CN102586404B - Kit for specifically detecting genotype of ApoE4 and application of kit - Google Patents

Abstract

The invention relates to a kit for specifically detecting the genotype of ApoE4 and application of the kit. The kit comprises sucrose water of 4 percent, a NaOH solution of 10mmol/L, a Tris-HCL (pH 7.5) solution of 1mol/L, DMSO (Dimethylsulfoxide), a reagent I, a reagent II, Hha I incision enzyme and 10*M Buffer. The application of the kit comprises the following steps of: preparing and preserving a sample; extracting genome DNA; carrying out PCR (Polymerase Chain Reaction) amplification; and identifying the genotype of ApoE4. Comparing with the prior art, the reagent provided by the kit can noninvasively obtain individual genome DNA; the ApoE4 gene can be efficiently amplified, the genotype of the ApoE4 can be quickly and accurately determined, and the genotype determination of a large quantity of individuals can be simultaneously carried out; and the kit has the advantages of short determination time and high accuracy and is suitable for large-scale screening of molecular epidemiology and preventive medicine.

Description

The genotypic test kit of a kind of specific detection ApoE4 and application thereof

Technical field

The invention belongs to molecular biological field, specifically a kind of fast, the genotypic test kit of specific detection ApoE4 and application thereof.

Background technology

Human apo E (Apolipoprotein E, ApoE) is coded by 19q11.2, and its gene is common 3 kinds of phenotypes, and namely 3 kinds of coded Apolipoprotein E2s, E3 and E4 of ε 2, ε 3 and ε 4 allelotrope consist of 6 kinds of different genotype.Comprise 3 kinds of homozygotes (ε 2/ ε 2, ε 3/ ε 3, ε 4/ ε 4) and 3 kinds of heterozygotes (ε 2/ ε 3, ε 3/ ε 4, ε 2/ ε 4).Allelic change causes ApoE4 larger to ldl receptor avidity, i.e. ApoE4＞ApoE3＞ApoE2, thus affecting lipoprotein metabolism and blood lipid level, the ApoE4/4 genotype is as the independent hazard factor of cardiovascular and cerebrovascular disease morbidity.Because the ApoE gene is relevant with insulin resistant (IR), obesity etc., therefore detect the ApoE genotype significant to the research of medical science Molecular Epidemic.

At present, it mainly is PCR-RFLP that the medical science Molecular Epidemic detects the genotypic method of ApoE4, at first from peripheral blood extracting genomic dna, select ApoE exon 4 flanking sequences design primer to carry out the amplification of ApoE4, Hha I carries out enzyme and cuts, then enzyme is cut product and carry out vertical non-sex change PAGE and separate, to determine its genotype.But this kind method sampling request is high, detects loaded down with trivial detailsly, is unfavorable for the use of epidemiology generaI investigation, and traumatic blood drawing to obtain genomic dna be not to be accepted by each individuality, the individuality of Normal group especially.ApoE exon 4 sequence GC content are very high, are difficult for amplification, and genotypic detected result is had a significant impact, and are necessary amplification condition is optimized.Therefore, set up a kind of simple, AT extracting genomic dna, efficient specific amplification ApoE 4 gene fragments, the genotypic method of rapid detection ApoE4 has important meaning to the molecular epidemiology of ApoE4 genotypic correlation and the research of preventive medicine.

Summary of the invention

Purpose of the present invention is exactly to provide a kind of judgement time short for the defective that overcomes above-mentioned prior art existence, the genotypic test kit of the specific detection ApoE4 that accuracy is high and application thereof.

Purpose of the present invention can be achieved through the following technical solutions:

The genotypic test kit of a kind of specific detection ApoE4, it is characterized in that, this test kit is comprised of following reagent: the Tutofusin tris (Tris) of the NaOH solution of 4wt% sucrose water, 10mmol/L, 1mol/L-HCl (pH 7.5) solution, dimethyl sulfoxide (DMSO) (DMSO), reagent I, reagent II, restriction nuclease (Hha I) restriction endonuclease, 10 * M Buffer.

Described test kit is comprised of the reagent of the described proportioning of table 1,

Table 1

Sequence number	Reagent name	Concentration/purity/explanation	Volume (μ L)
				1	Sucrose water	4wt％	10000
2	NaOH solution	10mmol/L	2000
				3	Tris-HCl (pH 7.5) solution	1mol/L	500
4	DMSO	Analytical pure	12.5
				5	Reagent I	See table 2 for details	192.5
6	Reagent II	See table 3 for details	20
				7	Hha I restriction endonuclease (TaKaRa company)	4～12U/μL	10
8	10×M Buffer	See table 4 for details	10

The composition of described reagent I is as shown in table 2,

Table 2

Sequence number	Moiety	Cumulative volume (μ L)
			1	2.0Taq premix (TaKaRa company)	125
2	Aqua sterilisa	67.5
			Add up to		192.5

The composition of described reagent II is as shown in table 3,

Table 3

The composition of described 10 * M Buffer is as shown in table 4,

Table 4

Sequence number	Moiety	Concentration (mM)
			1	Tris-HCl，pH7.5	100
2	MgCl 2	100
			3	Dithizone	10
4	NaCl	500

The application of the genotypic test kit of a kind of specific detection ApoE4 is characterized in that, the step of the application of test kit is as follows:

(1) preparation of sample and storage: the 10mL 4% sucrose water centrifugal 5min of 3000r/min that gargles behind the 10s collects and contains the epithelial precipitation of buccal mucosa, if the sample that obtains detects in the short period of time and can be stored in-20 ℃, if can be stored in-80 ℃ or the liquid nitrogen for subsequent use in long-time detection;

(2) carry out the pcr amplification of ApoE4 gene;

(3) the ApoE4PCR product is after Hha I enzyme is cut, and enzyme is cut product and carried out the 15%PAGE electrophoresis;

(4) namely can simplify judgement ApoE4 genotype with reference to the Richard method.

Described ApoE4 gene is utilizing pcr amplification, adopts the dimethyl sulfoxide (DMSO) of 5wt% that the PCR response procedures is optimized, and the annealing temperature of amplification is 70 ℃.

Compared with prior art, the present invention adopts non-invasive methods to pass through simple alkaline lysis and obtains quickly and easily the oral mucosa genomic dna, utilize 5%DMSO optimize PCR program that the ApoE4 gene order is efficiently increased, obtain rapidly the genotype of ApoE4, the judgement time is short, accuracy is high, for the molecular epidemiology of ApoE4 genotypic correlation disease and the research of preventive medicine provide high efficiency method.

Description of drawings

The 9 routine ApoE4 genotype electrophorograms of Fig. 1 for using present method to obtain.

Wherein: swimming lane 1 is dna molecular amount standard, and that use in this example is 20bp DNA Ladder Maker; Swimming lane 2,3,4,5,8 ApoE4 genotype are ε 3/ ε 3 types, totally 5 routine Different Individual; The ApoE4 genotype of swimming lane 6 is ε 4/ ε 4 types, totally 1 example; The ApoE4 genotype of swimming lane 7 is ε 2/ ε 2 types, totally 1 example; The ApoE4 genotype of swimming lane 9 is ε 3/ ε 4 types, totally 1 example; The ApoE4 genotype of swimming lane 10 is ε 2/ ε 3 types, totally 1 example.

Embodiment

The present invention is described in detail below in conjunction with the drawings and specific embodiments.

Embodiment

One, the reagent and the explanation that comprise of test kit

Test kit of the present invention comprises 8 kinds of reagent, is respectively that 4% sucrose water, 10mmol/L NaOH are molten, 1mol/L Tris-HCl (pH 7.5) solution, DMSO, reagent I, reagent II, Hha I restriction endonuclease, Buffer.Wherein, 4% sucrose water is used for the epithelial collection of buccal mucosa, 10mmol/L NaOH is molten, 1mol/L Tris-HCl (pH 7.5) solution is the extraction reagent of genomic dna, reagent I is the PCR of TaKaRa company operation reagent, DMSO is the optimization reagent of PCR program, reagent II is the PCR primer of Gene A poE4, and Hha I restriction endonuclease, Buffer are used for the enzyme of Gene A poE4PCR amplified production and conscientiously test.

Specifically as table 1 ' shown in, wherein the concrete component of reagent I, reagent II and Buffer sees Table 2 '-4 '.

Table 1 ': the reagent that test kit of the present invention comprises

Sequence number	Reagent name	Concentration/purity/explanation	Volume (μ L)
				1	4% sucrose water	Self-control	10000
2	NaOH solution	10mmol/L	2000
				3	Tris-HCl (pH 7.5) solution	1mol/L	500
4	DMSO	Analytical pure	12.5
				5	Reagent I	See table 2 for details	205
6	Reagent II	See table 3 for details	20
				7	Hha I restriction endonuclease (TaKaRa company)	4～12U/μL	10
8	Buffer	See table 4 for details	10

Annotate: this shows listed amount of reagent is 10 pattern detection

Table 2 ': the component of reagent I

Sequence number	Moiety	Cumulative volume (μ L)
			1	2.0Taq premix (TaKaRa company)	125

2	Aqua sterilisa	67.5
			Add up to		192.5

Table 3 ': the component of reagent II

The said gene primer can be synthetic with synthetic by existing gene by those skilled in the art.

Table 4 ': the component of Buffer

Sequence number	Moiety	Concentration (mM)
			1	Tris-HCl，pH7.5	100
2	MgCl 2	100
			3	Dithiothreitol	10
4	NaCl	500

One, operation steps

This step comprises the preparation of sample and storage, extracting genomic dna, the pcr amplification of ApoE4 gene, the genotypic evaluation of ApoE4.

(1) preparation of sample and storage

Sample derives from individual sucrose collutory, and namely the 10mL 4% sucrose water centrifugal 5min of 3000r/min that gargles behind the 10s collects and contains the epithelial precipitation of buccal mucosa.If the sample that obtains detects in the short period of time and can be stored in-20 ℃, if on 3rd afterwards detection can be stored in-80 ℃ or the liquid nitrogen for subsequent use.

(2) extracting genomic dna

Present method is applicable to the processing of a sample, and a plurality of samples are parallel running simultaneously.

1. the epithelial precipitation of buccal mucosa that also has that will collect moves in the EP pipe of 1.5mL.

2. in precipitation, add 200 μ L 10mmol/L NaOH solution, 100 ℃ of alkaline lysis 5min behind the concussion 15s.The centrifugal 5min of 3000r/min under the room temperature.

3. supernatant is transferred in the EP pipe of a clean 1.5mL, adds 50 μ L 1mol/L Tris-HCl (pH7.5) neutralization ,-20 ℃ frozen for subsequent use.

(3) pcr amplification of ApoE4 gene

The supernatant 2.5 μ L that get above-mentioned acquisition are used for the pcr amplification of ApoE4 gene, and the PCR system is configured according to component shown in the table 5.

Table 5:PCR reaction system component

Reagent name	Volume (μ L)
		Reagent I	19.25
Reagent II	2
		DMSO	1.25
Genomic dna	2.5
		Add up to	25

After pressing table 5 preparation PCR reaction system, mixing carries out the PCR reaction simultaneously according to condition shown in the table 6 gently, carries out the reaction of HhaI endonuclease digestion after reaction finishes.

The PCR reaction conditions of table 6:ApoE4 gene

(4) endonuclease reaction of ApoE4 gene PCR product

Get above-mentioned PCR product 8 μ L, according to the configuration of component shown in the table 7 endonuclease reaction system.

Table 7: endonuclease reaction system

Reagent name	Volume (μ L)
		The PCR product	8
Hha I restriction endonuclease (TaKaRa)	1
		Buffer	1

Mixing gently after reaction system prepares, 37 ℃ of reaction 1h, product carries out 15% native polyacrylamide gel electrophoresis.

(5) 15%PAGE electrophoresis

1, reagent preparation and preparation

The native polyacrylamide gel electrophoresis damping fluid of conventional configuration 15% etc., specific as follows:

(1) configuration 5 * TBE solution: dissolving 54g Tris alkali in 0.6L distilled water, add 27.5ml glacial acetic acid and 20m L 0.5moL/L EDTA solution (p H8.0), adding distil water is settled to 1L again, room temperature preservation.

(2) preparation of electrophoretic buffer (TBE).Get above-mentioned 5 * TBE, the adding distil water dilution is that 1 * TBE is as electrophoretic buffer.

The preparation of (3) 15% non-denaturing polyacrylamide gels.Prepare according to ratio shown in the table 8.

Table 8: prepare 15% polyacrylamide gel agents useful for same volume ratio

With the abundant mixing of the coagulant liquid for preparing, pour in the Bio-Rad vertical gel electrophoresis instrument that has assembled, insert the suitably comb of size, carry out electrophoresis after at room temperature placing about 30min for subsequent use.

(4) preparation of sample loading buffer (Loading Buffer): conventional configuration 6 * Loading Buffer.

2, step

(1) prepares in proportion 15% the polyacrylamide gel mixed solution of 10mL, pour in the glass guide channel of vertical gel electrophoresis instrument, insert the suitably comb of size, after gel polymerisation is complete, the paper handkerchief that soaks with 1 * TBE holds comb and gel, preservative film sealing monoblock gel, 4 ℃ of preservations, until use (can preserve 1-2 days, and also can buy commercial gel).

When (2) preparing electrophoresis, gel is put into the gel vertical electrophoresis apparatus, assemble completely, prepare electrophoretic buffer, and wash well with electrophoretic buffer.

(3) 20bp DNA Ladder Maker 4 μ L add in the hand-hole as reference.Enzyme is cut product and is mixed with an amount of 6 * Loading Buffer, adds to carry out the electrophoresis operation in the loading hole.

(4) electrode is linked to each other with power supply, opening power is carried out electrophoresis.Electrophoresis to the standard reference fuel migration to desired location, stop electrophoresis.

(5) gel is separated, ethidium bromide staining, ultraviolet lamp is observed and is taken pictures, and the judgement of ApoE genotype is simplified with reference to methods such as Richard.

Two, method evaluation

Test the described method of sampling with this respectively and collect the individual buccal mucosa epithelial cell of 9 examples, the peripheral blood sample 200 μ L of the same individuality of simultaneously parallel collection, draw the drawer genomic dna that uses the same method, pcr amplification ApoE4 gene, Hha I enzyme is cut, and 15% native polyacrylamide gel electrophoresis is analyzed the genotype of individual ApoE4.The result shows that the result of determination in two kinds of sample sources is in full accord, always distinguishes that coincidence rate is 100%, distinguishes that alternately rate of load condensate is 100%, and sensitivity is 100%, and specific degree is 100%, result such as Fig. 1.

Claims (3)

1. genotypic test kit of specific detection ApoE4, it is characterized in that, this test kit is comprised of following reagent: the NaOH solution of 4wt% sucrose water, 10mmol/L, the Tutofusin tris of 1mol/L-HClpH7.5 solution, analytical pure dimethyl sulfoxide (DMSO) DMSO, reagent I, reagent II, restriction nuclease Hha I restriction endonuclease, 10 * M Buffer; Described test kit is comprised of the reagent of the described proportioning of table 1,

Table 1

The composition of described reagent I is as shown in table 2,

Table 2

The composition of described reagent II is as shown in table 3,

Table 3

The composition of described 10 * M Buffer is as shown in table 4,

Table 4

。

2. the application of the genotypic test kit of specific detection ApoE4 as claimed in claim 1 is characterized in that, the step of the application of test kit is as follows:

(1) preparation of sample and storage: the 10mL4% sucrose water centrifugal 5min of 3000r/min that gargles behind the 10s collects and contains the epithelial precipitation of buccal mucosa, if the sample that obtains detects in the short period of time and can be stored in-20 ℃, if can be stored in-80 ℃ or the liquid nitrogen for subsequent use in long-time detection;

(2) carry out the pcr amplification of ApoE4 gene;

(3) the ApoE4PCR product is after Hha I enzyme is cut, and enzyme is cut product and carried out the 15%PAGE electrophoresis;

(4) namely can simplify judgement ApoE4 genotype with reference to the Richard method.

3. the application of the genotypic test kit of a kind of specific detection ApoE4 according to claim 2, it is characterized in that, described ApoE4 gene is utilizing pcr amplification, adopts the dimethyl sulfoxide (DMSO) of 5wt% that the PCR response procedures is optimized, and the annealing temperature of amplification is 70 ℃.

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