amplification and sensitive detection in a PCR technology, and not only has the advantages of good stability, a good fluorescence effect and high sensitivity. Through use of the one-step real-time fluorescent RT-PCR reaction buffer for preparing the fluorescent RT-PCR reaction system, the real-time fluorescent PCR method has the advantages ofa reliable, accurate and sensitive result, simple operation, time saving, labor saving, reduction in the detection cost, improvement on the detection efficiency and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a one-step real-time fluorescent RT-PCR reaction buffer solution, a reaction system thereof and a PCR method. Background technique [0002] Real-time fluorescent PCR technology is the most widely used real-time online detection technology in recent years. It can be both qualitative and quantitative. It is a new "gold standard" for detection in recent years. [0003] Real-time fluorescent PCR can be divided into probe method and dye method according to different chemical principles. Among them, Taqman real-time fluorescence technology is based on Taqman hydrolysis probe. When the probe is intact, the fluorescent signal emitted by the fluorophore at the 5' end is absorbed by the quencher at the 3' end without emitting light; when the probe is degraded by the Taq enzyme with 5'-3' exonuclease activity, the fluorescence The group and the quencher group are separated, an...

Claims

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Application Information

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IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2527/125C12Q2561/101
Inventor 于洪波
Owner SHENZHEN ZIJIAN BIOTECH
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