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Method and probe for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S

A technology of EGFR-C797S and EGFR-T790M, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problems of long time consumption, drug resistance, high detection sensitivity, etc., and achieve specificity Effects of High, Reduced Sensitivity, and High Sensitivity

Active Publication Date: 2020-09-15
ZHUHAI LIVZON CYNVENIO DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The third-generation EGFR-TKI Tagrisso / Tagrisso (also known as Osimertinib / Osimertinib, AZD9291) is an irreversible selective TKI, a new generation of targeted drugs that can effectively deal with the T790M mutation, and is aimed at overcoming the secondary drug resistance of T790M Here comes hope, but eventually drug resistance
[0008] Under the existing technology, next-generation gene sequencing technology (NGS) is the only high-sensitivity and high-specificity detection method that can directly provide sequence information to clearly distinguish the cis-trans mutation configuration of C797S and T790M, while other PCR technologies , such as ARMS PCR or digital PCR can only infer the mutation configuration indirectly through the fluorescent signal detected by a single point, and cannot provide sequence information, and cannot make an accurate and uncontroversial direct judgment on the configuration
However, although NGS technology has the characteristics of large throughput, high accuracy and rich information, it is difficult to meet the detection needs of all patients due to the time-consuming, complicated experimental equipment and process, and high cost.
Therefore, there is still a lack of a method and product that has high detection sensitivity, short detection cycle, low cost, and can directly obtain T790M and C797S mutation sequence information to accurately determine the cis-trans mutation configuration.

Method used

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  • Method and probe for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S
  • Method and probe for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S
  • Method and probe for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] This embodiment provides a kit for detecting cis-trans mutation types of EGFR-T790M and EGFR-C797S, the composition of which is shown in the following table:

[0140] Table 2

[0141]

[0142]

[0143] (2) The experimental procedure of this kit is as follows

[0144] table 3

[0145]

[0146] (1) Comparison of blocking performance of Blocker 1:

[0147] Use plasmid DNA with different mutation information as a template, and the plasmid DNA information is shown in the table below:

[0148] Table 4

[0149] name mutation type Plasmid 001 wild type (WT) Plasmid 002 T790M C>T Plasmid 003 C797S T>A Plasmid 004 C797S G>C Plasmid 005 T790M C>T&C797S T>A Plasmid 006 T790M C>T&C797S G>C

[0150] The system was prepared according to the formula in the table below, and the PCR program was shown in Table 3.

[0151] table 5

[0152]

[0153]

[0154] The result is as Figure 3-8 As shown, Blocker 1 block...

Embodiment 2

[0196] This embodiment provides a kit for detecting cis-trans mutation types of EGFR-T790M and EGFR-C797S. The difference from the embodiment is that the following probes are used:

[0197] Table 11

[0198]

[0199] Use plasmid DNA with different mutation information as a template, and the plasmid DNA information is shown in the table below:

[0200] Table 12

[0201] name mutation type Plasmid 001 wild type (WT) Plasmid 002 T790M C>T Plasmid 003 C797S T>A Plasmid 004 C797S G>C

[0202] The system was prepared according to the formula in the table below, and the PCR program was shown in Table 3.

[0203] Table 13

[0204]

[0205] The product is sent for inspection and next-generation sequencing: the results are as follows

[0206] Plasmid 001 (wild type) Blocker 3 product sequencing results are as follows Figure 55 Shown; Blocker 4 product sequencing results are as follows Figure 56 Shown: Blocker 3 product has T790M C>...

Embodiment 3

[0212] This embodiment provides a kit for detecting cis-trans mutation types of EGFR-T790M and EGFR-C797S. The difference from the embodiment is that the following probes are used:

[0213] Table 14

[0214]

[0215] The system was prepared according to the formula in the following table, the DNA samples are shown in Table 4, and the PCR program is shown in Table 3.

[0216] Table 15

[0217]

[0218] Blocker5 amplification results are as follows Figures 63-68 Shown: Blocker 5 blocks wild-type and single-point C797S mutation DNA amplification, does not block or less blocks single-point T790M mutation and cis mutation T790M C>T&C797S T>A or G>C DNA amplification.

[0219] Blocker6 amplification results are as follows Figures 69-74 As shown, Blocker 6 blocks wild-type and single-point T790M mutant DNA amplification, does not block or less blocks single-point C797S T>A or G>C and cis-mutant T790M C>T&C797S T>A or G>C DNA amplification.

[0220] Comparison of blocking...

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Abstract

The invention provides a method and probe for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S, and relates to the technical field of gene detection. The method comprises the steps of respectively using a probe system selectively restraining T790 wild types, and a probe system selectively restraining C797 wild types for PCR amplification on samples, then sequencing amplification products, and according to the sequencing result, determining the mutation configurations of T790 and C797 in the samples. By the method, the cis-trans mutation configurations of the T790M and the C797S can be effectively distinguished, the specificity is quite high, and sequence information of the samples can be directly given; besides, the method and the probe also have the advantage of being high in sensitivity; two-stage type PCR amplification of deviation amplification and non-deviation amplification is adopted, and then low-cost Sanger sequencing is adopted, so that mutation as lowas 0.1% can be detected; and NGS sequencing can guarantee high sensitivity, and besides, the cost can be reduced through reducing sequencing depth.

Description

Technical field [0001] The present invention relates to the field of gene detection technology, and in particular, to a method and probe for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S. Background technique [0002] EGFR mutations mainly occur in the first four exons (18-21) of the intracellular tyrosine kinase (TK) region. Currently, there are more than 30 mutations in the TK region discovered. Among them, T790M and C797S mutations on exon 20 are the most troublesome types of genetic mutations found in tumor patients. EGFR gene T790M specifically refers to the mutation from threonine (T) to methionine (G) at the 790th amino acid position of exon 20 of the epidermal growth factor receptor (EGFR), and the gene level is expressed as c .2369C>T; EGFR gene C797S specifically refers to the mutation of the 797th amino acid position of EGFR exon 20 from cysteine ​​(C) to serine (S). The gene level is c.2389T>A or c.2390G. >C. When T790M and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6806C12N15/11
CPCC12Q1/6886C12Q1/6806C12Q2600/156C12Q2600/106C12Q2600/118C12Q2531/113C12Q2537/163C12Q2535/101
Inventor 唐东江赵计昌黄雅菁
Owner ZHUHAI LIVZON CYNVENIO DIAGNOSTICS
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