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Breast tumor specific methylation detection kit

A detection kit and methylation technology, applied in biochemical equipment and methods, microbial determination/examination, DNA/RNA fragments, etc., can solve the problem of high sensitivity requirements for assay, achieve stable diagnostic ability, improve sensitivity and specificity sex, improve survival

Active Publication Date: 2021-11-30
ANCHORDX MEDICAL CO LTD
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  • 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

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  • Breast tumor specific methylation detection kit
  • Breast tumor specific methylation detection kit
  • Breast tumor specific methylation detection kit

Examples

Experimental program
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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...

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Abstract

The invention discloses a detection kit for breast cancer-related methylation biomarkers, which includes: primers and probes selected from any of the following groups for the biomarker cg23035715: SEQ ID NO.1‑SEQ ID NO.3, SEQ ID NO.70-SEQ ID NO.72, SEQ ID NO.139-SEQ ID NO.141. The present invention first uses the unique multiplex PCR technology and designed primers and probes to target and enrich the multi-gene target region, avoiding the problem of low amount of original cfDNA in multi-gene detection, and combining the low cost and rapidity of MethyLight PCR detection , which improves the detection throughput, and also improves the sensitivity and specificity of model detection. The detection result of the primer probe of molecular marker cg16304215 alone has good sensitivity, and its combination with any one of the primer probes of other molecular markers has stronger and more stable diagnostic ability. Through the detection kit of the present invention, the sensitivity and specificity of breast cancer detection can be well improved, the detection rate of early breast cancer can be effectively improved, early treatment and intervention can be performed, and the survival rate of patients can be improved.

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

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Application Information

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Patent Type & Authority Patents(China)
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
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