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Methylation detection kit for nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3

A detection kit and technology for nasopharyngeal carcinoma, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve problems such as non-specific amplification, PCR pollution, etc., to improve specificity, eliminate PCR pollution, improve The effect of sensitivity

Active Publication Date: 2020-01-03
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Therefore, problems such as PCR pollution and non-specific amplification when using PCR technology for gene methylation detection have yet to be resolved.
In addition, there are currently no kits for methylation detection of nasopharyngeal carcinoma-related genes DACT1, NFAT1 and SHISA3 on the market

Method used

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  • Methylation detection kit for nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3
  • Methylation detection kit for nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3
  • Methylation detection kit for nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Preparation of kits for detection of methylation of nasopharyngeal carcinoma-related genes DACT1, NFAT1 and SHISA3

[0052] Kits for detection of methylation of nasopharyngeal carcinoma-related genes DACT1, NFAT1 and SHISA3, including fluorescent PCR reaction system, negative quality control and positive quality control. The preparation of the kit includes the following steps:

[0053] (1) Preparation of alkaline solution: Take an appropriate amount of NaOH to prepare a solution with a concentration of 75 mM and a pH value of 12.5, which is an alkaline solution, and save it for future use.

[0054] (2) Design and preparation of primers and probes: Design several sets of primers and probes for the DACT1, NFAT1, and SHISA3 gene CpG islands and ACTB genes, respectively, and conduct pre-experiments on each primer and probe to compare sensitivity, specificity and other properties Finally, four sets of LNA modified primers and specially designed competitive fluore...

Embodiment 2

[0070] Example 2: Validation experiment of fluorescent PCR reaction system for detection of non-desulfurized samples

[0071] (1) Purpose of the experiment

[0072] In this example, by comparing the detection results of the desulfurized samples and non-desulfurized samples with the conventional fluorescent PCR system and the fluorescent PCR system of the present invention without UDG enzyme, to verify the effectiveness of the fluorescent PCR reaction system of the present invention in the detection of non-desulfurized samples sex.

[0073] (2) Experimental method

[0074] In this example, methylated human genomic DNA was used, and after bisulfite conversion treatment, it was divided into two groups, one of which was desulfurized by NaOH and purified to obtain desulfurized DNA samples, and the other group was directly desulfurized without desulfurization. Purified, that is, non-desulfurized DNA samples. The above-mentioned two groups of DNA samples were used as templates, re...

Embodiment 3

[0086] Embodiment 3: Effect Analysis Experiment of Fluorescent PCR Reaction System Eliminating PCR Contamination

[0087] (1) Purpose of the experiment

[0088] In this example, the effect of the fluorescent PCR reaction system of the present invention on eliminating PCR contamination was analyzed by comparing the detection results of samples containing or not containing PCR pollutants with the fluorescent PCR system of the present invention without UDG enzyme.

[0089] (2) Experimental method

[0090] In this example, non-methylated human genomic DNA that has been converted by bisulfite but not desulfurized, methylated human genomic DNA that has been converted by bisulfite but not desulfurized, and PCR pollutants are selected to prepare 4 kinds Samples with or without PCR contamination. Among them, PCR pollutants are amplification products of methylated human genomic DNA. See the table below for the preparation of the 4 samples with or without PCR contaminants.

[0091] ...

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Abstract

The present invention provides a methylation detection kit for nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3. The kit comprises an alkaline solution and a fluorescent PCR reaction solution, and the alkaline solution and the fluorescent PCR reaction solution are packaged in a same PCR amplification tube by a hot-melt material in a layering manner; the alkaline solution is a NaOH solution with pH value of 12.5-13.3 and concentration of 50-100 mmol / L, and volume of the alkaline solution is less than or equal to 50% of the volume of the PCR reaction solution; and the fluorescent PCR reaction solution comprises primers and probes aiming at the DACT1, NFAT1 and SHISA3 genes and UDG enzymes. A systematic and step-by-step heat treatment is conducted before the fluorescent PCR reaction, the UDG enzymes are effectively applied, and the kit effectively solves a problem of PCR pollution in methylation detection, and also has higher detection sensitivity, specificity and detectionflux.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection kit for the methylation of nasopharyngeal carcinoma-related genes DACT1, NFAT1 and SHISA3. [0002] technical background [0003] Nasopharyngeal carcinoma is a malignant tumor originating from the nasopharyngeal epithelium, which has strong local invasion and can produce distant metastasis, and its incidence rate is high in southern China and Southeast Asia (Torre LA et al., CA Cancer J Clin , 2015, 65, 87-108). In recent years, the application of intensity-modulated radiotherapy and chemotherapy has greatly improved the prognosis of nasopharyngeal carcinoma, especially the prognosis of early nasopharyngeal carcinoma, but about 30% of nasopharyngeal carcinoma patients eventually develop recurrence and / or distant metastasis (Lai SZ et al., Int J Radiation Oncology Biol Phys, 2011, 80, 661-668). Therefore, the major challenge in the treatment of NPC remains the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686
CPCC12Q1/6886C12Q1/686C12Q2600/154C12Q2521/531C12Q2525/117C12Q2563/107C12Q2537/143
Inventor 许嘉森吴诗扬彭璨璨刘志明刘芳
Owner SUREXAM BIO TECH
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