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Colorectal cancer gene methylation detection primer probe combination and kit and application thereof

A detection kit and technology for colorectal cancer, applied in the biological field, can solve the problems of only targeting a single gene, unsatisfactory detection accuracy, and limited diagnostic effect, achieving high accuracy, early screening and diagnosis, and high The effect of sensitivity

Pending Publication Date: 2022-02-25
广州达健生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few methods for detecting DNA methylation in colorectal cancer based on methylation fluorescence quantitative method. At the same time, relevant detection is often only for a single gene. The detection accuracy is not ideal and the diagnostic effect is limited.

Method used

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  • Colorectal cancer gene methylation detection primer probe combination and kit and application thereof
  • Colorectal cancer gene methylation detection primer probe combination and kit and application thereof
  • Colorectal cancer gene methylation detection primer probe combination and kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Sample DNA Extraction and Bisulfite Conversion

[0068] 1. Sample processing and DNA extraction

[0069] The nucleic acid extraction kit of Guangzhou Dajian Biotechnology Co., Ltd. was used for extraction, and the operation was as follows:

[0070] 1) Preprocessing of samples to be tested:

[0071] (a) The pretreatment operation of the stool sample is as follows: take 15mL of fresh stool and put it into a 50mL centrifuge tube containing the stool preservation solution, place the 50ml stool preservation tube containing the sample in a rotary mixer and mix for 0.5 hours; Put the uniform sample in a centrifuge at 4200rpm for 30min, take the supernatant, and divide it into three 5mL cryopreservation tubes, each with 3mL; take a 1.5mL centrifuge tube, add 1mL of fecal cell suspension, 100μL of digestive solution LP , mix well and digest at 37°C for 20 minutes, centrifuge at 4000rpm for 10min, transfer the supernatant to a 2mL centrifuge tube for later use.

[0...

Embodiment 2

[0094] Example 2: Screening of colorectal cancer tissue hypermethylation candidate genes and specific primers and probes

[0095] 1. Screening of candidate genes for fecal hypermethylation in patients with colorectal cancer

[0096] Through the comprehensive analysis of literature research results, TCGA methylation chip database and transcriptome sequencing expression profiles, methylation sites with significant differences were screened, and through multiple data filtering analysis, NDRG4, SPG20, 3OST2, and ING1 were finally screened and determined as colorectal cancer cells. Candidate loci of hypermethylation in cancer.

[0097] 2. Screening of specific primers and probes for colorectal cancer methylation detection

[0098] 1) Design and screening of specific primers and probes

[0099] According to the nucleic acid sequences of NDRG4, SPG20, 3OST2, and ING1 mentioned above, methylation primers and probes were designed on the Methyl primer Express v1.0 software. After repe...

Embodiment 3

[0115] Example 3: Clinical sample detection and verification kit effect

[0116] 1. Interpretation of colorectal cancer gene methylation detection kit results

[0117] 1) Threshold setting

[0118] It can be output automatically according to the instrument, or manually adjust the baseline according to the instruction of the instrument, set the threshold in the linear part of the logarithmic graph of the fluorescence value, export the data from the software and read the CT value.

[0119] 2) Judgment of the effectiveness of the kit

[0120] The internal reference gene of the negative quality control product is amplified and the CT value is ≤25, and the gene methylation detection site is not amplified; the internal reference gene and the gene methylation detection site of the positive quality control product are both amplified and the CT value is ≤25.

[0121] 3) Judgment of sample validity

[0122] a) If the internal reference gene is amplified and the CT value is ≤25, the a...

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Abstract

The invention provides a colorectal cancer gene methylation detection primer probe combination and a kit and application thereof, the kit takes an important biological index DNA methylation abnormity of cancer diagnosis, early screening and prognosis as a detection object, and methylation chip data related to colorectal cancer is acquired for analysis by utilizing a fluorescent quantitative PCR technology and combining TCGA data, four gene methylation detection sites such as NDRG4, SPG20, 3OST2 and ING1 are screened out, and colorectal cancer methylation detection based on fluorescent quantitative PCR is established, so that the colorectal cancer detection kit with higher sensitivity and better specificity is obtained, early screening and diagnosis of colorectal cancer are realized, and early diagnosis and early treatment of colorectal cancer are facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a composition and its application in disease detection, in particular to a colorectal cancer gene methylation detection primer-probe combination and its kit and application. Background technique [0002] Colorectal cancer (CRC) causes approximately 600,000 deaths per year, and despite significant advances in treatment options for CRC, survival rates for patients with CRC remain poor due to a lack of effective early diagnostic tools and limited ability to make optimal treatment decisions . According to data from the National Cancer Center, there were 429,200 new cases of colorectal cancer in mainland China in 2015, and 281,400 deaths due to colorectal cancer that year, with an average daily incidence and death of 11,759 and 7,710, respectively. Colorectal cancer often occurs in men over 40 years old, and its incidence is related to factors such as age, region, gender, etc. The early cli...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/154C12Q2563/107C12Q2545/114
Inventor 邵琦
Owner 广州达健生物科技有限公司
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