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Quick detecting gene mutation correlative to curative effect of non small-cell carcinoma of the lung

A technology for non-small cell lung cancer and curative effect, applied in the fields of biotechnology and medicine, can solve the problems of long detection cycle, difficulty in meeting the requirements of clinical drug timeliness, and detection failure.

Active Publication Date: 2010-07-14
庶安永康(厦门)健康产业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing problems: (1) The detection period is long, and it takes 15-21 hours continuously from receiving the sample to be tested to successfully completing the test and issuing a report
If the test is carried out during normal working hours, it will actually take at least 3 working days to issue the test result report. Therefore, it is difficult to meet the timeliness requirements for guiding clinical medication
(2) The detection process is complicated, and the technical requirements for the detection personnel are high. If the detection personnel lack rich DNA sequencing knowledge and experience, it is easy to cause detection failure
(3) The instruments and equipment used in the sequencing method are expensive, and it is difficult to apply them widely in clinical practice.

Method used

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  • Quick detecting gene mutation correlative to curative effect of non small-cell carcinoma of the lung
  • Quick detecting gene mutation correlative to curative effect of non small-cell carcinoma of the lung
  • Quick detecting gene mutation correlative to curative effect of non small-cell carcinoma of the lung

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1 Determination of the EGFR mutation site to be detected

[0105]According to relevant research reports on EGFR mutations in NSCLC patients (Mu XL et al., Gefitinib-sensitive mutations of the epidermal growth factor receptor tyrosine kinase domain in Chinese Patients with non small cell lung cancer. Clin Cancer Res 2005; 11(12): 4289-4294; ZhangXT et al., The EGFR mutation and its correlation with response of gefitinib in previously treated Chinese patients with advanced non-small-cell lung cancer. Annals of Oncology Advance Access, 2005. doi: 10.1093 / annonc / mdi340; Qin BM et al., Identification of EGFRkinase domain Among lung cancer patients in China: implications for targeted cancer therapy. Cell Research, 2005.15(3): 212-217; Huang SF et al., High Frequency of Epidermal Growth Factor Receptor Mutations with Complex Patterns in Non-Small Cell Lung Cancers Related to Gefitinib Responsive in Taiwan .Clinical Cancer Research, 2004.10 (12): 8195-8203) and the inve...

Embodiment 2

[0111] Embodiment 2 detection method design and the synthesis of primer and probe

[0112] For the selected 7 EGFR mutation sites, use Primer Express V2.0 software (product of Applied Systems, USA) to design 3 pairs of PCR primers (respectively amplify the mutation regions in the three exons) and 10 TaqMan Probes (combined into 7 pairs, wherein the wild-type detection probe in the exon 19 mutation region is shared by the 5 deletion mutations). The 5' end of the wild-type allele-specific probe was labeled as FAM, the 5' end of the mutant allele-specific probe was labeled as VIC, and the 3' end quencher of all probes was TAMRA. All primers and probes were synthesized by Shanghai Jikang Biotechnology Co., Ltd.

[0113] See Table 2 for details.

Embodiment 3

[0114] Example 3 Construction and preparation of various genotype standards for mutation sites to be detected

[0115] Using the normal human genomic DNA sequence as a template, design a pair of outer primers and a pair of mutation primers (introducing point mutations or deletion mutations) that can amplify the target exon region for each mutation site or deletion site to be tested. The method of overlapping PCR (over-lap PCR) was used for PCR amplification.

[0116] The specific mutation primers and related information are shown in Table 3.

[0117] After the final amplified product with the correct sequence verified by sequencing was ligated with the pMD18-T vector (purchased from TaKaRa Company) (that is, the sequence was ligated into the multiple cloning site of the pMD18-T vector by conventional means), it was transformed into DH5α Competent cells, after incubation, pick monoclonal colonies and continue to culture, amplify, extract plasmids, and perform sequencing verifi...

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Abstract

This invention discloses a test kit for detecting EGFR gene mutation associated with curative effects of non-small cell lung cancer. This invention also discloses a method for rapidly detecting EGFR gene mutation associated with curative effects of non-small cell lung cancer. The method is rapid and precise, and also has such advantages as easy operation, low requirement for apparatus, and high detection rate (higher than 90%).

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, and more specifically, the invention relates to the rapid detection of EGFR gene mutations related to the curative effect of non-small cell lung cancer. Background technique [0002] Lung cancer is the most common primary malignant tumor of the lung. In recent decades, the morbidity and mortality of lung cancer have risen rapidly in countries all over the world, especially in industrialized countries. Male lung cancer accounts for the first cause of death from various cancers, while female Second only to breast cancer. According to the traditional histopathological classification, lung cancer can be roughly divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Among them, NSCLC accounts for about two-thirds of the total number of lung cancer patients. So far, the method of treating NSCLC is still based on surgery, but due to the difficulties in early diagn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 朱冠山赵翊均曾骥孟
Owner 庶安永康(厦门)健康产业有限公司
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