CN109112202A - α is detected for Genotyping0The primer sets and kit of deletion form thalassemia - Google Patents

Abstract

The present invention provides one kind to detect α for Genotyping⁰The primer sets and kit of deletion form thalassemia.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.1 and SEQ ID NO.3 respectively；The upstream and downstream primer sequence THAIF/THAIR as shown in SEQ ID NO.4 and SEQ ID NO.5 respectively.Primer sets of the present invention and kit are in detection α⁰Ground it is poor (--^THAIWith --^SEA) aspect has many advantages, such as quick, sensitive, safe, it can be widely applied to α⁰Ground it is poor (--^THAIWith --^SEA) detection.

Description

α is detected for Genotyping0The primer sets and kit of deletion form thalassemia

Technical field

The invention belongs to molecular biology field of medicaments, are related to a kind of detection α⁰Deletion form thalassemia technology, especially It is to be related to a kind of single tube Direct PCR (Direct PCR) technology while detecting Southeast Asia deletion form and Thailand's type alpha Thalassemia Kit.

Background technique

α-thalassemia (abbreviation α-ground is poor) is one of most common hematological system hereditary disease in the world, the disease be by In the gene delection or dysfunction of α-globin, causes the synthesis of a globin skin chain to reduce or do not synthesize and cause.The disease it is high-incidence in From the wide geographic area of the Italy of Mediterranean, Greece, Malta, Cyprus to Southeast Asian countries.South China is also The hotspot of this disease, in Guangxi, Guangdong, the Population carriage of the poor gene in α-ground is up to 17.55%, 8.53% respectively.Due to α-ground is poor to have the characteristics that generality, large population base, coverage are wide, the thalassemia major including south China The birth of infant is universally acknowledged public health problem.

Under normal circumstances, it preserves due proportion between α and beta globin chain expressed by mankind's globin gene (1:1), shape At having functional tetrameric hemoglobin body, seldom or without remaining α-chain or beta chain.When globin gene occurs in vivo When the gene defects such as missing, mutation, causes the peptide chain of globin one or more in tetrameric hemoglobin body to synthesize and lack or subtract It is few, and cause internal hemoglobin alpha-chain and beta chain proportional imbalance, and then cause hemoglobin unstable, the globin chain of redundancy It is deposited on erythrocyte membrane, the permeability and brittleness of erythrocyte membrane change, and lead to hemolytic anemia, and clinical signs go out disease The chronic progressive hemolytic anemia that shape differs in weight.Clinically, α chain is synthesized and is reduced and β chain relative surplus is then classified as α- Ground is poor.

It can be classified as according to molecular defect: the non-deletion type of the insertion of several nucleotide and missing or single base replacement The poor deletion form α-poor two major classes in ground with large fragment gene in α-ground.And for a monoploid, subtracted according to α-globin synthesis Few degree, poor α-ground is two class defects: α⁰(poor 1) in α-ground, feature are that the synthesis of α-globin skin chain is complete to thalassemia It is obstructed, lacks two α genes (--/α α)；α⁰The poor gene in ground has apparent geographical distribution and racial traits, and China is most common For --^SEA, it is rare --^THAI, and --^FILIt is then rarer；②α⁺(poor 2) in the ground α show as the synthesis of α-globin skin chain to thalassemia Part is obstructed, and usually lacks a α gene or the missing of point mutation or a few base occurs for a α gene or insertion causes (--/- α, α^Tα/α α), 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 and Hb The point mutation such as CS.

It is typical Gene Deletion hereditary disease that α-ground is poor, research data show 95% or more α-ground it is poor be due to α-pearl Caused by (deletion form) is lost in protein gene sheet breakthrough, missing number is the foundation of α-thalassemia clinical manifestation and parting. It is classified as silent oscillation (- α/α α, α again according to clinical manifestation^Tα/α α, standard type (--/α α ,-α/- α, α^Tα/α^Tα,-α/α^Tα, blood Lactoferrin H disease (--/α^Tα, --/- α and Hb Bart's hydrops fetalis (--/--) four classes.Wherein clinical symptoms are most heavy For Hb Bart's hydrops fetalis, infant generally can not survive, and data shows that Hb Bart's Hb Ballt's hydtops fetails Hb Bart's are comprehensive Levying genotype is --^SEAHomozygous, --^THAIHomozygous and --^SEAIt is compound --^THAIType it is commonly reported that；It secondly is Hb H Sick (HbH), clinical manifestation often differs in weight, and the clinical manifestation of usual nondeletion Hb H disease people is often than deletion form HbH disease People is even more serious.The disease there is no ideal treatment method at present, research shows that being poor to Mr. and Mrs both sides in the poor district occurred frequently in ground The pregnant woman of gene carrier is examined in First Trimester screening or mid-term application corresponding analysis technology by genetic screening and prenatal gene Disconnected selectively superseded severe α-poor fetus in ground is then to control the pathogenetic primary approach and effective precautionary measures.Therefore, right In α⁰The poor detection in 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 Southeast Asia patent type relevant to the detection of Thailand's deletional α-thalassemia have it is several, in addition to patent Application No. is 201110098786.1 patents of invention to provide ELISA method by detection of specific antibody SEA type poor specificity The method of globin does not need to extract outside nucleic acid DNA, other patents are miscellaneous based on notch PCR (Gap-PCR), oligonucleotide probe Friendship, the analysis of TaqMan probe technology, high-resolution solubility curve, Denaturing high performance liquid chromatography, what multiple ligase relied on The technologies such as probe amplification technology (MLPA), genetic chip are required to extract in peripheral blood, embryo villi tissue and/or amniotic fluid DNA just can be carried out later experimental implementation, and the quality of DNA is the successful very crucial factor of PCR, and is extracted It is easily polluted with purifying DNA process, so that false negative or false positive easily occurs in experiment.

Currently on the market deletion form poor detection patent and product mostly use Gap-PCR combination electrophoresis method or gene core The technologies such as piece detect deletion form --^SEAWith --^THAI, it is necessary to extraction and purification DNA could continue subsequent experimental, sample extraction time About 1-2h, the detection of PCR amplification time are up to 3-4h, electrophoresis about 50min；If with the reverse hybridization longer time, subsequent operation Need 4h.Whole flow process needs 5-10h.

In addition, required sample size is more simultaneously for these patents and product, cold chain is needed for the sample transport of outlying area acquisition Equipment, needs three-level packaging system, and specimen storage space is big；Bio-safety risk factor is high.Currently, this field is to whole blood, sheep The technology that water and DBS directly detect poor aspect there is no, and there is no such product in the market.

Summary of the invention

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) Technology provides one kind and does not need DNA extracting directly amplification, directly detection α⁰The poor kit in ground, i.e. single tube directly detect Southeast Asia With Thailand deletion form α poor method and its kit.The kit can it is easy to operate, parting is quick, low-cost, as a result sentence It reads intuitive.

α is detected for Genotyping⁰The primer sets of deletion form thalassemia, including 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.1 and SEQ ID NO.3 respectively；

The upstream and downstream primer sequence THAIF/THAIR as shown in SEQ ID NO.4 and SEQ ID NO.5 respectively.

The detection refers to using human body fluid, and/or blood, and/or human genome DNA as template, with the primer sets After carrying out PCR amplification, gained amplified production is subjected to electrophoresis you can learn that result.

For quickly detecting α⁰The kit of deletion form thalassemia genotype, including the primer sets.

The quick detection refers to, with blood of human body, and/or body fluid, and/or the inhereditary material, and/or human body gene of embryo Group DNA is template, and after carrying out PCR amplification with the primer sets, gained amplified production is carried 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: template 1~10ng/ μ L；Each primer: 0.05~1 μm of ol/L；MgCl₂Concentration is 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 is preferred are as follows: each component comprising following volume parts: 0.1-1 parts of MightyAmp Taq, 10 parts of 2 × MightyAmp Buffer, 0.1-1 parts of SEAF/APF, 0.1-1 parts of SEAR, 0.1-1 parts of APR, THAIF 0.1- 1 part, 0.1-1 parts of THAIR, template+4-9.4 parts of ultrapure water；

The PCR reaction system is further preferred are as follows: each component comprising following volume parts: MightyAmp Taq 0.5 part, 10 parts of 2 × MightyAmp Buffer, 0.5 part of SEAF/APF, 0.3 part of SEAR, 0.2 part of APR, THAIF 0.3 Part, THAIR0.3 parts, 7.9 parts of template+ultrapure water；

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

SEAF/APF: SEAR: APR: THAIF: THAIR=5: 3: 2: 3: 3；

The response procedures of the PCR are as follows: 95 DEG C 3-5 minutes；With 94 DEG C 15-30 seconds, 62-66 DEG C 25~35 seconds, 72 DEG C 35 Second is 1 circulation, carries out 30~36 circulations；72 DEG C 5 minutes；12 DEG C 12 seconds

The response procedures of the PCR, preferably are as follows: 95 DEG C 5 minutes；With 94 DEG C 30 seconds, 64 DEG C 30 seconds, 72 DEG C 35 seconds be 1 Circulation carries out 34 circulations；72 DEG C 5 minutes；12 DEG C 12 seconds；

The electrophoresis refers to, the PCR reaction product is placed in and is added to the Ago-Gel that mass ratio is 0.8~1.5% In, electrophoresis 40~55 minutes under 5V/cm voltage；The nucleic acid dye of volume ratio 0.005% is 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 liquid sample acquisition 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 examination Agent box is preparing deletion form α⁰Purposes in terms of deletion form thalassemia detection reagent, which is characterized in that indicating α⁰Deletion form The primer sets are put into the packaging of thalassemia detection applications, and/or, α is indicated on the container equipped with the primer sets⁰ Deletion form thalassemia detection applications.

Quick detection Southeast Asia of the present invention and Thailand's deletion form α⁰The kit of deletion form thalassemia, including Amplimer, by a large amount of Practical adjustments primer sequence of the invention, preferred primer composite sequence is as follows: one group can expand In α-globin gene cluster --^THAIWith --^SEAThe primer of the characteristic sequence of allele: primer SEAF, SEQ ID NO.1；Primer SEAR, SEQ ID NO.4；Primer THAIF, SEQ ID NO.5；Primer THAIR, SEQ ID NO.6.

In order to α⁰Ground it is poor (--^THAIWith --^SEA) Genotyping assessment is carried out, in the PCR system, introduces internal reference and expand Increase the primer of normal people's gene (NG_000006.1) sequence in α-globin gene cluster: APF:SEQ ID NO.1；APR:SEQ ID NO.2.The product segment of PCR amplification is 334bp.

Quick detection α of the present invention⁰Ground it is poor (--^THAIWith --^SEA) kit further include in some available reagent boxes Conventional and necessary component, such as buffer, enzyme solution, MgCl₂With dNTP etc..Specifically, enzyme solution is Taq polymerase system, including It can be used for the thermal starting enzyme system etc. of direct PCR method；The buffer is that can be used for blood Direct PCR buffer；When enzyme solution selects It selects using treasured bioengineering (Dalian) Co., Ltd MightyAmp produced^TMWhen DNA Polymerase, buffer is then preferred For with MightyAmp^TMThe matched buffer of DNA Polymerase.

Preferably, above-mentioned α⁰In the gene detecting kit of thalassemia, the reaction solution for multiple Direct PCR is pressed Every person-portion content is made of the following components formula, and (primer working concentration 10 μm of ol/L, MightyAmp DNA Polymerase are 1.25U/ μ L):

Table 1PCR reacts formula of liquid and reaction condition

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

Quick detection α of the kit of the present invention based on Direct PCR method⁰Ground it is poor (--^THAIWith --^SEA) equipotential base Cause quickly detects α using the kit⁰Ground it is poor (--^THAIWith --^SEA) allele method the following steps are included:

1) sample collection: anticoagulation cirumferential blood, DBS sample, embryo villi tissue, amniotic fluid, Cord blood, peripheral blood, similar DBS The samples such as the sample of processing, multiple cells or individual cells can also use above-mentioned mark directly as template, embryo villi tissue This DNA through nucleic acid extraction is as template.

2) reaction system is prepared, specifically:

By primer THAIF, THAIR, SEAF, SEAR, APF and APR；And PCR buffer, enzyme solution, MgCl₂, dNTP, water Reaction system is configured to peripheral blood；Preferably, table 1.

3) pattern detection: directly will be that the reaction system that template is prepared is carried out with reaction condition with anticoagulation cirumferential blood respectively PCR amplification obtains amplified production.Reaction system is preferred with reaction condition, table 1.

4) electrophoresis detection identifies PCR product: taking 4.0~5.0 μ L of product in PCR amplification；Point sample is in 2~2.5% agar Sugared additional 0.005% nucleic acid dye of gel；Electrophoresis about 30~35 minutes under 5V/cm voltage are taken out in gel imaging system Observe result and preservation of taking pictures.

5) data analysis and result judgement: interpretation result is analyzed according to PCR product agarose gel electrophoresis, is only had to When one band 334bp band, result is normal wild type；Obtaining result when two band of 478bp and 334bp is carrying --^THAIDeng Position gene；Obtaining result when two band of 537bp and 334bp is carrying --^SEAAllele；Only have to a band 478bp item When band, result is --^THAIAllele genic homozygote；When only having to a band 537bp band, result is --^SEAAllele Homozygote；Result is when obtaining two band of 478bp and 537bp --^THAIIt is compound --^SEAAllele.

In the step 1) of the above method, using fresh acquisition anticoagulation cirumferential blood or (- 20 DEG C or -80 DEG C save not The oldness anticoagulation cirumferential blood of multigelation) cultivate amniocyte or DBS sample either as template.Detect embryo villi group Knit, amniotic fluid or when Cord blood must avoid parent blood stains from contaminating, 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 in reaction system are as follows: peripheral blood template: 1~4 μ l is (to be detected Body fluid sample or DNA:1~4ng/ μ L)；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 20~40 μ L.

In the step 3) of the above method, PCR amplification condition are as follows: 95 DEG C initial denaturation 3~5 minutes, then 94 DEG C 15~30 seconds, 62-66 DEG C is annealed 25~35 seconds, 30~36 circulations, all to carry out agarose gel electrophoresis analysis after circulation terminates.

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 α⁰Ground it is poor (--^THAIWith --^SEA) equipotential The detection of gene carries out α⁰Ground it is poor (--^THAIWith --^SEA) entirely testing process only needs about 2h, and its traditional technology needs 5- 8h.With quick succinct, the stability of height, sensitivity and accuracy, specificity.

2, when kit of the present invention is applied in Direct PCR method to detect α⁰Ground it is poor (--^THAIWith --^SEA) (i.e. Quick detection α based on Direct PCR method⁰Ground it is poor (--^THAIWith --^SEA) kit) when, without open pipe operate, greatly limit Degree ground reduces the possibility of laboratory PCR product pollution；On the other hand, using agarose gel electrophoresis method, this method is to make at present With experimental system construction method most cheap in PCR molecular diagnostic techniques, cost is lower, does not need expensive instrument and probe and sets Meter, laboratories at different levels and hospital all easily carry out, it is easier to promote.

α is carried out using detection kit of the invention⁰Ground it is poor (--^THAIWith --^SEA) detection only needs about 2h, and the present invention Detection kit testing result it is consistent with the normal conventional Gap-PCR method testing result through nucleic acid extraction DNA, and nucleic acid mentions It takes the normal conventional Gap-PCR method of DNA to detect and then needs 5h or more, and the cost is higher, cumbersome, DNA is easily mutual Pollution and degradation.Therefore, Direct PCR method detects α⁰Ground it is poor (--^THAIWith --^SEA) aspect have it is quick, sensitive, safe etc. excellent Point, can be applied to α⁰Ground it is poor (--^THAIWith --^SEA) detection.

Detailed description of the invention

Agarose gel electrophoresis results in Fig. 1 embodiment of the present invention 1: where M:DL500DNA Marker (Takara Code No.3590A)；1: negative control；2: normal wild type (whole blood)；3:--^THAIHeterozygote (whole blood)；4:--^SEAHeterozygote (whole blood)；5:--^SEAHomozygote (purification DNA).

Agarose gel electrophoresis results in Fig. 2 embodiment of the present invention 2: where M:DL500DNA Marker (Takara Code No.3590A)；1: normal wild type (DBS)；2:--THAI heterozygote (whole blood)；3:--^SEAHeterozygote (DBS)；4:- -^SEAHomozygote (purification DNA).

Agarose gel electrophoresis results in Fig. 3 embodiment of the present invention 3: where M:DL500DNA Marker (Takara Code No.3590A)；1: normal wild type；2:--^THAIHeterozygote；3:--^SEAHeterozygote；4:--^SEAHomozygote.

Fig. 4 is agarose gel electrophoresis results in the embodiment of the present invention 4: where M:DL500DNA Marker (Takara Code No.3590A)；1:--^SEAHomozygote (amniotic fluid)；2:--^SEAHeterozygote (whole blood)；3:--^THAIHeterozygote (whole blood)；4: just Normal wild type (whole blood)；5: negative control；；6: normal wild type (DBS)；7:--^THAIHeterozygote (DNA)；8:--^SEAHeterozygote (bleeding of the umbilicus).

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:

α in α-globin gene cluster can be expanded⁰During ground is poor --^THAIWith --^SEAAllele and normal gene (NG_ 000006.1) primer sets THAIF, THAIR, SEAF, SEAR, APF and APR of characteristic sequence, base sequence are shown in Table 2:

Table 2 detects α⁰The primer sets of the poor characteristic sequence in ground

Preferably, PCR reaction system (primer working concentration 10 μm of ol/L, MightyAmp DNA are prepared by following Table 3 Polymerase is 1.25U/ μ L).

The reaction system of the PCR are as follows: template 1~10ng/ μ L；Each primer: 0.05~1 μm of ol/L；MgCl₂Concentration is 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 is preferred are as follows: each component comprising following volume parts: 0.1-1 parts of MightyAmp Taq, 10 parts of 2 × MightyAmp Buffer, 0.1-1 parts of SEAF/APF, 0.1-1 parts of SEAR, 0.1-1 parts of APR, THAIF 0.1- 1 part, 0.1-1 parts of THAIR, template+4-9.4 parts of ultrapure water；

The PCR reaction system is further preferred are as follows: each component comprising following volume parts: MightyAmp Taq 0.5 part, 10 parts of 2 × MightyAmp Buffer, 0.5 part of SEAF/APF, 0.3 part of SEAR, 0.2 part of APR, THAIF 0.3 Part, THAIR0.3 parts, 7.9 parts of template+ultrapure water；

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

SEAF/APF: SEAR: APR: THAIF: THAIR=5: 3: 2: 3: 3；

The response procedures of the PCR are as follows: 95 DEG C 3-5 minutes；With 94 DEG C 15-30 seconds, 62-66 DEG C 25~35 seconds, 72 DEG C 35 Second is 1 circulation, carries out 30~36 circulations；72 DEG C 5 minutes；12 DEG C 12 seconds

The response procedures of the PCR, preferably are as follows: 95 DEG C 5 minutes；With 94 DEG C 30 seconds, 64 DEG C 30 seconds, 72 DEG C 35 seconds be 1 Circulation carries out 34 circulations；72 DEG C 5 minutes；12 DEG C 12 seconds；

It using orthogonal test method, compares, is compared by many experiments through a large number of experiments, finally determined optimal PCR reaction solution formula and system are shown in Table 18 μ l of 3:PCR reaction solution, the samples such as peripheral blood (embryo villi tissue, amniotic fluid or Cord blood) 2 μ l of this sample-adding amount, reaction total volume are 20 μ l.

Table 3PCR reaction solution formula system and reaction condition.

2, implementation method:

The samples such as sample collection, anticoagulation cirumferential blood or 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.It takes 4.0~5.0 μ L of product in PCR amplification；Point sample is in additional 0.005% nucleic acid dye of 2~2.5% Ago-Gels；In 5V/cm Electrophoresis about 30~35 minutes under voltage take out and observe result and preservation of taking pictures in gel imaging system.

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: interpretation result is analyzed according to PCR product agarose gel electrophoresis, is only had to When one band 334bp band, result is normal wild type；Obtaining result when two band of 478bp and 334bp is carrying --^THAIDeng Position gene；Obtaining result when two band of 537bp and 334bp is carrying --^SEAAllele；Only have to a band 478bp item When band, result is --^THAIAllele genic homozygote；When only having to a band 537bp band, result is --^SEAAllele Homozygote；Result is when obtaining two band of 478bp and 537bp --^THAIIt is compound --^SEAAllele.

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 system 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), and reaction is total Volume is 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).1., (or common using Whatman903 filter paper 4, filter paper dried blood spot sample (DBS) acquisition and preparation: Filter paper).Subject's number is marked on filter paper and is collected the date.2., peripheral blood and anticoagulation be used equally for filter paper dry Blood spot sample production.If preparing the dry blood cake of filter paper from peripheral blood, acquisition position is arch of foot, ear-lobe or finger.With 70% second Alcohol or alcohol swab sterilize blood sampling site, carefully puncture skin with sterilized syringe needle.Discard the first drop peripheral blood.Second is bled Point needs 50~80 μ l whole bloods in the circle in filter paper center, every about hole, guarantees to drip full circle.Each sample should put full one Open 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 At the center of filter paper print circle, by blood sample drop on filter paper.Each hole of a full filter paper should be dripped.3., blood filter paper in It spontaneously dries at room temperature at least 4 hours (at least 24 hours under humid climate), not heat Blood piece or they are stacked on one It rises, 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, Avoid 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 examines not in time The DBS sample storage refrigerator of survey is to be checked at -20 DEG C or less.

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

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, the testing result of the present embodiment is as shown in Figure 4.

The properties of product test of the invention of embodiment 6.

Product performance index

1 measurement accuracy

10 parts of negative samples and 20 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 Shenzhen YiShengTang Biology Enterprise 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 to α⁰Deletion form thalassemia (--^THAIWith --^SEA) the poor detection site in 2 kinds of α-ground carries out Sensitivity analysis, each sample include 7 concentration gradients, determine each genotype can stablize detection genomic DNA it is minimum dense Degree 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, 1 whole blood sample, 2 G-6-PD for infecting Chlamydia face Bed 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 for Genotyping detection 0 deletion form thalassemia of α

<130> P170158/CZZ

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> APF/SEAF

<400> 1

aatggatgag gacggagcga t 21

<210> 2

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> APR

<400> 2

agttccctga gccccgacac 20

<210> 3

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> SEAR

<400> 3

gagtctcgct ctgtctccta ggct 24

<210> 4

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> THAIF

<400> 4

tgccctcaac ccctgacaat 20

<210> 5

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> THAIR

<400> 5

cttggatctg cacctctggg tag 23

Claims (10)

1. detecting α for Genotyping⁰The primer sets of deletion form thalassemia, including 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.1 and SEQ ID NO.3 respectively；

The upstream and downstream primer sequence THAIF/THAIR as shown in SEQ ID NO.4 and SEQ ID NO.5 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, and/or human genome DNA of embryo is 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. for quickly detecting α⁰The kit of deletion form thalassemia genotype, including primer sets described in claim 1.

4. kit according to claim 2, which is characterized in that the quick detection refers to, directly with blood of human body and/body Liquid or and/or the inhereditary material of embryo be template, after carrying out PCR amplification with the primer sets, gained amplified production is carried out electric Swimming is 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~ 10ng/μL；Each primer: 0.05~1 μm of ol/L；MgCl₂Concentration is 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 is preferred are as follows: each component comprising following volume parts: 0.1-1 parts of MightyAmp Taq, 2 × 10 parts of MightyAmp Buffer, 0.1-1 parts of SEAF/APF, 0.1-1 parts of SEAR, 0.1-1 parts of APR, THAIF 0.1-1 Part, 0.1-1 parts of THAIR, template+4-9.4 parts of ultrapure water；

The PCR reaction system is further preferred are as follows: each component comprising following volume parts: 0.5 part of MightyAmp Taq, 10 parts of 2 × MightyAmp Buffer, 0.5 part of SEAF/APF, 0.3 part of SEAR, 0.2 part of APR, 0.3 part of THAIF, 0.3 part of THAIR, 7.9 parts of template+ultrapure water；

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

SEAF/APF: SEAR: APR: THAIF: THAIR=5: 3: 2: 3: 3；

The response procedures of the PCR are as follows: 95 DEG C 3-5 minutes；With 94 DEG C 15-30 seconds, 62-66 DEG C 25~35 seconds, 72 DEG C 35 seconds be 1 A circulation carries out 30~36 circulations；72 DEG C 5 minutes；12 DEG C 12 seconds；

The response procedures of the PCR, preferably are as follows: 95 DEG C 5 minutes；It is recycled with 94 DEG C for 1 within 35 seconds within 30 seconds, 72 DEG C within 30 seconds, 64 DEG C, Carry out 34 circulations；72 DEG C 5 minutes；12 DEG C 12 seconds；

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

8. the kit according to claim 4 or 7, which is characterized in that the template further includes fixing on a solid carrier Blood of human body, and/or body fluid, and/or genomic DNA, and/or embryo genetic substance；

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, and/or filter paper are dry The genetic material samples of embryo.

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 described in claim 1, and/or, kit described in claim 2-9 is preparing deletion form α⁰Deletion form Purposes in terms of thalassemia detection reagent, which is characterized in that indicating α⁰The packet of deletion form thalassemia detection applications The primer sets are put into dress, and/or, α is indicated on the container equipped with the primer sets⁰The detection of deletion form thalassemia is used On the way.