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Double-probe composition for fluorescent quantitative PCR, kit, application and method thereof

A fluorescent quantitative and compositional technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., to achieve the effect of improving signal-to-noise ratio, low signal, and high sensitivity

Active Publication Date: 2020-11-13
湖南圣维尔医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in practical tests, two complementary probes will produce non-specific fluorescent signals under the action of Taq enzyme 5' exonuclease activity

Method used

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  • Double-probe composition for fluorescent quantitative PCR, kit, application and method thereof
  • Double-probe composition for fluorescent quantitative PCR, kit, application and method thereof
  • Double-probe composition for fluorescent quantitative PCR, kit, application and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1, the scheme design composition of the present invention detects EBV virus

[0073] Aiming at the EBV virus sequence, a composition designed according to the scheme of the present invention was prepared, and its specific sequence is shown in Table 1 below (the wavy underline represents area B, the same below).

[0074]

[0075] Use the above primers and probes to construct a 50 μL reaction system, including 400 nM primers, 200 nM probes, 10 mM Tris-HCl, 50 mM KCl, 3.5 mM MgCl 2 , 0.01% (w / v) gelatin, 0.02% (w / v) Tween 20, 1% (w / v) glycerol, 1.5 mM dATP, 1.5 mM dTTP, 1.5 mM dCTP, 1.5 mM dGTP, 10 U Taq DNA aggregate enzyme.

[0076] Thermal cycling was performed using the following reaction program: 95°C for 1 minute heat activation; 94°C for 15 seconds, 60°C for 30 seconds and fluorescence acquisition (45 cycles). 3 replicates were detected in each group, and the detection results were as follows Figure 3A and 3B shown.

Embodiment 2

[0092] Embodiment 2, composition of the present invention detects human B2M gene mRNA

[0093] Aiming at the B2M gene sequence, a composition designed according to the scheme of the present invention was prepared, and its specific sequence is shown in Table 3 below.

[0094]

[0095] Use the above primers and probes to construct a 50 μL reaction system, including 400 nM primers, 200 nM probes, 10 mM Tris-HCl, 50 mM KCl, 3.5 mM MgCl 2 , 0.01% (w / v) gelatin, 0.02% (w / v) Tween 20, 1% (w / v) glycerol, 1.5 mM dATP, 1.5 mM dTTP, 1.5 mM dCTP, 1.5 mM dGTP, 10 U Taq DNA aggregate Enzyme, 100U mMLV reverse transcriptase.

[0096] The following reaction program was used for thermal cycling: reverse transcription at 50°C for 30 minutes; heat activation at 95°C for 1 minute; 94°C for 15 seconds, 60°C for 30 seconds and fluorescence collection (cycle 45 times). Each group was tested with 4 replicates. The test results are as follows: Figure 7A and 7B shown.

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Abstract

The invention relates to the field of molecular biological detection, in particular to a detection composition for fluorescent quantitative PCR. The invention discloses a double-probe composition forfluorescent quantitative PCR, which comprises a first probe and a second probe, the first probe and the second probe are respectively used for detecting different chains of a template to-be-detected,and the probes can be sequentially divided into three regions from 5'end to 3 'end: a region A, a region B and a region C. The composition improves the signal-to-noise ratio of a detection result andhas higher sensitivity.

Description

technical field [0001] The invention relates to the field of molecular biology detection, in particular, the invention relates to a detection composition for fluorescent quantitative PCR. Background technique [0002] PCR is the English abbreviation of polymerase chain reaction (Polymerase Chain Reaction), which is a well-known technology. PCR technology has been widely used in biology, molecular biology, biochemistry, biomedicine and other disciplines, especially in molecular diagnosis of clinical medicine. In the PCR reaction, the basic reaction components include: double-stranded DNA template (DNA fragment to be amplified, also called target or target gene), DNA polymerase, 4 kinds of single nucleotides (ATP, CTP, GTP, TTP) , two DNA primers located at both ends of the template, and a reaction buffer. These components are mixed in an appropriate ratio, and under certain temperature cycle conditions, DNA will be amplified and replicated at an exponentially increasing spe...

Claims

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Application Information

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IPC IPC(8): C12Q1/6876C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6876C12Q2531/113C12Q2563/107
Inventor 颜进刘佳邓中平戴立忠
Owner 湖南圣维尔医学检验所有限公司
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