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Nucleic acid and kit for detecting human ROS1 fusion genes and application method of kit

A technology that integrates genes and kits, and is used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Short, low-quality, easy-to-use effects

Inactive Publication Date: 2017-05-10
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity of direct sequencing is low, the detection sensitivity is only 20-30%, and false negatives are prone to occur; the detection operation is complicated and the efficiency is low, and the detection result usually takes 1-2 days; long time
In addition, FISH detection requires expensive special instruments and equipment, the cost of reagents is high, and the operation is complicated, which limits the widespread use of clinical detection.
FISH testing requires that the test samples should not be stored for too long. Using samples with a long storage time for testing will lead to false negatives and affect the accuracy of the test.
Therefore, direct sequencing and fluorescence in situ hybridization (FISH) methods cannot meet the actual needs of clinical detection, and there is an urgent need to develop a method for rapid detection of fusion gene mutations in order to achieve rapid and accurate detection of fusion gene mutations.

Method used

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  • Nucleic acid and kit for detecting human ROS1 fusion genes and application method of kit
  • Nucleic acid and kit for detecting human ROS1 fusion genes and application method of kit
  • Nucleic acid and kit for detecting human ROS1 fusion genes and application method of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1 Primer, probe, verification template

[0086] The present invention is designed to design detection primers for 12 different fusion gene types of human ROS1 fusion genes. The detection primers designed by the present invention can be used in ordinary PCR to amplify the specific products of the corresponding fusion gene types, and design detection probes for For the detection of real-time fluorescent RT-PCR, the types of specific fusion genes are shown in Table 1.

[0087] Table 1 The types of fusion genes detectable by the present invention

[0088]

[0089] Through the screening of the detection primer pair and the detection probe and the optimization of the system, the final determined detection primer pair and detection probe of each gene and the sequence of the corresponding amplification product of each gene that can be used as a positive control are as follows, wherein the detection primer pair The nucleotide sequence of the probe and the probe is...

Embodiment 2

[0110] Embodiment 2: The kit for detecting human ROS1 fusion gene

[0111] The kit for detecting human ROS1 fusion gene includes detection primer pairs for detecting 12 different ROS1 fusion gene variant types in Example 1, which can be used for ordinary PCR amplification to detect corresponding ROS1 fusion gene variants .

[0112] Preferably, on the basis of the above kit, when used for real-time fluorescent RT-PCR detection, each gene detection probe is added, the 5' end of the detection probe is labeled with a fluorescent group, and the 3' end is labeled with a quencher group, The corresponding ROS1 fusion gene variants can be detected using real-time fluorescent RT-PCR. The detection efficiency is high, the time-consuming is short, and the detection result can be obtained without running electrophoresis. Wherein, the fluorescent group is any one of FAM, JOE, CY3, HEX, and VIC, and the quenching group is any one of MGB, BHQ1, TAMRA, and BHQ2.

[0113] In order to facilit...

Embodiment 3

[0120] Embodiment 3 Real-time fluorescent RT-PCR method amplifies clinical sample RNA

[0121] 1. Extraction of RNA from clinical samples

[0122] In this example, RNA was extracted and quantified from paraffin-embedded tissue sections of 4 patients with non-small cell lung adenocarcinoma obtained from Tongji Hospital, and used as a detection template. Cat.NO.73504QIAamp paraffin-embedded tissue extraction kit from Qiagen was used. For details, refer to the instruction manual. Finally, the extracted RNA was quantified with a UV spectrophotometer, and the RNA concentration was diluted to 1 ng / μL.

[0123] 2. use the kit described in table 4 in embodiment 2 to detect, wherein, the 5 ' end of the detection probe is labeled as a fluorescent group and is FAM, and the 3 ' end label quenching group is the 5 of the MGB quality control probe. The 'end is marked with JOE, and the 3' end is marked with a quencher group BHQ1. The detection steps are as follows:

[0124] (1) First, take ...

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Abstract

The invention discloses nucleic acid and kit for detecting human ROS1 fusion genes and an application method of the kit. ROS1 fusion genes capable of being detected herein include 12 types of ROS1 fusion genes, with specified fusion types. The kit is applicable to various clinical sample types, and has the advantages of capability of detecting many types, high sensitivity, high specificity, good experimental operational simplicity, short cycle, good safety and zero toxicity, low cost and the like.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a nucleic acid, a kit and a method for human ROS1 fusion gene. Background technique [0002] Lung cancer is a malignant tumor with the highest morbidity and mortality in the world. Histopathologically, it can be divided into two categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC includes squamous cell carcinoma, adenocarcinoma, and adenosquamous carcinoma. , large cell carcinoma, carcinoid, etc. NSCLC accounts for about 85% of the total cases of lung cancer. The 5-year survival rate of lung cancer patients in developed countries can reach about 20%, while the 5-year survival rate of lung cancer patients in my country is only 13%, and about 70% of NSCLC are already in the advanced stage when they are diagnosed. The main reason is that most lung cancer patients miss the best time for treatment due to lack of highly sensitive gene ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/106C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 李存耀王秀娟梁惠段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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