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The kit for screening colorectal cancer and advanced adenoma and its application

A technology of colorectal cancer and kits, applied in biochemical equipment and methods, microbiological determination/testing, sugar derivatives, etc., can solve the problem of lack of detailed comprehensive research on hypermethylated CpG sites and unhelpful identification of methylated sites points, limited sample size, etc.

Pending Publication Date: 2021-05-04
HANGZHOU NEW HORIZON HEALTH TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] Although several studies have been performed on methods to detect methylation of BMP3 and NDRG4 genes in fecal samples from patients with CRC and AA, there are no detailed studies on hypermethylated CpG sites in BMP3 and NDRG4 genes in Asian populations. Comprehensive research (ONCOLOGY LETTERS 2014; 8:1751-1756; ONCOLOGY LETTERS 2015; 9:1383-1387)
Furthermore, previous studies on BMP3 and NDRG4 gene methylation in Asian patients were not helpful in identifying the methylation sites most associated with CRC and AA due to limited sample size

Method used

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  • The kit for screening colorectal cancer and advanced adenoma and its application
  • The kit for screening colorectal cancer and advanced adenoma and its application
  • The kit for screening colorectal cancer and advanced adenoma and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0270] Methylated CpG sites in the promoter regions of BMP3 and NDRG4 genes were found in Chinese CRC and AA populations, respectively.

[0271] (1) Sample collection

[0272] A total of 191 colonic FFPE tissue samples were collected from patients with CRC and AA confirmed by colonoscopy, including 50 colorectal cancer tissues and 49 paired adjacent normal tissues, 46 adenoma cancer tissues and 46 paired Subsidiary normal organization.

[0273] (2) DNA extraction

[0274] Genomic DNA was extracted from FFPE samples with the TaKaRa MiniBEST FFPE DNA Extraction Kit (Catalogue #: 9782). The detailed operation steps are described as follows:

[0275] i. Cut 30 mg of paraffin section tissue with a sterile scalpel and remove excess paraffin.

[0276] ii. Place the paraffin section tissue in a 1.5 mL centrifuge tube and add 500 μL buffer DP, mix and incubate in water at 80° C. for 1 minute, then vortex for 10 seconds. Add 180 μL of Buffer GL and vortex.

[0277] iii. The mixtur...

Embodiment 2

[0350] Comparison of differentially methylated CpG sites of CRC-associated BMP3 and NDRG4 genes in different ethnic groups.

[0351] We analyzed the methylation microarray data of BMP3 and NDRG4 genes in the TCGA database (Illumina Human Methylation 450 data), and found that there were five significantly different methylated CpG sites (Figure 2 and Table 9). To further validate differentially methylated CpG sites, tissue and blood samples were collected from 106 Chinese CRC and AA patients. DNA was extracted and treated with bisulfate. The promoter regions of the BMP3 and NDRG4 genes were amplified and sequenced. We analyzed the sequencing data and indeed found that, in the TCGA database, the hypermethylated CpG sites of the Asian population were different from those of the Caucasian population, and the hypermethylated CpG sites were different between CRC and AA tissue samples. According to these different methylated CpG sites, we have developed a detection kit specifically...

Embodiment 3

[0356]Screening of primers and probes for BMP3 and NDRG4 genes

[0357] (1) Design and selection of primers and probes for BMP3 and NDRG4 genes.

[0358] qPCR primers and probes were designed based on the methylated CpG sites of BMP3 and NDRG4 genes. Three pairs of preferred primers and probes were identified. The preferred primers and probes were compared to several other candidate primers and probes and positive and negative controls for the BMP3 and NDRG4 genes. The information of primers and probes is shown in Table 10.

[0359] Table 10 Preferred and remaining primer and probe information

[0360]

[0361]

[0362] (2) Comparison of preferred primers and probes with controls and positive and negative controls for BMP3 and NDRG4 genes.

[0363] Standard samples with different methylation ratios were formed by spiking positive control DNAs of BMP3 and NDRG4 genes into negative control DNAs at different ratios, respectively (Table 11). Analytical sensitivity was c...

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Abstract

The present inventions provides combinations of primers and probes for performing quantitative PCRs that can be used to determine the methylation state and level of BMP3 gene and NDRG4 genes in a patient in need thereof, which leads to surprisingly high diagnostic specificity and sensitivity for diagnosing the presence or the absence of colorectal cancer (CRC) and / or advanced adenoma (AA) in a patient in need therefore. Compositions and methods for performing the diagnosis are provided.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to Chinese Patent Application Serial No. 201810502359.7 filed May 23, 2018 and Chinese Patent Application Serial No. 201810502387.9 filed May 23, 2018, which are incorporated by reference in their entirety for all purposes into this article. technical field [0003] The present invention relates to compositions and methods for screening for colorectal cancer and advanced adenomas, among other applications. [0004] Instructions for Electronically Submitted Text Files [0005] The contents of the text file electronically filed herewith are hereby incorporated by reference in their entirety: Copy of Sequence Listing in Computer Readable Format (File Name: NEWH-017_01WO_SeqList_ST25.txt, Date of Record: May 22, 2019, File size: 22 kilobytes). Background technique [0006] Colorectal cancer (CRC) is the fourth most common cancer in the world, with a mortality rate lower only than lung, l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C12N15/09C12N15/11C12P19/34C12Q1/02C12Q1/6886
CPCC12Q1/6886C12Q2600/154C12Q2600/16C12Q2600/112C12Q2600/166C12Q2561/113C12Q2600/156G01N33/721
Inventor 陈一友李存耀李慧郑伟贤杨蛟刘刚吕宁
Owner HANGZHOU NEW HORIZON HEALTH TECH CO LTD
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