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Kit for quantitatively detecting EGFR (Epidermal Growth Factor Receptor) gene mutation and application thereof

A kit and gene technology, applied in the biological field, can solve problems such as insufficient determination of weakly positive patients with mutations, inability to give the proportion of EGFR gene mutations, inability to detect blood samples, etc.

Active Publication Date: 2014-04-09
GENOSABER BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The sensitivity of the sequencing method is about 20% lower, and the operation is complicated, the detection time is longer, and the false negative rate is higher
The ARMS-PCR method can achieve a sensitivity of 1%, which can meet the detection requirements for tumor tissue samples, but for blood samples that are convenient to sample, such as circulating tumor cells in plasma or blood, the tumor DNA content is often lower than 1%, ARMS-PCR Method not adequate for testing blood
In addition, the latest research shows that the proportion of EGFR gene sensitive mutations is closely related to the efficacy of targeted drugs. For patients with a low proportion of EGFR sensitive mutations who take targeted drugs, the effectiveness of the drug will be significantly reduced, similar to the population without mutations
Sequencing and ARMS-PCR methods are both qualitative tests, which cannot give the proportion of EGFR gene mutations in the patient's tumor tissue, which is not enough to judge whether patients with weakly positive mutations can benefit from TKI treatment
[0006] Traditional qualitative detection can only give information about the presence or absence of gene mutations, but cannot determine the sensitive mutation ratio of tumors, and cannot effectively detect blood samples, which limits clinicians' judgment on the efficacy of targeted drugs
In addition, the drug resistance mutations produced in the course of clinical drug use cannot be detected by previous methods due to insufficient sensitivity

Method used

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  • Kit for quantitatively detecting EGFR (Epidermal Growth Factor Receptor) gene mutation and application thereof
  • Kit for quantitatively detecting EGFR (Epidermal Growth Factor Receptor) gene mutation and application thereof
  • Kit for quantitatively detecting EGFR (Epidermal Growth Factor Receptor) gene mutation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1, the selection of amplified fragment length

[0073] Formaldehyde-fixed, paraffin-embedded (FFPE) tumor tissue samples are often used as the first choice for research because they are easier to store for a long time. In addition, studies have shown that the DNA content in plasma of tumor patients is significantly higher than that of blood samples from normal healthy people, and plasma, serum, etc. are easier to obtain in clinical practice and less traumatic to patients, so blood samples are also an ideal source of clinical samples . However, studies have shown that genomic DNA extracted from FFPE samples and plasma samples is fragmented, most of which are less than 200bp. Fragmented DNA brings certain difficulties to subsequent PCR-based detection.

[0074] Taking the detection of exon No. 4 of the EGFR gene as an example, in order to investigate the influence of DNA fragmentation extracted from FFPE samples and plasma samples, the inventors designed 3 se...

Embodiment 2

[0088] Embodiment 2, the selection of amplification primer length

[0089] In this embodiment, the detection of the L858R mutation of the EGFR gene is taken as an example, and the mutant plasmid T-L858R and the wild plasmid T-exon20-21 are used as templates.

[0090] In the amplification detection primers involved in the method, the inventors designed detection sites on the front primers, so the front primers are the key to distinguish mutant and wild-type genes. The present inventors designed amplification detection pre-primers of different lengths, and designed the last base of the pre-primers at the base mutation site of L858R to investigate the ability of pre-primers of different lengths to distinguish mutant genes from wild-type genes. Taking the detection of the L858R mutation of the EGFR gene as an example, the front primer sequences of different lengths are shown in Table 8, the back primer used is R858, and the probe used is P858. respectively for 1.0×10 5 The copie...

Embodiment 3

[0097] Embodiment 3, the effect of competitive Block oligonucleotide

[0098] Templates used in this example: mutant plasmids: T-T790M, T-L858R; wild-type plasmids: T-exon20-21.

[0099] The primers used in this example are F-858-5 and R-858 for L858R, the probe is P-858; the primers F-790 and R-790 for T790M, and the probe P-790.

[0100] For the L858R and T790M mutation sites of the EGFR gene, the inventors designed competitive Block oligonucleotides B-858-1 and B-790-1 (Table 3), respectively. Block oligonucleotides and wild-type The gene is completely complementary and partially complementary to the mutant gene. In the presence of the wild-type gene, the Block oligonucleotide can block the wild-type gene and prevent the amplification of the wild-type gene from causing false positives, thus improving the specificity of detection. sex. Select the mutant plasmid and the wild-type plasmid for comparison, and perform PCR amplification detection, in which the competitive Block...

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Abstract

The invention relates to a high-sensitivity kit for quantitatively detecting EGFR (Epidermal Growth Factor Receptor) gene mutation and application thereof. The application comprises the following steps: respectively designing a forward primer and a backward primer in a sequence with the length of 50-100bp in a specific amplification gene mutation site area aiming at a to-be-detected gene template, designing the mutation sites at corresponding positions of the forward primer, and designing competitive Block oligonucleotides for inhibiting non-mutant amplification nearby the mutation sites. The detection is performed by utilizing the specificity aiming at a probe of the amplification product, and the gene mutation conditions are obtained by detecting the detectable markers on the probe. The invention provides a gene mutation analysis method which is stable in result, high in sensitivity and high in repeatability.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a kit for quantitatively detecting EGFR gene mutation with high sensitivity and its application. Background technique [0002] Lung cancer is one of the most common malignant tumors, as its five-year survival rate is only 15%. There are two main types of lung cancer: non-small cell lung cancer and small cell lung cancer. Non-small cell lung cancer (NSCLC) accounts for 75-80% of lung cancers. Most patients with advanced NSCLC are treated with systemic chemotherapy. Some patients with early NSCLC can be treated with surgery or local radiotherapy and chemotherapy. However, the efficacy of traditional chemotherapy regimens in the treatment of advanced NSCLC is not satisfactory, and the adverse reactions of chemotherapy are large, which are difficult for patients to tolerate. Therefore, new treatments are urgently needed. With the development of biotechnolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q1/6858C12Q2531/113C12Q2537/163C12Q2561/101
Inventor 杨国华何伟李英辉郭志伟
Owner GENOSABER BIOTECH CO LTD
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