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Kit for detecting methylation of promoter region of fragile X mental retardation 1 (FMR1) gene through fluorescent quantitative method and application thereof

A technology of fluorescence quantification and kit, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc. It can solve the problems of poor specificity and difficulty in screening a large number of samples, and achieve the effect of reducing non-specificity and pollution

Inactive Publication Date: 2018-12-07
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

However, MS-PCR ultimately needs to detect the presence of methylation by electrophoresis, which makes it difficult to screen a large number of samples
And MS-PCR only relies on the specificity of PCR primers to distinguish methylated and unmethylated DNA, the specificity is not good

Method used

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  • Kit for detecting methylation of promoter region of fragile X mental retardation 1 (FMR1) gene through fluorescent quantitative method and application thereof
  • Kit for detecting methylation of promoter region of fragile X mental retardation 1 (FMR1) gene through fluorescent quantitative method and application thereof
  • Kit for detecting methylation of promoter region of fragile X mental retardation 1 (FMR1) gene through fluorescent quantitative method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1. Primers and probes for detecting methylation of FMR1 gene by qPCR method

[0016] In order to overcome the above-mentioned defects in the MS-PCR method, the present invention provides a kit for detecting the methylation of the promoter region of the FMR1 gene based on a fluorescence quantitative method. The sequences of primers and probes involved in the present invention are listed in Table 1 below.

[0017] Table 1. Primers and probes for detecting methylation of FMR1 gene by qPCR method

[0018]

[0019] The underlined and bold bases are designed for the methylation sites of the FMR1 gene, and the size of the qPCR product is 143bp. The probe has a fluorescent reporter group FAM at its 5' end and a fluorescent quencher group MGB at its 3' end.

[0020] in the CG of the upstream primer in the GC in the downstream primer Both target methylated sequences that remain after sulfite treatment or The characteristics of the design; if no methylation oc...

Embodiment 2

[0025] Embodiment two. the application of kit of the present invention

[0026] The type of sample detected by the present invention is a clinical sample of peripheral whole blood. After the genomic DNA is extracted from the blood sample, PCR+capillary electrophoresis method is used to confirm that the tested sample method is Fragile X positive and Fragile X negative.

[0027] The extracted DNA was treated with a sulfite treatment kit of Jiangsu Jingshan Biotechnology Co., Ltd. in an amount of 400 ng to recover the converted DNA, and the eluted DNA was 60 μl. The reaction was carried out through the following fluorescent quantitative PCR reaction system. The qPCR reaction system used the product of Promega Company in the United States, as shown in Table 3, and the reaction conditions were shown in Table 4.

[0028] Table 3. Reaction system for detection of methylation in the promoter region of FMR1 gene by qPCR

[0029] name

Volume (20μl reaction system)

Fina...

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Abstract

The invention relates to a kit for detecting the methylation of a promoter region of a fragile X mental retardation 1 (FMR1) gene through a fluorescent quantitative method. The kit comprises the following PCR (Polymerase Chain Reaction) primers and probes for detecting the methylation of the promoter region of the FMR1 gene, wherein basic groups designed aiming at methylation sites of the FMR1 gene are designed in the primers and the probes. Compared with a PCR capillary electrophoresis method, the fluorescent quantitative PCR method adopted by the kit performs the detection aiming at the FMR1gene methylation and is not limited by the large number of repetitions of (CGG)n, so that missed diagnosis is not easy to happen; and compared with an MS (Methylation Specific)-PCR method, the fluorescent quantitative PCR method has the advantages that the specificity aiming at the detection of the methylation sites is provided on the primers and the specificity aiming at the detection of the methylation sites is further increased on the probes.

Description

technical field [0001] The invention relates to the field of molecular diagnosis, in particular to a kit for detecting the methylation of the FMR1 gene promoter region by a fluorescent quantitative method and an application thereof. Background technique [0002] Fragile X Syndrome (FXS) is the second most common hereditary mental retardation after Down syndrome, and it is an incompletely dominant X-linked genetic disease. Fragile X mental retardation gene I (Fragile XMental Retardation I, FMR1) is located on chromosome Xq27.3, with a genome sequence of about 38Kb, consisting of 17 exons and 16 introns. The molecular genetic basis of FXS is that the number of non-coding regions (CGG) n at the 5' end of the FMR1 gene is different, which can be divided into normal, premutation and full mutation. The length of (CGG)n repeat sequence is polymorphic in the population. In FXS patients, (CGG)n repeats more than 230 times, even up to thousands of times, this type is called full mut...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883
CPCC12Q1/6883C12Q2600/154
Inventor 胡锦张凤笑
Owner 广东辉锦创兴生物医学科技有限公司
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