Primers, probes, detection system and kit for one time detection of lung cancer multiple genes

A one-time, lung cancer technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of difficult material collection, small sample size, and long time-consuming for advanced patients

Active Publication Date: 2015-08-05
上海厦维医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at present, most of them can only detect one mutation or a mutation of a gene at a time, the detection throughput is low, and they can only be screened one by one, which takes a long time
At the same time, due to the difficult

Method used

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  • Primers, probes, detection system and kit for one time detection of lung cancer multiple genes
  • Primers, probes, detection system and kit for one time detection of lung cancer multiple genes
  • Primers, probes, detection system and kit for one time detection of lung cancer multiple genes

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0088] Example 1

[0089] Human EGFR, KRAS, BRA, ALK and ROS1 published according to Cosmic data are wild-type gene sequences. Based on the driving mutation sites and fusion sites of EGFR, KRAS, BRA, ALK and ROS1, specific primers and And new probes (see Table 1).

[0090] This example uses EML4exon13; ALK exon20 (ALK fusion gene), SLC34A2exon14del; ROS1exon32 (ROS1 fusion gene), TPM3exon8; ROS1exon35 (ROS1 fusion gene), E746_A750del (2235_2249del15) (EGFR gene mutation), L858R (EGFR gene mutation), T790M (EGFR gene mutation), G719A (EGFR gene mutation), G12S (KRAS gene mutation), G12C (KRAS gene mutation), G13D (KRAS gene mutation), V600E (BRAF gene mutation) as examples to illustrate the fluorescent PCR of the present invention Detection of the above-mentioned lung cancer genes.

[0091] The experiment uses the armored RNA containing the above-mentioned fusion gene and each mutant plasmid template, and the fluorescent PCR detection includes the following steps:

[0092] (1) Plasmi...

Example Embodiment

[0123] Example 2

[0124] Take 65 cases of clinical lung cancer paraffin-embedded tissue samples sent to our company from December 2013 to April 2014 for one-time multiple lung cancer gene mutation detection.

[0125] 1. Extraction of DNA and RNA from test samples: DNA and RNA can be extracted using the FFPE sample DNA / RNA co-separation kit of Xiamen Aide Biomedical Technology Co., Ltd. (Minxia Food and Drug Administration (quan) No. 1400070) ), Cat No. ADx-FF03 or QIAGEN Paraffin Tissue DNA Extraction Kit, Cat NO.56404; follow the instructions of the extraction kit. After the extraction of DNA and RNA, immediately use an ultraviolet spectrophotometer to detect the concentration and purity of DNA and RNA. DNA and RNA OD260 / OD280 should be between 1.8~2.1; RNA concentration should be between 50~500ng / μL. The DNA and RNA extracted above are used as the template for fluorescent PCR amplification of lung cancer genes.

[0126] 2. Perform PCR amplification on each of the DNA and RNA me...

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Abstract

The present invention discloses primers, probes, a detection system and a kit for one time detection of lung cancer multiple genes, wherein the primers, the probes, and the distribution way for detecting 18 EGFR gene mutations, 7 KRAS gene mutations, BRAF V600E gene mutation, 5 fusion genes of ALK5, and 9 fusion genes of ROS1 are provided. According to the present invention, the detection kit adopts the 12 linking PCR reaction strip design, each 12 linking PCR strip detects multiple genes of a sample, the corresponding detection reagents and the internal control reagents are filled in the pipes 1-11 of the 12 linking PCR strip, the mutation is indicated by the FAM signal, and the internal control is indicated by the HEX (or VIC) signal; the pipe 12 is adopted as the DNA extraction quality external control detection pipe and is indicated by the FAM; and with the primers, the probes, the detection system and the kit, the one-time detection of the lung cancer EGFR/KRAS/BRAF/ALK/ROS1 gene can be achieved, such that the detection time is substantially shortened, the sensitivity is high, the specificity is strong, the operation is simple and rapid, and the reference for selection of tumor targeting drug therapy on lung cancer patients can be provided for clinician.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to primers, probes and a detection system for multiple genes of lung cancer. Background technique [0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality among malignant tumors, among which non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer. my country is the country with the highest incidence of lung cancer, and about 600,000 people are diagnosed with lung cancer every year. In the past 30 years, the mortality rate of lung cancer has increased by 464.84%. According to the World Health Organization (WHO), by 2025, the number of lung cancer deaths in my country will reach 1 million each year. At present, chemotherapy is still the most important treatment for lung cancer. Although with the emergence of a new generation of chemotherapy drugs, the efficacy of lung cancer has improved to a certain extent, but the overall...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 黄蛤目林清华施伟杰江风阁宋庆涛阮力郑立谋
Owner 上海厦维医学检验实验室有限公司
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