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crRNA, detection kit and detection method for gene mutation of colon cancer related molecular marker

A technology of molecular markers and detection kits, which is applied in the field of detection kits for colon cancer-related molecular marker gene mutations, can solve the problems that large-scale clinical sample research cannot be applied, only one gene can be detected at a time, and the detection cost is high.

Active Publication Date: 2020-03-20
江苏博嘉生物医学科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • crRNA, detection kit and detection method for gene mutation of colon cancer related molecular marker
  • crRNA, detection kit and detection method for gene mutation of colon cancer related molecular marker
  • crRNA, detection kit and detection method for gene mutation of colon cancer related molecular marker

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Effect test

Embodiment 1

[0048] Embodiment 1 Design and acquisition of crRNA targeting gene mutation site

[0049] 1. Discovery of colon cancer detection sites based on the CRISPR-cpf1 system

[0050] According to the latest "NCCN Guidelines", we obtained molecular marker genes commonly used in colon 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.1-4 is used as the colon cancer mutation detection site based on the CRISPR-cpf1 system (the bold part is the mutation region), which has a good detection effect.

[0051] Table 1

[0052]

[0053] 2. Design of crRNA targeting gene mutation site

[0054] (1) Design principles of crRNA targeting gene mutation sites

[0055] Since the CRISPR-cpf1 system is a novel targe...

Embodiment 2

[0071] Example 2 Detection kit and detection method for gene mutation of colon cancer-related molecular markers

[0072] 1. The composition of the kit

[0073] This kit includes 4 crRNAs for colon cancer detection (crRNAs with 4 mutation sites are obtained as shown in Example 1) or crDNA with 4 mutation sites (when crDNA is used in the kit, the operator needs to first mix the crDNA The fragments were respectively generated RNA under the action of T7 RNA polymerase, recovered and purified to obtain crRNA, see Example 1), a specific fluorescent probe (see Table 3), cpf1 protein, enzyme-free water, DNase inhibitor;

[0074] table 3

[0075] fluorescent probe Sequence (5'-3') Probe 1 HEX-(CH2)6-CTCACTACAGACGCACGCTA-BHQ1 (as shown in SEQ ID NO.23) Probe 2 HEX-CTCACTACAGACGCACGCTA-BHQ1 (as shown in SEQ ID NO.24) Probe 3 HEX-CACATCAGCAGCCTACAGCA-BHQ1 (as shown in SEQ ID NO.25)

[0076] Preferably, the kit can also include an amplification system...

Embodiment 3

[0086] Embodiment 3 mutant crRNA is to the specific detection of wild-type and mutant sequence

[0087] The target sequences of the wild strain and the mutant strain were synthesized, and the specificity was detected by using the above four mutant crRNAs respectively. The crRNAs of the four mutation sites prepared in Example 1 were respectively constructed into CRISPR-cpf1 systems to verify the cutting effectiveness in vitro.

[0088] 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 a crRNA, a detection kit and a detection method for gene mutation of a colon cancer related molecular marker. The crRNA includes crRNA (with a sequence shown as any one of SEQ ID NO. 14-16) at a KRAS-exon 2 mutant site, crRNA (with a sequence shown as any one of SEQ ID NO. 17-18) at a KRAS-exon 3 mutant site, crRNA (with a sequence shown as any one of SEQ ID NO. 19-20) at anNRAS-exon 2 gene mutation site and crRNA (with a sequence shown as any one of SEQ ID NO. 21-22) at an BRAF gene mutation site. For the crRNA provided by the invention, 4 crRNA at mutation targets ofthe colon cancer related molecular marker are designed and performs mutation detection in combination with a CRISPR-cpf1 system. The method has the characteristics of high speed, high sensitivity andstrong 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 colon 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] Colorectal cancer is one of the most common malignant tumors in human beings, and its incidence rate ranks fourth among all malignant tumors in the world. Although colorectal polyps and cancers may not cause symptoms early on, simple screening can find many growths and polyps. Colorectal polyps can be found and removed with sigmoidoscopy and colonoscopy, reducing the risk of them developing into cancer. However, there are still risks in late discovery. Therefore, accurate knowledge of tumor biological infor...

Claims

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Application Information

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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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