CN103468820B - Kit for detecting common mutations of CYP4V2 gene - Google Patents

Abstract

The invention relates to the field of gene detection,in particular to a kit used for detecting mutations of a CYP4V2 virulence gene of BCD,The kit can rapidly, economically and accurately judge gene types of a c.802-8_810del17insGC mutation site, a c.992A>C mutation site and a c.1091-2A>mutation site, through the joint detection on the three high-frequency mutation sites of the CYP4V2 gene, early-stage definite diagnosis, early-stage warning and early-stage intervention can be carried out on a BCD carrier and a BCD patient, and the early-stage warning time of BCD related to the three high-frequency excludability mutations of the CYP4V2 gene can be brought forward to a foetal period, so that a BCD child gets early-stage treatment, and the kit is beneficial to BCD good birth and good care and improving the prevention and cure level of the BCD; the detection method provides the basis for diagnosis and treatment of the BCD patient and the BCD carrier.

Description

Detect the test kit of CYP4V2 gene common mutations

Technical field

The present invention relates to field of gene detection, particularly for detecting test kit and the application thereof of BCD Disease-causing gene CYP4V2 gene common mutations.

Background technology

Fluorescent angiography in patients with crystalline retinitis pigmentosa (Bietti crystalline corneoretinal dystrophy, BCD) be a kind ofly be dispersed in eyeground the retina degenerative change that the little shining point of fractional crystallisation sample is feature, choroidal vessels sclerosis can be had, Progressive symmetric erythrokeratodermia yctalopia and constriction of visual field simultaneously.Fluorescent angiography in patients with crystalline retinitis pigmentosa (crystallineretinal degeneration) is first reported in nineteen thirty-seven by Bietti, has another name called Bietti crystalline malnutrition (Bietti's crystalline dystrophy).The age of onset of BCD is generally at 20 ~ 40 years old, and eyes pathology is roughly symmetrical, and synchronized development, afterwards Progressive symmetric erythrokeratodermia visual loss, can occur total blindness during some patients 50 ~ 60 years old.This disease China and Japan more common, more rare in American-European countries, the male sex is slightly more than women, and the ratio of men and women is about 4:3.

BCD is a kind of recessive hereditary disease, its Disease-causing gene is CYP4V2, the homozygous mutation of CYP4V2 gene or two assorted and sudden change can cause morbidity, an allelic mutation is only had to be then the healthy carrier of BCD, in China's Chinese Han Population, the frequency of CYP4V2 gene pathogenic mutation is about 7/1000, and Prevalence is about 1/24000.

The normal production social activity of eyesight to the mankind is extremely important, and current China blind person reaches more than 500 ten thousand, causes serious burden to economic society.BCD is the larger blinding disease of a kind of harm, and age of onset is generally at 20 ~ 40 years old, and therefore BCD patient has longer " window phase ", therefore to the early diagnosis of " window phase " BCD patient and early intervention significant.BCD is a kind of recessive hereditary disease, though an allelic mutation is the healthy carrier of BCD, but the marriage of two carrier can make its filial generation BCD risk up to 25%, therefore, the examination of CYP4V2 gene pathogenic mutation carrier is also seemed extremely important.

Gene diagnosis, as a kind of novel diagnostic techniques, has that susceptibility is good, specificity high, and the application in disease especially heredopathia is more and more extensive.Newborn infant's eyesight gene screening technology is extensively carried out in countries in the world, can early discovery neonatal period inpairment of vision carry out early intervention, with the visual disorder avoiding newborn infant to bring due to inherited disease by this examination.BCD is more rare in American-European countries, and is more common in China and Japan, still lacks industrialization, normalized fetus or neonatal B CD gene screening technology at present, is difficult to accomplish early diagnosis to BCD infant.The Disease-causing gene of BCD is CYP4V2, is positioned rice chromosome long-armed, CYP4V2 full length gene 19kp, comprises 12 exons, and coded product is 525 amino acid whose protein, and this albumen belongs to CYP450 family, plays a significant role in fatty acid metabolism.CYP4V2 transgenation form differs, and feature is different.The detection of BCD Disease-causing gene CYP4V2 high frequency sudden change can carry out early warning to the patient that there is BCD onset risk principle, can reach the object of early detection, treatment in conjunction with other eye examinations.

Summary of the invention

The present inventor is by the research of 110 routine BCD clinical samples, find in Chinese han population, in numerous pathogenic mutations of CYP4V2 gene c.802-8_810del17insGC, c.992A>C and c.1091-2A>G three sudden changes are the most common, wherein, 9 Nucleotide GTCATCGCT that 8 Nucleotide TCATACAG at described expression the 6th intron end that c.802-8_810del17insGC suddenlys change and the 7th exon start lack and insert two each and every one Nucleotide GC in the position of disappearance, thus cause the 7th Exon deletion.Described c.992A>C sudden change represents that 992nd encoding base of CYP4V2 gene in the 8th exon sports C by A, thus causes its 331st coded amino acid to be proline(Pro) by Histidine mutagenesis.Described c.1091-2A>G sudden change represents that CYP4V2 gene the 8th intron second-to-last base sports G by A, causes shearing acceptor AG to become GG, thus causes the 9th Exon deletion.Show the Mutation Screening of the BCD patient statistical information in conjunction with other area, the occurrence frequencies of these three sudden changes are about 76%, are the modal BCD mutational sites found at present; And proved by research, three the high frequency exclusiveness sudden changes of CYP4V2 gene c.802-8_810del17insGC, and c.1091-2A>G closely related with BCD c.992A>C, it is the important high frequency sudden change of Chinese BCD crowd, account for 76% of sum, risk assessment and the gene early warning of BCD can be carried out as early molecule mark.

In view of this, c.802-8_810del17insGC three high frequencies that the invention provides for detecting BCD Disease-causing gene CYP4V2 gene suddenly change, c.992A>C test kit and c.1091-2A>G, its detected result specificity is high, simple to operate.

For achieving the above object, technical scheme of the present invention is:

For detecting the test kit of CYP4V2 transgenation, described test kit comprises the PCR reaction reagent for amplified sample DNA, described PCR reaction reagent comprises the specificity amplification primer I for detecting c.802-8_810del17insGC mutational site, the upstream primer sequence of described specificity amplification primer I is as shown in SEQ IDNO:4, and the downstream primer sequence of described specificity amplification primer I is as shown in SEQ ID NO:5.Described specificity amplification primer I correspondence detects the c.802-8_810del17insGC sudden change of CYP4V2 gene, and the target fragment that described specificity amplification primer I increases is CYP4V2-7, and sequence is as shown in SEQ ID NO:1, and size is 246bp.

Further, described test kit, described PCR reaction reagent also comprises the specificity amplification primer II for detecting c.992A>C mutational site, the upstream primer sequence of described specificity amplification primer II is as shown in SEQ IDNO:6, and the downstream primer sequence of described specificity amplification primer II is as shown in SEQ ID NO:7.Described specificity amplification primer II correspondence detects the c.992A>C sudden change of CYP4V2 gene, and the target fragment that described specificity amplification primer II increases is CYP4V2-8, and sequence is as shown in SEQ ID NO:2, and size is 220bp.

Further, described test kit, described PCR reaction reagent also comprises the specificity amplification primer III for detecting c.1091-2A>G mutational site, the upstream primer sequence of described specificity amplification primer III is as shown in SEQID NO:8, and the downstream primer sequence of described specificity amplification primer III is as shown in SEQ ID NO:9.Described specificity amplification primer III correspondence detects the c.1091-2A>G sudden change of CYP4V2 gene, and the target fragment that described specificity amplification primer III increases is CYP4V2-9, and sequence is as shown in SEQ ID NO:3, and size is 215bp.

In test kit of the present invention, c.802-8_810del17insGC specificity amplification primer I correspondingly can only detect sudden change; C.992A>C specificity amplification primer II correspondingly can only detect sudden change; C.1091-2A>G specificity amplification primer III correspondingly can only detect sudden change; C.992A>C and c.1091-2A>G when three pairs of primer pairs are all selected, three high frequencies sudden changes of CYP4V2 gene could corresponding be detected c.802-8_810del17insGC.Therefore, preferably, test kit of the present invention, described PCR primer comprises above-mentioned three pairs of specificity amplification primers.

The sudden change detecting CYP4V2 gene can be undertaken by the method for any check point sudden change of this area, such as PCR(polymerase chain reaction)-sequencing, adopt mark CYP4V2 gene DNA probe hybridization method or by method of restriction fragment length polymorphism method or sequence specific primers etc.Carry out with test kit of the present invention PCR reaction product that pcr amplification reaction obtains to detect by the method for direct Sequencing and whether exist by the sudden change determined, also can adopt hybridization probe to detect, hybridization probe used can be hybridize with normal CYP4V2 gene nucleotide series, or with the nucleotide sequence hybridization of sudden change, or the probe of complementary sequence hybridization with them.These probes can use radio isotope, chromonic material or fluorescent substance to mark, and especially can utilize allelic specific probe.In one embodiment of the invention, pcr amplification is adopted to detect sample in conjunction with the method (PCR-RFLP) of digestion with restriction enzyme.

Therefore, further, described test kit also comprises the reagent of endonuclease reaction, the reagent of described endonuclease reaction comprises restriction enzyme, restriction enzyme corresponding to described c.802-8_810del17insGC mutational site is CdiI, restriction enzyme corresponding to described c.992A>C mutational site is ApaI, and restriction enzyme corresponding to described c.1091-2A>G mutational site is CdiI.

Analysis of clinical in conjunction with BCD patient finds, the penetrance of three high frequency pathogenic mutations of CYP4V2 gene is 100%, by the joint-detection to CYP4V2 gene three high frequency mutational sites, can early warning BCD patient and carrier very accurately, having existing BCD patient visual and detect the advantage do not possessed, is very effective molecular marked compound.

Therefore, another object of the present invention is to provide the method utilizing test kit of the present invention to detect the sudden change of CYP4V2 gene high frequency, described method is the method for non-diagnostic and therapeutic purpose, comprises the step of carrying out as follows:

A gets sample to be detected, extracts complete genome DNA;

B for template with the complete genome DNA in step a, adds described PCR reaction reagent, carries out pcr amplification reaction, obtain pcr amplification reaction product;

The pcr amplification reaction product obtained in the step b reagent of described endonuclease reaction is carried out endonuclease reaction by c, obtains digestion products;

According to the digestion products of step c gained, d judges whether sample to be detected exists the sudden change of CYP4V2 gene.

Further, described method, is characterized in that, in step b, in described PCR reaction reagent, the upstream primer of PCR primer and the mol ratio of downstream primer are 1:1.

Further, described method, in step b, the condition of PCR reaction is: react and carry out on ABI9700 thermal cycler, 95 DEG C of denaturation 5min, then according to 95 DEG C of sex change 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 30sec is a circulation, after carrying out 34 circulations, 95 DEG C of sex change 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 5min, preserve PCR primer after completion of the reaction at 4 DEG C.

Further, described method, in step c, the ratio of the restriction enzyme in described PCR reaction product and described endonuclease reaction reagent is 1ug:5U, and the condition of described endonuclease reaction is: react and carry out in 37 DEG C of thermostat water baths, and the reaction times is 12h.

Further, described method, in steps d, carries out agarose gel electrophoresis to the digestion products of step c gained, judges whether sample to be detected exists the sudden change of CYP4V2 gene according to electrophoresis result.Also can check order to the pcr amplification product of step b gained, the result according to order-checking judges whether sample to be detected exists the sudden change of CYP4V2 gene.When adopting the method for agarose gel electrophoresis, that digestion products and DNA standard molecular weight are carried out constant voltage electrophoresis on same sepharose, after electrophoresis terminates, gel is put into gel imaging system and carries out image scanning, contrast according to the band of digestion products on image and DNA standard molecular weight and identify.

Another object of the present invention is to provide described test kit in the application for detecting in fluorescent angiography in patients with crystalline retinitis pigmentosa disease.

Test kit of the present invention can detect the sudden change of fluorescent angiography in patients with crystalline retinitis pigmentosa disease (BCD) Disease-causing gene CYP4V2 gene, for BCD gene test provides basis, and can early discovery BCD case and BCD Disease-causing gene carrier; In addition, c.802-8_810del17insGC test kit of the present invention can also suddenly change to three high frequencies of BCD Disease-causing gene CYP4V2 gene simultaneously, c.992A>C and c.1091-2A>G carry out joint-detection, the joint-detection of three high frequencies sudden change can early discovery the BCD case of early warning more than 76% and carrier.

BCD Disease-causing gene CYP4V2, the individuality merely with a sudden change is the carrier of BCD, and the individuality with homozygous mutant or two heterozygous mutant is then BCD patient.

The detection method of test kit of the present invention is used to be come from individual sample to be tested whether there is CYP4V2 gene c.802-8_810del17insGC by detecting, c.992A>C and c.1091-2A>G suddenly change, can fast, economical, judge the individual genotype in these three mutational sites accurately, thus individuality is carried out to risk assessment and the gene early warning of BCD.By the joint-detection to CYP4V2 gene three high frequency mutational sites, can accomplish to make a definite diagnosis in early days to BCD carrier and patient, early warning and early intervention, having existing BCD patient visual and detect the advantage do not possessed, is very effective molecular marked compound.Wherein, 9 Nucleotide GTCATCGCT that 8 Nucleotide TCATACAG at described expression the 6th intron end that c.802-8_810del17insGC suddenlys change and the 7th exon start lack and insert two each and every one Nucleotide GC in the position of disappearance, thus cause the 7th Exon deletion.Described c.992A>C sudden change represents that 992nd encoding base of CYP4V2 gene in the 8th exon sports C by A, thus causes its 331st coded amino acid to be proline(Pro) by Histidine mutagenesis.Described c.1091-2A>G sudden change represents that CYP4V2 gene the 8th intron second-to-last base sports G by A, causes shearing acceptor AG to become GG, thus causes the 9th Exon deletion.C.992A>C and c.1091-2A>G c.802-8_810del17insGC, the schematic diagram in mutational site is shown in Fig. 1, Fig. 2 and Fig. 3 to CYP4V2 gene respectively.

Fluorescent angiography in patients with crystalline retinitis pigmentosa (Bietti crystallinecorneoretinal dystrophy, the BCD) disease that CYP4V2 transgenation is relevant is autosomal recessive inheritance disease.Contriver, by ophthalmology outpatient service, have collected the China Han BCD patient of 110 routine clinical definites altogether.Clinical definite case definition is: 1) Progressive symmetric erythrokeratodermia yctalopia and constriction of visual field, 2) there is eyeground and be dispersed in the retina degenerative change that the little shining point of fractional crystallisation sample is feature.CYP4V2 detection in Gene Mutation is undertaken by pcr amplification and two-way sanger order-checking, and Mutation Screening primer and reaction conditions are in table 1.Result shows, three the exclusiveness sudden changes of CYP4V2 gene c.802-8_810del17insGC, c.992A>C the total incidence and c.1091-2A>G in confirmed cases group and case relatives group is all about 70%, closely related with BCD, it is the important high frequency sudden change of Chinese BCD crowd, statistical information in conjunction with other area shows, these three sudden change occurrence frequencies add up to and are about 76%, can carry out risk assessment and the gene early warning of BCD as early molecule mark.

Table 1PCR primer sequence and annealing temperature

Primer	Product length (bp)	Annealing temperature (DEG C)	Primer sequence
				BCD1F1	719	55	CCTCCAGTGCAATCACTAC
BCD1F2			AGAAAATGCTCACTTTGGGA
				BCD2F1	353	60	ACCTGGCTTCCTCTAACAGTAACA
BCD2F2			TTTTTGTGCTGAAATGGCTGAA
				BCD3F1	598	60	AGATTCGCCTCCTCCCACCTCAC
BCD3F2			ACCTGGACTCTTGGCCTCTTGACG
				BCD4F1	467	60	TGCCAAAAGCATTTGAGAACCTGT
BCD4F2			CGCGCTGAAGAGCCCGTCAC
				BCD5F1	335	60	AGGAAGAACAGGAACAGGGAGTAG
BCD5F2			CAACGCAGAAATTGTTAGCAATAA
				BCD6F1	445	60	GCTTCATGGGATGCGTAATAGC
BCD6F2			GAAATGAACGGTGGGGATGGT
				BCD7F1	501	60	CCTATGTTGTCGAAATGTTGAAAT
BCD7F2			TCTGAAGAAGTTGAGCTGGTACTT
				BCD8F1	418	60	TTGCAGTCACAGTGCAGTCATCA
BCD8F2			CCAGCATCCGGCCTAGTACAGTC
				BCD9F1	595	60	ATGCCATGCCTTGATCCACCTG
BCD9F2			TGGGCAATGTCACATCACATCTCA
				BCD11F1	500	60	CTCTTCATCTTTAACAGGTGTTCC
BCD11F2			CAAAACTCAAAACTTTTTCTTTGT

Note: F1 is forward primer; F2 is reverse primer.The DNA sequence data of BCD Disease-causing gene CYP4V2 gene is see NG_007965.1.

Beneficial effect of the present invention: (1) test kit of the present invention can be quick, economical, judge CYP4V2 gene accurately c.802-8_810del17insGC, c.992A>C and the c.1091-2A>G genotype in three mutational sites, by the joint-detection to CYP4V2 gene three high frequency mutational sites, can accomplish to make a definite diagnosis in early days to BCD carrier and patient, early warning and early intervention, the pre-warning time of the relevant BCD that suddenlyd change by CYP4V2 gene three high frequency exclusiveness can be advanced to fetus period, thus make BCD infant obtain early treatment, and be conducive to the prenatal and postnatal care of BCD, that improves BCD prevents and treats level.(2) test kit of the present invention is with low cost, using method is simple to operate, the antenatal diagnosis of BCD patient, early detection can be widely used in, make a definite diagnosis, the examination etc. of early intervention and BCD carrier, be conducive to the prenatal and postnatal care of BCD, that improves BCD prevents and treats level, has existing BCD patient visual and detects the advantage do not possessed.(3) detection method of the present invention is that the Diagnosis and Treat of BCD patient and carrier provides foundation.

Accompanying drawing explanation

C.802-8_810del17insGC the mutational site schematic diagram of Fig. 1 CYP4V2 gene.

C.992A>C the mutational site schematic diagram of Fig. 2 CYP4V2 gene.

C.1091-2A>G the mutational site schematic diagram of Fig. 3 CYP4V2 gene.

Fig. 4 restriction endonuclease at CYP4V2 gene c.802-8_810del17insGC, the restriction enzyme site schematic diagram c.992A>C and c.1091-2A>G in sudden change.

The electrophoretic effects figure of Fig. 5 pcr amplification product.

The electrophoretic effects figure of Fig. 6 restriction endonuclease endonuclease reaction product, wherein A is the electrophoretic effects figure of different states individual restriction endonuclease endonuclease reaction product of c.802-8_810del17insGC suddenling change; B is the electrophoretic effects figure of different states individual restriction endonuclease endonuclease reaction product of c.992A>C suddenling change; C is the electrophoretic effects figure of different states individual restriction endonuclease endonuclease reaction product of c.1091-2A>G suddenling change.

Fig. 7 is the DNA sequencing result in the c.802-8_810del17insGC mutational site of CYP4V2 gene in the inventive method, homozygous mutation (patient's sequence), heterozygous mutant (patient's sequence) and wild-type sample (normal sequence).

Fig. 8 is the DNA sequencing result in the c.992A>C mutational site of CYP4V2 gene in the inventive method, homozygous mutation (patient's sequence), heterozygous mutant (patient's sequence) and wild-type sample (normal sequence).

Fig. 9 is the DNA sequencing result in the c.1091-2A>G mutational site of CYP4V2 gene in the inventive method, homozygous mutation (patient's sequence), heterozygous mutant (patient's sequence) and wild-type sample (normal sequence).

Embodiment

Illustrated embodiment is to be described content of the present invention better, but is not that content of the present invention is only limitted to illustrated embodiment.So those of ordinary skill in the art carry out nonessential improvement and adjustment according to foregoing invention content to embodiment, still belong to protection scope of the present invention.In order to make the object, technical solutions and advantages of the present invention clearly, below in conjunction with drawings and Examples, the invention will be further described.

The experimental technique of unreceipted actual conditions in preferred embodiment, usual conveniently condition, the such as Molecular Cloning: A Laboratory guide (third edition, J. the work such as Pehanorm Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or to carry out according to the condition that manufacturer advises.The test materials that the present invention is used, if no special instructions, is commercially available purchase product.

The base sequence that restriction endonuclease CdiI identifies is CATCG, and it to the shearing site of CYP4V2-7 gene as shown in Figure 4.

The base sequence that restriction endonuclease ApaI identifies is GGGCCC, and it to the shearing site of CYP4V2-8 gene as shown in Figure 4.

The base sequence that restriction endonuclease CdiI identifies is CATCG, and it to the shearing site of CYP4V2-9 gene as shown in Figure 4.

In addition, the sequential write of involved in embodiment nucleotide sequence is defaulted as from 5 ' end to 3 ' end.

Embodiment 1 extracting genome DNA

The centrifugal column type poba gene group DNA extraction kit (TIANampBlood DNA Kit) adopting Beijing Tian Gen company to produce extracts the complete genome DNA in venous blood.Reagent set prejudice table 2.

Table 2 Kit components

Reagent name	Specification (50 times)
		Cell pyrolysis liquid	60ml
Damping fluid GS	15ml
		Damping fluid GB	15ml
Damping fluid GD	13ml

Rinsing liquid PW	15ml
		Elution buffer TB	15ml
Proteinase K	1
		Adsorption column CB3	50
Collection tube (2ml)	50
		Centrifuge tube (1.5ml)	50

The step extracting the complete genome DNA in venous blood is as follows:

1, blood sampling 200ul is in 1.5ml sterile centrifugation tube, adds 20ul Proteinase K Solution, mixing.

2, add 200ul damping fluid GB, fully put upside down mixing, 56 degree of water-bath 10min, put upside down therebetween for several times.

3, add 200ul dehydrated alcohol, fully put upside down mixing.

4, previous step gained solution is added (CB3 first puts into collection tube) in adsorption column CB3, the centrifugal 30sec of 12000rpm, discards waste liquid, CB3 is put into collection tube.

5, add in adsorption column CB3 before 500ul damping fluid GD(uses and first add dehydrated alcohol), the centrifugal 30sec of 12000rpm, discards waste liquid, CB3 is put into collection tube.

6, add in adsorption column CB3 before 700ul rinsing liquid PW(uses and first add dehydrated alcohol), the centrifugal 30sec of 12000rpm, discards waste liquid, CB3 is put into collection tube.

7, add in adsorption column CB3 before 500ul rinsing liquid PW(uses and first add dehydrated alcohol), the centrifugal 30sec of 12000rpm, discards waste liquid, CB3 is put into collection tube.

8, put back in collection tube by adsorption column CB3,12000 centrifugal 2min, discard waste liquid, adsorption column are placed in room temperature number minute thoroughly to dry rinsing liquid.

9, adsorption column CB3 is put into clean 1.5ml centrifuge tube, unsettled dropping 200ul elution buffer TB in the middle of adsorption film, room temperature places several minutes, and the centrifugal 2min of 12000rpm, obtains Whole Blood Genomic DNA.

10, utilize detection of nucleic acids instrument to carry out quantitatively and purity check (result is as shown in table 3) genomic dna ,-20 degree save backup.

Table 3 genomic dna quantitatively and purity check result

Sample number	Concentration of specimens (ng/ul)	Sample purity (OD260/OD280)
			1	112	1.87

2	103	1.84
			3	158	1.89
4	135	1.88
			5	128	1.81
6	119	1.85
			7	133	1.80
8	208	1.88
			9	144	1.82
10	121	1.87

Embodiment 2 pcr amplification goal gene fragment

1, primer sequence

According to goal gene fragment design specific PCR amplimer, primer sequence is as shown in table 4.

Table 4 PCR primer sequence

Primer	Primer sequence
		CYP4V2-7 F1	AGCCTATGTTGTCGAAATGT
CYP4V2-7 F2	AAAAGCAAGTCAAGAAAGGC
		CYP4V2-8 F1	CACAGTGCAGTCATCAAATC
CYP4V2-8 F2	CCAAACATACCAAACACGTC
		CYP4V2-9 F1	CTTTTTAGATGTCTGCACCC
CYP4V2-9 F2	AGCATACTTACCCACTTCAC

Note: F1 is forward primer; F2 is reverse primer.The DNA sequence data of CYP4V2 gene is see NG_007965.1.

C.802-8_810del17insGC primer pair CYP4V2-7 F1 and CYP4V2-7 F2 correspondence detects dashes forward, and product is CYP4V2-7 gene, and size is 246bp; C.992A>C primer pair CYP4V2-8 F1 and CYP4V2-8 F2 correspondence detects sudden change, and product is CYP4V2-8 gene, and size is 220bp; C.1091-2A>G CYP4V2-9 F1 and CYP4V2-9 F2 correspondence detects sudden change, and product is CYP4V2-9 gene, and size is 215bp.

2, the foundation of pcr amplification reaction system

When carrying out pcr amplification reaction, preparation 50ul PCR reaction system, the PCR primer described in table 4 carries out pcr amplification reaction to template DNA respectively.Three kinds of primer amplification reaction systems are identical, in table 5.

Table 5PCR amplification reaction system

Composition	Volume (ul)
		Mastermix	25
Template DNA (100ng/ul)	1
		Forward primer (F1) (10uM)	0.5
Reverse primer (F2) (10uM)	0.5
		ddH2O	23
Amount to	50

Note: what template DNA adopted is the genomic dna extracted in embodiment 1, has carried out quantitatively and purity check.

When carrying out PCR test, the reagent needed for the various PCR reaction such as buffer, dNTP, DNA enzymatic, primer, template, water often will be added in PCR pipe, if and operate lack of standardization being easy to when adding these reagent and pollute, cause PCR result inaccurate, therefore, all ingredients is repeatedly added just by first for the reagent such as buffer, dNTP, DNA Polymerase admixed together in order to simplify, namely be mixed with Mastermix and then use, namely simplify PCR operation steps, decrease pollution by way of.In the present embodiment, pcr amplification reagent adopts the concrete composition of Mastermix(of Beijing Tian Gen biotech firm to comprise Taq archaeal dna polymerase, dNTPs, MgCl ₂, reaction buffer, PCR reaction toughener and optimize agent and stablizer, concentration is 2 ×).

3, reaction conditions

PCR reaction completes on ABI9700 thermal cycler, and the amplification condition of three PCR reactions is identical: 95 DEG C of denaturation 5min, 95 DEG C of sex change 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 30sec, 35 circulations, last 72 DEG C extend 5min, preserve PCR primer for 4 DEG C.

4, the qualification of PCR reaction product

The qualification of PCR reaction product adopts the method for agarose gel electrophoresis, negative control be in step 2 above the setting up of pcr amplification reaction system time do not add DNA profiling.

(1) glue: take 1g agar powder, adds in 50mlTAE damping fluid the sepharose being made into 2%, pours in glue bed after mixing heating, for subsequent use after cooling.

(2) application of sample: add in well after the Sybgreen fluorescence dye of 1ul tetrabromophenol sulfonphthalein and 1ul mixes with the PCR primer of 4ul, the Marker I of 4ul is as DNA standard molecular weight application of sample simultaneously, and each electrophoresis adds negative control (not adding DNA profiling during PCR reaction) to judge whether PCR exists pollution simultaneously;

(3) electrophoresis: the gel after loading is put into electrophoresis apparatus, carry out constant voltage electrophoresis with 100V voltage, electrophoresis time is about 30min.

(4) detect: after electrophoresis terminates, gel is put into gel imaging system and carry out image scanning, the results are shown in Figure shown in 5, pcr amplification successfully shows as the single bright band that correct molecular weight size appears in corresponding position.

(5) utilize detection of nucleic acids instrument to carry out quantitatively (PCR primer concentration is good to reach 1ug/ul) PCR primer ,-20 degree save backup.

As can be seen from Figure 5, pcr amplification successfully shows as the single bright band that correct molecular weight size appears in corresponding position, in figure, upper row is depicted as electrophoresis loading wells, 1st swimming lane is electrophoresis standard substance Marker I, and after electrophoresis, its stripe size is followed successively by 600bp, 500bp, 400bp, 300bp, 200bp and 100bp; 2nd swimming lane is PCR primer CYP4V2-7, and stripe size is 246bp; 3rd swimming lane is PCR primer CYP4V2-8, and stripe size is 220bp; 4th swimming lane is PCR primer CYP4V2-9, and stripe size is 215bp.

The digestion with restriction enzyme reaction of embodiment 3PCR reaction product

The PCR primer obtained in embodiment 2 and CYP4V2-7, CYP4V2-8 and CYP4V2-9 are carried out digestion with restriction enzyme reaction respectively, and restriction enzyme used is respectively CdiI, ApaI and CdiI.C.802-8_810del17insGC PCR primer CYP4V2-7 gene pairs should detect sudden change, and c.992A>C CYP4V2-8 gene pairs should detect sudden change, and c.1091-2A>G CYP4V2-9 gene pairs should detect sudden change.The base sequence that restriction enzyme CdiI identifies is the base sequence that CATCG, ApaI identify is GGGCCC, and in recognition sequence, whether the change of any one base all can cause the identification of restriction enzyme.Digestion with restriction enzyme reaction system is set up according to shown in table 6.

Table 6 digestion with restriction enzyme reaction system

Composition	Volume (ul)
		Damping fluid (10 ×)	2
Restriction enzyme (5U/ul)	1
		PCR primer (1ug/ul)	1
ddH 2O	16
		Amount to	20

Damping fluid (CutSmart Buffer) main component used in the present embodiment comprises: 50mM Potassium ethanoate, 20mM Tris-acetate, 10mM magnesium acetate, and 100 μ g/ml bovine serum albumins (BSA), 25 DEG C time, pH value is 7.9.

The digestion with restriction enzyme reaction of PCR reaction product is carried out in 25 DEG C of thermostat water baths, and the reaction times is 12h, and reaction terminates rear gained digestion products and carries out agarose gel electrophoresis qualification or 4 DEG C of preservations immediately.

The qualification of embodiment 4 digestion with restriction enzyme reaction product

1, agarose gel electrophoresis detects

Carry out agarose gel electrophoresis detection to embodiment 3 digestion with restriction enzyme reaction gained digestion products, step is as follows:

(1) glue: take 1g agar powder, adds in 50mlTAE damping fluid the sepharose being made into 2%, pours in glue bed after mixing heating, for subsequent use after cooling.

(2) application of sample: add in well after 1ul tetrabromophenol sulfonphthalein and 1ul Sybgreen fluorescence dye mix with the digestion products of 4ul, the Marker I of 4ul is as DNA standard molecular weight application of sample simultaneously, described DNA standard molecular weight Marker I is produced by Beijing Tian Gen biotech firm, after electrophoresis, have 6 bands visible, fragment length is respectively 600bp, 500bp, 400bp, 300bp, 200bp and 100bp.

(3) electrophoresis: the gel after loading is put into electrophoresis apparatus, carry out constant voltage electrophoresis with 100V voltage, electrophoresis time is about 30min.

(4) detect: after electrophoresis terminates, gel is put into gel imaging system and carry out image scanning, result as shown in Figure 6.

Wherein Fig. 6-A is the c.802-8_810del17insGC different states individual restriction endonuclease endonuclease reaction product electrophoretic effects figure that suddenlys change.In figure, upper row is depicted as electrophoresis loading wells, and the 1st swimming lane is electrophoresis standard substance Marker I, and after electrophoresis, its stripe size is followed successively by 600bp, 500bp, 400bp, 300bp, 200bp and 100bp; 2nd swimming lane is the c.802-8_810del17insGC heterozygous carriers's digestion products that suddenlys change, and has 3 bands, is followed successively by 246bp, 140bp and 106bp; 3rd swimming lane is the c.802-8_810del17insGC homozygote patient digestion products that suddenlys change, and only has 1 band, 246bp; 4th swimming lane is not containing the individual digestion products of c.802-8_810del17insGC wild-type that suddenlys change, and has 2 bands, is followed successively by 140bp and 106bp.

Insert because c.802-8_810del17insGC sudden change causes 17 base TCATACAGGTCATCGCT to lack 2 bases G C, the base sequence that restriction endonuclease CdiI identifies is that CATCG is included in 17 bases of this mutation deletes, therefore use this restriction endonuclease to carry out endonuclease reaction to CYP4V2-7 and just there will be three kinds of possible results: homozygous mutation all lacks this restriction enzyme site due to two allelotrope, and therefore enzyme only has the band of a 246bp after cutting; Heterozygous mutant is owing to only having this restriction enzyme site of deletion allele, and therefore enzyme also has the band of 140bp and 106bp after cutting except the band of 246bp after cutting; And wild-type all contains this restriction enzyme site due to two allelotrope, therefore enzyme there will be 140bp and 106bp two bands after cutting.

Fig. 6-B is the c.992A>C different states individual restriction endonuclease endonuclease reaction product electrophoretic effects figure that suddenlys change.In figure, upper row is depicted as electrophoresis loading wells, and the 1st swimming lane is electrophoresis standard substance Marker I, and after electrophoresis, its stripe size is followed successively by 600bp, 500bp, 400bp, 300bp, 200bp and 100bp; 2nd swimming lane is the c.992A>C heterozygous carriers's digestion products that suddenlys change, and has 2 bands, is followed successively by about 220bp and 110bp (the band agarose gel electrophoresis of 112bp and 108bp size cannot be distinguished); 3rd swimming lane is not containing the individual digestion products of c.992A>C wild-type that suddenlys change, and only has 1 band, 220bp; 4th swimming lane is the c.992A>C homozygote patient digestion products that suddenlys change, and only has 1 band, about 110bp.

Because c.992A>C sudden change causes A base mutation to be C, the base sequence that restriction endonuclease ApaI identifies is GGGCCC, therefore use this restriction endonuclease to carry out endonuclease reaction to CYP4V2-8 and just there will be three kinds of possible results: homozygous mutation is because two allelotrope are all containing restriction enzyme site, and therefore enzyme there will be 112bp and 108bp band (the band agarose gel electrophoresis of 112bp and 108bp size cannot distinguish the band being therefore shown as about 110bp) after cutting; Heterozygous mutant contains restriction enzyme site owing to only having an allelotrope, therefore the 220bp band that do not cut in addition except a band of about 110bp after cutting of enzyme; And wild-type does not comprise this restriction enzyme site due to two allelotrope, therefore only has the band of 220bp.

Fig. 6-C is the c.1091-2A>G different states individual restriction endonuclease endonuclease reaction product electrophoretic effects figure that suddenlys change.In figure, upper row is depicted as electrophoresis loading wells, and the 1st swimming lane is electrophoresis standard substance Marker I, and after electrophoresis, its stripe size is followed successively by 600bp, 500bp, 400bp, 300bp, 200bp and 100bp; 2nd swimming lane is the c.1091-2A>G heterozygous carriers's digestion products that suddenlys change, and has 3 bands, is followed successively by 215bp, 148bp and 67bp; 3rd swimming lane is not containing the individual digestion products of c.1091-2A>G wild-type that suddenlys change, and only has 1 band, 215bp; 4th swimming lane is the c.1091-2A>G homozygote patient digestion products that suddenlys change, and has 2 bands, 148bp and 67bp.

Because c.1091-2A>G sudden change causes A base mutation to be G, the base sequence that restriction endonuclease CdiI identifies is CATCG, therefore use this restriction endonuclease to carry out endonuclease reaction to CYP4V2-9 and just there will be three kinds of possible results: homozygous mutation is because two allelotrope are all containing restriction enzyme site, and therefore enzyme there will be 67bp and 148bp two bands after cutting; Heterozygous mutant contains restriction enzyme site owing to only having an allelotrope, therefore the 215bp band that do not cut in addition except 67bp and 148bp two bands after cutting of enzyme; And wild-type does not comprise this restriction enzyme site due to two allelotrope, therefore only has the band of 215bp.

2, enzyme cuts result verification

For three mutational sites, choose homozygous mutation, heterozygous mutant and the wild-type sample identified through restriction endonuclease reaction respectively, adopt DNA sequencing to verify, sequencing result adopts Chrome software to analyze.Order-checking mutational site is analyzed and is seen Fig. 7,8,9, Fig. 7 is homozygous mutation, heterozygous mutant and wild-type sequencing result c.802-8_810del17insGC, Fig. 8 is homozygous mutation, heterozygous mutant and wild-type sequencing result c.992A>C, and Fig. 9 is homozygous mutation, heterozygous mutant and wild-type sequencing result c.1091-2A>G.

C.802-8_810del17insGC embodiment 5 detects BCD disease related gene CYP4V2 gene, the test kit c.992A>C and c.1091-2A>G suddenlyd change and application thereof

1, the composition of test kit

(1) for the PCR reaction reagent of amplified sample goal gene

The DNA sequence data of CYP4V2 gene is see NG_007965.1.

PCR reaction reagent comprises the reagent needed for the various PCR reaction such as specific PCR amplimer and buffer, dNTP, DNA enzymatic, primer, template, water; When carrying out PCR reaction, except primer, also has the reagent needed for the various PCR reaction such as buffer, dNTP, DNA enzymatic, primer, template, water, general in order to simplify PCR operation steps, by first admixed together for the reagent needed for various PCR reaction, be mixed with Mastermix and then use, namely simplifying PCR operation steps, decrease pollution by way of.In the present embodiment, pcr amplification reagent adopts the concrete composition of Mastermix(of Beijing Tian Gen biotech firm to comprise Taq archaeal dna polymerase, dNTPs, MgCl ₂, reaction buffer, PCR reaction toughener and optimize agent and stablizer, concentration is 2 ×).

Specific PCR amplimer comprises specificity amplification primer I, specificity amplification primer II and specificity amplification primer III.

Specificity amplification primer I:

CYP4V2-7F1：agcctatgttgtcgaaatgt，

CYP4V2-7F2：aaaagcaagtcaagaaaggc；

C.802-8_810del17insGC primer pair CYP4V2-7F1 and F2 correspondence detects sudden change, and product is CYP4V2-7, and size is 246bp;

Specificity amplification primer II:

CYP4V2-8F1：cacagtgcagtcatcaaatc，

CYP4V2-8F2：ccaaacataccaaacacgtc；

C.992A>C primer pair CYP4V2-8F1 and CYP4V2-8F2 correspondence detects sudden change, and product is CYP4V2-8, and size is 220bp;

Specificity amplification primer III:

CYP4V2-9F1：ctttttagatgtctgcaccc，

CYP4V2-9F2：agcatacttacccacttcac；

C.1091-2A>G primer pair CYP4V2-9F1 and CYP4V2-9F2 correspondence detects sudden change, and product is CYP4V2-9, and size is 215bp.

(2) reagent of endonuclease reaction

The reagent of endonuclease reaction mainly comprises restriction enzyme and damping fluid.

Restriction enzyme: CdiI and ApaI;

The restriction enzyme that described specificity amplification primer I is corresponding is CdiI, and the restriction enzyme of described specificity amplification primer II correspondence is ApaI, and the restriction enzyme of described specificity amplification primer III correspondence is CdiI.

Damping fluid (CutSmart Buffer): 50mM Potassium ethanoate, 20mM Tris-acetate, 10mM magnesium acetate, 100 μ g/ml bovine serum albumins (BSA), concentration is 10 ×, 25 DEG C time, pH value is 7.9.

2, using method

Mainly comprise the steps:

A gets blood to be measured, extracts complete genome DNA;

B for template with the complete genome DNA in step a, carries out pcr amplification reaction with above-mentioned specificity amplification primer I, specificity amplification primer II and specificity amplification primer III for primer respectively, obtains pcr amplification reaction product;

The pcr amplification reaction product that c will obtain in step b, carries out endonuclease reaction with the reagent of described endonuclease reaction respectively, obtains digestion products;

D carries out contrast to the digestion products obtained in step c and DNA standard molecular weight to be identified, determines whether there is the sudden change of CYP4V2 gene.

Concrete grammar is see embodiment 1,2,3,4.

What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.

Claims (6)

1. for detecting the test kit of CYP4V2 transgenation, it is characterized in that, described test kit comprises the PCR reaction reagent for amplified sample DNA, described PCR reaction reagent comprises the specificity amplification primer I for detecting c.802-8_810del17insGC mutational site, the upstream primer sequence of described specificity amplification primer I is as shown in SEQ ID NO:4, and the downstream primer sequence of described specificity amplification primer I is as shown in SEQ ID NO:5; Described PCR reaction reagent also comprises the specificity amplification primer II for detecting c.992A>C mutational site, the upstream primer sequence of described specificity amplification primer II is as shown in SEQ ID NO:6, and the downstream primer sequence of described specificity amplification primer II is as shown in SEQ ID NO:7; Described PCR reaction reagent also comprises the specificity amplification primer III for detecting c.1091-2A>G mutational site, the upstream primer sequence of described specificity amplification primer III is as shown in SEQ ID NO:8, and the downstream primer sequence of described specificity amplification primer III is as shown in SEQ ID NO:9; Described test kit also comprises the reagent of endonuclease reaction, the reagent of described endonuclease reaction comprises restriction enzyme, the described corresponding restriction enzyme that c.802-8_810del17insGC suddenlys change is CdiI, the described corresponding restriction enzyme that c.992A>C suddenlys change is ApaI, and the described corresponding restriction enzyme that c.1091-2A>G suddenlys change is CdiI.

2. test kit according to claim 1 is for detecting the method for CYP4V2 transgenation, and described method is the method for non-diagnostic and therapeutic purpose, it is characterized in that, comprises the step of carrying out as follows:

A gets sample to be detected, extracts complete genome DNA;

B for template with the complete genome DNA in step a, adds described PCR reaction reagent, carries out pcr amplification reaction, obtain pcr amplification reaction product;

The pcr amplification reaction product obtained in the step b reagent of described endonuclease reaction is carried out endonuclease reaction by c, obtains digestion products;

According to the digestion products of step c gained, d judges whether sample to be detected exists the sudden change of CYP4V2 gene.

3. method according to claim 2, is characterized in that, in step b, in described PCR reaction reagent, the upstream primer of PCR primer and the mol ratio of downstream primer are 1:1.

4. method according to claim 2, is characterized in that, in step b, the condition of PCR reaction is: react and carry out on ABI9700 thermal cycler, 95 DEG C of denaturation 5min, then according to 95 DEG C of sex change 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 30sec is a circulation, after carrying out 34 circulations, 95 DEG C of sex change 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 5min, preserve PCR primer after completion of the reaction at 4 DEG C.

5. method according to claim 2, it is characterized in that, in step c, the ratio of the restriction enzyme in described PCR reaction product and described endonuclease reaction reagent is 1ug:5U, the condition of described endonuclease reaction is: react and carry out in 37 DEG C of thermostat water baths, and the reaction times is 12h.

6. method according to claim 2, is characterized in that, in steps d, carries out agarose gel electrophoresis to the digestion products of step c gained, judges whether sample to be detected exists the sudden change of CYP4V2 gene according to electrophoresis result.