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Nucleic acid quantitative detection reagent kit of multiple digital PCR based on dual-fluorescence probe

A detection kit and nucleic acid quantification technology, applied in the field of digital PCR, can solve problems such as different fluorophores, and achieve the effects of increasing detection throughput, wide application and shortening detection time.

Pending Publication Date: 2020-06-02
TARGETINGONE TECH (BEIJING) CORP +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, most of the digital PCR systems are dual fluorescent channel detection systems, which can detect two kinds of fluorescent signals emitted in the reaction unit at the same time, so at most two targets can be quantified at the same time, and the detection probes corresponding to each target are labeled with different fluorescent signals. group

Method used

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  • Nucleic acid quantitative detection reagent kit of multiple digital PCR based on dual-fluorescence probe
  • Nucleic acid quantitative detection reagent kit of multiple digital PCR based on dual-fluorescence probe
  • Nucleic acid quantitative detection reagent kit of multiple digital PCR based on dual-fluorescence probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Quantitative detection of novel coronavirus dual genes based on dual fluorescent probes

[0022] 1. Plasmid reference products

[0023] In this experiment, the feasibility of the method was verified by using plasmid reference products, including orf1ab gene plasmids and N gene plasmids. At the same time, normal human genome ABL1 was used as a control to prepare two sets of viral gene plasmid reference products with different copy numbers (10 4 and 10 2 orders of magnitude copy number).

[0024] 2. Primers and probes

[0025] The probes used in this experiment were labeled with FAM or VIC fluorophore at the 5' end and MGB at the 3' end. The specific primers and probes used are shown in Table 1:

[0026] Table 1. Sequences of novel coronavirus-specific primers and probes

[0027]

[0028] 3. Preparation of PCR reaction system

[0029] Prepare the PCR reaction system according to Table 2:

[0030] Table 2. Digital PCR reaction system

[0031]

[003...

Embodiment 2

[0043] Example 2 Quantification of HER2 gene copy number based on dual fluorescent probes with different concentration ratios

[0044] 1. Template DNA

[0045] This experiment uses the human normal genome as a template.

[0046] 2. Primers and probes

[0047] The probes used in this experiment were labeled with FAM or VIC fluorophore at the 5' end and MGB at the 3' end. The specific primers and probes used are shown in Table 4:

[0048] Table 4. Specific Primer and Probe Sequences

[0049]

[0050] 3. Preparation of PCR reaction system

[0051] Prepare the PCR reaction system according to Table 5:

[0052] Table 5. Digital PCR reaction system

[0053]

[0054]

[0055] Among them, four groups of experimental groups with different concentration ratios were set up, EFTUD2 probe-FAM: EFTUD2 probe-VIC were 1:2, 1:3, 1:4, 1:6 respectively, and the corresponding probe volumes and final concentrations were as follows: Table 6 shows:

[0056] Table 6. Dual probe volumes...

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Abstract

The invention provides a nucleic acid quantitative detection reagent kit of multiple digital PCR based on a dual-fluorescence probe. The reagent kit comprises a first probe for detecting a first target, a second probe for detecting a second target and a third probe for detecting a third target, wherein the first probe is labelled with a first fluorophore, the second probe is labelled with a secondfluorophore, one part of the third probe is labelled with the first fluorophore, the other part of the third probe is labelled with the second fluorophore, and the first fluorophore and the second fluorophore are two different fluorophores; and the nucleic acid quantitative detection reagent kit comprises a two-dimensional result figure obtained based on multiple digital PCR of three targets to realize respective quantitation of the three targets to be tested. Based on a conventional dual-fluorescence passage detecting system, development of a dual-fluorescence probe multiple digital PCR method is great in significance, so that a digital PCR technique conforms to clinical requirements, and is wide to apply.

Description

technical field [0001] The invention relates to the technical field of digital PCR, in particular to a nucleic acid quantitative detection kit based on double fluorescent probe multiplex digital PCR. Background technique [0002] Digital PCR is a nucleic acid quantitative technology. Its basic principle is to dilute the nucleic acid and evenly distribute it to countless independent reaction units, perform PCR amplification on the PCR system in the reaction unit, and then pass the PCR terminal signal "with or without" To achieve absolute quantification of single molecules independent of standard curves. Digital PCR has the advantages of high sensitivity, absolute quantification, and strong anti-interference, so it is more and more widely used in many fields such as medical care and biological research. [0003] At present, most of the digital PCR systems are dual fluorescent channel detection systems, which can detect two kinds of fluorescent signals emitted in the reaction ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2600/16Y02A50/30
Inventor 陈芊如王芳王楠祝令香郭永杨文军
Owner TARGETINGONE TECH (BEIJING) CORP
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