Detection kit and detection method for gene mutation of lung cancer-related molecular markers

A molecular marker and detection kit technology, applied in the field of detection kits for lung cancer-related molecular marker gene mutations, can solve the problems of high detection cost, inability to apply large-scale clinical sample research, and only one gene can be detected at a time.

Active Publication Date: 2020-03-24
江苏博嘉生物医学科技有限公司
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  • Abstract
  • Description
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Problems solved by technology

[0004] However, in the current clinical stage, quantitative real-time polymerase chain reaction (Quantitative realtime polymerase chain reaction, Q-PCR), non-radioactive in situ hybridization (fluorescence insitu hybridization, Fish), multiple primer PCR (multiplex PCR), etc. For the detection of gene mutations at relevant sites in patients, these technologies have problems such as only one gene can be detected at a time or the detection cost is high, and they cannot be applied to large-scale clinical sample research. Therefore, it is urgent to provide a simple temperature control with high sensitivity , Specific fragment detection method with high specificity

Method used

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  • Detection kit and detection method for gene mutation of lung cancer-related molecular markers
  • Detection kit and detection method for gene mutation of lung cancer-related molecular markers
  • Detection kit and detection method for gene mutation of lung cancer-related molecular markers

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 Design and acquisition of crRNA targeting gene mutation site

[0053] 1. Discovery of lung cancer detection sites based on the CRISPR-cpf1 system

[0054] According to the latest "NCCN Guidelines", we obtained molecular marker genes commonly used in lung cancer mutation detection. According to the gene sequence and a large amount of clinical test data, the sequence of the common mutation site of the gene is determined, and the crRNA is designed for these different regions and the CRISPR / cpf1 system is constructed for research. The results show that the region sequence shown in SEQ ID NO.1-5 is used as the detection site for lung cancer mutations based on the CRISPR / cpf1 system (the bold part is the mutation region), which has a good detection effect.

[0055] Table 1

[0056]

[0057]

[0058] 2. Design of crRNA targeting gene mutation site

[0059] (1) Design principles of crRNA targeting gene mutation sites

[0060] Since the CRISPR-cpf1 system ...

Embodiment 2

[0078] Example 2 Detection Kit and Detection Method for Gene Mutations of Lung Cancer Related Molecular Markers

[0079] 1. The composition of the kit

[0080] This kit includes 5 crRNAs related to the detection of lung cancer gene mutations (crRNA of 5 mutation sites is obtained as shown in Example 1) or crDNA of 5 mutation sites (when the kit is crDNA, the operator needs to first The crDNA fragments generate RNA under the action of T7 RNA polymerase respectively, recover and purify to obtain crRNA, see Example 1 for details), a specific fluorescent probe (see Table 3 for the sequence, any one shown in SEQ ID NO.26-28 kind of probe), cpf1 protein, enzyme-free water, DNase inhibitor;

[0081] table 3

[0082]

[0083] Preferably, the kit can also include an amplification system, which includes a pair of isothermal amplification primers, wherein the sequence of the pair of isothermal amplification primers for the EGFR-T790M mutation site is shown in SEQ ID NO.29-30 The se...

Embodiment 3

[0092] Embodiment 3 mutant crRNA is to the specific detection of wild-type and mutant sequence

[0093] The target sequences of the wild strain and the mutant strain were synthesized, and the specificity was detected by using the above five mutant crRNAs respectively. The crRNAs of the five mutation sites prepared in Example 1 were respectively constructed into CRISPR-cpf1 systems to verify the cutting effectiveness in vitro.

[0094] Use 100-250nM purified cpf1, 250-500nM crRNA, 1-5μl synthetic fluorescent probe, 2μL DNase inhibitor, target DNA at different dilution concentrations, incubate in detection buffer (NEBuffer 3) at 37°C for 1-3 Hour. At the same time, a blank control was established. The blank control group is the signal group without crRNA and without cpf1 corresponding to each experimental group. Several groups of reaction mixtures were simultaneously reacted in a portable detector (the temperature was set at 37° C., and kinetic detection was performed every 1...

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Abstract

The invention provides crRNAs for gene mutation of lung cancer-related molecular markers, a detection kit and a detection method thereof. The crRNAs comprise: a crRNA at the EGFR-T790M mutation site:as shown in any one of SEQ ID NO.16-17; a crRNA at the EGFR-L858R mutation site: as shown in any one of SEQ ID NO.18-19; a crRNA at the BRAF gene mutation site: as shown in any one of SEQ ID NO.20-21;a crRNA at the ALK gene mutation site: as shown in any one of SEQ ID NO.22-23; and a crRNA at the ROS1 gene mutation site: as shown in any one of SEQ ID NO.24-25. According to the invention, the 5 crRRNAs related to gene mutation targets of lung cancer-related molecular markers are designed, and the crRNAs are adopted in combination with a CRISPR-cpf1 system to carry out mutation detection. The method of the invention has the characteristics of simplicity, fast speed, high sensitivity and strong specificity.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a detection kit and a detection method for gene mutations of lung cancer-related molecular markers. Background technique [0002] Tumor is a new organism formed by the abnormal proliferation of cells in certain or certain parts of the body under the action of various adverse factors in the human body. The abnormally proliferating cells are tumor cells. [0003] Primary lung cancer is the highest morbidity and mortality among all kinds of malignant tumors in my country, which seriously threatens human health. Incidence and mortality of lung cancer worldwide About 80%-85% of lung cancer patients are non-small cell lung cancer (NSCL), and about 50% of patients are diagnosed at advanced stage with poor prognosis. Accurately knowing the biological information of tumors, so as to carry out individualized treatment under the guidance of genotyping is very important for gui...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 姚杰程诚王恩慧赵洪友
Owner 江苏博嘉生物医学科技有限公司
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