CN105018579A

CN105018579A - Biological reagent used for detecting Alzheimer disease gene mutation

Info

Publication number: CN105018579A
Application number: CN201410175252.8A
Authority: CN
Inventors: 钟建; 方平科
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-04-28
Filing date: 2014-04-28
Publication date: 2015-11-04

Abstract

The invention provides a biological reagent used for detecting Alzheimer disease gene mutation, a preparing method thereof and application to detection. The reagent comprises primers, a probe, a DNA polymerase, a polymerase buffer, dNTP, DMSO and sterile deionized water. The preparing method for the reagent includes the steps that a gene segment to be detected is determined, the primers are designed and produced according to the gene segment to be detected, blood genomic DNA is extracted, PCR amplification conditions are determined, repeated detection is carried out to finally determine the reaction condition, and the reagent is prepared according to the reaction condition. By the adoption of the reagent, Alzheimer disease gene mutation can be detected through a PCR gene amplification method to achieve Alzheimer disease screening and clinical diagnosis. The reagent is high in detection efficiency, the steps of using the reagent for gene detection are simple and convenient, and results are reliable.

Description

For the biological reagent of Alzheimer's disease gene test

Technical field

The present invention relates to the technique of gene detection of biology field, particularly relating to for the biological reagent of Alzheimer's disease gene test, its preparation method and the method for detecting.

Background technology

The syndrome of dementia to be one group with Cognitive function damage be core symptom.Usually there is the progressive impairments of the multiple senior function of cortex such as memory, thinking, orientation, understanding, calculating, language in patient, these infringements can disturb the work of patient, daily life and social activity to some extent, finally make patient lose self-care ability.Dementia patients accounts for 5.4% in 65 years old and above crowd, and morbidity constantly raises with age growth, wherein based on Alzheimer disease (Alzheimer ' s disease, AD).Along with the aging of world population aggravates, AD patient grows with each passing day.In the U.S., estimate at 4,500,000 AD patients, Chinese AD patient numbers, also more than 5,000,000, accounts for 1/4 of all numbers of patients in the whole world.Along with the prolongation of human longevity, estimate that Future 30 Years dementia patients number will be doubled and redoubled.Alzheimer's disease mostly is gene genetic factor, environmental factors and old and feeble common pathogenetic, and inherited genetic factors impact accounts for 60% to 80% of AD all risk.

Making a definite diagnosis of Alzheimer's disease is carried out mainly through clinical diagnosis and some auxiliary diagnosis measures, generally first checks medical history, and carries out neurologic examination and brief mechanical aptitude test.Basic examination has neuropsychological test, blood routine, biochemical analysis (hepatic and renal function), vitamin B12 concentration, thyroid function, serum inspection and brain computerized tomography or magnetic to shake radiography etc., and Special Circumstances also have other inspect-types.If have to lose the memory of and cause worried symptom, just Alzheimer's disease may be suffered from,

But the mechanical aptitude test and the brain tomoscan that have to pass through doctor could be determined.

The methods for the treatment of of current Alzheimer's disease mainly by drug effect in different neurotransmitter systems, strengthen the high-grade movable of central nervous system, the various symptoms occurred in the process that palliates a disease, delay dull-witted further developing.Therapeutic strategy conventional clinically has: (1) increases the medicine of vagusstoff (Ach) concentration in brain; (2) promote the survival of brain Cholinergic Neurons or improve the medicine of its Nerve conduction; (3) generation reducing amyloid beta or the medicine promoting it to degrade.

The detection methods of Alzheimer's disease is consuming time longer, and diagnosis costly, and can not carry out early diagnosis to Alzheimer disease.Once make a definite diagnosis, the later stage nurse fees of Alzheimer's disease is quite high, and treatment aspect is merely able to delay the state of an illness, can not effect a radical cure.

Summary of the invention

The object of the invention is to the conventional means-fluorescent quantitative PCR technique of applied molecular biology aspect-judge ApoE gene type quickly and efficiently, for examination and the early diagnosis of Alzheimer disease high risk population, the morning realizing Alzheimer disease finds, early prevention, early treatment, reduces the misery after patient and alleviates the economical load of patient home.

For achieving the above object, the invention provides a kind of biological reagent for the gene test of Alzheimer's disease, its preparation method and the method for detecting.

The invention provides a kind of biotinylation kit for the gene test of Alzheimer's disease.The component of described test kit comprises: primer, probe, archaeal dna polymerase, polysaccharase buffering, dNTP, DMSO and aseptic deionized water.

The preparation method of the test kit of Alzheimer's disease gene test mainly comprises the steps:

1) determine testing gene fragment: according to the preliminary selected candidate gene segment of the directivity of scientific and technical literature report, then in R&D process with the comparing of sample Correlation with Pathology to be checked, finally confirm the testing gene segment with clinical disease most dependency;

2) according to described testing gene fragment design primer and probe, and polynucleotide synthesizer is adopted to produce described primer;

3) poba gene group DNA is extracted: employment poba gene group extracts the genomic dna that test kit extracts patient's group and control group;

4) fluorescent quantitative PCR condition is determined, it comprises enzyme, primer, probe: the primer segment of various combination, the experimentally specificity of result, the technical characteristics such as susceptibility, repetition test, carries out the detection reaction of many described testing gene segments simultaneously, finally determines the reaction conditions comprising described primer, probe, described enzyme, wherein, the result software of fluorescent quantitative PCR carrys out automatic analysis;

5) carry out repeatability to detect to determine reaction conditions further: by great many of experiments, comparative experiments result repeatedly comprises the factor of sharpness and susceptibility, finally determines reaction conditions;

6) according to the described reaction conditions finally determined, prepare test kit, its component comprises: primer, probe, archaeal dna polymerase, polysaccharase buffering, dNTP, DMSO and aseptic deionized water.

Another aspect, present invention also offers the method adopting test kit of the present invention to carry out the gene test of Alzheimer's disease.

Adopt test kit of the present invention to carry out the gene test of Alzheimer's disease, the component of wherein said test kit comprises: primer, probe, archaeal dna polymerase, polysaccharase buffering, dNTP, DMSO and aseptic deionized water.

The present invention is to extract the poba gene group DNA of the patient that test kit extracts for template with genome, the components such as the archaeal dna polymerase provided with test kit, primer, probe, 20 μ L reaction systems, add respectively according to specific ratio, setting program, carry out increasing and obtaining detected result with quantitative real time PCR Instrument, thus realize the gene test to Alzheimer's disease.

Key problem in technology of the present invention relates to the design of probe and primer, the production of primer, the determination etc. of quantitative fluorescent PCR reaction system and reaction conditions.

DNA synthesizer used is German polygen, and the preparation method of primer adopts solid phase phosphoramidite triester method, phosphoramidite triester method synthetic DNA fragment, has efficiently, coupling fast and the more stable feature of initial reactant.Phosphoramidite triester method DNA is fixed on the synthesis that solid phase carrier completes DNA chain, and synthesis is held by the 3' of primer to be synthesized to hold synthesis to 5', and adjacent Nucleotide is connected by 3' → 5' phosphodiester bond.

The present invention adopts the method for quantitative fluorescent PCR to detect ApoE gene type by gene amplification, method design and process CIMS simple; Easy to use, to cut without the need to specific apparatus and enzyme, the step such as leakage of electricity swimming; Consuming time shorter, only need can complete detection in one and a half hours; Detection efficiency is high, once can detect dozens of clinical sample, reliable results, effectively simultaneously, primary first-order equation detects somatotype (the homozygous genotypes:E2/E2 of six class ApoE, E3/E3, E4/E4, heterozygous genotypes:E2/E3, E2/E4, E3/E4), can be used for examination and the clinical diagnosis of Alzheimer disease, there is sizable market potential.

Accompanying drawing explanation

Fig. 1 is the diagram that a kind of embodiment according to the present invention carries out gene test.

Fig. 2 is Normal group and Alzheimer's disease pcr gene amplification contrast figure.

Embodiment

Before further describing the invention, be to be understood that the present invention is not limited to the embodiment of following Invention.Also should be appreciated that term as used herein is just for being described for specific embodiment simultaneously, instead of be used for limiting.

The determination of embodiment 1. combination of primers

Design and synthesis primer sees the following form.

Title	Sequence
		112-F	GGGCGCGGACATGGA
112-R	CCTCGCCGCGGTACTG
		158-F	CCGCGATGCCGATGA
158-R	CCCCGGCCTGGTACACT

The determination of embodiment 2. probe combinations

Design and synthesis MGB probe combinations sees the following form.

Title	Sequence	Fluorescent mark
			AZC A1	ACGTGTGCGGCCG	VIC
AZC B1	ACGTGCGCGGCC	FAM
			AP E	CAGAAGCGCCTGGC	FAM
AP F	CAGAAGTGCCTGGCAG	VIC

The preparation of embodiment 3. primer

Adopt German polygen polynucleotide synthesizer to synthesize required primer, its step comprises:

1) instrument and software is opened;

2) calibrating reagent selects the slide block of slide block and calibration 10 pillars;

3) open automatic flushing function and liquid is full of each pipe;

4) hole in described slide block is filled with CPG; And determine that in the correct and described pillar of CPG of filling before synthesis starts, populated CPG contains 3 ' the corresponding Nucleotide held, because the order of instrument synthetic oligonucleotide has been held from 3 ' end to 5 '; It should be noted that add in CPG process can not the filter membrane of striking suddenly in addition, otherwise may have leak out existing;

5) described slide block is installed on described instrument;

6), before synthesis starts, the sequence required for input, as master routine, is pressed beginning key and is brought into operation;

7) after having run, described slide block is unloaded down from described instrument, with the thin head end of stamp, the material in described pillar and filter membrane are got rid of; Described pillar slide block is attached on model stand by operator, and installs firm; With little collection tube, CPG is gathered, be for further processing;

8) oligonucleotide is separated with CPG, by ammoniacal liquor pyroprocessing, be connected to primer on CPG cut come, purify primer by PAGE method, after dilute with water is complete, measure its content by micro-ultraviolet spectrophotometer.

The establishment of embodiment 4. Alzheimer's disease test kit

Test kit comprises following component: (1) primer; (2) archaeal dna polymerase; (3) polysaccharase buffering; (4) dNTP; (5) probe; (6) aseptic deionized water; (7) DMSO.

The determination of embodiment 5. quantitative fluorescent PCR reaction system

Quantitative fluorescent PCR reaction system sees the following form, and cumulative volume is 20 μ L.

The determination of embodiment 6. quantitative fluorescent PCR reaction conditions

Through repeatedly testing, determine that quantitative fluorescent PCR reaction conditions sees the following form:

The operation of embodiment 7. test kit and detected result

Key step is as follows:

(1) extract by poba gene group the poba gene group DNA that test kit extracts sample, measure the concentration of DNA with ultraviolet spectrophotometer.

(2) with poba gene group DNA for template, the system provided according to embodiment 5, adds the component such as archaeal dna polymerase, primer that test kit provides respectively.

(3) open quantitative real time PCR Instrument, the reaction conditions setting program provided according to embodiment 6, and Loading sequence (see Fig. 1) is set.

(4) working procedure, after EP (end of program), reads result from quantitative real time PCR Instrument, carries out analyzing (see Fig. 2).

All technology used herein and scientific terminology, unless otherwise defined, have the implication that those skilled in the art can understand usually.Although similar with described herein or that be equal to any method, device and material can be used to enforcement of the present invention or detection, described herein is preferred method, device and material.

Be described for some preferred implementations in above-mentioned specification sheets, and for illustrate object and provide many details, but those skilled in the art should be understood that the present invention can have various change and more how different embodiments, and details described herein can have suitable change and can not depart from the spirit and scope of the present invention.

Claims

1. for a biological reagent for Alzheimer's disease gene test, it comprises: primer, probe, archaeal dna polymerase, polysaccharase buffering, dNTP, DMSO and aseptic deionized water, and wherein, described primer comprises following primer combination:

2. reagent according to claim 1, wherein said probe comprises following probe combinations:

3. reagent according to claim 2 is for carrying out the purposes of ApoE genotype detection.

4. reagent according to claim 1 and 2, wherein said primer adopts polynucleotide synthesizer to synthesize, and comprises the steps:

1) instrument and software is opened;

3) open automatic flushing function and liquid is full of each pipe;

4) fill the hole in described slide block with CPG, and to determine that before synthesis starts the correct and CPG of the CPG that fills contains 3 ' the corresponding Nucleotide held, in addition, to fill in CPG process can not the filter membrane of striking suddenly with non-leaking;

5) described slide block is installed on described instrument;

6), before synthesis starts, the sequence required for input as master routine, and brings into operation;

7) after having run, described slide block is unloaded down from described instrument, remove the material in described pillar and filter membrane, described pillar slide block is attached on model stand and also installs firmly, and CPG is collected in little collection tube, be for further processing;

8) oligonucleotide is separated with CPG, by ammoniacal liquor pyroprocessing, be connected to primer on CPG cut come, purify primer by PAGE method, after dilute with water is complete, measure content by micro-ultraviolet spectrophotometer.

5. the preparation method of the reagent described in claim 1 or 2, it comprises the steps:

2) design described primer and probe according to described testing gene fragment, and adopt polynucleotide synthesizer to produce described primer;

6. adopt reagent according to claim 1 to carry out the method for Alzheimer's disease gene test, it comprises:

Extract the poba gene group DNA of sample by poba gene group, measure the concentration of DNA with ultraviolet spectrophotometer;

With described poba gene group DNA for template, add according to quantitative fluorescent PCR reaction system the component such as archaeal dna polymerase, primer that test kit provides respectively;

Open quantitative real time PCR Instrument, according to predetermined quantitative fluorescent PCR reaction conditions setting program, and Loading sequence is set;

Working procedure, after EP (end of program), reads result from quantitative real time PCR Instrument, analyzes.