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

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 invention relates to the technical field of gene detection, in particular to a method and a 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, and more than 30 mutations in the TK region have been discovered so far. Among them, the T790M and C797S mutations on exon 20 are the most difficult types of gene mutations found in tumor patients. The EGFR gene T790M specifically refers to the mutation of threonine (T) to methionine (G) at the 790th amino acid site 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 cysteine ​​(C) to serine (S) at the 797th amino acid site of exon 20 of EGFR, and the gene level is c.2389T>A or c.2390G >C. When T790M and C797S are mutat...

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.

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