CN109112205A - The primer sets and kit of 3 kinds of deletional α-thalassemias are detected for Genotyping - Google Patents

Abstract

The present invention provides a kind of primer sets and kit that 3 kinds of deletional α-thalassemias are detected for Genotyping, belongs to molecular Biological Detection field.The primer sets include following primer pair: the upstream and downstream primer sequence APF/APR as shown in SEQ ID NO.1 and SEQ ID NO.2 respectively；The upstream and downstream primer sequence SEAF/SEAR as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively；The upstream and downstream primer sequence 4.2F/4.2R as shown in SEQ ID NO.5 and SEQ ID NO.6 respectively；The upstream and downstream primer sequence 3.7F/3.7R as shown in SEQ ID NO.1 and SEQ ID NO.7 respectively.The present invention also provides the kits based on the primer sets.Primer sets and kit of the invention can be applied to the detection of common deletion type thalassemia, have many advantages, such as quick, sensitive, safe.

Description

The primer sets and kit of 3 kinds of deletional α-thalassemias are detected for Genotyping

Technical field

The invention belongs to molecular biology field of medicaments, and in particular to one kind is for 3 kinds of deletion form α of Genotyping detection The primer sets and kit of middle sea anaemia.

Background technique

Thalassemia (thalassemia, referred to as poor), also known as " globin dyssynthesis anaemia " etc. are a kind of Recessive gene hereditary disease can lead to hereditary hemolytic anemia, and molecular pathology basis is mainly due to globin gene Defect (gene mutation or missing) leads to one or more of globin skin chain dyssynthesis, and causes α-chain of hemoglobin/non- α-chain proportional imbalance, and then cause hemoglobin unstable, the globin chain of redundancy is deposited on erythrocyte membrane, erythrocyte membrane Permeability and brittleness change, and lead to hemolytic anemia, and clinical signs go out the chronic progressive haemolysis of symptoms of varying severity Property anaemia.And according to the type of the peptide chain of globin of involvement with being divided into α, β, γ, δ and δ β types such as poor.It is wherein with α, β poor Most commonly seen, harm is also maximum.Perenniporia martius country is betided extensively, and disease incidence is high on the south China the Changjiang river, without obvious disease The carrier of shape is more than crowd 20%.

Clinically, α chain is synthesized reduce and β chain relative surplus to be then classified as α-ground poor, be since alpha globin gene lacks Or defect causes caused by alpha globin peptide chain dyssynthesis.Globin gene is located at alpha globin gene cluster, the dyeing of the gene cluster Body is positioned as 16p13.3, has 2 alpha globin genes on No. 16 chromosomes of normal person every.α it is poor can be divided into deletion form and Non-deletion type dpd gene type .95% is due to caused by alpha globin gene large fragment deletion the above are deletion form.The poor tool in ground The genetic heterogeneity of height.Missing or 1 α gene of defect on item chromosome are α⁺Ground is poor, shows as the conjunction of α-globin skin chain At being partially obstructed, usually lacks a α gene or the missing of point mutation or a few base occurs for a α gene or insertion is drawn Play (- α/α α, α α/α^Tα), the most common α in China⁺The poor gene in ground is-α^3.7、-α^4.2Deletion form mutation and Hb WS, Hb QS with The point mutation such as Hb CS.2 α genes are lacked, are α⁰Ground is poor, and feature is that the synthesis of α-globin skin chain is obstructed completely, China is most normal See and be --^SEA, it is rare --^THAI, and --^FILIt is then rarer.α⁺Ground is poor or α⁰There is the next generations blood red egg occurs by the poor carrier in ground White H sick (Hb H) (--/- α or --/α^Tα) or the risk of heavy Hb Bart's oedema syndrome fetus.Hb H gene type with The disease clinical symptoms severity is related, can be anemia, is also possible to anemia.Most commonly α⁰Ground is poor multiple Close α⁺The poor deletion form Hb H disease in ground, however the Hb H disease of non-deletion type also occupies considerable proportion, this kind of patient lacks than those The clinical symptoms that 3 alpha globin genes are showed are more serious, and anaemia is more serious.

α it is poor at present the disease there is no ideal treatment method, research shows that being ground to Mr. and Mrs both sides in the poor district occurred frequently in ground The pregnant woman of poor gene carrier passes through genetic screening and prenatal gene in First Trimester screening or mid-term application corresponding analysis technology It is to control the pathogenetic primary approach and effective precautionary measures that diagnosis, which selectively eliminates severe α-poor fetus in ground then,.Therefore, The detection poor for deletion form α-ground be badly in need of it is a kind of simple, can quick and precisely Genotyping technological means.

At present to the poor screening method in α-ground such as blood routine parameter detection and analysis, erythrocyte osmotic fragility test, hemoglobin The specificity such as electrophoresis tests is not high, is relatively also easy to produce false negative.For the molecular diagnosis of α-thalassemia, Southern Blot Hybridization technique is goldstandard, and accuracy is high, but it is cumbersome, sample dosage is big, time-consuming and laborious, detection flux is small and unsuitable often Rule detection；Other TaqMan probe technologies, denaturing high-performance chromatography (DHPLC), DNA direct Sequencing and DNA microarray technology Deng.But these method detection process are cumbersome, detection time is long, use reagent or expensive equipment and special labeled primer Deng, thus it is unfavorable for clinical extensive popularization and application.

Existing deletional α-thalassemia detect relevant patent type have it is several, in addition to number of patent application is 201110098786.1 patent of invention provides by detection of specific antibody SEA type the poor specific globin of ELISA method Method does not need to extract outside nucleic acid DNA, other patents are based on notch PCR (Gap-PCR), high-resolution solubility curve is analyzed, TaqMan probe technology, oligonucleotide probe hybridization, Denaturing high performance liquid chromatography, the probe amplification that multiple ligase relies on The technologies such as technology (MLPA), genetic chip are required to extract peripheral blood, DNA in villus and/or amniotic fluid, just can be carried out later Experimental implementation, and the quality of DNA is the successful very crucial factor of PCR, and extraction and purification DNA process is easily dirty Dye easily causes missing inspection or erroneous detection so that experiment easily false negative or false positive occurs.

Currently on the market deletion form poor detection method and product mostly use Gap-PCR combination electrophoresis method or gene core The technologies such as the piece detection common three kinds of deletion form α of China it is poor (--^SEA、-α^3.7With-α^4.2), it is necessary to extraction and purification DNA, ability Continue subsequent experimental.

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.This field is related at present To whole blood, amniotic fluid and the direct augmentation detection of DBS poor technology there is no, and there is no such product in the market.

Summary of the invention

The technical problems to be solved by the present invention are: provide a kind of directly stopped pipe can detect 3 kinds and lack simultaneously without purifying DNA Mistake type gene of alpha thalassemia (- α^3.7、-α^4.2、--^SEA) kit.

In view of this, object of the present invention is to be directed to the defect of existing detection method, using Direct PCR (Direct PCR) Method, provide one kind do not need DNA extract the most common three kinds of deletion forms α-ground of direct augmentation detection China it is poor (--^SEA、-α^3.7 With-α^4.2) method and its kit.The reaction of single tube single stopped pipe may be implemented using the kit to complete in Thailand's type α-ground The detection of extra large anaemia allele has easy, quick, sensitivity, stability and the accuracy of height and higher special Property.

Technical scheme is as follows:

The primer sets of 3 kinds of deletional α-thalassemias, including following primer pair are detected for Genotyping:

The upstream and downstream primer sequence APF/APR as shown in SEQ ID NO.1 and SEQ ID NO.2 respectively；

The upstream and downstream primer sequence SEAF/SEAR as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively；

The upstream and downstream primer sequence 4.2F/4.2R as shown in SEQ ID NO.5 and SEQ ID NO.6 respectively；

The upstream and downstream primer sequence 3.7F/3.7R as shown in SEQ ID NO.1 and SEQ ID NO.7 respectively.

The detection refers to the inhereditary material with blood of human body, and/or body fluid, and/or human genome DNA, and/or embryo For template, after carrying out PCR amplification with the primer sets, gained amplified production is subjected to electrophoresis you can learn that result.

For quickly detecting the kit of 3 kinds of deletional α-thalassemia genotype, including the primer sets.

The quick detection refers to, is directly PCR reaction with blood of human body, and/or body fluid, and/or the inhereditary material of embryo Template, after carrying out PCR amplification with the primer sets, by gained amplified production carry out electrophoresis you can learn that result.

The kit further includes the reagent for carrying out PCR and/or electrophoresis.

It is described to be used to carry out the reagent of PCR to include dNTP, archaeal dna polymerase, buffer, distilled water, MgCl₂；

It is described to be used to carry out the reagent of electrophoresis to include agarose, distilled water, nucleic acid dye；

The nucleic acid dye is selected from: EB, GelGreen, SYBR Green I, GoldView, GelRed and GelGreen.

The reaction system of the PCR are as follows: 1~4 μ l of template；Each primer: 0.1~1 μm of ol/L；MgCl₂Concentration be 1.5~ 5mM/L；The concentration of dNTP is 100~500 μM/L；The concentration of archaeal dna polymerase is 1~4U/ reaction；

The PCR reaction system preferably comprises each component of following volume parts: 0.1-1 parts of MightyAmp Taq, 2 × 10 parts of MightyAmp Buffer, 0.1-1 parts of APF/3.7F, 0.1-1 parts of APR, 0.1-1 parts of 4.2F, 4.2R

0.1-1 parts, 0.1-1 parts of 3.7R, 0.1-1 parts of SEA-F, 0.1-1 parts of SEA-R, template+0-7.2 parts of ultrapure water；

The PCR reaction system further preferably includes each component of following volume parts: MightyAmp Taq 0.3,2 ×MightyAmp Buffer 10、APF/3.7F 0.5、APR 0.3、4.2F 0.2、4.2R 0.2、3.7R

0.2, SEA-F 0.2, SEA-R 0.2, template+ultrapure water 7.1；

Preferably, each primer molar concentration ratio of the primer sets is as follows:

APF/3.7F: APR: 4.2F: 4.2R: 3.7R: SEA-F: SEA-R=5: 3: 2: 2: 2: 2: 2；

The response procedures of the PCR are as follows: 95 DEG C 3~5 minutes；With 94 DEG C 30 seconds, 61-66 DEG C 45~60 seconds, 72 DEG C 130 seconds It is recycled for 1, carries out 30-36 circulation；72 DEG C 8 minutes；12 DEG C 12 seconds.

The response procedures of the PCR are preferred are as follows: 95 DEG C 5 minutes；With 94 DEG C 30 seconds, 63-65 DEG C 45~60 seconds, 72 DEG C 130 Second is 1 circulation, carries out 32 circulations；72 DEG C 8 minutes；12 DEG C 12 seconds；

The electrophoresis refers to that it is in 0.8~1.5% Ago-Gel, in 5V/ that the PCR reaction product, which is placed in mass ratio, Electrophoresis about 40~55 minutes under cm voltage；0.005% nucleic acid dye of volume ratio is also added in the Ago-Gel.

The template further include fixed blood of human body, and/or body fluid, and/or genomic DNA on a solid carrier and/ Or the inhereditary material of embryo；

Preferably, the template can be selected from filter paper dried blood spot sample, the dry amniotic fluid samples of filter paper, filter paper dry pulp film The dry joint fluid sample of chamber liquid sample, filter paper, filter paper dried saliva sample, the dry genome DNA sample of filter paper, and/or filter paper The genetic material samples of the dry embryo of piece.

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.

The human body fluid is selected from the one or more of following substances: amniotic fluid, embryo villi tissue, serous cavity liquid, joint Liquid, saliva；The blood includes peripheral blood, Cord blood, peripheral blood；The inhereditary material of the embryo includes gamete such as sperm or ovum Son, the blastomere of cleavage stage embryo, blastaea Trophectoderm cells, i.e. blastomere.

The primer sets, and/or, the kit is in terms of preparing 3 kinds of deletional α-thalassemia detection reagents Purposes, which is characterized in that the primer sets are put into the packaging for indicating 3 kinds of deletional α-thalassemia detection applications, And/or 3 kinds of deletional α-thalassemia detection applications are indicated on the container equipped with the primer sets.

The kit of quick detection deletion form α-thalassemia of the present invention, including amplimer, by a large amount of Practical adjustment primer sequence of the invention, preferred primer composite sequence are as follows: one group can expand in α-globin gene cluster Three kinds of deletion form α-ground it is poor (--^SEA、-α^3.7With-α^4.2) allele characteristic sequence primer, base sequence is as follows: primer 3.7F, SEQ ID NO.1；Primer 3.7R, SEQ ID NO.7；Primer SEAF, SEQ ID NO.3；Primer SEAR, SEQ ID NO.4；Primer 4.2F, SEQ ID NO.5；Primer 4.2R, SEQ ID NO.6.

In order in the PCR system, introduce internal reference and expand α-pearl egg to the poor progress Genotyping assessment in deletion form α-ground The primer of normal people's gene (NG_000006.1) sequence in white gene cluster.The product segment of PCR amplification is about 1.8kb.

Primer APF:SEQ ID NO.1；Primer APR:SEQ ID NO.2.

The kit of quick detection deletion form thalassemia allele of the present invention further includes some existing examinations Conventional and necessary component in agent box, such as PCR buffer, enzyme solution, MgCl₂With dNTP etc..Specifically, enzyme solution is Taq polymerase System, including the thermal starting enzyme system etc. that can be used for direct PCR method；The buffer is that can be used for blood Direct PCR buffer； When enzyme solution selects to use treasured bioengineering (Dalian) Co., Ltd MightyAmp produced^TMWhen DNA Polymerase, delay Fliud flushing then preferably and MightyAmp^TMThe matched buffer of DNA Polymerase.

Preferably, in the gene detecting kit of above-mentioned 3 kinds of deletion form α-thalassemias, the reaction for Direct PCR Liquid is made of the following components formula (primer working concentration 10 μm of ol/L, MightyAmp DNA by every person-portion content Polymerase is 1.25U/ μ L):

PCR reaction solution formula table 1

Kit of the present invention is suitable for agarose gel electrophoresis method.

The 3 kinds of common deletion type equipotential bases of quick detection thalassemia of kit of the present invention based on Direct PCR Cause, using the kit quickly detect deletion form thalassemia allele method the following steps are included:

1) sample collection, anticoagulation cirumferential blood, DBS sample, villus, amniotic fluid, Cord blood, and/or inhereditary material of embryo etc. Sample, can also be using DNA of the above-mentioned sample through nucleic acid extraction as template directly as template.

2) PCR reaction system is prepared, specifically:

By primer SEAF, SEAR, 4.2F, 4.2R, 3.7F, 3.7R, APF and APR；And PCR buffer, enzyme solution, MgCl₂, dNTP, water and peripheral blood be configured to reaction system；

3) pattern detection: anticoagulation cirumferential blood, DBS sample, villus, amniotic fluid, Cord blood, and/or inhereditary material of embryo etc. Sample can also be carried out directly as template using the reaction system that DNA of the above-mentioned sample through nucleic acid extraction is prepared as template PCR amplification obtains amplified production.

4) electrophoresis detection identifies PCR product: taking 1.0~5.0 μ L of product in PCR amplification；Point sample is in 0.8~1.5% fine jade Additional 0.005% nucleic acid dye of sepharose；Electrophoresis about 40~55 minutes under 5V/cm voltage are taken out in gel imaging system In observe result and preservation of taking pictures.

5) interpretation result, only 1.8kb data analysis and result judgement: are analyzed according to PCR product agarose gel electrophoresis When band, result is normal wild type；Result is SEA allele heterozygosis when having two band of 1.8kb and 1.3kb simultaneously；Together When having two band of 1.8kb and 1.5kb result be 4.2 allele heterozygosis；Knot when having two band of 1.8kb and 2.1kb simultaneously Fruit is 3.7 allele heterozygosis；When only band 1.3kb or 1.5kb or 2.1kb a band, result is that SEA allele is pure Zygote or 4.2 allele genic homozygotes or 3.7 allele genic homozygotes；Result when having two band of 1.3kb and 1.5kb simultaneously For 4.2 allele of SEA allele heterozygosis, while result is SEA allele heterozygosis when having two band of 1.3kb and 2.1kb 3.7 allele, while result is 4.2 allele heterozygosis, 3.7 allele when having two band of 1.5kb and 2.1kb.Above-mentioned side In the step 1) of method, using fresh acquisition anticoagulation cirumferential blood or (- 20 DEG C or -80 DEG C save the outmoded of non-multigelation Property anticoagulation cirumferential blood) either to cultivate amniocyte or DBS sample be template.It must be avoided when detection villus, amniotic fluid or Cord blood Parent blood stains dye is excluded using other related identification experiments.

Filter paper dried blood spot sample acquisition in the step 1) of the above method is saved with preparation: 1., using Whatman 903 Filter paper (or ordinary filter paper piece).Subject's number is marked on filter paper and is collected the date.2., peripheral blood and anticoagulation it is equal It can be used for the production of filter paper dried blood spot sample.If preparing the dry blood cake of filter paper from peripheral blood, acquisition position be arch of foot, ear-lobe or Finger.Blood sampling site is sterilized with 70% ethyl alcohol or alcohol swab, carefully punctures skin with sterilized syringe needle.Discard the first drop tip Blood.Second is bled a little in the circle in filter paper center, needs 50~80 μ l whole bloods every about hole, guarantees to drip full circle.Often A sample should put each hole of a full filter paper.The dry blood cake of filter paper such as is prepared from anticoagulation, pipettor draws 50~80 μ l Anticoagulation blood sample, alignment filter paper print 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 room temperature at least 4 hours (at least 24 hours under humid climate), not heat Blood piece or They are stacked, it should not be with other interfacial contacts in drying process.After filter paper blood cake is sufficiently dry, by filter paper It is put into hermetic bag, avoids the mutual pollution of sample between filter paper.4., the DBS sample of integral packaging can be protected from light storage at room temperature It deposits 2 weeks.The DBS sample storage refrigerator detected not in time is to be checked at -20 DEG C or less.

In the step 2) of the above method, the concentration of each component is preferred in reaction system are as follows: peripheral blood (amniotic fluid) template: 1~4 μ l (DNA:1~4ng/ μ L to be detected)；Each primer: 0.1~1 μm of ol/L, MgCl₂Concentration is 1.5~5mM/L, the concentration of dNTP For 100~500 μM/L, the concentration of archaeal dna polymerase is 1~4U/ reaction.The final volume of reaction system is preferably 20 μ L.Above-mentioned side In the step 3) of method, 95 DEG C 3~5 minutes；With 94 DEG C 30 seconds, 61-66 DEG C 45~60 seconds, 72 DEG C 130 seconds for 1 circulation, carry out 30-36 circulation；72 DEG C 8 minutes；12 DEG C 12 seconds.Preferred PCR amplification condition is table 1, all to carry out agar after circulation terminates Sugared gel electrophoresis analysis.

Compared with prior art, present invention is characterized in that

1, kit of the present invention may be implemented the reaction of single tube single stopped pipe and complete deletion form thalassemia equipotential base The detection of cause, carries out the detection of deletion form thalassemia and only needs about 3h, saves 2-3h than other methods overall flow, has fast Prompt succinct, the stability of height, sensitivity and accuracy, specificity.

2, when kit of the present invention is applied in Direct PCR method to detect deletion form thalassemia equipotential base Because when, without open pipe operate, to the utmost reduce laboratory PCR product pollution possibility；On the other hand, solidifying using agarose Gel electrophoresis method, this method are at present using experimental system construction method most cheap in PCR molecular diagnostic techniques, and cost is lower, Expensive instrument and probe design are not needed, laboratories at different levels and hospital all easily carry out, it is easier to promote.

Detection kit of the invention carries out the detection of deletion form thalassemia using Direct PCR method, and stopped pipe operates, Without extracting DNA operation, DNA cross contamination and degradation are avoided.Whole flow process only needs about 3h, and detection kit of the invention Testing result it is consistent with the normal conventional Gap-PCR method testing result through nucleic acid extraction DNA, and nucleic acid extraction DNA's is common Conventional Gap-PCR method detection then needs 4.5h or more, and the cost is higher, cumbersome, and DNA is easily mutually polluted and dropped Solution.Therefore, Direct PCR method detection deletion form thalassemia aspect has many advantages, such as quick, sensitive, safe, can be applied to The detection of common deletion type thalassemia.

Detailed description of the invention

Fig. 1 is agarose gel electrophoresis results in the embodiment of the present invention 1；Wherein, M:1kb DNA Ladder (Dye Plus)(Takara Code No.3426A)；1: normal wild type；2:- α^4.2Heterozygote；3:- α^4.2Homozygote；4:- α^3.7Heterozygosis Son；5:- α^3.7Homozygote；6:--SEA heterozygote: 7:--SEA homozygote；8: negative control.

Fig. 2 is agarose gel electrophoresis results in the embodiment of the present invention 2；Wherein, M: Shenzhen Asia can the limited public affairs of biotechnology It takes charge of thalassemia and detects dedicated marker；1: negative control；2:- α^4.2Homozygote；3:--SEA homozygote；4:- α^4.2/--SEA Double heterozygote；: 5:- α^3.7/ -- SEA double heterozygote；3.7 heterozygote of 6:- α；7:- α^4.2Heterozygote；8: normal wild type；9:-- SEA heterozygote.

Fig. 3 is agarose gel electrophoresis results in the embodiment of the present invention 3；Wherein, M:1kb DNA Ladder (Dye Plus)(Takara Code No.3426A)；1: negative control；2: normal wild type；4.2 heterozygote of 3:- α；4:- α^3.7Heterozygosis Son；5:- α^3.7Homozygote；6:--SEA heterozygote.

Fig. 4 is agarose gel electrophoresis results in the embodiment of the present invention 4；Wherein, M:1kb DNA Ladder (Dye Plus)(Takara Code No.3426A)；1:- α^4.2Heterozygote (whole blood)；2:- α^3.7Heterozygote (whole blood)；3:--SEA heterozygosis Sub (whole blood)；4:--SEA heterozygote (amniotic fluid)；5:--SEA heterozygote (whole blood purifies DNA)；6:--SEA heterozygote (DBS)； 7:--SEA heterozygote (bleeding of the umbilicus)；8: negative control.

Specific embodiment

The present invention is described in further detail combined with specific embodiments below, content to better understand the invention, but The present invention is not limited to following embodiments.No special explanation, reagent used in the embodiment of the present invention are commercial goods, the present invention The database that embodiment uses is disclosed online database.

Instrument and equipment

PCR react instrument: PCR thermal cycler, as ABI thermal cycler, Bio-Rad thermal cycler C1000 or T100 or Hangzhou lattice gene magnification PCR instrument etc..

Reagent and consumptive material

DNA Ladder (Dye Plus) is purchased from Takara company, article No.: Takara Code No.3424A；

MightyAmp^TMDNA Polymerase and MightyAmp^TMThe matched buffer of DNA Polymerase is purchased From precious bioengineering (Dalian) Co., Ltd；

The source of biomaterial

All blood/body fluid samples relevant to human body used in the embodiment of the present invention, such as: anticoagulation cirumferential blood, umbilical cord The biological samples such as blood, amniotic fluid, bleeding of the umbilicus or embryo villi tissue, which are all from, accepts for medical treatment in the ground of The People's Hospital, Guangxi Zhuang Autonomous Region Extra large Anemic patients and normal health person, patient's informed consent.

Embodiment 1: using testing result of the kit of the present invention in known pattern sheet

1, the composition of kit:

3 kinds of common deletion type allele and normal gene (NG_000006.1) in α-globin gene cluster can be expanded Characteristic sequence primer sets SEAF, SEAR, 4.2F, 4.2R, 3.7F, 3.7R, APF and APR, base sequence is shown in Table 2:

2 deletion form α-thalassemia detection primer sequence of table

The reaction system of the PCR are as follows: 1~4 μ l of template；Each primer: 0.1~1 μm of ol/L；MgCl₂Concentration be 1.5~ 5mM/L；The concentration of dNTP is 100~500 μM/L；The concentration of archaeal dna polymerase is 1~4U/ reaction；

The PCR reaction system preferably comprises each component of following volume parts: 0.1-1 parts of MightyAmp Taq, 2 × 10 parts of MightyAmp Buffer, 0.1-1 parts of APF/3.7F, 0.1-1 parts of APR, 0.1-1 parts of 4.2F, 4.2R

0.1-1 parts, 0.1-1 parts of 3.7R, 0.1-1 parts of SEA-F, 0.1-1 parts of SEA-R, template+0-7.2 parts of ultrapure water；

The PCR reaction system further preferably includes each component of following volume parts: MightyAmp Taq 0.3,2 ×MightyAmp Buffer 10、APF/3.7F 0.5、APR 0.3、4.2F 0.2、4.2R 0.2、3.7R

0.2, SEA-F 0.2, SEA-R 0.2, template+ultrapure water 7.1；

Preferably, each primer molar concentration ratio of the primer sets is as follows:

APF/3.7F: APR: 4.2F: 4.2R: 3.7R: SEA-F: SEA-R=5: 3: 2: 2: 2: 2: 2；

The response procedures of the PCR are as follows: 95 DEG C 3~5 minutes；With 94 DEG C 30 seconds, 61-66 DEG C 45~60 seconds, 72 DEG C 130 seconds It is recycled for 1, carries out 30-36 circulation；72 DEG C 8 minutes；12 DEG C 12 seconds.

The response procedures of the PCR are preferred are as follows: 95 DEG C 5 minutes；With 94 DEG C 30 seconds, 63-65 DEG C 45~60 seconds, 72 DEG C 130 Second is 1 circulation, carries out 32 circulations；72 DEG C 8 minutes；12 DEG C 12 seconds it is preferred, by following Table 3 preparation PCR reaction system:

It using orthogonal test method, compares, is compared by many experiments through a large number of experiments, finally determined optimal PCR reaction solution formula (primer working concentration 10 μm of ol/L, MightyAmp DNA Polymerase are 1.25U/ μ L) is shown in Table 3: 18 μ l of PCR reaction solution, 2 μ l of peripheral blood (villus, amniotic fluid or Cord blood) sample-adding amount, reaction total volume are 20 μ l.

Table 3 prepares PCR reaction system and condition

2, implementation method:

Preferably, PCR response procedures are shown in Table 3, all progress agarose gel electrophoresis analyses after circulation terminates.

Sample collection, anticoagulation cirumferential blood, the samples such as anticoagulant Cord blood.

Pattern detection: by the known type sample to be examined (whole blood) determining with conventional method detection, according to above-mentioned reaction System and response procedures, it is all to carry out agarose gel electrophoresis analyses after circulation terminates in carrying out augmentation detection in PCR instrument.

3, samples sources: all sample standard deviations determine the anticoagulation cirumferential blood sample of genotype to be originated from through conventional Gap-PCR technology This.

4) data analysis and result judgement:

Determination method: when analyzing interpretation result, only 1.8kb band according to PCR product agarose gel electrophoresis, result is Normal wild type；Result is SEA allele heterozygosis when having two band of 1.8kb and 1.3kb simultaneously；Simultaneously have 1.8kb and Result is 4.2 allele heterozygosis when two band of 1.5kb；Result is 3.7 equipotentials when having two band of 1.8kb and 2.1kb simultaneously Genetic heterozygosis；When only band 1.3kb or 1.5kb or 2.1kb a band, result is SEA allele genic homozygote or 4.2 Allele genic homozygote or 3.7 allele genic homozygotes；Result is SEA equipotential base when having two band of 1.3kb and 1.5kb simultaneously Because of 4.2 allele of heterozygosis, while when having two band of 1.3kb and 2.1kb, result is 3.7 allele of SEA allele heterozygosis, Result is 4.2 allele heterozygosis, 3.7 allele when having two band of 1.5kb and 2.1kb simultaneously.

The testing result of the present embodiment is as shown in Figure 1.

Detection sample in the present embodiment can also acquire human body fluid, the inhereditary material of embryo or human genome DNA As PCR reaction template, the human body fluid is selected from the one or more of following substances: amniotic fluid, embryo villi tissue, serous cavity Liquid, joint fluid, saliva；The inhereditary material of the embryo includes gamete such as sperm or ovum, the blastomere of cleavage stage embryo, blastaea Trophectoderm cells, i.e. blastomere.Using in any one of above-mentioned humoral sample or the genetic material samples of embryo It is any, or use human genome DNA, through the invention provided by primer sets or kit, using identical PCR Method can obtain same experimental result, reach same testing goal, no longer repeat one by one herein.

Embodiment 2: detection effect of the kit of the present invention in DBS sample.

1, the composition of kit:

With embodiment 1.

Preferably, PCR reaction system is prepared by table 3:

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, DBS sample 1-3 piece (diameter 1-2mm) of 3:PCR reaction solution (2 μ l of moisturizing), reacts total volume For 20 μ l.

2, implementation method:

With embodiment 1.

3, samples sources:

All sample standard deviations determine the anticoagulation cirumferential blood sample of genotype to be originated from through conventional Gap-PCR technology, through filter paper The acquisition of piece dried blood spot sample and preparation.It is detected using double blind experiment.A sequin is intercepted (directly in sample area with punch Diameter: 1-2mm).

4, filter paper dried blood spot sample (DBS) acquisition and preparation: 1., (or the common filter of Whatman903 filter paper is used The scraps of paper).Subject's number is marked on filter paper and is collected the date.2., peripheral blood and anticoagulation be used equally for the dry blood of filter paper Spot sample making.If preparing the dry blood cake of filter paper from peripheral blood, acquisition position is arch of foot, ear-lobe or finger.With 70% ethyl alcohol Or alcohol swab sterilizes blood sampling site, carefully punctures skin with sterilized syringe needle.Discard the first drop peripheral blood.Second is bled a little In the circle in filter paper center, 50~80 μ l whole bloods are needed every about hole, guarantee to drip full circle.Each sample should be put one full Each hole of filter paper.The dry blood cake of filter paper such as is prepared from anticoagulation, pipettor draws 50~80 μ l anticoagulation blood samples, alignment filter At the center of scraps of paper print circle, by blood sample drop on filter paper.Each hole of a full filter paper should be dripped.3., blood filter paper is in room Temperature is lower to spontaneously dry at least 4 hours (at least 24 hours under humid climate), not heat Blood piece or they are stacked, It should not be with other interfacial contacts in drying process.After filter paper blood cake is sufficiently dry, filter paper is put into hermetic bag, is avoided The mutual pollution of sample between filter paper.4., the DBS sample of integral packaging can be protected from light storage 2 weeks at room temperature.It detects not in time It is to be checked at -20 DEG C or less that DBS sample stores refrigerator.

5, data analysis and result judgement: for determination method with embodiment 1, testing result is as shown in Figure 2.

DBS sample in the present embodiment can also be substituted for: the dry amniotic fluid samples of filter paper, filter paper dry pulp membrane cavity liquid sample The dry joint fluid sample of product, filter paper, filter paper dried saliva sample, the dry embryo of filter paper genetic material samples, and/or filter paper Dry genome DNA sample, above-mentioned each filter paper stem body liquid/DNA sample preparation method with DBS sample conventional preparation method, and And identical experimental result can be obtained using identical primer sets, identical PCR method, it no longer repeats one by one herein.

Embodiment 3: detection effect of the kit of the present invention in amniotic fluid sample (or amniotic fluid sample through cultivating).

1, the composition of kit:

With embodiment 1.

PCR reaction system is prepared by table 3:

2, implementation method:

With embodiment 1.

3, samples sources:

All sample standard deviations determine the amniotic fluid sample of genotype to be originated from through conventional Gap-PCR technology.

4, data analysis and result judgement: for determination method with embodiment 1, testing result is as shown in Figure 3.

Embodiment 4: double blind experiment, detection effect of the kit of the present invention in different directly template types are used.

1, the composition of kit:

With embodiment 1.

PCR reaction system is prepared by table 3.

2, implementation method:

Using double blind experiment, other are the same as embodiment 1.

3, samples sources:

All sample standard deviations determine genotype to be originated from the conventional Gap-PCR technology provided through third party (researcher does not know) Different type sample.

4, data analysis and result judgement: for determination method with embodiment 1, testing result is as shown in Figure 4.

The properties of product test of the invention of embodiment 5.

Product performance index

1 measurement accuracy

10 parts of negative samples and 24 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 deletion form alpha-mediterranean anemia gene diagnosis kit (PCR method) kit of Yaneng Biotechnology (Shenzhen) Co., Ltd. It surveys result to comply fully with, product positive coincidence rate and negative match-rate are all up to 100%.

2 sensitivity for analysis

Using kit of the present invention it is to three kinds of deletion form α poor (--^SEA、-α^3.7With-α^4.2) detection site progress sensitivity Analysis, each sample include 7 concentration gradients, determine that each genotype can stablize the genomic DNA minimum concentration of detection and be 10ng/μL；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 infection Chlamydia it is complete 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 above content is specific embodiment is combined, further detailed description of the invention, and it cannot be said that this hair Bright specific implementation is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, it is not taking off Under the premise of from present inventive concept, a number of simple deductions or replacements can also be made.It is all to utilize description of the invention and attached drawing Equivalent structure or equivalent flow shift made by content is applied directly or indirectly in other relevant technical fields, similarly It is included within the scope of the present invention.

SEQUENCE LISTING

<110>Chen Zhizhong

<120>primer sets and kit of 3 kinds of deletional α-thalassemias are detected for Genotyping

<130> P170162/CZZ

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> APF/ 3.7F

<400> 1

cccctgtcct ttccctaccc 20

<210> 2

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> APR

<400> 2

tccattgttg gcacattccg 20

<210> 3

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> SEAF

<400> 3

ccttcaccct cccacagttc c 21

<210> 4

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> SEAR

<400> 4

cgtcaccctc agagccatca c 21

<210> 5

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> 4.2F

<400> 5

tgcttttgtg agtgctgtgt tgac 24

<210> 6

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> 4.2R

<400> 6

aggcggagtt tcgctgttgt 20

<210> 7

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> 3.7R

<400> 7

ggagtgggac ttctctgacc tacc 24

Claims (10)

1. detecting the primer sets of 3 kinds of deletional α-thalassemias, including following primer pair for Genotyping:

The upstream and downstream primer sequence APF/APR as shown in SEQ ID NO.1 and SEQ ID NO.2 respectively；

The upstream and downstream primer sequence SEAF/SEAR as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively；

The upstream and downstream primer sequence 4.2F/4.2R as shown in SEQ ID NO.5 and SEQ ID NO.6 respectively；

The upstream and downstream primer sequence 3.7F/3.7R as shown in SEQ ID NO.1 and SEQ ID NO.7 respectively.

2. primer sets according to claim 1, which is characterized in that it is described detection refer to blood of human body, and/or body fluid and/ Or the inhereditary material of human genome DNA, and/or embryo are that template expands gained after carrying out PCR amplification with the primer sets Increase production object and carries out electrophoresis you can learn that result.

3. the kit for quickly detecting 3 kinds of deletional α-thalassemia genotype, including primer described in claim 1 Group.

4. kit according to claim 2, which is characterized in that the quick detection refers to, directly with blood of human body and/ Or body fluid, and/or the inhereditary material of embryo are that the template that PCR reacts expands gained after carrying out PCR amplification with the primer sets Increase production object and carries out electrophoresis you can learn that result.

5. kit according to claim 2 or 3, which is characterized in that further include the examination for carrying out PCR and/or electrophoresis Agent.

6. according to any kit of claim 2-4, which is characterized in that the reagent for carrying out PCR includes DNTP, archaeal dna polymerase, buffer, distilled water, MgCl₂；

It is described to be used to carry out the reagent of electrophoresis to include agarose, distilled water, nucleic acid dye；

The nucleic acid dye is selected from: EB, GelGreen, SYBR Green I, GoldView, GelRed and GelGreen.

7. according to any kit of claim 2-5, which is characterized in that the reaction system of the PCR are as follows: template 1~4 μl；Each primer: 0.1~1 μm of ol/L；MgCl₂Concentration is 1.5~5mM/L；The concentration of dNTP is 100~500 μM/L；DNA polymerization The concentration of enzyme is 1~4U/ reaction；

The PCR reaction system preferably comprises each component of following volume parts: 0.1-1 parts of MightyAmp Taq, 2 × 10 parts of MightyAmp Buffer, 0.1-1 parts of APF/3.7F, 0.1-1 parts of APR, 0.1-1 parts of 4.2F, 0.1-1 parts of 4.2R, 0.1-1 parts of 3.7R, 0.1-1 parts of SEA-F, 0.1-1 parts of SEA-R, template+0-7.2 parts of ultrapure water；

The PCR reaction system further preferably includes each component of following volume parts: MightyAmp Taq 0.3,2 × MightyAmp Buffer 10、APF/3.7F 0.5、APR 0.3、4.2F 0.2、4.2R 0.2、3.7R 0.2、SEA-F 0.2, SEA-R 0.2, template+ultrapure water 7.1；

Preferably, each primer molar concentration ratio of the primer sets is as follows:

APF/3.7F: APR: 4.2F: 4.2R: 3.7R: SEA-F: SEA-R=5: 3: 2: 2: 2: 2: 2；

The response procedures of the PCR are as follows: 95 DEG C 3~5 minutes；With 94 DEG C 30 seconds, 61-66 DEG C 45~60 seconds, 72 DEG C 130 seconds be 1 A circulation carries out 30-36 circulation；72 DEG C 8 minutes；12 DEG C 12 seconds；

The response procedures of the PCR are preferred are as follows: 95 DEG C 5 minutes；With 94 DEG C 30 seconds, 63-65 DEG C 45~60 seconds, 72 DEG C 130 seconds be 1 A circulation carries out 32 circulations；72 DEG C 8 minutes；12 DEG C 12 seconds；

The electrophoresis refers to that it is in 0.8~1.5% Ago-Gel, in 5V/cm electricity that the PCR reaction product, which is placed in mass ratio, Pressure electrophoresis about 40~55 minutes；0.005% nucleic acid dye of volume ratio is also added in the Ago-Gel.

8. the kit according to claim 4 or 7, which is characterized in that

The template further include fixed blood of human body, and/or body fluid, and/or genomic DNA on a solid carrier, and/or And/or the inhereditary material of embryo；

Preferably, the template can be selected from filter paper dried blood spot sample, the dry amniotic fluid samples of filter paper, filter paper dry pulp membrane cavity liquid The dry joint fluid sample of sample, filter paper, filter paper dried saliva sample, the dry genome DNA sample of filter paper, filter paper it is dry, and/or Embryo genetic material sample.

9. the kit according to claim 4 or 8, which is characterized in that the human body fluid is selected from one kind of following substances It is or a variety of: amniotic fluid, embryo villi tissue, serous cavity liquid, joint fluid, saliva；The blood includes peripheral blood, Cord blood, tip Blood；The inhereditary material of the embryo includes 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.

10. primer sets of any of claims 1 or 2, and/or, any kit of claim 3-9 is lacked at 3 kinds of preparation Purposes in terms of mistake type alpha Thalassemia detection reagent, which is characterized in that used indicating 3 kinds of deletional α-thalassemia detections The primer sets are put into the packaging on way, and/or, to indicate 3 kinds of Mediterranean deletion form α poor on the container equipped with the primer sets Blood detection applications.