Kit for detecting methylation status of Septin 9 genes and application of kit

A state detection and kit technology, which is applied in the field of Septin9 gene methylation state detection kits, can solve problems such as false positives and misjudgments, and achieve the effects of preventing non-specific amplification and improving detection efficiency.

Pending Publication Date: 2019-12-24
广东辉锦创兴生物医学科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the similarity of sequences, primers and probes designed to detect methylated sequences by fluorescent quantitative PCR are prone to non-sp

Method used

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  • Kit for detecting methylation status of Septin 9 genes and application of kit
  • Kit for detecting methylation status of Septin 9 genes and application of kit
  • Kit for detecting methylation status of Septin 9 genes and application of kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1. Septin9 gene methylation status detection kit of the present invention

[0021] 1. Design of primers and probes for detection of Septin9 gene methylation status

[0022] The present invention finds the relatively enriched region of CpG sequence by analyzing the sequence of the Septin9 gene promoter, and then uses a hairpin-shaped primer to specifically amplify the CG base sequence in the CpG island to prevent the amplification of the UG base sequence.

[0023] Primers and probes were designed according to the 158 bp region downstream of the transcription start point of the second transcript NM_001113493.2 of the Septin9 gene. The underline indicates the sequence of the primer, and the box indicates the position of the probe; the product is located in exon 1. The A base in the following sequence is the transcription start codon, from the A base in the following sequence to the last underlined base CAGCC There is a length of 158bp.

[0024]

[0025] ...

Embodiment 2

[0048] Embodiment 2. Application of the Septin9 gene methylation state detection kit of the present invention

[0049]Taking the analysis of blood samples from patients with colorectal cancer as an example, 5ml of peripheral blood from patients with colorectal cancer and patients with colorectal polyps confirmed by colonoscopy were collected, centrifuged to obtain plasma, and free DNA in the plasma was extracted by magnetic bead method. After the DNA was converted to sulfite, the above methylation fluorescence quantitative PCR reaction was carried out, and the fluorescence quantitative reaction curves of the hairpin-like primer strategy and the blocking probe strategy were compared in terms of specificity and sensitivity.

[0050] like figure 1 Shown, colonoscopy-confirmed methylation-based PCR reactions performed on DNA extracted from peripheral blood plasma of patients with colorectal polyps. The strategy of blocking probes in Document 1 is adopted. The long dashed line is...

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Abstract

The invention relates to a kit for detecting the methylation status of Septin 9 genes. The kit includes a hairpin-shaped primer, wherein the primer is applied special amplification detection of a CpGisland-rich sequence in an exon 1 of the second transcript NM_001113493.2 of the Septin 9 genes. Through screening and comparison, it is shown that the segment, of a length of 158 bp, in the backwardposition of a transcription initiation point of the second transcript NM_001113493.2 of the Septin 9 genes is a region with the most significant methylation change in the tumor tissue. Through the designed hairpin-shaped primer, the detection efficiency of methylated DNA in DNA can be improved significantly, and non-specific amplification of unmethylated DNA templates is prevented.

Description

technical field [0001] The invention relates to the field of molecular diagnosis, in particular to a Septin9 gene methylation state detection kit and application thereof. Background technique [0002] Existing studies have shown that methylation of the Septin9 gene occurs in tumor tissues such as colorectal cancer, gastric cancer, liver cancer, and cervical cancer, but does not occur in the corresponding normal tissues. Therefore, methylation of the Septin9 gene is an auxiliary diagnostic tool. Molecular markers of various tumors. At present, early diagnosis of colorectal cancer by detecting the methylation of Septin9 gene on free DNA in peripheral blood has been clinically applied, but it faces a problem of unsatisfactory sensitivity and specificity. This is mainly because the free DNA in peripheral blood plasma comes from various tissues and organs of the body, among which the DNA released by normal cells accounts for the vast majority, while the methylated DNA released b...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2525/301C12Q2523/125C12Q2531/113
Inventor 冯德峰陈敏
Owner 广东辉锦创兴生物医学科技有限公司
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