RASSF1A gene methylation state detection kit and application thereof

A state detection and kit technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc.

Pending Publication Date: 2019-09-27
广东辉锦创兴生物医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] When detecting gene methylation in tumor tissue, there is a problem of detection specificity

Method used

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  • RASSF1A gene methylation state detection kit and application thereof
  • RASSF1A gene methylation state detection kit and application thereof
  • RASSF1A gene methylation state detection kit and application thereof

Examples

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Effect test

Embodiment 1

[0017] Example 1. RASSF1A gene methylation status detection kit of the present invention

[0018] 1. Design of primers and probes for detection of RASSF1A gene methylation status

[0019] The present invention finds the relatively enriched region of CpG sequence by analyzing the sequence of the RASSF1A gene promoter, and then uses a stem-loop primer to specifically amplify the CG base sequence to prevent the amplification of the TG base sequence.

[0020] The inventors of the present application found a sequence rich in CpG islands at -1 to -187 bp upstream of the transcription initiation region of the RASSF1A gene. The following is a fragment of the transcription region of the RASSF1A gene. The starting point, the underline indicates the sequence where the primer acts, the box indicates the position of the probe, and the sequence from CCGG above ATG to the beginning sequence is exactly 187 bases.

[0021] The present application designed the following primers and probe seq...

Embodiment 2

[0038] Example 2. Application of the RASSF1A gene methylation status detection kit of the present invention

[0039] Taking the analysis of nasopharyngeal carcinoma as an example, the nasopharyngeal swabs of nasopharyngeal carcinoma patients confirmed by pathology and chronic nasopharyngitis were taken respectively, and DNA was extracted, and after the DNA was converted to sulfite, the above methylation fluorescent quantitative PCR reaction was carried out. Compare the specificity and sensitivity of the fluorescent quantitative response curves of the stem-loop upstream primers and common upstream primers.

[0040] Such as figure 1 As shown, DNA extracted from nasopharyngeal swabs confirmed pathologically as patients with nasopharyngitis was subjected to methylation fluorescent quantitative PCR reaction. Common upstream primers participated in the reaction, the long dashed line is the reaction curve of ACTB gene; the short dashed line is the reaction curve of RASSF1A gene, th...

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Abstract

The invention relates to an RASSF1A gene methylation state detection kit. The kit comprises a stem-loop primer, and the stem-loop primer is used for performing specific amplification detection on CpG island-rich sequences in the upstream -1bp to -187bp of the transcriptional initiation region of an RASSF1A gene. The stem-loop primer designed by the invention can significantly improve the detection efficiency of methylated DNA in DNA and prevent non-specific amplification of an unmethylated DNA template; and the kit improves the sensitivity of detection while the specificity of tumor detection is improved.

Description

technical field [0001] The invention relates to the field of molecular diagnosis, in particular to a kit for detecting the methylation state of RASSF1A gene and its application. Background technique [0002] Gene methylation is an early event in tumorigenesis. Gene methylation occurs first, which affects the expression of genes, and eventually leads to abnormal expression of proteins, leading to cancerous cells. In recent years, the early diagnosis of tumors through gene methylation, or the assessment of the prognosis of tumor patients, has moved from the laboratory to clinical application. RASS1A gene (Ras-associated domain family 1A, Ras-associated domain family 1A) has been widely considered as a tumor suppressor gene. The normal expression of RASSF1A gene plays an important influence and regulation role in the normal growth and differentiation of cells and the process of apoptosis. The RASSF1A gene is inactivated or silenced in a variety of tumor cells, and the main m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/154
Inventor 陈敏汪莉冯德峰
Owner 广东辉锦创兴生物医学科技有限公司
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