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Primer probe composition, kit and method for detecting KRAS and BRAF gene mutations through 3D (Three-dimensional) digital PCR (Polymerase Chain Reaction)

A technology of primer probes and kits, applied in the field of 3D-PCR detection, can solve the problems of low detection sensitivity and achieve high detection efficiency and intuitive and clear results

Active Publication Date: 2017-12-19
GENE CRAB BIOTECH CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The main purpose of the present invention is to provide a kind of primer probe composition, test kit and method for detecting KRAS and BRAF gene mutations by 3D digital PCR, to solve the problem of low detection sensitivity when detecting intestinal cancer gene mutations in the prior art question

Method used

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  • Primer probe composition, kit and method for detecting KRAS and BRAF gene mutations through 3D (Three-dimensional) digital PCR (Polymerase Chain Reaction)
  • Primer probe composition, kit and method for detecting KRAS and BRAF gene mutations through 3D (Three-dimensional) digital PCR (Polymerase Chain Reaction)
  • Primer probe composition, kit and method for detecting KRAS and BRAF gene mutations through 3D (Three-dimensional) digital PCR (Polymerase Chain Reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1: Detection of the effect of the primers and probes of the kit of this application

[0092] 1. Prepare the primer probe of the application kit

[0093] The primers and probes of KRAS and BRAF shown in Table 1 of this application are selected for use;

[0094] The primers and probes of other synthetic KRAS and BRAF are selected for comparison (referred to as comparison primers and probes), and the sequence information of other synthetic primers and probes is shown in Table 6:

[0095] Table 6:

[0096]

[0097]

[0098] 2. Amplification Reagent Preparation

[0099] Take out the corresponding PCR reaction solution from the kit, melt and mix at room temperature, centrifuge at 2000rpm for 10s, and prepare the PCR premix for each test according to Table 7 below. The preparation system is shown in Table 7 below:

[0100] Table 7:

[0101]

[0102] 3. Add sample

[0103] Load 14.5ul of the reaction system onto the digital PCR chip.

[0104] 4.PCR ampli...

Embodiment 2

[0113] Example 2: The minimum detection limit detection of the kit of this application

[0114] 1. Preparation of minimum detection limit quality control products

[0115] The DNA of KRAS gene G12A, G12C, G12D, G12S, G12V, G13D mutants was mixed with the KRAS-negative cell line genomic DNA in proportion to prepare the minimum quality control products for each mutant.

[0116] The DNA of the BRAF gene V600E mutant and the genomic DNA of the BRAF-negative cell line were mixed in proportion to prepare the minimum quality control product for the BRAF gene mutation.

[0117] A sample with a DNA template amount of 30ng and a mutation ratio of 0.2% was used as a detection limit quality control product in this embodiment. The minimum detection limit quality control products are specifically shown in Table 8:

[0118] Table 8:

[0119]

[0120] 2. The reaction system and operation steps are the same as in Example 1.

[0121] 3. The test was repeated 20 times, and the experimenta...

Embodiment 3

[0125] Example 3: Repeatability detection of the kit of the present application

[0126] 1. Preparation of duplicate quality control products

[0127] Select KRAS gene G12A, G12C, G12D, G12S, G12V, G13D mutant DNA and KRAS-negative cell line genomic DNA in proportion to prepare samples with a mutation ratio of 1% for each mutant, and select BRAF gene V600E mutation Somatic DNA and BRAF-negative cell line genomic DNA were mixed in proportion to prepare a sample with a mutation ratio of 1%, which was used as a repeated quality control product in this embodiment. Specifically as shown in Table 10:

[0128] Table 10:

[0129]

[0130] 2. Reaction system and operating steps are the same as in Example 1

[0131] 3. Repeat the test 10 times, and the experimental results obtained from the test are shown in Table 11-Table 17:

[0132] Table 11:

[0133]

[0134] It can be seen from the experimental results in Table 11 that the positive coincidence rate of the test results is...

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Abstract

The invention discloses a primer probe composition, a kit and a method for detecting KRAS and BRAF gene mutations through 3D (Three-dimensional) digital PCR (Polymerase Chain Reaction). The primer probe composition comprises primers and probes for detecting the six amino acid mutations on a No.2 exon of the KRAS gene, and primers and probes for detecting a V600E amino acid mutation on a No.15 exon of the BRAF gene, wherein the six amino acid mutations are respectively G12A, G12C, G12D, G12V, G12S and G13D. The primer probe composition comprises the seven common mutation sites related to intestinal cancer, and the mutation sites are respectively detected through the primers and the probes of carried different fluorescence signals according to the different mutation sites, so that the multiple mutations can be detected, the detection efficiency is high, and a result is more intuitive and clearer since the primer probe composition is combined with a 3D-PCR detection method for detecting.

Description

technical field [0001] The invention relates to the field of 3D-PCR detection, in particular to a primer probe composition, a kit and a method for detecting KRAS and BRAF gene mutations by 3D digital PCR. Background technique [0002] According to the forecast of the International Cancer Research Center (IARC), the number of cancer patients will increase at a rate of 3% to 5% per year in the future, and research shows that the incidence of cancer in low- and middle-income countries is much higher than that in developed countries. In 2020, there will be 20 million new cases and 12 million deaths worldwide. Therefore, it is urgent for countries to take preventive measures against cancer. Every year, the medical expenditure for cancer patients in my country is as high as 80 billion yuan, accounting for about 20% of the total health expenditure, which is much higher than the medical expenses for other chronic diseases. In recent years, the incidence of cancer in my country has...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/156C12Q2563/159C12Q2563/107
Inventor 蔡畅闫慧婷王海波
Owner GENE CRAB BIOTECH CO
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