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Kit and method for detecting DNA methylation by triplex qPCR assay

A kit and methylation technology, applied in the field of medicine and biology, can solve problems such as misdiagnosis

Active Publication Date: 2019-01-01
深圳市新合生物医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This detection method requires a relatively large amount of cfDNA to detect the methylation status of SEPT9, and at the same time, the insufficient rigorous qPCR detection will produce a large number of false positive results, these factors may lead to misdiagnosis

Method used

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  • Kit and method for detecting DNA methylation by triplex qPCR assay
  • Kit and method for detecting DNA methylation by triplex qPCR assay
  • Kit and method for detecting DNA methylation by triplex qPCR assay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1 SEPT9 gene methylation triple detection method sensitivity test

[0100] Experimental Materials

[0101] 1. Methylated human genomic DNA (purchased from NEB Company, item number N4007)

[0102] 2. EZ DNA Methylation-Lightning Kit (purchased from Zymo Research, Cat. No. D5031)

[0103] 3. Primers and probes corresponding to SEPT9 amplicon 1 (SEPT9_1) and amplicon 2 (SEPT9_2) (see the table below)

[0104] 4. ACTB amplification primers and probes (see the table below)

[0105] 5. AmpliTaq Gold TM DNA polymerase and buffer reagent (purchased from Thermo Fisher, Cat. No. 4311806)

[0106]

[0107] Wherein, the two nucleotides at the 3' end of each of the above primers are modified by thionucleotide; the DNA probe contains a TaqMan fluorophore at the 5' end, and a fluorescent quencher is attached to the 3' end.

[0108] experimental method

[0109] 1. DNA fragmentation treatment

[0110] In order to simulate the length of free DNA fragments, this experime...

Embodiment 2

[0123] Example 2 Comparison of SEPT9 gene methylation triple detection method with epiProcolon v2.0 high-sensitivity PCR kit

[0124] Experimental Materials

[0125] 1. Unmethylated human genomic DNA (purchased from Promega, Cat. No. G3041)

[0126] 2. Methylated human genomic DNA (purchased from NEB Company, item number N4007)

[0127] 3. EZ DNA Methylation-Lightning kit (purchased from Zymo Research, Cat. No. D5031)

[0128] 4. SEPT9 amplicon 1 (SEPT9_1) and amplicon 2 (SEPT9_2) corresponding primers and probes (same as Example 1)

[0129] 5.ACTB amplification primer and probe (with embodiment 1)

[0130] 6. AmpliTaq Gold TM DNA polymerase and buffer reagent (purchased from Thermo Fisher, Cat. No. 4311806)

[0131] 7. epiProcolon v2.0 High Sensitive PCR Kit (purchased from Epigenomics, Cat. No. M5-02-002)

[0132] experimental method

[0133] 1. DNA fragmentation treatment

[0134] The non-methylated human genomic DNA and the methylated human genomic DNA are ultraso...

Embodiment 3

[0151] Example 3 Detection of normal control samples using SEPT9 methylation triple qPCR detection and epiProcolon v2.0 high-sensitivity PCR kit

[0152] Experimental Materials

[0153] 1. Healthy human plasma (purchased from Bloodworks NW company)

[0154] 2. QIAamp Circulatory System Nucleic Acid Extraction Kit (purchased from Qiagen, Cat. No. 55114)

[0155] 3. EZ DNA Methylation-Lightning kit (purchased from Zymo Research, Cat. No. D5031)

[0156] 4. SEPT9 amplicon 1 (SEPT9_1) and amplicon 2 (SEPT9_2) corresponding primers and probes (same as Example 1)

[0157] 5.ACTB amplification primer and probe (with embodiment 1)

[0158] 6. AmpliTaq Gold TM DNA polymerase and buffer reagent (purchased from Thermo Fisher, Cat. No. 4311806)

[0159] 7. epiProcolon v2.0 High Sensitive PCR Kit (purchased from Epigenomics, Cat. No. M5-02-002)

[0160] experimental method

[0161] 1. DNA extraction from plasma

[0162] Cell-free DNA was extracted from 10 plasma samples using the ...

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Abstract

The present disclosure provides a kit and method for detecting DNA methylation by a triplex qPCR assay. The kit includes a combination of three sets of primers and probes, wherein two sets of primersand probes are combined to target two regions selected from the sequence of the human SEPT9 gene. Another set of primers and probes targets an internal reference. The kit described in the present disclosure is capable of more sensitive and accurate determination of the DNA methylation level or status in a particular region.

Description

technical field [0001] The disclosure belongs to the field of medical biology, specifically, it mainly relates to a DNA methylation detection kit and method, especially a DNA methylation detection method of the target gene Septin9 (SEPT9) related to colorectal cancer in human liquid biopsy And a kit for detecting DNA methylation of the target gene SEPT9. Background technique [0002] Colorectal cancer (CRC) is the third most common cancer worldwide, killing 600,000 people each year. For CRC, early recognition and early intervention are the best ways to reduce mortality. The current screening method is colonoscopy with fecal occult blood and immunohistochemical testing (gFOBT / FIT). These screening methods are underutilized due to the discomfort of colonoscopy and the distaste for bowel products. Therefore, assessment of CRC risk using reliable non-invasive blood-based tests may be valuable for early detection of CRC. [0003] Altered DNA methylation is one of the earliest...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2537/143C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 韩艳霍华德·霍约恩·章余涛万季宋麒
Owner 深圳市新合生物医疗科技有限公司
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