CN106834437A

CN106834437A - A kind of fragile X mental retardation quick detection kit

Info

Publication number: CN106834437A
Application number: CN201611214263.8A
Authority: CN
Inventors: 刘淑园; 陈华云; 肖湘文; 丁渭; 张天海; 刘孝礼; 邓利琼; 陆同山
Original assignee: GUANGZHOU HESHI BIOTECHNOLOGY Co Ltd
Current assignee: GUANGZHOU HESHI BIOTECHNOLOGY Co Ltd
Priority date: 2016-12-26
Filing date: 2016-12-26
Publication date: 2017-06-13

Abstract

The invention discloses a kind of fragile X mental retardation quick detection kit.A kind of fragile X mental retardation quick detection kit, including：1）The design gene 5' ends non-translational regions of FMR 1（CGG duplicate blocks）Sense primer and anti-sense primer；2）Upstream and downstream primer and high-fidelity enzyme, 10 × PCR buffer etc. mix, and enter performing PCR amplification；3）Amplified production is carried out into agarose electrophoresis, the substantially copy number of CGG is judged by judging the clip size of amplified production, so as to judge whether tester is fragile X syndrome patient or carrier.The amplified production fragment of normal person<300bp, if in the presence of>The amplified production of 700bp, then be premutation person or full mutation person.The design of primers difficulty used in detection method of the invention is low, significantly reduces the cost of primer synthesis；The agarose electrophoresis method judged result of use, further reduces testing cost, substantially increases Clinical practicability.

Description

A kind of fragile X mental retardation quick detection kit

Technical field

The present invention relates to biological technical field, more particularly to a kind of fragile X mental retardation quick detection kit.

Background technology

Fragile X syndrome causes patient's amentia（Martin-Bell syndromes）, it is that a kind of clinical manifestation is mainly The x linked recessive genetic disease of feeblemindedness, most of mechanism of causing a disease is FMR-1 gene 5' ends non-translational region（CGG）n Three nucleic acids repeat sequence transmission extremely and cause.Normal person's（CGG）Repeat copy number and be about 30 copies（90bp）, and women The copy number that repeats of carrier and normal male transmitter increases to 150 ~ 500bp, is referred to as premutation, and adjacent CpG islands are not It is methylated, this premutation（premutation）Nothing or only light symptoms.Full mutation is converted into by premutation mother only occurs Parent is in offspring's transmittance process：The CGG areas of female carrier are unstable, can cause fragile site high in offspring's transmittance process The female carrier and male patient of expression increase to 1000 ~ 3000bp, and adjacent CpG islands are also methylated, this full mutation The expression of neighboring gene can be closed, so as to clinical symptoms occur.

The incidence of disease of fragile X syndrome is about 1/1200 ~ 1/2500 in males, in women population About 1/2000.And the performance of the complicated clinical manifestation of fragile X syndrome, particularly female carrier is not obvious, hold Easily increase newborn infant.Presently mainly by genetic counselling and the reduction of pre-natal diagnosis means, the birth of the newborn infant of prevention, from And reduce the incidence of disease of fragile X mental retardation.

To reduce the incidence of disease of fragile X mental retardation, according to the understanding to fragile site DNA sequence dna, can be connected with RFLP The methods such as lock analysis, DNA hybridization analysis, real-time fluorescence quantitative PCR amplification detect Disease-causing gene, but all because costly, or Time-consuming etc. that reason limits its application for person.A kind of fragile X mental retardation quick detection kit of the invention avoids prior art Shortcoming, it can be by detecting the copy number of the CGG of FMR-1 genes, so as to quickly, accurately judge whether tester is fragility X chromosome syndrome patient or carrier, reduce Newborn Birth-defects rate, are that the prenatal and postnatal care engineering in the whole nation contributes.

The content of the invention

It is an object of the invention to provide a kind of fragile X mental retardation quick detection kit.

The technical solution used in the present invention is：

1) design is directed to FMR-1 gene 5' ends non-translational region（CGG duplicate blocks）Sense primer and anti-sense primer.

2) upstream and downstream primer and high-fidelity enzyme, 10 × PCR buffer etc. are mixed, is expanded.

3) amplified production is carried out into agarose electrophoresis, CGG is judged substantially by judging the clip size of amplified production Copy number, so as to judge whether tester is fragile X syndrome patient or carrier.The amplified production fragment of normal person <300bp, if in the presence of>The amplified production of 700bp, then be premutation person or full mutation person.

As the further improvement of the above method, PCR synergist is with the addition of in PCR system, promote primer-masterplate annealing And aid in archaeal dna polymerase to extend through secondary structure area.

Particularly, a kind of sequence of the primer of above-mentioned fragile X mental retardation quick detection kit is as follows：

F：5'-GCTCAGCTCCGTTTCGGTT-3'

R:5'-ATTGGAGCCCCGCACTTC-3'

Brief description of the drawings

Fig. 1 is the agarose electrophoresis figure of the DNA cloning product of normal person and fragile X mental retardation patient.Distinguish from left to right It is passage 1 ~ 7.Passage 1~3 is the DNA cloning product of fragile X syndrome patient；Passage 4~6 is normal person's DNA cloning product； Passage 7 is 100bp Marker.

Specific embodiment

Embodiment 1：A kind of compound method of fragile X mental retardation quick detection kit

1st, primer：Repeat to go to design primer, including sense primer F and anti-sense primer R in the CGG of FRM-1 genes.

2、10×PCR buffer：Its component mainly includes the Tris-AC of 50mM（pH9.2）, the KAC of 50mM, 20mM's (NH₄)₂SO₄, the MgCl of 10mM₂。

Embodiment 2：Using QIAamp DNA Blood Mini Kit (the 50)-reagent of article No. 51104 of QIAGEN companies Box carries out nucleic acid extraction, obtains sample to be checked.

Embodiment 3：Detection sample

Use the PCR amplification system of embodiment 1：

Tris-AC（pH9.2）	50mM
		KAC	50mM
	20mM
			10mM
dNTPs	300μM
		High-fidelity enzyme	4U
DMSO	6%
		Glycine betaine	5M
F	10pmol
		R	10pmol
DNA	50ng

Its PCR amplification condition：

After reaction terminates, PCR primer is carried out into agarose electrophoresis, it can be seen that normal person exists<There is clear band at 300bp（Figure 1）, and fragile X mental retardation patient exists>There is clear band at 700bp（Fig. 1）.This result illustrates that this kit can be with quick detection Fragile X mental retardation.

SEQUENCE LISTING

<110>Guangzhou two kinds of substance synthesis into another Technology Co., Ltd.

<120>A kind of fragile X mental retardation quick detection kit

<130> 2016

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 19

<212> DNA

<213>Artificial sequence

<400> 1

gctcagctcc gtttcggtt 19

<210> 2

<211> 18

<212> DNA

<213>Artificial sequence

<400> 2

attggagccc cgcacttc 18

Claims

1. a kind of fragile X syndrome quick detection kit, including：Expand the sense primer F of the CGG duplicate blocks of FMR-1 genes With anti-sense primer R；High-fidelity enzyme；10 × PCR buffer etc..

2. it is respectively according to the sense primer F and anti-sense primer R described in claim 1：

F：5'-GCTCAGCTCCGTTTCGGTT-3'

R:5'-ATTGGAGCCCCGCACTTC-3'。

3. PCR amplification system according to claim 1 is：

。

4. a kind of PCR amplification conditions of fragile X syndrome quick detection kit according to claim 1 are：

。

5. a kind of judged result method of fragile X syndrome quick detection kit according to claim 1 is agarose Electrophoresis.