Septin 9 gene methylation detection method and kit

A detection kit and methylation technology, which is applied in the field of human gene methylation detection, can solve the problems of reducing the sensitivity of PCR and subsequent analysis techniques, single Septin9 gene fragments, severe bisulfite conversion conditions, etc., and achieve detection results Accurate and reliable, prevent false positive results and false negative results, and improve the effect of detection specificity

Pending Publication Date: 2019-10-08
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the kit only detects the methylation status of a region of the Septin 9 gene, and the popularity of its applicable detection models is low, which limits its clinical promotion and application
[0007] In addition, it is worth noting that although the bisulfite conversion method is considered to be the "gold standard" for DNA methylation analysis, it has two major disadvantages: 1) The conditions of bisulfite conversion are too severe, and DNA may 2) Bisulfite conversion completely converts unmodified cytosine into thymine (unmodified cytosine

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  • Septin 9 gene methylation detection method and kit
  • Septin 9 gene methylation detection method and kit
  • Septin 9 gene methylation detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: A method and kit for Septin 9 gene methylation detection

[0055] A method and kit for detecting the methylation of Septin 9 gene, the detection method involved is to use TET enzyme to oxidize 5caC in 5mC and 5hmC in the DNA to be tested, then reduce 5caC to dihydro DHU with pyridine borane, and then use Specific primers and probes designed for gene methylation establish a multiplex fluorescent PCR system for PCR amplification, during which DHU is converted into T, and the methylation status of the Septin 9 gene is judged according to the fluorescent signal of the multiplex fluorescent PCR reaction; The kit includes TET oxidation buffer, TET enzyme, fluorescent PCR pre-reaction solution, polymerase, negative quality control, and positive quality control. The preparation of the Septin 9 gene methylation detection kit includes the following steps:

[0056] (1) Prepare TET oxidation buffer solution according to the formula of TET oxidation buffer solution of th...

Embodiment 2

[0093] Embodiment 2: sensitivity analysis experiment

[0094] (1) Selection of samples to be tested

[0095] In this embodiment, methylated human genomic DNA is selected as the sample to be tested. In order to simulate the length of free DNA fragments, the methylated human genomic DNA as the sample to be tested was ultrasonically treated in this experiment to fragment it.

[0096] (2) Transformation of DNA samples

[0097] Using a method and kit for detecting methylation of the Septin 9 gene obtained in Example 1, perform TET-assisted pyridine borane treatment on the fragmented methylated human genomic DNA according to the detection steps in Example 1 to obtain converted DNA .

[0098] (3) Multiplex fluorescent PCR detection

[0099] The converted DNA concentration obtained according to the method described in Example 1 was adjusted to 10ng / ml, 1ng / ml, 100pg / ml, 50pg / ml, 25pg / ml, 10pg / ml, and 1pg / ml, and used as DNA templates in Examples The method and kit for detecting t...

Embodiment 3

[0103] Embodiment 3: specificity analysis experiment

[0104] (1) Selection of samples to be tested

[0105] In this embodiment, unmethylated human genomic DNA, methylated human genomic DNA, 8 DNA samples from healthy individuals, and 8 DNA samples that were positive for Septin 9 methylation determined by bisulfite sequencing were selected as Sample to be tested. Wherein, non-methylated human genome DNA and methylated human genome DNA are fragmented by ultrasonic treatment.

[0106] (2) Transformation of DNA samples

[0107] Using the method and kit for detecting the methylation of the Septin 9 gene described in Example 1, according to the detection steps in Example 1, the above-mentioned samples to be tested were treated with TET-assisted pyridine borane to obtain converted DNA.

[0108] (3) Multiplex fluorescent PCR detection

[0109] The transformed DNA obtained above is used as a DNA template to perform multiple fluorescent PCR detection according to the detection step...

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Abstract

The invention provides a Septin 9 gene methylation detection kit. The kit comprises at least one of the following three groups of specific primers and probes aiming at the detection of Septin 9-v2 exon methylation, SEQ ID NO.5-SEQ ID NO.7, SEQ ID NO.8-SEQ ID NO.10, and SEQ ID NO.11-SEQ ID.13. The invention further provides a Septin 9 gene methylation detection method and a sample pretreatment method. The Septin 9 gene methylation detection method comprises the following steps of TET enzyme oxidation; pyridine borane reaction; and multiple fluorescence PCR reaction. The kit and the detection method have the advantages of high detection sensitivity, good specificity and high detection flux.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and a kit for detection of human gene methylation. Background technique [0002] The Septin9 gene is one of the members of the Septin gene family discovered by Hartwell et al., and has guanosine triphosphatase (GTPase) activity. The Septin9 gene is located on human chromosome 17q25.3, contains 17 exons, and is about 2.40×10 5 bp, encoding Septin 9 protein. However, because its transcripts contain multiple translation origins and variant splicing, Septin9 can mutate to produce 18 variant splicing forms and encode 15 polypeptides. The subtypes of Septin9 have 75% nucleotide sequence similarity and 89%-96% amino acid identity. In addition, the distribution of these variant splicing bodies is tissue-specific. For example, the variant splicing body Septin 9-v1 of the Septin9 gene is distributed in tissues other than the brain and thymus, and the variant splicing bod...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6806
CPCC12Q1/6858C12Q1/6806C12Q2531/113C12Q2537/143C12Q2563/107C12Q2527/125
Inventor 许嘉森吴诗扬彭璨璨刘志明刘芳
Owner SUREXAM BIO TECH
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