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

A kit, a one-time technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of small sample size, unable to meet one-by-one screening, time-consuming, etc., and achieve cost savings Effect

Active Publication Date: 2015-08-05
AMOY DIAGNOSTICS CO LTD
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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 diffi

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

Embodiment 1

[0077] According to the wild-type gene sequences of human EGFR, KRAS, BRA, ALK and ROS1 published by Cosmic data, specific primers and New probes (see Table 1 and Table 8).

[0078] Table 8 Distribution of each tube

[0079]

[0080] In this example, EML4 exon 13; ALK exon 20 (ALK fusion gene), CD74 exon 6; ROS1 exon 32 (ROS1 fusion gene), KIF5B exon 16; ROS1 exon 12 (RET fusion gene), L861Q (EGFR gene mutation), G12D (KRAS gene mutation), V600E1 (BRAF gene mutation), G776>VC (HER2 gene mutation), Q61R (NRAS gene mutation), H1047R (PIK3CA gene mutation) are examples to illustrate the detection of the above lung cancer genes by fluorescent PCR of the present invention.

[0081] The experiment uses the armored RNA containing the above-mentioned fusion gene and the plasmid template of each mutation respectively, and its fluorescent PCR detection includes the following steps:

[0082] (1) Treatment and extraction of plasmids and armored RNA:

[0083] The extraction of each p...

Embodiment 2

[0131] Using the present invention to detect clinical samples, from December 2013 to October 2014, multiple lung cancer gene mutations were detected on 1,100 clinical lung cancer paraffin-embedded tissue samples.

[0132] 1. Extraction of DNA and RNA from test samples: DNA and RNA can be extracted using the FFPE sample DNA / RNA co-separation kit from Xiamen Aide Biomedical Technology Co., Ltd. ), Cat No.ADx-FF03 or QIAGEN Paraffin Tissue DNA Extraction Kit, Cat No.56404; follow the instructions of the extraction kit. Immediately after DNA and RNA extraction, use a UV spectrophotometer to detect the concentration and purity of DNA and RNA. DNA and RNA OD260 / OD280 should be between 1.8 and 2.1; RNA concentration should be between 50 and 500 ng / μL. The DNA and RNA extracted above were used as templates for fluorescent PCR amplification of lung cancer genes.

[0133] 2. Carry out PCR amplification with the above-mentioned DNA and RNA respectively according to the following amplif...

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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 25 EGFR gene mutations, 7 KRAS gene mutations, 6 BRAF gene mutations, 9 NRAS gene mutations, 5 HER2 gene mutations, 2 PIK3CA gene mutations, 5 fusion genes of ALK5, 13 fusion genes of ROS1, and 6 fusion genes of RET 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, and the corresponding fusion detection reagents and the internal control reagents are filled in the pipes 1-4 of the 12 linking PCR strip; and with the primers, the probes, the detection system and the kit, the one-time detection of the 24 fusions and the 54 mutations of the lung cancer 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. With the deepening of research on tumor pathogenesis and its biological behavior, more and more people are focusing on molecular targeted therapy characterized by high specificity and mild adverse reactions. In recent years, in the field of molecular targeted therapy for lung cancer, research hotspots have mainly focused on EGFR, ALK, ROS1, KRAS, BRAF and other targets. The mutation or fusion status of these genes is related to the efficacy of targeted drugs. The combination of multiple genes Testing can further improve the accuracy of predicting prognosis. However, at present,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/16
Inventor 江风阁林清华黄蛤目施伟杰宋庆涛阮力
Owner AMOY DIAGNOSTICS CO LTD
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