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Fluorescent quantitative PCR reaction system, PCR reaction kit and nucleic acid quantitative detection method

A fluorescent quantitative and reaction system technology, applied in the field of fluorescent quantitative PCR reaction system, can solve the problems of high experimental cost and low detection sensitivity of critical samples, and achieve the effects of high sensitivity, high accuracy of detection results, and improved signal strength

Pending Publication Date: 2022-03-29
WUHAN AIMISEN LIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention provides a fluorescent quantitative PCR reaction system, a kit and a nucleic acid quantitative detection method to improve the problems of high experimental cost and low critical sample detection sensitivity in the existing TaqMan probe fluorescent quantitative PCR

Method used

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  • Fluorescent quantitative PCR reaction system, PCR reaction kit and nucleic acid quantitative detection method
  • Fluorescent quantitative PCR reaction system, PCR reaction kit and nucleic acid quantitative detection method
  • Fluorescent quantitative PCR reaction system, PCR reaction kit and nucleic acid quantitative detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1: Utilize fluorescent quantitative PCR reaction system one to detect ACTB' gene

[0060] This embodiment provides a fluorescent quantitative PCR reaction system one for detecting the ACTB' gene, the nucleotide sequence of the ACTB' gene is shown in SEQ ID NO.5, and the fluorescent quantitative PCR reaction system one includes: Flap endonuclease 1 (FEN1), buffer, dNTP, Taq enzyme, luminescent probe FP shown in SEQ ID NO.1, the first nucleotide sequence shown in SEQ ID NO.2, SEQ ID NO. The upstream probe P1 shown in 3, the downstream probe P2 shown in SEQ ID NO.4, the ACTB' gene template shown in SEQ ID NO.5, and the ACTB' gene template shown in SEQ ID NO.6 are used to amplify the ACTB' gene template The forward primer F1, wherein, the reverse primer R1 used to amplify the ACTB' gene template (as shown in SEQ ID NO.3, is the same as the upstream probe P1).

[0061] Wherein, the ACTB' gene template is a nucleotide sequence obtained by C-T conversion after comp...

Embodiment 2

[0076] Embodiment 2: Fluorescent quantitative PCR reaction system one detects the ACTB' gene in the mixed DNA solution sample

[0077] In this embodiment, the ACTB' gene template and the ACTB" gene template are configured as a mixed DNA solution to serve as the amplification template of the fluorescent quantitative PCR reaction system 1, wherein the ACTB" gene template is unmethylated Nucleotide sequence obtained by C-T transformation of ACTB gene template. In the mixed DNA solution, the total concentration of the ACTB' gene template and the ACTB "gene template is 10 4 copies / μL, wherein the copy number of the ACTB' gene template accounts for 0.05% of the total copy number of the ACTB' gene template and the ACTB" gene template.

[0078] The template in Table 2 was replaced with the mixed DNA solution, and 5 parallel samples were set. In addition, five NTC control groups were set up, and the components and configuration of the NTC control group were referred to the NTC contro...

experiment example

[0083] Experimental example: Comparing the amplification performance and detection sensitivity of the fluorescent quantitative PCR reaction system of Example 1 and Comparative Example 1

[0084] The ACTB' gene was used as a template for fluorescent quantitative PCR to compare the amplification performance and detection sensitivity of the fluorescent quantitative PCR reaction systems provided in Example 1 and Comparative Example 1.

[0085] Prepare a concentration of 10 4 Copies / μL of the ACTB' gene template, the fluorescent quantitative PCR reaction system 1 of Example 1 was used to amplify the ACTB' gene template respectively, and 2 parallel samples were taken, and 2 NTC control groups were set. Similarly, the fluorescent quantitative PCR reaction system 2 of Comparative Example 1 was used to amplify the ACTB' gene template, and 2 parallel samples were also taken, and 2 NTC control groups were set. The amplification performance and detection sensitivity of the fluorescent qu...

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Abstract

The invention discloses a fluorescent quantitative PCR reaction system, a kit and a nucleic acid quantitative detection method, the fluorescent quantitative PCR reaction system utilizes endonuclease to specifically generate a Flap sequence, the Flap sequence can be used as a primer for amplification of a nucleotide sequence, and the nucleotide sequence has a plurality of luminescent probe binding segments, so that the fluorescent quantitative PCR reaction system can be used for detecting the fluorescence of the fluorescent quantitative PCR reaction system. When the nucleotide sequence is amplified, the luminescent probe is cleaved by DNA polymerase to generate a fluorescence signal. As the nucleotide sequence is combined with a plurality of luminous probes, and the number of Flap sequences is related to the number of target genes to be subjected to fluorescent quantitative PCR detection, the fluorescent quantitative PCR reaction system can realize specific amplification of the target genes, and the signal intensity is greatly improved, so that the detection sensitivity of a critical sample is improved.

Description

technical field [0001] The invention relates to the technical field of fluorescent quantitative PCR, in particular to a fluorescent quantitative PCR reaction system, a PCR reaction kit and a nucleic acid quantitative detection method. Background technique [0002] Real-time fluorescent quantitative PCR (Quantitative Real-time PCR) technology refers to: add fluorescent groups to the PCR reaction system, use the accumulation of fluorescent signals to achieve the purpose of real-time monitoring of the entire PCR process, and combine the corresponding software for quantitative analysis of the product , to obtain the initial concentration of the starting template. Real-time fluorescence quantitative PCR technology greatly simplifies the process of quantitative detection, and truly realizes absolute quantification. It can be applied to disease diagnosis, food safety detection, scientific research and other fields, and has broad application prospects. [0003] The real-time fluore...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2521/301C12Q2531/113C12Q2561/101C12Q2563/107Y02A50/30
Inventor 张良禄李婷婷董兰兰
Owner WUHAN AIMISEN LIFE TECH CO LTD
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