Liquid phase chip for detecting EGFR (epidermal growth factor receptor) gene mutation

Abstract

The invention belongs to biochip technical field and discloses a liquid chip, for detecting EGFR gene mutations, comprising: (i) allele-specific primer extension (ASPE) primer pair(s) designed for both wild-type and mutant EGFR gene, where each ASPE primer comprises a tag sequence at 5' end and a specific primer part at 3' end, which is aiming at the mutant site of target gene; (ii) microspheres coated separately with a specific anti-tag sequence and encoded with different colors, where the said anti-tag sequence matches correspondingly with the said tag sequence; and (iii) amplification primers for amplifying target sequence(s) with mutant site(s) of Exon 19, Exon 20 and / or Exon 21. The detection method provided by the present invention has a coincidence rate up to 100% with sequencing methods. The liquid chip for detecting EGFR gene mutations prepared is excellent in signal to noise ratio, and cross reaction between designed probes and anti-tag sequences is basically free.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a liquid phase chip for detecting EGFR gene mutation. technical background [0002] Epidermal growth factor receptor (EGFR) is a multifunctional glycoprotein widely distributed in the cell membranes of various human tissues. It has tyrosine kinase activity and is one of the four members of the ErbB family, so it is also known as HER1 or ErbB1. After being activated by ligands, EGFR initiates intracellular signal transduction, and through the cascade reaction of adapter proteins and enzymes in the cytoplasm, it regulates the transcription of transcription factor-activated genes and guides cell migration, adhesion, proliferation, differentiation and apoptosis. Studies have shown that there is high or abnormal expression of EGFR in many solid tumors, which provides a theoretical basis and experimental basis for EGFR-targeted tumor therapy and ...

AI Technical Summary

Problems solved by technology

The PCR-sequencing method has the advantage of being able to determine the range and type of mutations, and is currently a widely used detection method, but the sensitivity of the sequencing method is only 20%-25%, which is far from meeting the needs of practical applications, especially for heterogeneity of tumor somatic mutations, low sensitivity will lead to a large number of missed detections

At the same time, the detection operation of the sequencing method is complicated and the timeliness is poor. For the detection of practical applications that require high timeliness and high sensitivity, the limitations of the sequencing method have long been highlighted

Real-time fluorescence quantitative PCR detection technology has high detection efficiency and strong timeliness, but its high false positive rate is also criticized by practical applications.

Based on the problems of the above-mentioned detection technology, the detection method of "Detection Probe for EGFR Gene Mutation Site, Liquid Chip and Detection Method" (Application No.: 200810027613.9) successfully developed by the applicant in the early stage is simple to operate and enriched by enzyme digestion. The interference caused by a large number of wild-type sequences has good specificity, high sensitivity, and an accuracy of more than 99%. However, this method involves two rounds of PCR operations, which is easy to cause pollution, and the probes in the hybridization detection step are relatively close (only the mutant position different points), it is not convenient for parallel detection of multiple gene mutation sites

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