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Specific primer, probe and kit for detecting EGFR gene L858R mutation based on digital PCR technology

A technical detection and kit technology, which is applied in the field of probes and kits for detecting EGFR gene L858R mutation, and specific primers, can solve the problems of restricting the application of peripheral blood samples, and achieve guidance for treatment strategies and prognosis analysis, with high specificity , the effect of high detection sensitivity

Pending Publication Date: 2019-07-30
DELUTONG SHIJIAZHUANG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for some patients, the proportion of circulating tumor DNA (ctDNA) in blood to free blood DNA (cfDNA) is only 0.01%, which limits the application of peripheral blood samples in tumor molecular detection, so it is necessary to develop a highly sensitive Detection method to be applied to the gene mutation detection

Method used

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  • Specific primer, probe and kit for detecting EGFR gene L858R mutation based on digital PCR technology
  • Specific primer, probe and kit for detecting EGFR gene L858R mutation based on digital PCR technology
  • Specific primer, probe and kit for detecting EGFR gene L858R mutation based on digital PCR technology

Examples

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Embodiment 1

[0055] This embodiment relates to the amplification efficiency and amplification specificity verification of a specific primer for detecting EGFR gene L858R mutation based on digital PCR technology.

[0056] Four upstream primers and three downstream primers were combined into 12 sets of primer pairs, and the nucleotide sequences of each primer pair are shown in Table 1.

[0057] Table 1 Nucleotide sequences of primer pairs of Examples 1.1-1.12

[0058]

[0059]

[0060] In this example, real-time quantitative PCR is used to verify the amplification efficiency and specificity of the 12 sets of primer pairs in Examples 1.1 to 1.12.

[0061] 1. Amplification efficiency verification of 12 sets of primer pairs in Examples 1.1 to 1.12

[0062] (1) Extract genomic DNA from the sample to be tested and use it as a template.

[0063] (2) Preparation of PCR reaction system: The reaction system is 20 μL, including:

[0064] 2 μL (450 nM) of any set of primer pairs in Examples 1....

Embodiment 2

[0072] This embodiment relates to the performance verification of specific primers and probes for detecting EGFR gene L858R mutation based on digital PCR technology.

Embodiment 21

[0073] Example 2.1 Detection accuracy

[0074] (1) The L858 locus wild-type plasmid standard and L858R mutant plasmid standard of the full sequence of EGFR gene exon21 were synthesized respectively; according to the mass ratio, the mutation ratios were 50%, 10%, 5%, 1%, The 0.5% and 0.1% samples are recorded as Examples 2.11, 2.12, 2.13, 2.14, 2.15 and 2.16, respectively.

[0075] (2) Prepare a digital PCR reaction system: the reaction system is 20 μL, including:

[0076] 1.8 μL (450 nM) of primer pairs in Example 1.3,

[0077] Wild-type probe 0.5 μL (250 nM),

[0078] Mutant probe 0.5 μL (250 nM)

[0079] Template DNA 2μL (~1000copies / μL),

[0080] PCR 2×supermix 10 μL and

[0081] ddH 2 O 5.2 μL.

[0082] (3) Digital PCR amplification reaction procedure: pre-denaturation at 95°C for 10 min, for 1 cycle; denaturation at 94°C for 30 seconds, annealing and extension at 58°C for 1 min, for 40 cycles; 98°C for 10 min, for 1 cycle.

[0083] Figure 3 to Figure 8 The two-d...

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Abstract

The invention provides a specific primer, probe and kit for detecting an EGFR gene L858R mutation based on a digital PCR technology, wherein the specific primer used for detecting the EGFR gene L858Rmutation based on the digital PCR technology comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.1-4, and the nucleotide sequenceof the downstream primer is shown as SEQ ID No.5-7. The specific primer of the EGFR gene L858R mutation has good amplification specificity and amplification efficiency; meanwhile, the kit for detecting the EGFR gene L858R mutation based on the digital PCR technology can carry out absolute quantification on the L858R mutation frequency, and has high specificity and higher detection sensitivity. The method is not only suitable for detecting DNA derived from pathological tissues or sections, but also suitable for detecting the EGFR gene L858R mutation in blood free DNA.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a specific primer for detecting L858R mutation of EGFR gene based on digital PCR technology. At the same time, the invention also relates to a probe and a kit for detecting EGFR gene L858R mutation based on digital PCR technology. Background technique [0002] Lung cancer is one of the most common malignant tumors in the world, and its incidence is still increasing year by year, and it has become the most lethal cancer among malignant tumors. Among them, Non-Small Cell Lung Cancer (NSCLC) accounts for more than 80% of the total number of lung cancers, and because of the slow growth and division of cancer cells and the relatively late spread and metastasis, about 70% of patients diagnosed with non-small cell lung cancer are diagnosed with non-small cell lung cancer. For the advanced stage, it is difficult to achieve effective treatment through surgery, radioth...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156C12Q2600/118
Inventor 许宝芝秦颖
Owner DELUTONG SHIJIAZHUANG BIOTECH CO LTD
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