CN103602751B - Gene chip and kit for detecting rare Mediterranean anemia genes - Google Patents
Gene chip and kit for detecting rare Mediterranean anemia genes Download PDFInfo
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Abstract
The invention relates to a gene chip and kit for detecting rare Mediterranean anemia genes. The gene chip for detecting rare Mediterranean anemia genes comprises a substrate and oligonucleotide probes fixed on the substrate, wherein the oligonucleotide probes comprise normal control oligonucleotide probes disclosed as SEQ ID NO:1-2 corresponding to non-deletion alpha-Mediterranean anemia gene, normal control oligonucleotide probes disclosed as SEQ ID NO:3-4 corresponding to point mutant beta-Mediterranean anemia gene, oligonucleotide probes disclosed as SEQ ID NO:5-13 for detecting non-deletion alpha-Mediterranean anemia gene, and oligonucleotide probes disclosed as SEQ ID NO:14-40 for detecting point mutant beta-Mediterranean anemia gene. The gene chip and kit can detect 9 rare non-deletion alpha-Mediterranean anemia genes and 27 rare point mutant beta-Mediterranean anemia genes at one time, and create conditions for more comprehensive screening of point mutant Mediterranean anemia genes.
Description
Technical field
The present invention relates to thalassemic diagnositc equipment, particularly relate to a kind of gene chip and test kit of rare thalassemia gene test.
Background technology
Thalassemia (Thalassemia, poor hereinafter referred to as ground) is because patient certain or some globin chain synthesis rate reduce, and causes some peptide chains to lack, and other peptide chains relatively too much, occur the imbalance of quantity of peptide chains resulted and the hemolytic anemia that causes.Ground poorly mainly contains two types, is respectively the synthesis of α-globin chain and subtracts and lack the poor and beta-globin synthesis in the α-ground caused and subtract that to lack the β-ground caused poor.
α-ground poor (comprising poor and non-deletion type α-ground, absence type α-ground poor) is one of modal single gene inheritance disease in the world, in China except the poor (-α in common absence type α-ground
3.7,-α
4.2with--
sEA) outside, also have small part to be because α-globin point mutation causes, this kind of α-ground is poor poor also referred to as non-deletion type α-ground.
α-globin point mutation type causes the poor one of the main reasons of failing to pinpoint a disease in diagnosis in clinical α ground.Because the transgenation great majority that non-deletion type α-ground is poor occur in (α 2 or α 1) on 1 gene, another 1 α-gene function can not be made impaired, therefore be commonly defined as α
+-ground is poor, and heterozygote generally shows as light-duty or silent oscillation, but compound α
0time Hb H can be caused sick, seriously also can cause Hb H fetus edema syndromes.In China, reported that 12 kinds of non-deletion type α-ground were poor at present, on existing market, main only non-deletion type α-ground that inspection 3 kinds is common is poor, (α
cS, α
qS, α
wS), all the other 9 kinds rare type non-deletion type α-ground are poor in table 1.
The rare non-deletion type α-ground of table 1 China Report 9 kinds of crossing is poor
Poor some areas being mainly distributed in (comprising point mutation β-ground poor poor with absence type β-ground) provinces and cities such as Guangdong, Guangxi, Yunnan, Hainan, Taiwan, Hong Kong and adjacent provinces and regions in China, β-ground.It is reported that in the poor carrying rate in β-ground, Guangdong be 1.83%-3.36%, Guangxi and Hainan are even higher.
β-ground is poor mainly caused by beta-globin point mutation.It is poor that the poor heterozygote in β-ground is usually expressed as light-duty β-ground, and the poor homozygote in β-ground or double heterozygote are usually expressed as heavyly poor.Up to the present, the whole world has found the generation that more than 200 kind of beta-globin gene mutation can cause β-ground poor, and wherein southern china report has 46 kinds, and existing main only inspection on the market 19 kinds of common point mutation β-ground are poor, all the other rare β point mutation 27 kinds, refer to table 2.
The rare point mutation β-ground of table 2 China Report 27 kinds of crossing is poor
Summary of the invention
The object of the present invention is to provide a kind of gene chip of rare thalassemia gene test and the rare non-deletion type α-ground of above-mentioned 9 kinds of test kit one-time detection is poor and 27 kinds rare saltant type β-ground poor.
Technical scheme of the present invention is for providing a kind of gene chip of rare thalassemia gene test, and comprise a substrate and be fixed on described suprabasil oligonucleotide probe, described oligonucleotide probe comprises:
Normal control oligonucleotide probe corresponding to non-deletion type gene of alpha thalassemia, its base sequence is as SEQ ID NO:1-2;
Normal control oligonucleotide probe corresponding to point mutation β-thalassemia gene, its base sequence is as SEQ ID NO:3-4;
For detecting the oligonucleotide probe of non-deletion type gene of alpha thalassemia, its base sequence is as SEQ ID NO:5-13;
For the oligonucleotide probe of check point sudden change β-thalassemia gene, its base sequence is as SEQ ID NO:14-40.
Preferably, in the gene chip of above-mentioned rare thalassemia gene test, described substrate is nylon membrane.
Another technical scheme of the present invention comprises for providing test kit described in a kind of test kit of rare thalassemia gene test: the gene chip of the rare thalassemia gene test described in any one of claim 1-2, and described test kit also comprises PCR reaction solution; Described PCR reaction solution comprises two pairs of primers of amplification non-deletion type gene of alpha thalassemia: primer A1F:SEQ ID NO:41; Primer A1R:SEQ ID NO:42; Primer A2F:SEQ ID NO:43; Primer A2R:SEQ ID NO:44;
Described PCR reaction solution also comprises two pairs of primers of amplification point mutation β-thalassemia gene: primer BF1:SEQ ID NO:45; Primer BR1:SEQ ID NO:46; Primer BF2:SEQ ID NO:47; Primer BR2:SEQ ID NO:48.
Preferably, in the test kit of above-mentioned rare thalassemia gene test, described oligonucleotide probe is fixed on nylon membrane.
Preferably, in the test kit of above-mentioned rare thalassemia gene test, described primer 5 ' holds mark vitamin H.
Preferably, in the test kit of above-mentioned rare thalassemia gene test, the described each component concentration of PCR reaction solution is: deionized water: 14.22 μ L; 10 × PCR buffer:2.5 μ L; 25mM MgCl
2: 0.5 μ L; 2.5mM dNTP:2 μ L; 10mM dUTP:0.5 μ L; 100 μMs of primer A1F:0.05 μ L; 100 μMs of primer A1R:0.05 μ L; 100 μMs of primer A2F:0.07 μ L; 100 μMs of primer A2R:0.07 μ L; 100 μMs of primer BF1:0.07 μ L; 100 μMs of primer BR1:0.07 μ L; 100 μMs of primer BF2:0.05 μ L; 100 μMs of primer BR2:0.05 μ L; 5U/ μ L Taq enzyme: 0.5 μ L; 1U/ μ L UNG enzyme: 0.3 μ L.
Beneficial effect of the present invention: can the rare non-deletion type α-ground of one-time detection 9 kinds poor and 27 kinds of rare point mutation β-ground are poor, in order to screen more comprehensively, point mutation ground is poor creates condition, the thalassemic loss that reduction normal blood examination method and common type gene screening cause, reduces or avoids the birth of major thalaseemia infant; Realize detecting the poor and poor genetic flaw in point mutation β-ground in Chinese population rare non-deletion type α-ground simultaneously, totally 36 kinds, make up the detection limitation of currently available products.
The present invention detects target according to DNA chip, chooses specific gene sequences as probe from genes involved database; Carry out probe sequence and topological design thereof according to selected nucleotide sequence, make that designed probe combinations is strong to the hybrid specificities of testing gene, cognation between probe is good, the optimization of probe array and space layout thereof.Present invention applicant is on basis reasonable in design, and constantly by a large amount of experiments adjustment, the probe specificity obtained is strong, and stability is high, and the multiple pcr amplification of the present invention can react under a condition, has greatly saved lab resources.
Embodiment
By describing technology contents of the present invention, structural attitude in detail, being realized object and effect, be explained in detail below in conjunction with embodiment.
Embodiment 1
1, the chip of the present invention's exploitation is based upon on the basis of membrane DNA chip
Gene chip is made up of sheet glass or nylon membrane and probe array fixed thereon, the two ultimate principle is similar, the complicated process of preparation of glass-chip, testing process is loaded down with trivial details, especially signal detection needs laser scanner, directly results in its use cost high, can not effectively be promoted in market particularly clinical detection, therefore its R&D direction is mainly for institution of scientific research; The exploitation of membrane DNA chip then has the clear superiorities such as preparation is relatively simple, easy and simple to handle, with low cost, is extremely conducive to the popularization in market, more can the industrialization of the Study of the Realization achievement quickly and efficiently.
2, the design of gene chip of the present invention
Detect target according to DNA chip, from genes involved database, choose specific gene sequences as probe; Carry out probe sequence and topological design thereof according to selected nucleotide sequence, make that designed probe combinations is strong to the hybrid specificities of testing gene, cognation between probe is good, the optimization of probe array and space layout thereof; Its major advantage can detect 9 kinds simultaneously non-deletion type α-ground is poor and 27 kinds of point mutation β-ground are poor, simple and easy to do, technical requirements is low, and chip is not by the restriction of probe molecule size kind, can make the chip meeting object easily according to the requirement of user.
Probe on the film bar of gene chip of the present invention puts in order as shown in table 3, according to the 9 kinds of non-deletion type α-ground selected are poor and 27 kinds of saltant type β-ground are poor, designing probe array (10 × 4).
Table 3 film bar site figure
| α13M | α30N | α31N | 19N | 30N | +32M | 13-14M | 40-41M | 89-93M | IVS-I-2M |
| α43-44M | α30M | α31M | 19M | 30M | 53M | 15-16M | 41-43M | Cap+8M | IVS-I-128M |
| α49M | α74M | α118M | -73M | -28M | 95M | 37M | 54-58M | Cap+39M | IVS-Ⅱ-2M |
| α59M | α78M | -31M | -50M | -90M | 112M | 38M | 71-72M | Poly?AM | IVS-Ⅱ-5M |
Note: 1, each grid represents a kind of probe, in order to the poor genotype in ground that checkout and diagnosis is different.That 2, marks " α " before probe is the poor probe in α-ground, and all the other are the poor probe in β-ground.
As shown in table 4, it is the meaning of each site representative and the relation with normal control thereof on film bar.
Table 4
3, primer of the present invention, probe design and screening
3.1, in Genebank database, α and beta-globin gene order is transferred, design pcr amplification with DNAStar and Oligo6.0 and obtain target DNA fragment, in these fragments, design specific recognition 36 kinds of poor probes in ground to be checked simultaneously, require that each probe Tm value difference is no more than 5 DEG C, there is optimum hybridization sensitivity and specificity at the same temperature.Primer and probe are synthetic oligonucleotide, are synthesized by Shanghai Ying Jun Bioisystech Co., Ltd.Checked by associate after sequent synthesis, be then diluted to desired concn as required.Being screened by great many of experiments can efficiently 4 pairs of primers of specific amplified and the probe of 36 energy specific hybridizations.The change of this primer and probe length or position all can reduce the sensitivity of test kit of the present invention, specificity and repeatability, and therefore primer and probe sequence are protection contents of the present invention.Primer and probe sequence are as shown in table 5 and table 6.
Table 5 α and β primer sequence
Table 6 α and β probe sequence
3.2, other concentration of component of primer, concentration and probe concentration and reaction system are determined
(1) utilize orthogonal test method, contrasted by great many of experiments, finally determine that optimum PCR reaction system is as shown in table 7.
Table 7 test kit PCR of the present invention reaction solution is filled a prescription
| Reagent | 1 person-portion (μ L) |
| Water | 14.22 |
| 10×PCR?buffer（Qiagen） | 2.5 |
| 25mM?MgCl2（Qiagen） | 0.5 |
| 2.5mM?dN（U）TP | 2 |
| 10mMdUTP | 0.5 |
| 100μM?A1F | 0.05 |
| 100μM?A1R | 0.05 |
| 100μM?A2F | 0.07 |
| 100μM?A2R | 0.07 |
| 100μM?BF1 | 0.07 |
| 100μM?BR1 | 0.07 |
| 100μM?BF2 | 0.05 |
| 100μM?BR2 | 0.05 |
| 5U/ μ L Hot Star Taq enzyme (Qiagen) | 0.5 |
| 1U/ μ L UNG enzyme | 0.3 |
| Total amount | 21 |
Note: DNA application of sample amount is 4 μ L, total reaction volume is 25 μ L.
(2) determination of PCR reaction conditions
Optimize through great many of experiments contrast, the optimum reaction condition finally determined is:
Annealing temperature and annealing time affect comparatively large on pcr amplification efficiency and specific amplification, the above-mentioned condition optimizing result display annealing temperature non-specific amplification signal that has on the low side causes false positive results; Temperature drift amplification efficiency is on the low side, and sensitivity declines.Specificity of the present invention is good, and amplification efficiency is high, and sensitivity can reach 2ng/ μ L.
(3) determination of hybridization conditions
The interpretation impact of hybridization temperature on end-result is very large, and meeting that hybridization temperature is on the low side causes the probe non-specific binding on PCR primer and film bar, thus is likely mistaken for the positive; The higher meeting of hybridization temperature causes the joint efficiency of object product and object probe to decline, and hybridization signal intensities weakens, thus is likely mistaken for feminine gender.The length of washing film time and developing time also has similar impact to results of hybridization.Tested by series of optimum, the hybridization finally determined, to wash the optimum condition such as film and colour developing as follows:
1) hybridize
Containing getting 15mL plastic centrifuge tube, putting into the film bar (should of film bar jiao with Pencil marks) indicating sample number into spectrum, add all PCR primer in A liquid (2 × SSC, 0.1%SDS) 6-7mL and PCR reaction solution, tighten pipe lid, then the circle that circles round slightly being unscrewed.Centrifuge tube is put into boiling water bath heating 10 minutes (guaranteeing that hybridization solution liquid level is positioned under boiling water bath liquid level completely), take out and tighten lid, put into hybridization 43 DEG C, case hybridization more than 1.5 hours, but be no more than 4 hours.
Get 50mL plastics tubing, add 40mL B liquid (0.5 × SSC, 0.1%SDS) and carry out being preheated to 43 DEG C in hybridization case.
2) film is washed
Take out film bar, move in the 50mL pipe that preheating B liquid is housed, wash 15 minutes (often pipe 40mL solution can wash 4 films at most) in 43 DEG C of jogs simultaneously.
3) develop the color
Prepare Incubating Solution (singly do two films and only need 4 μ LPOD mother liquors, be mixed with 8mL and use liquid, doing four films with 6 μ LPOD mother liquors, can be mixed with 12mL and use liquid) by A liquid: POD=2000:1, room temperature jog soaks 30 minutes, discards POD solution.Twice is washed, each 5 minutes with A liquid chamber temperature jog.Wash film 1-2 minute by C liquid (0.1mol/L Trisodium Citrate, pH5.0) room temperature, prepare nitrite ion (each component ratio: 19mL C liquid, 1mL TMB, the H of 2 μ L30% simultaneously
2o
2).Film bar is soaked in lucifuge colour developing in nitrite ion and gets final product observations in 5 to 10 minutes.
The result of use of embodiment 2 test kit of the present invention
The mutated-genotype that the poor detection reagent in ground in the market can detect is very limited, and thus some light-duty rare heterozygous mutation is difficult to realize.Such as have bibliographical information: Mr. and Mrs both sides, a side is rare CD37M(β
0) sudden change, the opposing party is common CD41-42M(β
0) sudden change, the first tire is the heavy β poor infant in ground (CD37/CD41-42), monthly needs lasting treatment of blood transfusion 1 time, and 3 years old is dead.Rear bosom second fetus requires that hospital diagnoses, but detects Mr. and Mrs both sides' genotype with the poor test kit in common ground, and can only detect a side is common CD41-42M, and to fail to detect the opposing party be rare CD37M.Therefore detecting rare type ground poor is do not detect Common genes type in poor examination over the ground, but has the patient of presumptive signs to carry out supplementing the effective ways detected.
Test kit of the present invention adopts PCR-revert dot blot hybridization, energy one-time detection 9 kinds rare non-deletion type α ground is poor and 27 kinds of rare β ground are poor, it is domestic that also do not have can the product of one-time detection these 36 kinds poor sudden change rarely, test kit of the present invention can reduce poor undetected risk greatly, create conditions for carrying out poor examination more comprehensively, for pre-marital, antenatal detection and the pregnancy period fetus thalassemia diagnosis the foundation of science is provided.
Utilize test kit of the present invention to detect and clinically have poor symptom, but be detected as negative sample 400 example by the poor detection kit in common ground, all sample standard deviation employing gold standard sequencing carry out sequence verification, and detected result is in table 8, and statistical study comparing result is in table 9.
Table 8 uses test kit of the present invention to detect, and poor in clinical sample a situation arises
Table 9 test kit detected result of the present invention and sequencing result contrast
Note: sequencing result is positives refers to that in test kit sensing range of the present invention, genotype is positive, and feminine gender refers to that other genotype samples outside test kit sensing range of the present invention are positive or negative.
Test kit of the present invention detects 400 routine clinical samples and the contrast of gold standard sequencing result, and accuracy is 100%; To other β ground of non-invention test kit sensing range are poor and non-deletion type α genotype positive and negative sample, test kit detected result of the present invention is feminine gender, and negative match-rate is 100%, and specificity is 100%.
Test kit of the present invention detects 400 routine clinical sample results and gold standard sequencing result comparative analysis statistics, in table 10.Table 10 is that in 400 routine clinical samples, each genotype sequencing result and test kit detected result of the present invention meet situation statistics.
Table 10
Can see that 400 examples have the poor genotype distribution situation in poor symptom sample various places from upper table 10,36 kinds of ground in test kit sensing range of the present invention are poor all has existence, and test kit detection accuracy of the present invention and specificity are 100%, illustrate that only detecting common 25 kinds or less genotype can not meet poor examination demand clinically, detect that 36 kinds of ground that test kit of the present invention comprises are poor to be necessary.
The performance index of test kit of the present invention:
Sensitivity for analysis: the minimum concentration that test kit of the present invention can stablize the genomic dna detected is 2ng/ μ L;
Measure accuracy: with the sample of Product checking known mutations of the present invention, result transgenation accuracy is 100%.；
Analyze specificity: detect non-thalassemic DNA sample, result display is all normal, and specificity is 100%.；
Stability: this product uses before the deadline and can meet above each index completely.
The use of embodiment 3 test kit of the present invention
1, test kit main component of the present invention is as table 11.
Table 11
Need other main agents (box) used
Whole blood genome extracts reagent, recommends to select: the whole blood DNA of QIAGEN company extracts test kit or phenol-chloroform reagent.
1.1 conditions of storage: test kit I is placed in less than-18 DEG C preservations; Test kit II is placed in 2-8 DEG C of preservation.If open packaging each component when separately preserving, except meeting respective temperature preservation condition, need pay special attention to TMB should keep in Dark Place.
Validity period: 6 months
1.2 are suitable for instrument
PCR instrument (unexpected rival 9600) Hybridization Oven (FinePCR Combi-H12)
1.3 sample requirements
Test kit samples sources of the present invention is anticoagulated whole blood, and antithrombotics used is Sodium Citrate or EDTA, can not use anticoagulant heparin.
Sample collection: venous blood samples 5mL, puts into the pipe containing antithrombotics, has marked name and the numbering equal samples information of sample.
Blood sample is preserved: anticoagulated whole blood is placed in room temperature and is no more than 24 hours, and 2-8 DEG C of preservation is no more than one month, and less than-18 DEG C Refrigerator stores are no more than 2 years, and-70 DEG C of refrigerators can be preserved for a long time, should avoid multigelation during freezen protective.Blood sample transports: need add ice bag sealing with curling stone or bubble chamber during anticoagulated whole blood transport, should ensure that ice bag does not thaw, and the time limit in transit be no more than 72 hours.
2, the method for inspection
2.1, DNA obtains
The extracting method of test kit of the present invention to human gene group DNA does not specify requirement, and general Available experimental room ordinary method (phenol-chloroform extraction process) or test kit extract human gene group DNA, and the whole blood DNA of test kit recommendation QIAGEN company extracts test kit.
The mensuration of PCR front template DNA concentration and purity can adopt nucleic acid quantification instrument or ultraviolet spectrophotometer.Test kit of the present invention requires that the concentration of genomic dna to be checked is 2-200ng/ μ L, and purity (A260/A280) is 1.7 ~ 2.0.
2.2, pcr amplification
Take out PCR reaction solution, tube wall carries out mark, the low-speed centrifugal several seconds, then add the testing sample DNA4 μ L extracted respectively, reaction is totally 25 μ L.
A pipe PCR reaction solution is separately got in each experiment, with 4 μ L pure water for template, makes blank.
PCR increases by following condition:
2.3, hybridize
Get 15mL plastic centrifuge tube, put into the film bar (should of film bar jiao with Pencil marks) indicating sample number into spectrum, add all in A liquid (2 × SSC, 0.1%SDS) 5-6mL and PCR reaction solution I, II (two pipe totally 50 μ L) PCR primer, tighten pipe lid, then the circle that circles round slightly is unscrewed.Centrifuge tube is put into boiling water bath heating 10 minutes (guaranteeing that hybridization solution liquid level is positioned under boiling water bath liquid level completely), take out and tighten lid, put into hybridization 43 DEG C, case hybridization more than 1.5 hours, but be no more than 4 hours.
Get 50mL plastics tubing, add 40mL B liquid (0.5 × SSC, 0.1%SDS) and carry out being preheated to 43 DEG C in hybridization case.4.4, film is washed
Take out film bar, move in the 50mL pipe that preheating B liquid is housed, wash 15 minutes (often pipe 40mL solution can wash 4 films at most) in 43 DEG C of jogs simultaneously.
2.4, develop the color
Prepare Incubating Solution (singly do two films and only need 4 μ LPOD mother liquors, be mixed with 8mL and use liquid, doing four films with 6 μ LPOD mother liquors, can be mixed with 12mL and use liquid) by A liquid: POD=2000:1, room temperature jog soaks 30 minutes, discards POD solution.Twice is washed, each 5 minutes with A liquid chamber temperature jog.Wash film 1-2 minute by C liquid (0.1mol/L Trisodium Citrate, pH5.0) room temperature, prepare nitrite ion (each component ratio: 19mL C liquid, 1mL TMB, the H2O2 of 2 μ L30%) simultaneously.Film bar is soaked in lucifuge colour developing in nitrite ion and gets final product observations in 5 to 10 minutes.
2.5, result interpretation
2.5.1 the probe, on film bar puts in order as shown in table 12.
Table 12
| α13M | α30N | α31N | 19N | 30N | +32M | 13-14M | 40-41M | 89-93M | IVS-I-2M |
| α43-44M | α30M | α31M | 19M | 30M | 53M | 15-16M | 41-43M | Cap+8M | IVS-I-128M |
| α49M | α74M | α118M | -73M | -28M | 95M | 37M | 54-58M | Cap+39M | IVS-Ⅱ-2M |
| α59M | α78M | -31M | -50M | -90M | 112M | 38M | 71-72M | Poly?AM | IVS-Ⅱ-5M |
Note: 1, above site the last letter " N " representative is normal, and " M " represents sudden change.That 2, marks " α " before probe is the poor probe in α-ground, and all the other are the poor probe in β-ground.
2.5.2, on film bar, corresponding relation that is normal and mutational site is as shown in table 4.
Test kit of the present invention carries out qualitative analysis to detected object, occurs whether signal judges with detection site, and the power of signaling point can not provide the reference of any quantitative aspect.
2.5.3, the explanation of assay
1). the film bar result of blank should be all sites and does not develop the color, otherwise this experiment may be polluted, and should all reform.
2). all clinical samples 4 normal locations should have at least 2 sites to have blue spot to occur, otherwise may test unsuccessful, and this sample should heavily be examined; If heavily examine result still so, then should contact solution with test kit manufacturer technician.
2.5.4, product performance
A, sensitivity for analysis: the minimum concentration that this product can stablize the genomic dna detected is 2ng/ μ L;
B, measure accuracy: with the sample of this Product checking known mutations or deletion type, result transgenation or deletion type, accuracy is 100%.；
C, analysis specificity: detect non-thalassemic DNA sample, result display is all normal, and specificity is 100%.；
D, stability: this product uses before the deadline and can meet above each index completely.
3, interpretation of result
According to the position that spot on film bar manifests, read the genotype information that corresponding position marks.See following Pictorial examples:
3.1, normal (N/N) is as table 13.
Table 13
3.2, the poor heterozygote in non-deletion type α-ground (routine α 30M/N) is as table 14.
Table 14
3.3, the poor homozygote in non-deletion type α-ground (routine α 31M/ α 31M) is as table 15.
Table 15
3.4, the poor heterozygote in β-ground (routine 19M/N) table 16.
Table 16
3.5, the poor homozygote in β-ground (routine 30M/30M) table 17.
Table 17
3.6, non-deletion type α ground poor compound β-ground poor (routine α 43-44M, 15-16M) table 18.
Table 18
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize description of the present invention to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (4)
1. a test kit for rare thalassemia gene test, is characterized in that, comprises gene chip and the PCR reaction solution of rare thalassemia gene test;
Described gene chip comprises a substrate and is fixed on described suprabasil oligonucleotide probe, and described substrate is nylon membrane, and described oligonucleotide probe comprises:
Normal control oligonucleotide probe corresponding to non-deletion type gene of alpha thalassemia, its base sequence is as SEQ ID NO:1-2;
Normal control oligonucleotide probe corresponding to point mutation β-thalassemia gene, its base sequence is as SEQ ID NO:3-4;
For detecting the oligonucleotide probe of non-deletion type gene of alpha thalassemia, its base sequence is as SEQ ID NO:5-13;
For the oligonucleotide probe of check point sudden change β-thalassemia gene, its base sequence is as SEQ ID NO:14-40;
Described PCR reaction solution comprises two pairs of primers of amplification non-deletion type gene of alpha thalassemia:
Primer A1F:SEQ ID NO:41;
Primer A1R:SEQ ID NO:42;
Primer A2F:SEQ ID NO:43;
Primer A2R:SEQ ID NO:44;
Described PCR reaction solution also comprises two pairs of primers of amplification point mutation β-thalassemia gene:
Primer BF1:SEQ ID NO:45;
Primer BR1:SEQ ID NO:46;
Primer BF2:SEQ ID NO:47;
Primer BR2:SEQ ID NO:48.
2. the test kit of rare thalassemia gene test according to claim 1, it is characterized in that, described oligonucleotide probe is fixed on nylon membrane.
3. the test kit of rare thalassemia gene test according to claim 1, is characterized in that, described primer 5 ' holds mark vitamin H.
4. the test kit of rare thalassemia gene test according to claim 1, is characterized in that, the described each component concentration of PCR reaction solution is:
Deionized water: 14.22 μ L;
10×PCR?buffer：2.5μL；
25mM?MgCl
2：0.5μL；
2.5mM?dNTP：2μL；
10mM?dUTP：0.5μL；
100 μMs of primer A1F:0.05 μ L;
100 μMs of primer A1R:0.05 μ L;
100 μMs of primer A2F:0.07 μ L;
100 μMs of primer A2R:0.07 μ L;
100 μMs of primer BF1:0.07 μ L;
100 μMs of primer BR1:0.07 μ L;
100 μMs of primer BF2:0.05 μ L;
100 μMs of primer BR2:0.05 μ L;
5U/ μ L Taq enzyme: 0.5 μ L;
1U/ μ L UNG enzyme: 0.3 μ L.
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| CN104372100B (en) * | 2014-11-28 | 2017-05-03 | 博奥生物集团有限公司 | Test kit for detecting thalassemia-related gene mutation and application thereof |
| CN104988218A (en) * | 2015-06-24 | 2015-10-21 | 深圳益生堂生物企业有限公司 | Nucleic acid membrane strip and reagent kit for non-deletion type alpha-thalassemia gene detection and detection method |
| CN104894279B (en) * | 2015-06-25 | 2016-10-19 | 北京嘉宝仁和医疗科技有限公司 | Gene of alpha thalassemia mutation detection kit |
| CN105420233B (en) * | 2015-12-08 | 2020-05-15 | 海南医学院附属医院 | HBB gene mutation and HLA typing detection kit |
| CN105803075B (en) * | 2016-04-14 | 2019-09-13 | 亚能生物技术（深圳）有限公司 | A kind of gene of alpha thalassemia detection kit |
| CN105861661B (en) * | 2016-04-14 | 2019-07-30 | 亚能生物技术（深圳）有限公司 | -α21.9Deletion form gene of alpha thalassemia detection kit |
| CN105755137B (en) * | 2016-04-14 | 2020-07-07 | 亚能生物技术（深圳）有限公司 | α -thalassemia gene detection kit |
| CN113846150B (en) * | 2020-06-28 | 2024-07-26 | 深圳华大生命科学研究院 | Nucleic acid molecules and uses thereof |
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| CN102943115B (en) * | 2012-11-28 | 2013-09-25 | 亚能生物技术（深圳）有限公司 | Nucleic acid membrane strip and kit for alpha and beta mediterranean anemia gene detection |
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