Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Kit for specific methylation detection of breast tumor

A detection kit and methylation technology, which can be used in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., and can solve the problem of high assay sensitivity

Active Publication Date: 2021-04-13
ANCHORDX MEDICAL CO LTD
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, the plasma cfDNA methylation detection of multiple genes in patients with breast tumors requires a low input amount for library construction due to the small amount of cfDNA, and the low input amount and damage affect the final bioinformatics analysis, and the sensitivity requirements for the entire assay advanced defects

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Kit for specific methylation detection of breast tumor
  • Kit for specific methylation detection of breast tumor
  • Kit for specific methylation detection of breast tumor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1 This example provides a kit for detecting breast cancer in plasma samples, which contains 23 detection genes TLR5, C1orf61, NR2F1-AS1, ZDHHC1, OTP, FEZF2, TSHZ3, FLJ45513, POU3F1, NKX2- 1-DNA methylation molecular marker Marker1 of 23 methylated regions in AS1, PNPLA1, TIFAB, RADIL, EFCAB10, MAST1, ZFHX4-AS1, HOXB13, LHX5-AS1, LECT1, ALX3, BMP7, PRDM13 and ULBP1 Detection primers and probes to Marker23.

[0098] The kit has designed three pairs of primers and three probes for each of the specific methylation sites in the 23 molecular markers Marker1 to Marker23 for the detection of breast cancer in plasma samples (probe fluorescent labels can be FAM, VIC and NED and other fluorophore labels), and labeled as combinations 1, 2, and 3, respectively. Wherein, the combination of primers and probes selected in each molecular marker can be arbitrarily selected to be combined with combinations 1, 2, and 3 of primers and probes in other methylation markers and detecte...

Embodiment 2

[0105] In this example, the kit described in Example 1 was used to detect the methylation levels of 23 biomarkers in plasma samples.

[0106] A method for detecting methylation levels of DNA methylation molecular markers, comprising the following steps:

[0107] 1. Extraction of cfDNA from samples:

[0108] The specific operation steps of plasma DNA extraction were carried out according to the instructions of MagMAX™ Cell-Free DNA Isolation Kit of Life Company.

[0109] 2. Sulphite conversion of extracted cfDNA

[0110] The extracted cfDNA (10ng) was subjected to bisulfite conversion to deaminate the unmethylated cytosine in the DNA and convert it to uracil, while the methylated cytosine remained unchanged to obtain the bisulfite converted The specific operation of the DNA transformation was carried out according to the instructions of the EZ DNA Methylation-Lightning Kit of Zymo Research. Deamination of unmethylated cytosine in DNA to uracil, while methylated cytosine rema...

Embodiment 3

[0134] This embodiment provides the detection of molecular markers in standard products, and the detection steps are as follows:

[0135] 1. Standard product preparation

[0136] 1) Preparation of 0% methylation standard: using Single Cell Kit (Qiagen, Cat#150343) and Mung Bean Nuclease (NEB, Cat#M0250L) were used to process NA12878 DNA to prepare 0% methylation standard;

[0137] 2) Preparation of 100% methylation standard:

[0138] The prepared 0% methylation standard was treated with CpG Methyltransferase (M.SssI) to obtain a 100% methylation standard.

[0139] 2. Preparation of standard products with different methylation ratios:

[0140] Mix 0% and 100% methylation standards according to the desired methylation ratio gradient to obtain 0.2%, 0.4%, and 1% methylation ratio standards.

[0141] 3. Perform bisulfite conversion on standard DNA with different methylation ratios: the steps are as in Example 2, and the conversion input amount is 10-50 ng, preferably 50 ng in...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a detection kit for a methylated biomarker related to breast cancer. The detection kit comprises a primer and probe which aim at a biomarker cg23035715 and are selected from any one of the following groups: SEQ ID NO.1-SEQ ID NO.3, SEQ ID NO.70-SEQ ID NO.72, and SEQ ID NO.139-SEQ ID NO.141. Firstly, a polygene targeting region is enriched in a targeting manner through a unique multiplex PCR technology and the designed primer and probe, the problem that the original cfDNA amount of polygene detection is relatively low, is solved, the detection flux is improved by combining low cost and quickness of MethyLight PCR detection, and besides, the sensitivity and specificity of model detection are also improved. The detection result of the single primer and probe of the molecular marker cg16304215 has very good sensitivity, and the primer and probe have stronger and more stable diagnostic capability when being combined with any one primer and probe in other molecular markers. By means of the detection kit, the sensitivity and specificity of breast cancer detection can be well improved, the detection rate of early breast cancer is effectively increased, treatment and intervention are conducted as soon as possible, and the survival rate of patients is increased.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for breast tumor-specific methylation detection. Background technique [0002] According to the China Cancer Registry Annual Report, the incidence of female breast cancer is at a low level in the 0-24 age group, gradually increases after the age of 25, reaches a peak in the 50-54 age group, and gradually decreases after the age of 55. A large number of studies have been done in Europe and the United States on the susceptibility genes of breast cancer. Currently, BRCA-1, BRCA-2, p53, PTEN, etc. are known. Breast cancers related to these gene mutations are called hereditary breast cancer. cancer, accounting for 5% to 10% of all breast cancers. Breast cancer 21-gene detection not only provides 1-5 years and 5-year recurrence risk prediction, but also serves as the only multi-gene detection to predict the benefit of chemotherapy and endocrine therapy for patients with ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2600/166C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 赵德志杨婷王军张显玉庞达陈志伟范建兵
Owner ANCHORDX MEDICAL CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com