CN109112200A - It is a kind of for detecting the genetic chip, amplifing reagent and kit of alpha Thalassemia - Google Patents
It is a kind of for detecting the genetic chip, amplifing reagent and kit of alpha Thalassemia Download PDFInfo
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Abstract
" for detecting the genetic chip, amplifing reagent and kit of α-thalassemia " of the invention, belong to molecular diagnostic techniques, combines hybridization (RDB) technology quickly to detect 3 kinds of non-deletion type (α of α-thalassemia simultaneously using Direct PCR (Direct PCR)CSα、αQSα、αWSα) and 4 kinds of deletion forms (--SEA、‑‑THAI、‑α4.2With-α3.7)。
Description
Technical field
The present invention relates to the molecular detection technologies of α-thalassemia, utilize Direct PCR (Direct more particularly to a kind of
PCR) 3 kinds of non-deletion type (α of α-thalassemia are quickly detected simultaneously in conjunction with hybridization (RDB) technologyCSα、αQSα、αWSα) and 4 kinds lack
Mistake type (--SEA、--THAI、-α4.2With-α3.7) kit.
Background technique
Thalassemia (thalassemia, referred to as poor), be due to globin gene defect (gene mutation or lack
Lose) make the peptide chain of globin of hemoglobin (hemoglobin, Hb) there are one or more of synthesis to reduce or cannot synthesize, and cause
α-chain/non-alpha-chain proportional imbalance of hemoglobin, and then lead to that hemoglobin is unstable, hematoclasis, hemoglobin yield
Reduce most common lethal, the inherited blood disorders disabled for seriously threatening human health in one group of caused whole world.With α and β
Middle sea anaemia is main Types.
α-thalassemia (hereinafter referred to as α-ground is poor), molecular basis are to be located at No. 16 the short arm of a chromosome ends
The congenital hereditary defect of alpha globin gene cluster on the site 16p13.3, according to the missing of gene or catastrophe and clinical table
Existing severity by α poor can be divided into silent oscillation (- α/α α), light-duty (standard type, --/α α or-α/- α), osculant (HbH
Disease, --/- α or --/αTα) and heavy (Hb Bart's schridde syndrome, --/--) 4 seed types.α-ground it is poor mainly due to
1 or simultaneously 2 alpha globin gene missing causes on item chromosome, small part is as caused by Nondeletion mutation.If lacked
Mistake type α-ground is poor be due on missing item chromosome two functional alpha-genes one of them, then the synthesis of alpha globin chain
Part is suppressed, such as the most common-α in asian population4.2With-α3.7；If two functional alpha genes all lack, such as
It is most common in the crowd of Southeast Asia --SEAAnd --THAI, then the composite part of alpha globin chain is more suppressed.--SEAItself or-
-THAIItself or with-α3.7Or-α4.2Mutual coinheritance can cause the Hb Bart's fetus of lethal or seriously affect life matter
The HbH disease of amount.Southeast hypotype (--SEA) homozygote (--SEA/--SEA) and Thailand's type (--THAI/--THAI) or Southeast Asia it is compound
Thailand's type (--SEA/--THAI), since its 4 alpha globin genes lack or defect, so that being generated entirely without α chain, in fetus
A large amount of γ chain synthesis γ 4 (Hb Bart ' s) occur for the phase, and Hb Bart ' s is high to the affinity of oxygen, causes histanoxia and draws
Schridde syndrome is played, fetus is often dead in miscarriage, stillborn foetus or half an hour after of giving birth to when 30~40 weeks.When Mr. and Mrs both sides are equal
For α when poor gene delection carrier, offspring has 25% chance to suffer from Hb Bart's or HbH disease.HbH patient is clinical
Show as osculant, can in anemia or even disability, seriously affect existence and quality of life；Hb Bart's
Oedema tire involvement infant fail before being carrying out because of severe depletion of oxygen or one birth i.e. death, caused by obstetric complication brought to puerpera
Great physical and mental injury；These are all exerted heavy pressures on to family and society.In China, at least six kinds of non-missings have been reported at present
Type α-poor the gene mutation in ground, wherein tri- kinds of point mutation of WS, QS and CS are most common mutation, highest in thalassemia disease incidence
There is the Hb H patient of 45.8-53.3% with carrying above-mentioned non-deletion type α poor gene in Guangxi, and this kind of patient is than those missings
3 alpha globin genes show more serious clinical symptoms, and anaemia is also more serious.
It is poor over the ground at present to there is no effective treatment method, therefore carried out on a large scale by approach such as pre-marital medical check-up and Prenatal Screenings
Mass screening and diagnosis, discovery carry the man and wife of poor gene, carry out prenatal gene diagnosis in First Trimester, are to pre-natal diagnosis
Poor fetus carries out effective prevention and terminal pregnancy medium and heavyly, is to prevent poor youngster's birth medium and heavyly, improve newborn population element
The effective measures of matter.
The screening of existing thalassemia relies primarily on routine analysis of blood and hemoglobin electrophoresis detection, and routine analysis of blood is used
There are hemoglobin (Hb), average volume of red blood cells (MCV), mean corpuscular hemoglobin to contain in the index of Screening for Thalassemia
Measure the value of (MCH), hemoglobin electrophoresis detection abnormal hemoglobin and HbA2.There was only the individual of apparent hematology phenotype at present
(i.e. MCV < 80fl, MCH < 27pg) and hemoglobin electrophoresis just can further carry out molecular diagnosis extremely, will lead to so big
Poor silent oscillation the carrier (- α in partial ground4.2With-α3.7), the MCV and MCH of these inactives carrier is mostly in normal value
It in range, is easy to miss inspection, so a kind of simple, energy rapid gene parting screening and diagnostic techniques are badly in need of in for α poor screening.
There are many detection method of gene mutation reported at present, including are prolonged based on single-strand conformation polymorphism (SSCP), primer
Stretch technology, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), TaqMan probe technology, denaturing high-performance liquid
Phase chromatography (DHPLC), high-resolution fusion curve detection technique (HRM), DNA direct Sequencing and DNA microarray technology etc..But these
Method detection process is cumbersome, detection time is long, using reagent or expensive equipment and special labeled primer etc., because without
Conducive to clinical extensive popularization and application.
However for the molecular diagnosis of α-thalassemia, Southern Blot hybridization technique is generally acknowledged classical standard
True property macromolecule diagnostic techniques, but sample dosage is big, cumbersome, time-consuming and laborious, detection flux is small, so being not suitable for conventional inspection
It surveys.The relevant patent type of existing non-deletion type alpha Thalassemia genetic test has several, mainly utilizes PCR reversed
In conjunction with Dot hybridization, gene chips, TaqMan probe fluorescent PCR method and high-resolution fusion curve detection technique (HRM).These
PCR diagnostic assays are all that DNA is extracted from blood using blood or amniotic fluid etc. as biomaterial；But it is both time-consuming in this way
The DNA pollution or degradation being easy to cause in extraction process again, cause missing inspection or erroneous detection.Time-consuming, and higher cost, the sample needed
This amount is big, it is difficult to meet that clinical detection is quick, inexpensive requirement.
Thalassemia is had in deletion form α-ground of the prebiotic hall in Shenzhen using the patent and testing product of gene tester
It is extra large anemia gene diagnostic kit and non-deletion type gene of alpha thalassemia detection kit, poor up to α-Mediterranean of peace gene
Blood gene detecting kit and non-deletion type gene of alpha thalassemia detection kit, sub- α-thalassemia base that can be biological
Because of detection kit and non-deletion type gene of alpha thalassemia detection kit, the triumphant general gene of alpha thalassemia inspection in Chaozhou
Test agent box (PCR+ flow hybridization method), these products are both needed to nucleic acid extraction, cannot be quick using Direct PCR (Direct PCR)
3 kinds of non-deletion type (α of α-thalassemia are detected simultaneouslyCSα、αQSα、αWSα) and 4 kinds of deletion forms (--SEA、--THAI、-α4.2With-
α3.7)。
In addition, normal conventional deletion form sample size needed for poor detection method it is more, the sample of outlying area acquisition is transported
It is defeated to need Basic characteristics, three-level packaging system is needed, specimen storage space is big；Bio-safety risk factor is high.To whole blood, amniotic fluid and
The direct augmentation detection of DBS the patent of poor technology there is no, there is no such product in the market.Up to now, clinically not yet occur
Direct PCR method detects 4 kinds of missings of detection and 3 kinds of non-missing α-thalassemia patents and product simultaneously.
There is missing inspection, the erroneous detection α-poor risk in ground in above product and method, lead to the birth of poor infant medium and heavyly,
Heavy burden is brought to patient and its family and society.There is presently no one kind being capable of directly fast and convenient while 4 kinds of detection
The kit of missing and 3 kinds of non-missings, use is very inconvenient, be easy to cause missing inspection or erroneous detection, endangers huge.
Summary of the invention
The technical problems to be solved by the present invention are: Direct PCR (Direct PCR) method combination RDB can be utilized by providing one kind
Technology quickly detects 3 kinds of non-deletion type (α of α-thalassemia simultaneouslyCSα、αQSα、αWSα) and 4 kinds of deletion forms (--SEA、--THAI、-
α4.2With-α3.7) kit.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention are as follows:
A kind of genetic chip of α-thalassemia, which is characterized in that be fixed on the chip:
For detecting 3 kinds of non-deletion type gene of alpha thalassemia αCSα、αQSα、αWSProbe corresponding to α, base sequence
Column are as shown in SEQ ID NO:11-16；And/or
For detecting 4 kinds of deletion form gene of alpha thalassemia --SEA、--THAI、-α4.2With-α3.7Probe, base sequence
Column are as shown in SEQ ID NO:17-20.
Preferably, it is further fixed on the chip:
Negative control probe (NC), base sequence such as SEQIDNO:21；
Positive control probe (AC), base sequence such as SEQIDNO:22,
Color development system controls probe (CC), base sequence such as SEQIDNO:23.
Preferably, the base material of the chip is selected from: nitrocellulose, cellulose acetate, sheet glass, silica gel chip,
The group of nylon membrane, polypropylene screen and miniature magnetic bead composition.
PCR- reverse dot blot hybridization (PCR-Reverse Dot Blot, PCR-RDB) technology, it and tradition ASO are not both film
Upper fixed target DNA is fixed probe substitution, and primary hybridization can carry out screening to multiple mutational sites of tested DNA, have spirit
The advantages that sensitivity is high, specificity is good and accuracy is high.
Based on said chip, the present invention provides a kind of amplification that the non-missing non-anemia gene in α-Mediterranean is detected for PCR
Reagent, it is characterised in that: the primer sets including following nucleotide sequence:
APF:SEQ ID NO.1,
APR:SEQ ID NO.2；
It is used to detect 3 kinds of non-deletion type α-thalassemias in its characteristic sequence and any of the above-described genetic chip for expanding
Gene αCSα、αQSα、αWSProbe specificity corresponding to α combines；
Other PCR amplification components are added in the amplifing reagent, by PCR reaction to sample to be tested or extract to
The DNA of sample directly carries out three kinds of non-missing α-Mediterranean poor gene QS, CS, WS；
The sample to be tested is selected from the anticoagulation cirumferential blood of fresh acquisition, -20 DEG C -- and 80 DEG C save the outmoded of non-multigelation
Property anticoagulation cirumferential blood, culture amniocyte, villus, amniotic fluid, Cord blood, peripheral blood, DBS sample and/or the sample of similar DBS processing
Sheet, saliva, embryo inhereditary material include gamete such as sperm or ovum, the blastomere of cleavage stage embryo, blastaea trophectoderm
Cell, i.e. blastomere, multiple cells or individual cells etc..
Filter paper is the excellent carriers of blood specimen collection and transport.The dry blood cake of filter paper is made in droplets of whole blood on filter paper
(Dried blood spots, DBS), there is good biological stability and safety, convenient for storage and transport.As is produced from China
Preceding screening, the continuous enhancing of neonatal screening dynamics, especially when screening range is expanded to, some regions are remote, medical condition phase
When to backward area, using Dried blood spots (DBS) for patient's collection of specimens, storage and transport and epidemiological survey then
Advantageously.
Preferably, in the PCR amplification reagent reacted for one, contain:
Based on said chip, another group provided by the invention is detected deletion form α-non-anemia gene in Mediterranean for PCR
Amplifing reagent, it is characterised in that: the primer sets comprising following nucleotide sequence:
SEAF:SEQ ID NO.3
SEAR:SEQ ID NO.4
THAIF:SEQ ID NO.5
THAIR:SEQ ID NO.6
4.2F:SEQ ID NO.7
4.2R:SEQ ID NO.8
3.7F:SEQ ID NO.9
3.7R:SEQ ID NO.10
It is used to detect 4 kinds of deletion forms in its characteristic sequence expanded and any genetic chip of claim 1-3
Gene of alpha thalassemia --SEA、--THAI、-α4.2With-α3.7Corresponding probe specificity combines；
Other PCR amplification components are added in the amplifing reagent, by PCR reaction to sample to be tested or extract to
The DNA of sample directly carries out 4 kinds of poor genes in missing α-Mediterranean --SEA、--THAI、-α4.2With-α3.7Thalassemia gene
Detection, the sample to be tested is selected from the anticoagulation cirumferential blood of fresh acquisition, -20 DEG C -- 80 DEG C save the outmoded of non-multigelation
Property anticoagulation cirumferential blood, culture amniocyte, villus, amniotic fluid, Cord blood, peripheral blood, DBS sample and/or the sample of similar DBS processing
Sheet, saliva, embryo inhereditary material include gamete such as sperm or ovum, the blastomere of cleavage stage embryo, blastaea trophectoderm
Cell, i.e. blastomere, multiple cells or individual cells etc..
Preferably, in the PCR amplification reagent reacted for one, contain:
Preferably, each primer molar concentration ratio of the primer sets is as follows:
Preferably, in the PCR amplification reagent reacted for one, contain:
Preferably, 5 ' ends of the primer are marked with marker, for making the band of amplification with the marker.
The present invention also provides a kind of kits for detecting α-thalassemia, it is characterised in that:
Including any genetic chip and any amplifing reagent.
The preparation method of any of the above-described genetic chip, it is characterised in that: steps are as follows:
(1) it prepares probe face liquid: synthetic specific probe is diluted to 10 μM of working solution, in case point sample is used；
(2) fixed probe: chip base film is soaked in 10% EDAC solution and activates 30 minutes, room temperature after pure water
It dries；The upper working solution, 0.5 μ L of every drop are put on the basilar memebrane being activated；After point film, film is placed in room temperature
It is reacted；
(3) it prepares genetic chip: stopping reaction using sodium hydroxide, genetic chip is made；
Wherein, for detecting 3 kinds of non-deletion type gene of alpha thalassemia αCSα、αQSα、αWSProbe corresponding to α, alkali
Basic sequence is as shown in SEQ ID NO:11-16；
For detecting 4 kinds of deletion form gene of alpha thalassemia --SEA、--THAI、-α4.2With-α3.7Probe, base sequence
Column are as shown in SEQ ID NO:17-20；
Preferably, the material of the basilar memebrane of the chip is selected from: nitrocellulose, cellulose acetate, sheet glass, silica gel are brilliant
The group of piece, nylon membrane, polypropylene screen and miniature magnetic bead composition.
The kit of quick detection alpha Thalassemia allele of the present invention further includes in some available reagent boxes
Conventional and necessary component, such as the primer of designed, designed, buffer, enzyme solution, MgCl2With dNTP etc..Specifically, enzyme solution Taq
Polymerase systems, including the thermal starting enzyme system etc. that can be used for direct PCR method；The buffer is blood Direct PCR buffer；
When enzyme solution selects to use treasured bioengineering (Dalian) Co., Ltd MightyAmp producedTMWhen DNA Polymerase, delay
Fliud flushing then preferably and MightyAmpTMThe matched buffer of DNA Polymerase.
α-globin gene order is obtained from GenBank database, and sudden change region is covered according to α-globin gene and is set
Multipair primer is counted, many experiments are carried out；It using orthogonal test method, compares through a large number of experiments, determines PCR amplification condition.
5 ' ends of the above-mentioned kit of the present invention, the primer are marked with the label of biotin or other groups, so that expanding
Containing corresponding label in the product of increasing, to analyze after hybridization results of hybridization.
The above-mentioned kit of the present invention, the genetic chip includes the position mark for positioning probe.
The above-mentioned kit of the present invention, the substrate for the nucleic acid hybond membrane item can be any suitable for immobilized oligonucleotide
Substrate of probe, such as sheet glass, silica gel chip, nylon membrane, nitrocellulose filter, polypropylene screen, miniature magnetic bead etc..The base
Because the probe of chip is fixed on film item.Preferably, the substrate of immobilized oligonucleotide probe is nylon membrane.
The above-mentioned kit of the present invention, the hybridizing reagent include hybridization I liquid, the hybridization II liquid, hybridization III for hybridization
Liquid, enzyme, developing solution composition.The reaction temperature of nucleic acid hybridization is 45 DEG C~54 DEG C, and optimal reaction temperature is 48 DEG C~52 DEG C.
Poor (the 2 kinds of deletion forms in α-ground of quick detection 5 kind common types of the kit of the present invention based on Direct PCR
With 3 kinds of Nondeletion mutations), i.e., deletion form gene of alpha thalassemia in α-thalassemia (--SEA、--THAI) and 3 kinds
Non-deletion type gene of alpha thalassemia (αCSα、αQSα、αWSα), α-thalassemia equipotential base is quickly detected using the kit
The method of cause the following steps are included:
1) sample collection and the preparation of pcr template: a) sample collection: Specimen origin is in anticoagulation cirumferential blood, DBS sample, suede
Hair, amniotic fluid, Cord blood, peripheral blood, saliva, embryo inhereditary material include the ovum of gamete such as sperm or ovum, cleavage stage embryo
Blastomere, blastaea Trophectoderm cells, i.e. blastomere, multiple cells or individual cells etc..Deng；B) preparation of pcr template: on
Sample is stated directly as template, DNA of the above-mentioned sample through nucleic acid extraction can also be used as template.
2) reaction system is prepared, specifically:
PCR reaction solution, water directly is added in sample (such as peripheral blood) or sample purification DNA and is configured to reaction system；
3) pattern detection: anticoagulation cirumferential blood, the sample that DBS sample, villus, amniotic fluid, Cord blood, peripheral blood, similar DBS are handled
Sheet, saliva, embryo inhereditary material include gamete such as sperm or ovum, the blastomere of cleavage stage embryo, blastaea trophectoderm
The samples such as cell, i.e. blastomere, multiple cells or individual cells, can also be using above-mentioned sample through nucleic acid directly as template
The reaction system and reaction condition progress PCR amplification that the DNA of extraction is prepared as template, obtain amplified production.Preferred reaction
System and reaction condition are shown in Table 2 and table 3.
4) production of membrane DNA chip
After activated liquid pre-processes nylon membrane 30 minutes, activated surface carboxyl；Meanwhile the probe for synthesizing designed, designed
(5 ' or 3 ' ends have amino labeled) is adjusted with probe dilution liquid to after concentration appropriate, is added on by DNA chip array order point
On nylon membrane corresponding position, at this point, amino and the carboxyl of activation react and generate stable covalent bond and fix probe in Buddhist nun
Imperial film surface；Finally, handling nylon membrane with confining liquid, the surface carboxyl groups of unbonded probe are closed, the preparation of completion membrane DNA chip is simultaneously low
Temperature saves.
5): hybridization
Amplified production is hybridized with the probe being fixed on membrane DNA chip, hybridization temperature and probe and pcr amplified fragment
Length, salinity, formamide, G/C content etc. are related.In system of the invention 48 DEG C be incubated for 1.5~4 hours after can get it is full
The results of hybridization of meaning.
6): the elution of non-specific binding thing
Non-specific hybridization is removed to greatest extent using different elution requirements, such as temperature, salinity.
7) chromogenic reaction
The biotin PCR product that 5 ' ends have and the Streptavidin (Streptavidin for being marked with peroxidase (POD)
Fibroin) specific bond, and chromogenic reaction occurs under the action of hydrogen peroxide and brown substrate TMB (tetramethyl benzidine),
Probe location is corresponded on film item, and blue spot is presented.
8) testing result interpretation
Testing result can directly interpretation by the naked eye.Can also after reading apparatus is scanned and is read, to signal and background by
Corresponding software system analysis obtains final results of hybridization, and result is saved and counted automatically.
Thalassemia gene detecting kit of the present invention directly can quickly detect the α-of 7 kinds of common types with stopped pipe
Ground is poor, combines RDB technology quickly to detect 3 kinds of non-deletion type (α of α-thalassemia simultaneously using Direct PCR (Direct PCR)CS
α、αQSα、αWSα) and 4 kinds of deletion forms (--SEA、--THAI、-α4.2With-α3.7), rapid gene diagnose point mutation caused by HbH disease and
Hb Bart's schridde syndrome.Without nucleic acid extraction, pollution is reduced, saves manpower and reagent cost.The PCR amplification time
2.5h, whole flow process save 3h or more than other methods, with other same analogies, save instrument and reagent cost.Therefore, of the invention
It is short, at low cost, easy to operate that kit has a detection time, stopped pipe operation, reduces pollution, and the split poor Mass screening in the site of an exhibition is lost
It passes consulting and pre-natal diagnosis is of great significance.
Detailed description of the invention
The picture of the specific distributing position of probe on genetic chip Fig. 1 of the invention
Each position represents different gene mutation or control point.
The detection of Fig. 2 agarose gel electrophoresis.M:50bp DNA Ladder (MD108) (TIANGEN)；Swimming lane 1: negative right
According to swimming lane；2:PCR amplified production (purpose band).
The detection of Fig. 3 agarose gel electrophoresis.M1:DNA Ladder (sub- energy biology)；M2:1kb DNA Ladder (Dye
Plus)(Takara Code No.3426A)；Swimming lane 1:--SEAPurpose band；Swimming lane 2:- α4.2Purpose band；Swimming lane 3:- α3.7Purpose
Band；Swimming lane 4: negative control；Swimming lane 4:--THAIPurpose band.PCR reaction solution II is separately added into different genotype template and expands through PCR
Increase, amplified production through 1.0% agarose gel electrophoresis detect, testing result referring to attached drawing 3, from electrophoresis result it can be observed that
Success amplifies purpose band respectively: amplified band fragment length respectively may be about 2.1kb (- α3.7)、1.5kb(-α4.2)、1.3kb(-
-SEA)、1.1kb(--THAIPurpose band).
Fig. 4 thalassemia testing result
(1)αα/αα；(2)αWSα/αα；(3)αWSα/αWSα；(4)αQSα/αα；(5)αQSα/αQSα；
(6)αCSα/αα；(7)αCSα/αCSα；(8)--SEA/αα；(9)--THAI/αα；(10)-α3.7/αα；
(11)-α4.2/αα；(12)--THAI/αCSα；(13) PCR negative control
Specific embodiment
Essence for a better understanding of the present invention, by the embodiment of the present invention technology that the present invention will be described in detail
Hold, the objects and the effects, be described in detail but do not limit the present invention.Component used in embodiment, unless otherwise specified,
Select the preferred embodiment in summary of the invention.
The most critical design of the present invention is: miscellaneous based on Direct PCR and/or Direct Multiple PCR, Gap-PCR and reversal point
The testing principle combined is handed over, corresponding amplimer and probe are designed according to the mutation of each genotype or deletion segment, with life
Probe is fixed in DNA chip, with amino labeled probe, and using genetic chip as substrate by special by object element labeled primer
Property the PCR product that comes out of primer amplification hybridized with probe, poor diagnosis is carried out with signal colour developing box interpretation.
Embodiment 1: kit Specimen origin of the present invention and pcr template type are used
1.1 sample collections and the preparation of pcr template:
A) sample collection: Specimen origin is in anticoagulation cirumferential blood, DBS sample, villus, amniotic fluid or Cord blood etc.；
B) preparation of pcr template: above-mentioned sample, can also be using above-mentioned sample through nucleic acid extraction directly as template
DNA is as template.
1.2 DBS sample preparations
Above-mentioned 1.1 filter paper dried blood spot sample (DBS) acquisition and preparation are as follows:
1., use Whatman903 filter paper (or ordinary filter paper piece).Subject's number is marked on filter paper and is received
Collect the date.2., peripheral blood and anticoagulation be used equally for filter paper dried blood spot sample to make.If it is dry to prepare filter paper from peripheral blood
Blood cake, acquisition position are arch of foot, ear-lobe or finger.Blood sampling site is sterilized with 70% ethyl alcohol or alcohol swab, with sterilized syringe needle
Carefully puncture skin.Discard the first drop peripheral blood.Second is bled a little in the circle in filter paper center, needs 50 every about hole
~80 μ l whole bloods guarantee to drip full circle.Each sample should put each hole of a full filter paper.Such as filter paper is prepared from anticoagulation
Dry blood cake, pipettor draw 50~80 μ l anticoagulation blood samples, and alignment filter paper prints at the center of circle, by blood sample drop on filter paper.
Each hole of a full filter paper should be dripped.3., blood filter paper spontaneously dry at least 4 hours at room temperature (under humid climate
At least 24 hours), it not heat Blood piece or they are stacked, it should not be with other interfacial contacts in drying process.It is filtering
After scraps of paper blood cake is sufficiently dried, filter paper is put into hermetic bag, the mutual pollution of sample between filter paper is avoided.4., complete packet
The DBS sample of dress can be protected from light storage 2 weeks at room temperature.Detect not in time DBS sample storage refrigerator -20 DEG C or less to
Inspection.
DBS sample in the present embodiment can also be substituted for: the dry amniotic fluid samples of filter paper, filter paper dried saliva sample, filter
The dry genome DNA sample of genetic material samples, filter paper of the dry embryo of the scraps of paper, the heredity of above-mentioned each filter paper stem body liquid/embryo
Substance/DNA sample preparation method with DBS sample conventional preparation method (as described above), also, using identical primer sets,
Identical PCR method can obtain identical experimental result, no longer repeat one by one herein.
Design and PCR reaction system of the embodiment 2. for the specific primer of PCR amplification determine
1.1 design of primers
α-globin gene order is obtained from GenBank database, and sudden change region is covered according to α-globin gene and is set
Multipair primer is counted, many experiments are carried out；Preferably, 1 pair of primer is used for the non-missing α of PCR amplification-poor gene in ground, it is preferred that 4 pairs
Primer combination, which is placed in the same reaction tube, carries out multiplex PCR, while expanding deletion form α-poor gene in ground.Primer is by professional biology
Company's synthesis.Primer sequence such as table 1.
The primer sets sequence of the detection poor gene in α-ground of table 1
The determination of 1.2 multi-PRC reaction systems
It using orthogonal test method, compares, is compared by many experiments through a large number of experiments, finally determined optimal
PCR reaction solution formula is shown in Table 18 μ l of 4-5:PCR reaction solution, 2 μ l, DBS sample of peripheral blood (villus, amniotic fluid or Cord blood) sample-adding amount
1-3 piece (diameter 1-2mm) (2 μ l of moisturizing), reaction total volume are 20 μ l.
Table 2 expands non-deletion type gene of alpha thalassemia PCR reaction system and condition
Table 3 expands deletion form gene of alpha thalassemia PCR reaction system and condition
The determination of 1.3 multiplexed PCR amplification programs
It compares and optimizes by many experiments, specific good, amplification efficiency can be accomplished by controlling annealing temperature and annealing time
Height, the best amplification program finally determined are shown in Table 2-3.
Template is added through PCR amplification in PCR reaction solution I, and amplified production is detected through 2% agarose gel electrophoresis, detection knot
Fruit is referring to attached drawing 2, and from electrophoresis result it can be observed that Successful amplification goes out purpose band: amplified band fragment length is 305bp.
PCR reaction solution II is separately added into different genotype template through PCR amplification, and amplified production is solidifying through 1.0% agarose
Gel electrophoresis detection, testing result is referring to attached drawing 3, from electrophoresis result it can be observed that successfully amplifying purpose band respectively: amplification item
Band fragment length respectively may be about 2.1kb (- α3.7)、1.5kb(-α4.2)、1.3kb(--SEA)、1.1kb(--THAI) purpose band.
Embodiment 3: design, point sample and the fixation of oligonucleotide probe
3.1. the design and screening of probe
α-globin gene order is obtained from GenBank database, is designed according to the poor gene mutation region in α-ground special
Property identification α-ground it is poor 7 kinds mutation (--SEA、--THAI、-α4.2、-α3.7、αCSα、αQSα、αWSOligonucleotide probe combination α)；And it sets
Meter feminine gender, positive and colour developing control probe and blank control (BC).It is synthesized by professional biotech firm and (is shown in Table 4).
The probe sequence group of the detection poor gene in α-ground of table 4
3.2. the preparation of genetic chip.
The preparation method of genetic chip of the present invention, includes the following steps:
(1) preparation of the working solution of oligonucleotide probe: by synthetic oligonucleotide probe probe dilution liquid (pH
8.4 0.5M Na2CO3With 0.5M NaHCO3Solution) mixed diluting is at working solution (10 μM), in case point sample is used.
(2) fixed probe: nylon membrane, which is soaked in 10% EDAC solution, activates 30 minutes, and pure water 3 times, every time 2
Minute, then room temperature is dried.The above-mentioned probe prepared is put respectively in the Buddhist nun being activated by micropipette equipment
On imperial film, 0.5 μ L of every drop.After point film, film is placed in room temperature 30 minutes, is reacted.Probe sequence on film item is such as
Shown in table 4.Being used to detect in specific probe there is no the site of mutation is nominal probe (being indicated with N), for detecting hair
The site of raw mutation is mutant probe (being indicated with M), and the probe on film item puts in order as shown in Figure 1, the position shown by spot
Setting can judging result.The site mutation of detection and normal control relationship are as shown in table 5.Detection site includes 4 kinds of deletion form α-
Thalassemia gene (--SEA、--THAI、-α4.2、-α3.7) and 3 kinds of non-deletion type gene of alpha thalassemia (αCSα、αQSα、αWS
α)。
The site mutation and normal control relationship that table 5 detects
(3) it prepares genetic chip: stopping reaction using sodium hydroxide, genetic chip is made.It is molten that film is transferred to 0.1M NaOH
It is impregnated 10 minutes in liquid, stops reaction.And sufficiently washed with distilled water, room temperature be stored in after drying 4 DEG C it is spare.Obtain gene
Chip.
Embodiment 4: determination, chromogenic reaction and the result judgement of hybridization conditions.
Sample to be tested or sample DNA are directly subjected to PCR reaction expansion with Direct PCR reaction solution and in corresponding amplification condition
Increase, amplified production is used for reverse dot blot hybridization.Reverse dot blot hybridization process including the following steps: nucleic acid hybridization combines
Nucleic acid with the elution of enzyme crosslinking and non-specific binding nucleic acid, in conjunction with zymolyte colour developing, results of hybridization analysis.In detail below
Illustrate the operation of each step.
4.1 hybridization
The film item merging 15ml plastic centrifuge tube for indicating sample number into spectrum, is added hybridization I liquid (2 × SSC, 0.1%SDS) 5-
Part or all (5-20 μ L) PCR products, tighten pipe lid in 6ml and PCR reaction solution I and PCR reaction solution II.Centrifuge tube is put
Enter in boiling water bath and heat 10 minutes, lid is tightened in taking-up, is put into 48 DEG C of case of hybridization hybridization 1.5 hours or more, but it is small to be no more than 4
When.
It takes 50ml plastic tube, 40ml hybridization II liquid (0.5 × SSC, 0.1%SDS) is added in (or the water bath with thermostatic control of hybridization case
Case) in carry out being preheated to 48 DEG C.
4.2 wash film
Film item is taken out, is moved in the 50ml pipe equipped with preheating hybridization II liquid, washs 15 minutes (every pipe 40ml in 48 DEG C of jogs
Solution can at most wash 4 films simultaneously).
4.3 colour developing
By hybridization I liquid: POD=2000:1 prepares Incubating Solution, and room temperature jog impregnates 30 minutes, discards POD solution.With hybridization
I liquid chamber temperature jog is washed twice, and 5 minutes every time.Room temperature washes film 1-2 minutes with C liquid, while Fresh developing solution.Film item is soaked
It steeps and is protected from light 6 to 12 minutes i.e. observable result of colour developing in developing solution.
4.4 result interpretations
(1.1) results of hybridization is analyzed
Developing solution is discarded, is washed with distilled water twice, it is 2-3 minutes each.Blotting paper blots the water on genetic chip surface, meat
Eye directly interpretation result or on the scanner scanning record result.
(1.2) result reads basic mode
The aobvious blue in correspondent probe position, illustrates that film item has corresponding signal on film item；Only normal locations on film item and corresponding
Quality Control control point has signal, illustrates that measuring samples are wild type；There is signal in mutational site on film item, illustrates that measuring samples contain
The type mutation；If the corresponding normal control in the mutational site also has signal, illustrate that measuring samples are the heterozygosis of the type mutation
Son；If the corresponding normal control in the mutational site does not have signal, illustrate that measuring samples are the type no mutant homozygote or merge scarce
Mistake type it is poor.
(1.3) quality controls
(1.31) negative control sample (replacing sample with water) is hybridized, if having in addition to the position CC is displayed in blue on film item
Hybridization spot, prompting for experiment has pollution, and experimental result is invalid.
(1.32) none spot on sample film item, the sample experiments are invalid.
(1.33) ordinary circumstance, the position BC, NC show colourless, and the position AC is displayed in blue, and the position CC is displayed in blue.The position NC
It is displayed in blue to prompt for testing and has pollution, experimental result is invalid.If the position BC, NC shows colourless, the position AC is not displayed in blue, CC
Position is displayed in blue, and needing to consider may the poor and/or sample experiments in other for homozygous deletion type poor and/or compound deletion forms ground
In vain, it needs further to analyze and/or further detect.The position CC is not displayed in blue, and illustrates that Color Appearance System is problematic, interrogates examination
Agent problem.
Embodiment 5: kit explanation and specific experiment method
5.1 kit main ingredient
(1) genetic chip, thereon with detection normally with mutational site probe and yin and yang attribute control probe and blank control；
(2) primer of the PCR reaction solution containing multiplex PCR for directly expanding；(3) hybridizing reagent such as POD, TMB and 30%H2O2。
This detection needs other main agents used
10%SDS:20g SDS 180ml pure water dissolves, and with 1N HCl tune pH value to pH 7.0, is finally settled to
200ml.It is stored at room temperature.
20 × SSC:175.3g NaCl, 88.2g sodium citrate 750ml pure water dissolve, with concentrated hydrochloric acid tune pH value to pH
7.0, it is finally settled to 1000ml, and high pressure sterilization saves.It is stored at room temperature.
1M sodium citrate: 294g sodium citrate is dissolved with 700ml, with dense HCl tune pH value to pH 5.0, is finally settled to
1000ml.It is stored at room temperature.
Hybridizing reagent is made of hybridization I liquid, hybridization II liquid, III liquid of hybridization, enzyme, developing solution, wherein the group of hybridization I liquid becomes
1~6 × SSC, 0.1%~1%SDS, wherein Preferable scheme is that 2 × SSC, 0.5%SDS；Hybridize II liquid group become 0.1~
1 × SSC, 0.1%~1%SDS, wherein Preferable scheme is that 0.5 × SSC, 0.5%SDS.Enzyme is horseradish peroxidase
(Streptavidin-POD), dosage is 0.005~1U/ml, and preferred dosage is 0.1U.Matched Color Appearance System,
The chromogenic substrate of horseradish peroxidase can be 3,3', 5,5'- tetramethyl benzidine (3,3', 5,5'-
Tetramethylbenzidine, abbreviation TMB), the preferred scheme of developing solution is 0.1M sodium citrate (pH4.9), 0.42mM
TMB, 0.004%H2O2(v/v)。
Hybridize I liquid: 100ml 20 × SSC, 10ml 10%SDS add pure water to be settled to 1000ml.It is stored at room temperature.
Hybridize II liquid: 25ml 20 × SSC, 10ml 10%SDS add pure water to be settled to 1000ml.It is stored at room temperature.
Hybridize III liquid: 100ml 1M sodium citrate adds pure water to be settled to 1000ml.It is stored at room temperature.
Developing solution: 19ml hybridizes III liquid and 1ml TMB and 2 μ l 30%H is added2O2。
5.2 PCR react instrument: Bio-Rad thermal cycler C1000 or T100 or Hangzhou lattice gene etc. are general
Logical PCR amplification instrument.
5.3 sample collections and pcr template requirement:
A) sample collection: Specimen origin is in anticoagulation cirumferential blood, DBS sample, villus, amniotic fluid, Cord blood or other body fluid marks
This etc.；
B) pcr template requirement: above-mentioned sample can also use DNA of the above-mentioned sample through nucleic acid extraction directly as template
As template.
C) blood sample saves: anticoagulated whole blood or Cord blood, villus, amniotic fluid or other humoral specimens etc. are no more than being placed at room temperature for
24 hours, DBS sample was placed at room temperature for no more than 2 weeks, and 2~8 DEG C saved no more than 7-15 days, and -20 DEG C or less preservations are no more than
1-2, -70 DEG C can long-term preservation, multigelation should be avoided in when freezen protective.
D) sample transports: when anticoagulated whole blood or Cord blood, villus, amniotic fluid or other humoral specimens etc. transport need to curling stone or
The bubble chamber of bag on the rocks seals, and should ensure that ice bag does not thaw, and in the way time limit no more than 72 hours.DBS sample can be with room temperature
The sealing of the bubble chamber of curling stone or bag on the rocks need to be used when (no more than 30 DEG C) transport or transport, and small no more than 72 in the way time limit
When.
5.4 the method for inspection
5.4.1 PCR amplification
Kit is taken out from refrigerator, and is taken out required PCR reaction solution, mark is carried out in equilibrium at room temperature dissolution on tube wall
Note is centrifuged 2-4 seconds in 5000-10000rpm, and is directly added into sample or extracted sample to be tested in backward PCR reaction solution
DNA1-3 μ L, reaction total system are 20 μ L.
Experiment separately takes a pipe PCR reaction solution every time, using 2 μ L pure water as template, makees blank control.
PCR is expanded by the following conditions, it is preferred that table 4 and 5:
5.4.2 hybridization
The film item merging 15ml plastic centrifuge tube for indicating sample number into spectrum, is added hybridization I liquid (2 × SSC, 0.1%SDS) 5-
Part or all (5-20 μ l) PCR products, tighten pipe lid in 6ml and PCR reaction solution.Centrifuge tube is put into boiling water bath and is heated
10 minutes, lid was tightened in taking-up, was put into 48 DEG C of case of hybridization hybridization 1.5 hours or more, but be no more than 4 hours.
It takes 50ml plastic tube, 40ml hybridization II liquid (0.5 × SSC, 0.1%SDS) is added in (or the water bath with thermostatic control of hybridization case
Case) in carry out being preheated to 48 DEG C.
5.4.3 film is washed
Film item is taken out, is moved in the 50ml pipe equipped with preheating hybridization II liquid, washs 15 minutes (every pipe 40ml in 48 DEG C of jogs
Solution can at most wash 4 films simultaneously).
5.4.4 colour developing
By hybridization I liquid: POD=2000:1 prepares Incubating Solution, and room temperature jog impregnates 30 minutes, discards POD solution.With hybridization
I liquid chamber temperature jog is washed twice, and 5 minutes every time.Hybridization III liquid chamber temperature laundering film 1-2 minutes is used again, while Fresh develops the color
Liquid.Film item is soaked in developing solution and is protected from light 6 to 12 minutes i.e. observable result of colour developing.
5.4.5 results of hybridization is analyzed
Developing solution is discarded, is washed with distilled water twice, it is 2-3 minutes each.Blotting paper blots the water on genetic chip surface, according to
According to table 5, determination method is the same as embodiment 4, naked eyes directly interpretation result or on the scanner scanning record result.
Embodiment 6: using testing result of the kit of the present invention in known pattern sheet
1, the composition of kit:
With embodiment 5.
2, implementation method:
Using double blind experiment, other are the same as embodiment 5.
3, samples sources:
All sample standard deviations provide fixed genotype (researcher does not know) different type sample through third party to be originated from.
4, data analysis and result judgement: according to table 5, determination method is with embodiment 4, according to 5 naked eyes of table directly interpretation knot
Fruit or on the scanner scanning record result.The result of the present embodiment is as shown in Figure 4.
The properties of product verifying of the invention of embodiment 7.
Product performance index
1 measurement accuracy
10 parts of negative samples and 21 parts of poor samples in ground to be collected, basic, normal, high 3 concentration is selected, each concentration is repeated 3 times, point
It is not detected with 3 batches of products, calculates separately negative and positive coincidence rate.Corresponding genotype as the result is shown, result of study with adopt
It is examined with the thalassemia gene of Shenzhen YiShengTang Biology Enterprise Co., Ltd and/or Yaneng Biotechnology (Shenzhen) Co., Ltd.
Disconnected kit (PCR method) kit test result complies fully with, and product positive coincidence rate and negative match-rate are all up to 100%.
2 sensitivity for analysis
Using kit of the present invention, poor detection site carries out sensitivity analysis over the ground, and each sample includes 7 concentration gradients,
The genomic DNA minimum concentration for determining that each genotype can stablize detection is 10ng/ μ L；
3 analysis specificity
By interfering Screening tests, EDTA, the sodium citrate of clinical normal dose are not the interfering substances of this product；Haemolysis sample
Originally this kit test result will not be interfered；Triglycerides (14.1mmol/L) and jaundice sample in piarhemia sample
(360.4mmol/L) detects this product noiseless.
The clinical sample outside 8 this product detection ranges, including the poor negative sample in 1 α-ground, 2 β-are detected with this product
Thalassemia clinical sample, 2 hypoferric anemia clinical samples, 2 G-6-PD clinical samples, 1 is infected the complete of Chlamydia
Blood sample, equal no cross reaction.
4 repeatability
Using kit difference lot number product of the present invention, different people (2 people) operation is done 2 times on the same day, does 2 days altogether, each
Reference material is tested be repeated 3 times detection every time.Stable detection α-poor genotype in ground can be repeated several times under different experimental conditions, as a result
Display is consistent.
The present invention using α-ground that the direct stopped pipe of Direct PCR method detects 7 kinds of common types it is poor (--SEA、--THAI、-
α4.2、-α3.7、αCSα、αQSα、αWSα), and under identical conditions RDB test is carried out, is intersected between avoiding nucleic acid extraction from causing sample
Pollution or DNA degradation reduce operating procedure, save time, manpower, instrument and reagent cost, reduce laboratory pollution, reduce leakage
Inspection and/or false detection rate.HbH disease and Hb Bart's schridde syndrome caused by energy rapid gene diagnosis of the present invention is mutated, it is right
Carry out poor Mass screening, genetic counselling and pre-natal diagnosis have good clinical value.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalents made by bright description are applied directly or indirectly in relevant technical field, are similarly included in this hair
In bright scope of patent protection.
Sequence table:
Claims (10)
1. a kind of for detecting the genetic chip of α-thalassemia, which is characterized in that be fixed on the chip:
For detecting 3 kinds of non-deletion type gene of alpha thalassemia αCSα、αQSα、αWSProbe corresponding to α, base sequence is such as
Shown in SEQ ID NO:11-16；And/or
For detecting 4 kinds of deletion form gene of alpha thalassemia --SEA、--THAI、-α4.2With-α3.7Probe, base sequence is such as
Shown in SEQ ID NO:17-20.
2. genetic chip according to claim 1, which is characterized in that be further fixed on the chip
Negative control probe (NC), base sequence such as SEQIDNO:21,
Positive control probe (AC), base sequence such as SEQIDNO:22,
Color development system controls probe (CC), base sequence such as SEQIDNO:23.
3. a kind of amplifing reagent for detecting the non-missing non-anemia gene in α-Mediterranean for PCR, it is characterised in that: including following core
The primer sets of nucleotide sequence:
APF:SEQ ID NO.1,
APR:SEQ ID NO.2；
It is used to detect three kinds of non-deletion type α-ground in its characteristic sequence and genetic chip of any of claims 1 or 2 for expanding
Middle sea anemia gene αCSα、αQSα、αWSProbe specificity corresponding to α combines；
Other PCR amplification components are added in the amplifing reagent, to sample to be tested or are extracted to test sample by a PCR reaction
The DNA of product directly carries out three kinds of non-missing α-Mediterranean poor gene QS, CS, WS；
The sample to be tested is selected from the anticoagulation cirumferential blood of fresh acquisition, -20 DEG C -- and 80 DEG C of oldness for saving non-multigelation are anti-
Solidifying peripheral blood, culture amniocyte, villus, amniotic fluid, Cord blood, peripheral blood, DBS sample and/or the sample of similar DBS processing,
Saliva, embryo inhereditary material include that gamete such as sperm or ovum, the blastomere of cleavage stage embryo, blastaea trophectoderm are thin
Born of the same parents, i.e. blastomere, multiple cells or individual cells etc..
4. amplifing reagent according to claim 3, which is characterized in that in the PCR amplification reagent reacted for one,
Contain:
5. a kind of detect deletion form α-non-anemia gene in Mediterranean amplifing reagent for PCR, it is characterised in that: include following core
The primer sets of nucleotide sequence:
SEAF:SEQ ID NO.3
SEAR:SEQ ID NO.4
THAIF:SEQ ID NO.5
THAIR:SEQ ID NO.6
4.2F:SEQ ID NO.7
4.2R:SEQ ID NO.8
3.7F:SEQ ID NO.9
3.7R:SEQ ID NO.10
It is used to detect in four kinds of deletion form α-ground in its characteristic sequence and genetic chip of any of claims 1 or 2 for expanding
Extra large anemia gene --SEA、--THAI、-α4.2With-α3.7Corresponding probe specificity combines；
Other PCR amplification components are added in the amplifing reagent, to sample to be tested or are extracted to test sample by a PCR reaction
The DNA of product directly carries out 4 kinds of poor genes in missing α-Mediterranean --SEA、--THAI、-α4.2With-α3.7The inspection of thalassemia gene
Survey, the sample to be tested is selected from the anticoagulation cirumferential blood of fresh acquisition, -20 DEG C -- 80 DEG C of oldness for saving non-multigelation are anti-
Solidifying peripheral blood, culture amniocyte, villus, amniotic fluid, Cord blood, peripheral blood, DBS sample and/or the sample of similar DBS processing,
Saliva, embryo inhereditary material include that gamete such as sperm or ovum, the blastomere of cleavage stage embryo, blastaea trophectoderm are thin
Born of the same parents, i.e. blastomere, multiple cells or individual cells etc..
6. amplifing reagent according to claim 5, which is characterized in that in the PCR amplification reagent reacted for one,
Contain:
7. amplifing reagent according to claim 5 or 6, which is characterized in that each primer molar concentration ratio of primer sets
It is as follows:
Preferably, in the PCR amplification reagent reacted for one, contain:
8. according to any amplifing reagent of claim 3-7, which is characterized in that 5 ' ends of the primer are marked with label
Object, for making the band of amplification with the marker.
9. a kind of kit for detecting α-thalassemia, it is characterised in that:
Including any amplifing reagent of genetic chip of any of claims 1 or 2 and claim 3-7.
10. the preparation method of genetic chip of any of claims 1 or 2, it is characterised in that:
Steps are as follows:
(1) it prepares probe face liquid: synthetic specific probe is diluted to 10 μM of working solution, in case point sample is used；
(2) fixed probe: chip base film is soaked in 10% EDAC solution and activates 30 minutes, and room temperature is dried in the air after pure water
It is dry；The upper working solution, 0.5 μ L of every drop are put on the basilar memebrane being activated；Point film after, by film be placed in room temperature into
Row reaction；
(3) it prepares genetic chip: stopping reaction using sodium hydroxide, genetic chip is made；
Wherein, for detecting 3 kinds of non-deletion type gene of alpha thalassemia αCSα、αQSα、αWSProbe corresponding to α, base sequence
Column are as shown in SEQ ID NO:11-16；
For detecting 4 kinds of deletion form gene of alpha thalassemia --SEA、--THAI、-α4.2With-α3.7Probe, base sequence is such as
Shown in SEQ ID NO:17-20；
Preferably, the material of the basilar memebrane of the chip is selected from: nitrocellulose, cellulose acetate, sheet glass, silica gel chip,
The group of nylon membrane, polypropylene screen and miniature magnetic bead composition.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110577990A (en) * | 2019-09-12 | 2019-12-17 | 南方医科大学 | A kit for detecting thalassemia gene mutation |
| CN111455039A (en) * | 2020-04-09 | 2020-07-28 | 广东凯普生物科技股份有限公司 | Nucleic acid composition for detecting α -thalassemia, gene chip thereof, kit thereof and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998023778A1 (en) * | 1996-11-27 | 1998-06-04 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | A RAPID METHOD FOR DIAGNOSING THE VARIOUS FORMS OF α-THALASSEMIA |
| CN101092647A (en) * | 2007-04-25 | 2007-12-26 | 亚能生物技术（深圳）有限公司 | Nucleic acid film tape and kit for diagnosing alpha mediterranean anemia |
| CN102181560A (en) * | 2011-04-29 | 2011-09-14 | 潮州凯普生物化学有限公司 | Joint detection kit for alpha,beta-thalassemia associated mutant genes |
| CN104789672A (en) * | 2015-04-14 | 2015-07-22 | 博尔诚（北京）科技有限公司 | Bar code magnetic bead liquid chip detection kit for thalassemia gene |
| CN105755137A (en) * | 2016-04-14 | 2016-07-13 | 亚能生物技术（深圳）有限公司 | Kit for detecting alpha-thalassemia genes |
| WO2017087961A1 (en) * | 2015-11-19 | 2017-05-26 | University Of Washington | Hematopoietic cell gene editing |
-
2017
- 2017-06-23 CN CN201710485206.1A patent/CN109112200A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998023778A1 (en) * | 1996-11-27 | 1998-06-04 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | A RAPID METHOD FOR DIAGNOSING THE VARIOUS FORMS OF α-THALASSEMIA |
| CN101092647A (en) * | 2007-04-25 | 2007-12-26 | 亚能生物技术（深圳）有限公司 | Nucleic acid film tape and kit for diagnosing alpha mediterranean anemia |
| CN102181560A (en) * | 2011-04-29 | 2011-09-14 | 潮州凯普生物化学有限公司 | Joint detection kit for alpha,beta-thalassemia associated mutant genes |
| CN104789672A (en) * | 2015-04-14 | 2015-07-22 | 博尔诚（北京）科技有限公司 | Bar code magnetic bead liquid chip detection kit for thalassemia gene |
| WO2017087961A1 (en) * | 2015-11-19 | 2017-05-26 | University Of Washington | Hematopoietic cell gene editing |
| CN105755137A (en) * | 2016-04-14 | 2016-07-13 | 亚能生物技术（深圳）有限公司 | Kit for detecting alpha-thalassemia genes |
Non-Patent Citations (3)
| Title |
|---|
| SITTICHAI PANYASAI ET AL.: ""A Simplified Screening for α-Thalassemia 1 (SEA Type) Using a Combination of a Modified Osmotic Fragility Test and a Direct PCR on Whole Blood Cell Lysates"", 《ACTA HAEMATOL》 * |
| TAKARA: ""MightyAmp DNA Polymerase Ver.2 Code NO.R071A"", 《MIGHTYAMP DNA POLYMERASE VER.2 CODE NO.R071A》 * |
| 郭柳薇: ""地中海贫血基因检测的研究进展"", 《医学综述》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110577990A (en) * | 2019-09-12 | 2019-12-17 | 南方医科大学 | A kit for detecting thalassemia gene mutation |
| CN111455039A (en) * | 2020-04-09 | 2020-07-28 | 广东凯普生物科技股份有限公司 | Nucleic acid composition for detecting α -thalassemia, gene chip thereof, kit thereof and application thereof |
| CN111455039B (en) * | 2020-04-09 | 2021-08-17 | 广东凯普生物科技股份有限公司 | Nucleic acid composition for detecting alpha-thalassemia, gene chip, kit and application thereof |
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