Neurofibromatosis type I virulence gene mutation and etiological diagnosis agent based on neurofibromatosis type I virulence gene mutation

A technology of neurofibromatosis and diagnostic reagents, which is applied in the field of molecular biology and gene detection, and can solve the problems of high experimental conditions, high price, and easy missed detection

Active Publication Date: 2017-05-31
杭州艾诺医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Since the NF1 gene contains 58 constitutive exons and 3 alternatively spliced ​​exons, the traditional sequencing analysis method for each exon takes a long time, and it usually takes six or seven days to complete the detection of each sequencing sample
Other detection methods such as NGS, FISH or MLPA, etc., need to be equipped with expensive large-scale instruments and equipment, and require high experimental conditio

Method used

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  • Neurofibromatosis type I virulence gene mutation and etiological diagnosis agent based on neurofibromatosis type I virulence gene mutation
  • Neurofibromatosis type I virulence gene mutation and etiological diagnosis agent based on neurofibromatosis type I virulence gene mutation
  • Neurofibromatosis type I virulence gene mutation and etiological diagnosis agent based on neurofibromatosis type I virulence gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Discovery of NF1 gene mutation in neurofibromatosis type I

[0118] The inventor has carried out gene mutation analysis to 13 routine patients, detailed steps are as follows:

[0119] 1) Detection by mutation analysis reagents: the reagents include 1) total RNA extraction system reagents; 2) RNA reverse transcription and cDNA amplification system reagents; 3) cDNA sequencing system reagents.

[0120] 2) Take 2ml of EDTA anticoagulated peripheral blood samples from suspected patients and normal controls, extract total RNA, perform reverse transcription PCR, and amplify cDNA to obtain 5 large fragments of cDNA1, 2, 3, 4, and 5 (for results, see Figure 4 ); Nested PCR was carried out to 5 large cDNA fragments, a total of 22 pairs of primers, PCR product agarose electrophoresis results ( Figure 5 ) and sequencing results ( Image 6 ).

[0121] Type I neurofibromatosis is an autosomal dominant inheritance. In this study, combining the actual situation of the family and ...

Embodiment 2

[0135] The usage method and application example of using the kit of the present invention for diagnosis.

[0136] 1. Obtain clinical samples and set up experimental and control groups

[0137] 1) Patient A

[0138] Patient A, a male, had samples from a tertiary hospital. He had 6 café-au-lait spots, axillary or inguinal freckles, and 9 neurofibromas. The initial diagnosis was type 1 neurofibroma.

[0139] 2) Patient B

[0140] Patient B’s sample came from a tertiary hospital. He had 8 or more café-au-lait spots, armpit or groin freckles, and 3 neurofibromas. The initial diagnosis was type 1 neurofibroma.

[0141] 3) Control group

[0142] Three normal subjects were selected as the control group.

[0143] 2. Experimental scheme and steps

[0144] The point mutation analysis kit of the present invention is used for detection: the kit includes 1) total RNA extraction system kit; 2) RNA reverse transcription and cDNA amplification system kit; 3) cDNA sequencing system kit.

...

Embodiment 3

[0180] The foregoing steps are basically the same as in Example 2, except that the detection of patient A and 3 normal persons with no detected mutation: c.7106G>A, p.W2369X continued as follows.

[0181] The NF1 whole gene large fragment deletion analysis kit of the present invention is used for detection, and the kit includes 1) a DNA extraction system kit; 2) an intragenic microsatellite polymorphic marker analysis kit.

[0182] The specific operation is as follows:

[0183] 5. Use the genomic DNA extraction kit to extract the peripheral blood DNA of suspected patient A and normal control (ensure that the extracted DNA storage concentration is >50ng / μl):

[0184] 5.1 Take 300 μl of anticoagulated whole blood, add 600 μl of cell lysate CL to the sample, mix by inversion, centrifuge at 10 000 rpm for 1 min, suck off the supernatant, and leave the cell nucleus pellet.

[0185] 5.2 Repeat the above steps once.

[0186] 5.3 Add 200 μl buffer GS to the cell nucleus pellet colle...

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Abstract

The invention relates to neurofibromatosis type I virulence gene mutation and an etiological diagnosis agent based on neurofibromatosis type I virulence gene mutation. The etiological diagnosis agent comprises point mutation analysis kits including a total RNA extraction system kit, a RNA reverse transcription and cDNA amplification system kit and a cDNA sequencing system kit. The total RNA extraction system kit is used for total RNA extraction of peripheral venous blood of a tested subject; the cDNA sequencing system kit is used for PCR amplification of cDNA obtained by reverse transcription and five primer pairs as shown in SEQ ID NO.7-16, nested PCR amplification and sequencing of cDNA five long segments obtained by amplification and primer pairs as shown in SEQ ID NO.17-60, and comparison with an original NF1 gene coding DNA sequence; if c.7106G>A and p.W2369X mutation exists in an amplified product, a neurofibromatosis type I patient is determined. The etiological diagnosis agent has advantages of time saving, accuracy and low test cost.

Description

technical field [0001] The invention relates to the fields of molecular biology and gene detection, in particular to a type I neurofibromatosis pathogenic gene mutation and an etiology diagnostic reagent based on the gene mutation. Background technique [0002] Neurofibromatosis type I (Neurofibromatosis type I, NF1) was first reported by von Recklinghausen in 1882, so it is also called von Recklinghausen disease. Type I neurofibromatosis is generally autosomal dominant and some are sporadic. Clinically, the main manifestations are skin coffee spots, multiple skin soft fibrous tumors; freckles and iris melanoma hamartomas in non-exposed parts (such as armpits and groin); multiple organs and systems can also be involved, such as eyes, bones, blood vessels , endocrine system, central and peripheral nervous system, etc., and may be accompanied by mental retardation, congenital dysplasia, and extremely rare malignant transformation. [0003] The disease is caused by mutations ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 俞萍
Owner 杭州艾诺医学检验所有限公司
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