Primer, probe and kit for detecting EGFR and/or K-ras genetic mutation

A probe and gene technology, applied in the field of devices for detecting gene mutations based on a digital PCR platform, can solve the problems of complex sample types, limited detection content, and inability to completely block the amplification of wild-type DNA

Active Publication Date: 2016-06-01
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] Patent CN103923973A discloses a method for detecting EGFR exon 19 deletion mutation based on a digital PCR platform. The principle of probe and primer design is: use peptide nucleic acids (peptidenucleic acids, PNA) to lock the wild-type detection site region, and use two A pair of primers and a pair of probes are used to detect the mutant type not locked by PNA and the total DNA template respectively, so as to calculate the mutation rate. This patent requires good specificity of PNA and high cost of reaction reagents
[0018] Patent CN103911427A discloses a method and kit for detecting EGFR20 (T790M) and EGFR21 (L858R) gene point mutations based on a digital PCR platform. detection needs
Use the amplification retardation mutation system (ampl

Method used

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  • Primer, probe and kit for detecting EGFR and/or K-ras genetic mutation
  • Primer, probe and kit for detecting EGFR and/or K-ras genetic mutation
  • Primer, probe and kit for detecting EGFR and/or K-ras genetic mutation

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Effect test

Embodiment 1

[0136] Embodiment 1: Composition and method of use of the kit of the present invention

[0137] The composition of the kit of the present invention is shown in Table 4 above, and its usage method is also described in the summary of the invention, and the detailed steps are shown in the following examples.

Embodiment 2

[0138] Embodiment 2: Simulation sample detection

[0139] (1) Prepare the sample: mix the DNA containing the wild-type EGFR gene with the positive quality control in a certain ratio. The source of DNA can be serum, plasma, peripheral blood, oral mucosa, pleural effusion, body fluid or tissue, etc.

[0140] In this example, samples A, B, and C were obtained by mixing different amounts of the above-mentioned positive plasmids with the same amount of wild-type DNA. Before mixing, the positive plasmids and wild-type DNA were respectively quantified using a digital PCR instrument to obtain a theoretical mutation rate. .

[0141] (2) Melt PCR primers and corresponding TaqMan probes at room temperature.

[0142] (3) Prepare the PCR reaction solution according to the ratio of the kit instructions.

[0143] (4) Add 20 μL of the prepared digital PCR mixture to the 8-lane droplet making plate, and then add 60 μL of droplet making oil to the making plate to prepare PCR micro-reaction d...

Embodiment 3

[0150] Embodiment 3: detection of paraffin-embedded standard substance

[0151] According to the usage method of the above-mentioned embodiment 1, the samples were taken as the standard product QuantitativeMultiplexFFPEReferenceStandard (purchased from Horizon Diagnostics Co., Ltd.). Test results such as Figure 4 shown. The analysis of the detection results is the same as in Example 1, the mutation rate=1-(Number wild type / Number total molecule)=1-(NumberFAM(+)VIC(+) / NumberVIC(+)), the specific results are derived, the wild-type signal The number and the total number of signals are as follows:

[0152]

[0153] The results of quantitative analysis show that the error is generally 0.3%-2.5%, not more than 5%. With the above method, it is possible to detect c.2235-c.2253 deletion mutations on exon 19 of EGFR, and various types of point mutations c.35, c.34, and c.38 on exon 2 of KRAS gene, and the detection limit reaches 2%.

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Abstract

The invention discloses a primer and probe for detecting a human epidermal growth factor receptor (EGFR) gene and/or a K-ras gene, a kit containing the primer and the probe and a device for detecting genetic mutation on the basis of a digital PCR platform.The method for detecting the genetic mutation by means of the primer and the probe comprises the steps that the prime and the probe are provided; DNA of a sample to be detected is extracted; a fluorescent PCR reaction system capable of amplifying a mutant gene sequence is prepared; a target probe and an internal reference probe are utilized to be hybridized with amplified products respectively, and fluorescent signals of corresponding fluorescent groups are detected; existence of the genetic mutation is judged and/or the mutation rate is calculated according to the strength and proportion of the fluorescent signals of the target probe and the internal reference probe.According to the method for detecting the genetic mutation, the needed primers and probes are small in number, the optimization procedure is simple, related mutation of EGFR and/or K-ras gene can be qualitatively or quantitatively detected, and the detection sensitivity is high; a DNA sample with low initial amount can also be detected stably.

Description

technical field [0001] The present invention relates to the field of gene mutation detection, in particular to primers and probes for detecting mutations in the human epidermal growth factor receptor (EGFR) gene and / or K-ras gene, a kit comprising the primers and probes, and a A device for detecting gene mutations based on a digital PCR platform. Background technique [0002] According to the estimates of the International Cancer Research Center (IARC), the number of cancer cases will increase at an average annual rate of 3% to 5% in the future. It is estimated that there will be 20 million new cases and 12 million deaths in the world in 2020. In terms of incidence, the incidence of cancer in low- and middle-income countries is much higher than that in developed countries. Therefore, it is emphasized that countries should take necessary precautionary measures. The annual medical expenses for cancer patients in our country are about 80 billion yuan, accounting for about 20%...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6827C12Q1/6886C12Q2600/156C12Q2563/107C12Q2545/114
Inventor 赵艳敏易吉
Owner SHENZHEN HUADA GENE INST
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