CN109112207A - For detecting rightward deletion α3.7The primer sets and kit of thalassemia - Google Patents

For detecting rightward deletion α3.7The primer sets and kit of thalassemia Download PDF

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CN109112207A
CN109112207A CN201710488631.6A CN201710488631A CN109112207A CN 109112207 A CN109112207 A CN 109112207A CN 201710488631 A CN201710488631 A CN 201710488631A CN 109112207 A CN109112207 A CN 109112207A
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陈治中
卿吉琳
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Abstract

The present invention provides one kind for detecting rightward deletion α3.7The primer sets and kit of thalassemia, belong to molecular biology 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.3 respectively；The upstream and downstream primer sequence 3.7F/3.7R as shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.The present invention also provides the kits based on the primer sets.Primer sets and kit of the invention have quick succinct, the stability of height, sensitivity and accuracy, specificity.

Description

For detecting rightward deletion α3.7The primer sets and kit of thalassemia
Technical field
The invention belongs to molecular biology field of medicaments, and in particular to one kind is for detecting rightward deletion α3.7Mediterranean is poor The primer sets and kit of blood.
Background technique
α-thalassemia (abbreviation α-ground is poor) is the gene delection or dysfunction due to α-globin, leads to a pearl egg White skin chain synthesis, which is reduced or do not synthesized, to be caused, and is the monogenic inheritance hemoglobin of mankind's one of the most common type incomplete dominance Disease.Nineteen twenty-five, Thomas Cooley and Pear Lee describe this hemolytic anemia for the first time in Italian children, extensively The general Perenniporia martius that betides is national, global about 1,800,000,000 carrier, is that the hotspot of this disease is for example wide on the south China the Changjiang river The ground such as east, Guangxi, Sichuan, Chongqing disease incidence is high, is widely present in the form of the carrier of non-evident sympton mostly.Research shows that Guangdong, Guangxi α-poor gene in ground Population carriage are up to 8.53-15.5% respectively.There is generality, population since α-ground is poor The birth of the features such as radix is big, coverage is wide, the thalassemia major infant including south China is universally acknowledged Public health problem.
α-globin gene is located in No. 16 the short arm of a chromosome end α-globin gene cluster of human genome (16p13.3).The gene cluster includes two α genes (α 1 and α 2), an embryonic period, embryonic phase α genoid (ζ 2), three pseudogenes (ψ ζ 1, ψ α 2, ψ α 1) and the unknown gene (θ 1) of a function, sequence order are as follows: 5'- ζ 2- ψ ζ 1- ψ α 2- ψ α 1- α 2- α 1- θ l-3', Overall length about 50kb.
α-thalassemia is due to caused by α-globin gene mutation or missing.It can be by its point according to molecular defect Are as follows: non-deletion type α-ground of the insertion of several nucleotide and missing or the replacement of single base is poor and the deletion form α-of large fragment gene Up to the present, at least oneself reports in 41 kinds of deletion form α-thalassemias and at least 32 kinds of α-ground the poor two major classes in ground in the whole world Extra large anaemia point mutation.In Chinese, oneself reports at least eight kinds of deletion form α-thalassemias, wherein the southeast hypotype missing (- -SEA), rightward deletion (- α3.7) and lefrward deletion (- α4.2) it is in the most common poor deletion type in α-ground in China.According to clinical table It is now classified as silent oscillation, four class of standard type, Hemoglobin H disease and Hb Bart's hydrops fetalis again.Wherein clinical condition Shape is most heavy for Hb Bart's hydrops fetalis, and infant generally can not survive；Secondly it is Hemoglobin H disease (HbH), Clinical manifestation often differs in weight, and the clinical manifestation of usual nondeletion Hb H disease people is often more tighter than deletion form patient HbH Weight.
The disease there is no ideal treatment method at present, research shows that being poor base to Mr. and Mrs both sides in the poor district occurred frequently in ground Because the pregnant woman of carrier passes through genetic screening and prenatal gene diagnosis in First Trimester screening or mid-term application corresponding analysis technology Severe α-poor fetus in ground is then to control the pathogenetic primary approach and effective precautionary measures in selectively eliminating.Therefore, right In α-ground poor detection 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.
It is existing to be based on notch PCR (Gap-PCR), few nucleotide probe hybridization, the analysis of high-resolution solubility curve, TaqMan Probe technique, Denaturing high performance liquid chromatography, the skills such as probe amplification technology (MLPA), genetic chip that multiple ligase relies on Art is 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 easily pollutes, so that easily there is false yin in experiment Property or false positive.
Currently on the market deletion form poor detection method mostly use the skills such as Gap-PCR combination electrophoresis method or genetic chip Art detects common three kinds of China --SEA、-α4.2With-α3.7, it is also 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.
In addition, sample size needed for simultaneously is more, Basic characteristics are needed for the sample transport of outlying area acquisition, need three are grade packaged System, specimen storage space are big；Bio-safety risk factor is high.This field is not yet found in the presence of in relation to whole blood, amniotic fluid and DBS Poor technology is directly detected, 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, and proliferation time is about 2.5h；It is easy, quick, the single tube of high sensitivity Direct detection-α3.7The poor method in ground and its kit.The kit can it is easy to operate, parting is quick, low-cost, as a result sentence It reads intuitive.
Technical scheme is as follows:
For detecting rightward deletion α3.7The primer sets of thalassemia, including following primer pair:
The upstream and downstream primer sequence APF/APR as shown in SEQ ID NO.1 and SEQ ID NO.3 respectively；
The upstream and downstream primer sequence 3.7F/3.7R as shown in SEQ ID NO.1 and SEQ ID NO.2 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 rightward deletion α3.7The kit of 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: 0.05~1 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 reaction system of the PCR preferably includes 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 3.7R, template+ultrapure water 6-9.6 part；
The reaction system of the PCR further preferably includes each component of following volume parts: MightyAmp Taq 0.5 Part, 10 parts of 2 × MightyAmp Buffer, 0.5 part of APF/3.7F, 0.3 part of APR, 0.2 part of 3.7R, template+ultrapure water 8.5 part；
Preferably, each primer molar concentration ratio of the primer sets is as follows:
APF/3.7F: APR: 3.7R=5: 3: 2；
The response procedures of the PCR are as follows: 95 DEG C 3~5 minutes；With 94 DEG C 30 seconds, 62~66 DEG C 60 seconds, 72 DEG C 140 seconds be 1 A circulation carries out 34 circulations；72 DEG C 8 minutes；12 DEG C 12 seconds；
The response procedures of the PCR are as follows: 95 DEG C 5 minutes；With 94 DEG C 30 seconds, 64 DEG C 60 seconds, 72 DEG C 140 seconds for 1 circulation, Carry out 34 circulations；72 DEG C 8 minutes；12 DEG C 12 seconds；The electrophoresis refers to, by the PCR reaction product be placed in mass ratio be 0.8~ In 1.5% Ago-Gel, electrophoresis about 40~55 minutes under 5V/cm voltage；Volume is also added in the Ago-Gel Than 0.005% nucleic acid dye.
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.
And filter paper is the excellent carriers of blood specimen collection and transport.The dry blood of filter paper is made in droplets of whole blood on filter paper Spot (Dried blood spots, DBS), there is good biological stability and safety, convenient for storage and transport.With China The continuous enhancing of Prenatal Screening, neonatal screening dynamics, especially when screening range is expanded to, some regions are remote, medical condition When the area fallen behind relatively, 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 preparing rightward deletion α3.7Thalassemia detection reagent side The purposes in face, which is characterized in that indicating rightward deletion α3.7The primer is put into the packaging of thalassemia detection applications Group, and/or, rightward deletion α is indicated on the container equipped with the primer sets3.7Thalassemia detection applications.
Quick detection-α of the present invention3.7The kit of deletional α-thalassemia, including amplimer, through excessive Practical adjustment primer sequence of the invention is measured, preferred primer composite sequence is as follows: a pair can expand α-globin gene cluster In-α3.7The primer 3.7-F and 3.7-R:3.7-F:SEQ ID NO.1 of the characteristic sequence of allele；3.7-R:SEQ ID NO.2.The product segment of PCR amplification is about 2.1kb.
In order to rightward deletion α-poor (- α in ground3.7) 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.3.The product segment of PCR amplification is about 1.8kb.
Quick detection-α of the present invention3.7The kit of deletion form thalassemia allele further includes some existing There is conventional and necessary component in kit, such as 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 producedTMWhen DNA Polymerase, delay Fliud flushing then preferably and MightyAmpTMThe matched buffer of DNA Polymerase.
Preferably, above-mentioned rightward deletion α-poor (- α in ground3.7) thalassemia gene detecting kit in, for direct The reaction solution of PCR 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):
1 PCR reaction solution formula of table and PCR 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 PCR3.7Deletional α-thalassemia equipotential base Cause, using the quick detection-α of the kit3.7The method of deletional α-thalassemia allele the following steps are included:
1) sample collection, anticoagulation cirumferential blood, the samples such as DBS sample, villus, amniotic fluid or Cord blood directly as template, It can be using DNA of the above-mentioned sample through nucleic acid extraction as template.
2) PCR reaction system is prepared, specifically:
By primer 3.7F, 3.7R, APF and APR；And PCR buffer, enzyme solution, MgCl2, dNTP, water and peripheral blood prepare At reaction system；Preferably, it is shown in Table 1.
3) pattern detection: respectively will be with sample (such as peripheral blood (EDTA-K2)；DBS sample, villus, amniotic fluid or Cord blood Deng) reaction system and reaction condition progress PCR amplification that (or purification DNA) directly prepares for template, amplified production is obtained, preferably , it is shown in Table 1.
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) data analysis and result judgement: interpretation result is analyzed according to PCR product agarose gel electrophoresis, is only had to When one band 1.8kb band, result is normal wild type；Obtaining result when two band of 1.8kb and 2.1kb is carrying-α3.7Equipotential Gene；When only having to a band 2.1kb band, result is-α3.7Allele genic homozygote or-α3.7Allele is homozygous Son is wait exclude.
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 villus, amniotic fluid or Parent blood stains must be avoided to contaminate when Cord blood, 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: sample (such as peripheral blood) template: 1 ~4 μ l (DNA:1~4ng/ μ L to be detected)；Each primer: 0.05~1 μm of ol/L, MgCl2Concentration is 1.5~5mM/L, dNTP's Concentration is 100~500 μM/L, and the concentration of archaeal dna polymerase is 1~4U/ reaction.The final volume of reaction system is preferably 20 μ L.
In the step 3) of the above method, the response procedures of the PCR are as follows: 95 DEG C 3~5 minutes；With 94 DEG C 30 seconds, 62~66 DEG C 60 seconds, 72 DEG C 140 seconds be 1 circulation, carry out 34 circulations；72 DEG C 8 minutes；12 DEG C 12 seconds.Preferably, PCR amplification condition It is all to carry out agarose gel electrophoresis analysis after circulation terminates for table 1.
Compared with prior art, present invention is characterized in that
1, single tube single stopped pipe reaction completion-α may be implemented in kit of the present invention3.7Deletion form thalassemia etc. The detection of position gene, progress-α3.7The detection of deletion form thalassemia only needs about 3.5h, has quick succinct, height stabilization Property, sensitivity and accuracy, specificity.
2, deletion form-α is quickly detected when kit of the present invention is based on Direct PCR method3.7Thalassemia equipotential Gene is operated without open pipe, reduces the possibility of laboratory PCR product pollution to the utmost；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 rightward deletion α using Direct PCR method3.7Thalassemia detection, stopped pipe Operation avoids DNA cross contamination and degradation without extracting DNA operation.Whole flow process only needs about 3h, and detection of the invention The testing result of kit is consistent with the normal conventional Gap-PCR method testing result through nucleic acid extraction DNA, and nucleic acid extraction DNA The detection of normal conventional Gap-PCR method then need 4.5h or more, and the cost is higher, and cumbersome, DNA easily mutually pollutes And degradation.Therefore, Direct PCR method detection-α3.7Have many advantages, such as in terms of deletion form thalassemia it is quick, sensitive, safe, It can be applied to-α3.7The detection of deletion form thalassemia.
Detailed description of the invention
Fig. 1 is the testing result of embodiment 1；Direct PCR method of the present invention quickly detects deletion form-α3.7Thalassemia Electrophoresis result；Wherein, M:1kb DNA Ladder (Dye Plus) (Takara Code No.3426A)；1: negative control；2: Normal wild type (whole blood)；3:- α3.7Heterozygote (whole blood)；4:- α3.7Homozygote (whole blood)；5: negative control；6:- α3.7It is homozygous Sub (the corresponding purification DNA of 3 whole blood of sample).
Fig. 2 is the testing result of embodiment 2；Direct PCR method of the present invention quickly detects deletion form-α3.7Thalassemia Electrophoresis result；Wherein, M:1kb DNA Ladder (Dye Plus) (Takara Code No.3426A)；1: negative control；2: Normal wild type (DBS)；3:- α3.7Heterozygote (DBS)；4:- α3.7Homozygote (DBS).
Fig. 3 is the testing result of embodiment 3；It is poor that Direct PCR method of the present invention quickly detects 3.7 Mediterranean deletion form-α Blood electrophoresis result；Wherein, M:1kb DNA Ladder (Dye Plus) (Takara Code No.3426A)；1: negative control； 2:- α3.7Heterozygote (amniotic fluid)；3: normal wild type (amniotic fluid)；4:- α3.7Heterozygote (bleeding of the umbilicus)；5: normal wild type (bleeding of the umbilicus).
Fig. 4 embodiment of the present invention 4 quickly detects 3.7 thalassemia electrophoresis result of deletion form-α；Wherein, M:1kb DNA Ladder(Dye Plus)(Takara Code No.3426A)；1: negative control；2: normal wild type (whole blood)；3:- α3.7It is miscellaneous Zygote (amniotic fluid)；3:- α3.7Homozygote (DBS)；6:- α3.7Homozygote (purification DNA).
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；
MightyAmpTMDNA Polymerase and MightyAmpTMThe 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:
Deletion form-α in α-globin gene cluster can be expanded3.7Thalassemia allele and normal gene (NG_ 000006.1) primer sets 3.7F, 3.7R, APF and APR of characteristic sequence, base sequence are shown in Table 2:
Table 2 detects deletion form-α3.7The primer sets of thalassemia trait sequence.
The reaction system of the PCR are as follows: 1~4 μ l of template；Each primer: 0.05~1 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 reaction system of the PCR preferably includes 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 3.7R, template+ultrapure water 6-9.6 part；
The reaction system of the PCR further preferably includes each component of following volume parts: MightyAmp Taq 0.5 Part, 10 parts of 2 × MightyAmp Buffer, 0.5 part of APF/3.7F, 0.3 part of APR, 0.2 part of 3.7R, template+ultrapure water 8.5 part；
Preferably, each primer molar concentration ratio of the primer sets is as follows:
APF/3.7F: APR: 3.7R=5: 3: 2；
The response procedures of the PCR are as follows: 95 DEG C 3~5 minutes；With 94 DEG C 30 seconds, 62~66 DEG C 60 seconds, 72 DEG C 140 seconds be 1 A circulation carries out 34 circulations；72 DEG C 8 minutes；12 DEG C 12 seconds；
The response procedures of the PCR are as follows: 95 DEG C 5 minutes；With 94 DEG C 30 seconds, 64 DEG C 60 seconds, 72 DEG C 140 seconds for 1 circulation, Carry out 34 circulations；72 DEG C 8 minutes；12 DEG C 12 seconds；
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 of 1:PCR reaction solution, 2 μ l of peripheral blood (villus, amniotic fluid or Cord blood) equal samples sample-adding amount, instead Answering total volume is 20 μ l.
Table 3PCR reacts formula system and condition: (primer working concentration 10 μm of ol/L, MightyAmp DNA Polymerase is 1.25U/ μ L)
2, implementation method:
Preferably, PCR response procedures are shown in Table 3, all progress agarose gel electrophoresis analyses after circulation terminates.
Sample collection, the samples such as anticoagulation cirumferential blood (EDTA-K2 or heparin) 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.
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 corresponds to purification DNA.
4, data analysis and result judgement:
Determination method: interpretation result is analyzed according to PCR product agarose gel electrophoresis, only has to a band 1.8kb When band, result is normal wild type；Obtaining result when two band of 1.8kb and 2.1kb is carrying-α3.7Allele；Only only When obtaining a band 2.1kb band, result is-α3.7Allele genic homozygote or-α3.7Allele genic homozygote waits excluding.
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.
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, the acquisition of filter paper dried blood spot sample and preparation: 1., Whatman903 filter paper (or ordinary filter paper piece) is used. Subject's number is marked on filter paper and is collected the date.2., peripheral blood and anticoagulation be used equally for filter paper dried 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% ethyl alcohol or alcohol Cotton sterilizes blood sampling site, carefully punctures skin with sterilized syringe needle.Discard the first drop peripheral blood.Second is bled a little in filter paper In the circle in piece center, 50~80 μ l whole bloods are needed every about hole, guarantee to drip full circle.Each sample should put a full filter paper Each hole.The dry blood cake of filter paper such as is prepared from anticoagulation, pipettor draws 50~80 μ l anticoagulation blood samples, 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 at room temperature from So dry at least 4 hours (at least 24 hours under humid climate), not heat Blood piece or they are stacked, dried It should not be with other interfacial contacts in journey.After filter paper blood cake is sufficiently dry, filter paper is put into hermetic bag, avoid filter paper it Between sample mutual pollution.4., the DBS sample of integral packaging can be protected from light storage 2 weeks at room temperature.The DBS sample detected not in time It is to be checked at -20 DEG C or less that product store 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 of the kit of the present invention in amniotic fluid sample (or amniotic fluid sample through cultivating) and/or bleeding of the umbilicus Effect.
1, the composition of kit:
With embodiment 1.
PCR reaction system is prepared by table 2.
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 15 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 it is to three kinds of deletion form α poor (--SEA、-α3.7With-α4.2) 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>for detecting the primer sets and kit of 3.7 thalassemia of rightward deletion α
<130> P170163/CZZ
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> 3.7F/APF
<400> 1
ctgtcctttc cctacccaga gc 22
<210> 2
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> 3.7R
<400> 2
cagccagttc ttggcacaag ag 22
<210> 3
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> APR
<400> 3
tccattgttg gcacattccg 20

Claims (10)

1. for detecting rightward deletion α3.7The primer sets of thalassemia, including following primer pair:
The upstream and downstream primer sequence APF/APR as shown in SEQ ID NO.1 and SEQ ID NO.3 respectively；
The upstream and downstream primer sequence 3.7F/3.7R as shown in SEQ ID NO.1 and SEQ ID NO.2 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. for quickly detecting rightward deletion α3.7The kit of 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: 0.05~1 μm of ol/L；MgCl2Concentration is 1.5~5mM/L；The concentration of dNTP is 100~500 μM/L；DNA is poly- The concentration of synthase is 1~4U/ reaction；
The reaction system of the PCR preferably includes 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 3.7R, template+ultrapure water 6- 9.6 part；
The reaction system of the PCR further preferably includes each component of following volume parts: 0.5 part of MightyAmp Taq, 2 10 parts of × MightyAmp Buffer, 0.5 part of APF/3.7F, 0.3 part of APR, 0.2 part of 3.7R, 8.5 parts of template+ultrapure water；
Preferably, each primer molar concentration ratio of the primer sets is as follows:
APF/3.7F: APR: 3.7R=5: 3: 2；
The response procedures of the PCR are as follows: 95 DEG C 3~5 minutes；With 94 DEG C 30 seconds, 62~66 DEG C 60 seconds, 72 DEG C are followed for 1 for 140 seconds Ring carries out 34 circulations；72 DEG C 8 minutes；12 DEG C 12 seconds；
The response procedures of the PCR are as follows: 95 DEG C 5 minutes；With 94 DEG C 30 seconds, 64 DEG C 60 seconds, 72 DEG C 140 seconds for 1 circulation, carry out 34 circulations；72 DEG C 8 minutes；12 DEG C 12 seconds；The electrophoresis refers to, by the PCR reaction product be placed in mass ratio be 0.8~ In 1.5% Ago-Gel, electrophoresis about 40~55 minutes under 5V/cm voltage；Volume is also added in the Ago-Gel Than 0.005% nucleic acid dye.
8. the kit according to claim 4 or 7, which is characterized in that
The template further includes fixed blood of human body, and/or body fluid, and/or genomic DNA, and/or embryo on a solid carrier The inhereditary material of tire；
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.
And 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.And this is very few in the poor aspect application in ground.
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 lacks on the right side of preparation Lose α3.7Purposes in terms of thalassemia detection reagent, which is characterized in that indicating rightward deletion α3.7Thalassemia detection The primer sets are put into the packaging of purposes, and/or, rightward deletion α is indicated on the container equipped with the primer sets3.7In ground Extra large anaemia detection applications.
CN201710488631.6A 2017-06-23 2017-06-23 For detecting rightward deletion α3.7The primer sets and kit of thalassemia Pending CN109112207A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105154579A (en) * 2015-10-26 2015-12-16 钦州市妇幼保健院 Reagent kit for rapidly detecting -alpha21.9 deletion type thalassemia alleles
CN106521023A (en) * 2016-11-23 2017-03-22 广东省妇幼保健院 Kit and method for detecting HPFH and delta beta-thalassemia of Chinese people

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105154579A (en) * 2015-10-26 2015-12-16 钦州市妇幼保健院 Reagent kit for rapidly detecting -alpha21.9 deletion type thalassemia alleles
CN106521023A (en) * 2016-11-23 2017-03-22 广东省妇幼保健院 Kit and method for detecting HPFH and delta beta-thalassemia of Chinese people

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Title
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Application publication date: 20190101