CN108570495A - A kind of the rapid amplifying diagnostic kit and amplification method of HLA high-resolution gene loci - Google Patents
A kind of the rapid amplifying diagnostic kit and amplification method of HLA high-resolution gene loci Download PDFInfo
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Abstract
The invention discloses a kind of rapid amplifying diagnostic kit of HLA high-resolution gene loci and amplification method, the amplification kit includes archaeal dna polymerase and amplimer, and HLA high-resolution gene locis are HLA DRB1, template sequence such as sequence table SEQ ID NO：Shown in 1, amplimer is used for linearized index PCR amplification, amplimer upstream sequence such as sequence table SEQ ID NO：Shown in 2, downstream sequence such as sequence table SEQ ID NO：Shown in 3.The present invention uses the target fragment amplification method of linearized index amplification, the amplimer amplification efficiency of Precise spraying is high and mispairing is few, not only considerably increase amplification efficiency, and it directly amplifies single stranded DNA and can be directly used for pyrosequencing, without amplified production is marked and double-strand separation, lengthy and tedious experimental procedure is avoided, damage of the strong basic reagent to amplified fragments is decreased.
Description
Technical field
The present invention relates to a kind of rapid amplifying diagnostic kit of HLA high-resolution gene loci and amplification methods, belong to raw
Object field, especially field of biological detection.
Background technology
Human leukocyte antigen (HLA) is human major histocompatibility's antigenic complex (MHC), is body internal specific
The main component of Immune discrimination and immune response is to influence one of successful key factor of hematopoietic stem cell transplantation, reliable HLA
Gene frequency especially high-resolution gene frequency data are the estimated important evidences for finding HLA matching donor possibilities, to HLA
With it is disease associated research, population genetic study and medical jurisprudence research etc. also have important application value.At present
Report in relation to Chinese population HLA between different regions are not agnate has very much, but majority is the low resolution gene with HLA
Parting is to be studied not over the high-resolution gene loci of HLA according to progress follow-up study with serotype
The method of a set of maturation.HLA is located at No. 6 chromosome of human body, and the high-resolution gene loci of HLA has 5：HLA-A、-B、-C、-
DRB1 and DQB1, wherein HLA-DRB1 are the gene locis that polymorphism is most abundant in HLA-II genoids, determine that DR antigens are polymorphic
Property.Therefore HLA-DRB1 gene pleiomorphisms are the important components of immunizing antigen polymorphism, may influence the hair of a variety of diseases
It is raw.
HLA-DRB1 genes all have single-gene polymorphism, the diagnosis of this gene pleiomorphism for disease generation with it is pre-
It is anti-to have important role.Existing gene diagnosis mainly uses traditional PCR sequencing PCR or genechip detection method, the inspection of this type
The amount that survey method needs detection sample is larger, and detection time is long, complex steps, and the economy for considerably increasing measured is negative
Load.Therefore, inventor has developed a HLA high-resolution gene loci DRB1's based on the research for many years to pyrosequencing
Rapid amplifying diagnostic kit and amplification method.
Invention content
In view of the above-mentioned problems existing in the prior art, the purpose of the present invention is based on linearized index round pcr, obtain
A kind of the rapid amplifying diagnostic kit and amplification method of HLA high-resolution gene loci.
One of for achieving the above object, the rapid amplifying diagnosis examination for the HLA high-resolution gene locis that the present invention uses
The technical solution of agent box is as follows：
The amplification kit includes archaeal dna polymerase and amplimer, and HLA high-resolution gene locis are HLA-DRB1,
It expands template sequence and is included in such as sequence table SEQ ID NO：In sequence shown in 1, amplimer expands for linearized index PCR
Increase, amplimer upstream sequence such as sequence table SEQ ID NO：Shown in 2, downstream sequence such as sequence table SEQ ID NO：Shown in 3.
Sequencing primer specificity height in the present invention, accuracy are good, can accurately detect the SNP site of HLA-DRB1,
Sequence can largely generate the single stranded DNA convenient for pyrosequencing for linearized index PCR (LATE-PCR), without to routine
Double-stranded DNA carries out uncoiling and separation.
It is Excess primer to have one in primer sequence, and one is restricted primer, Excess primer and restricted primer with just
To having no specific formulation relationship between primer and reverse primer, the fragment single-chain that can be expanded as needed selects to expand
Amount, it is Excess primer that the corresponding amplimer of big chain is measured in amplification, and the corresponding amplimer of another chain is restricted primer.
When design primer, since linearized index PCR is for the more demanding of primer, it is desirable that the difference of the Tm values between primer >=
5 DEG C, difference≤13~18 DEG C of the Tm values between amplified production and Excess primer.It can ensure best extension effect in this way, but
The temperature requirement is not absolute, as long as purpose product to be measured can be amplified by LATE-PCR, so that it may to use.
Preferably, such as sequence table SEQ ID NO in the present invention：Primer shown in 2 is Excess primer, such as sequence table SEQ ID
NO：Primer shown in 3 is restricted primer.
The single-stranded progress that the specific choice of Excess primer and restricted primer can expand according to actual needs in the present invention
It exchanges, that is to say, that such as sequence table SEQ ID NO：Primer shown in 2 is restricted primer, such as sequence table SEQ ID NO：3 institutes
The primer shown is Excess primer.The dosage of primer can be adjusted with common sense in the field according to actual needs, and one more than dosage
Primer is Excess primer, and a few primer of dosage is restricted primer, and protection scope of the present invention should not be by primer name
Claim the limitation with usage amount.
It is furthermore preferred that Excess primer is index times with additive amount of the restricted primer in system, minimum is 10 times.
Preferably, amplifing reagent further includes amplification buffer, dNTPs and Mg2+。
Amplified production in the present invention preferably uses pyrosequencing to carry out Sequence Detection, and Pyrosequencing primer is preferably such as
Sequence table SEQ ID NO：Shown in 4.But pyrosequencing is not unique sequencing mode, and any can detect gene order
The prior art can be used as detection method by the present invention use, therefore, pyrosequencing should not be used as be to the present invention
Limitation.
Second object of the present invention is to provide a kind of amplification method using any of the above-described a kit, the expansion
Increasing system uses 25 μ L systems, specially：
1×PCR buffer、3mmol/L MgCl2, 100 μm of ol/L dNTPs, 1.5U Taq DNA polymerases, 0.1 μ
The restricted primer P of mol/LL, 1 μm of ol/L Excess primers PE, 13% glycerine and 4%BSA 2mmol/L；1 μ L DNA profilings, add water
It is supplemented to 25 μ L.
Amplification condition is 95 DEG C of pre-degeneration 5min, 60 DEG C of amplification cycles, 72 DEG C of extension 10min, 4 DEG C of holdings, wherein every 87
DEG C 10s, 66 DEG C of 10s and 72 DEG C of 20s form an amplification cycles.
Pyrosequencing is preferably used after amplification, although the product after amplification is largely single-stranded, but still has subregion
Still amplified production is made annealing treatment, is prepared as single-stranded mould in order to make pyrophosphoric acid detection according to accurately and quickly for double-strand
Plate.
Preferably, two kinds of reagents are used when annealing：Reagent A is other pyrosequencings examination without Apyrase
Agent, including DTT, APS, PVP, ATP sulfurylase, Klenow, fluorescein and luciferase, reagent B be reagent A with
The mixed liquor of Apyrase.
Wherein, a kind of most preferred allocation plan of reagent A is：0.1mol/L Tris- acetic acid (pH 7.7), 2mmol/L
EDTA, 10mmol/L magnesium acetate, 0.1%BSA, 1mmol/L dithiothreitol (DTT)s (DTT), 2 μm of ol/L APS, 0.4g/L PVP,
200U/L ATP sulfurylase, 18U/mL Klenow, 0.8mmol/L D- fluoresceins, 6mg/L luciferases.
Most preferably a kind of allocation plans of reagent B are：The mixed liquor of reagent A and 3.2U/mL Apyrase.
When annealing, 1~2 μ L LATE-PCR products and 1.2 μ L APS (1mmol/L) are taken, be added 30 μ L reagent As, after mixing
10~15min is placed, then 40 μ L reagent B mixings is added to place 3~5min, annealing primer is then added, and (final concentration reaches 0.3 μ
Mol/L), 10~15min of annealing at room temperature.
The present invention is sequenced using the pyrosequencing instrument that our company voluntarily researches and develops, sequencing result and existing commercially available coke
Phosphoric acid sequenator is compared, and not only result is more accurate, and the high sample dosage of precision is small, and detection appearance is accurate, is not necessarily to repeated authentication
Experiment.
Due to the discovery of specific primer and the foundation of amplification system, the invention also discloses a kind of HLA-DQB1 genes
The application of specific primer and amplification system in rapid amplifying diagnostic kit, the kit using linearized index PCR with
The mode that pyrosequencing combines carries out amplification diagnosis to HLA-DQB1 genes.
Rapid amplifying diagnostic kit and amplification method in the present invention can be used for the clinic or medicine of HLA-DQB1 genes
Neo-Confucianism diagnosis, diagnostic sample source is unlimited, and diagnostic result can be used as gene screening, disease susceptibility, medication guide etc. multi-party
The diagnosis basis in face may be implemented pair using amplification kit, amplification method or other conventional amplification methods in the present invention
The efficient amplification of HLA-DQB1 genes diagnoses, and has great importance for the research and clinical development of the gene.
Compared with prior art, the present invention uses the target fragment amplification method of linearized index amplification, the expansion of Precise spraying
Increasing primer amplification is efficient and mispairing is few, not only considerably increases amplification efficiency, and directly amplify single stranded DNA and can be directly used for
Pyrosequencing, without amplified production is marked and double-strand separation, avoid lengthy and tedious experimental procedure, decrease highly basic
Damage of the property reagent to amplified fragments.Primer and experimental method in this kit are obtained by accurate design and test
, it is suitable for the SNP site detection of the HLA-DRB1 of various samples, and detection efficiency is high, testing result is accurate.Directly use
Amplification detection kit in the present invention and detection method, the operation of laboratory technician is required it is low, to detect environment tolerance it is strong,
Augmentation detection work can be rapidly carried out at any time, and convenient and efficient, testing cost is low, as a result accurate and show that the time is short, can be with
The economy and mental burden for farthest mitigating examinee, have good market potential value.
Description of the drawings
Fig. 1 is linearized index amplification product gel electrophoresis figure provided by the invention；
Fig. 2 is the gene sequencing figure of pyrosequencing provided by the invention.
Specific implementation mode
The rapid amplifying diagnostic kit to HLA high-resolution gene loci provided by the invention and expansion with reference to embodiment
Increasing method work is further detailed, completely illustrates.The embodiments described below is exemplary, and is only used for explaining the present invention, and
It is not considered as limiting the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.Reality as used in the following examples
It tests material unless otherwise specified, is that market is commercially available.
The instrument and reagent used in the present invention is as follows：
PTC-255PCR amplification instruments, MJ Research companies of the U.S.；
Pyrosequencing instrument, the Wuhan bio tech ltd Fei Site；
Taq DNA polymerase, dNTPs (10mmol/L), 10 × Buffer and MgCl2(25mmol/L) (Dalian TaKaRa is public
Department)；AmpliTaqGold polymerases (AppliedBiosystems companies)；KlenowFragment (no 5 prime excision enzyme activity), second
Vinyl pyrrolidone (PVP) and Luciferase (Promega companies), bovine serum albumin(BSA) (BSA), APS and Apyrase
(Sigma companies), α-vulcanization deoxy adenosine triphosphate (dATP α S), dGTP, dTTP and dCTP (Amersham Pharmacia
Biotech companies)；ATPsulfurylase is expressed by our company laboratory；Other reagents are that analysis is pure, and all reagents are used
Sterile deionized water dilutes.Primer is synthesized by Shanghai Invitrogen companies in kit, specific Primer and sequence see
Sequence table.
Rapid amplifying diagnostic kit in the present invention includes：Primer amplification group and pyrosequencing group, wherein primer expand
Increasing group includes amplimer, archaeal dna polymerase, amplification buffer, dNTPs and Mg2+, pyrosequencing group includes annealing primer, DNA
Polymerase (AmpliTaqGold polymerases), ATP sulfurylases (ATPsulfurylase), luciferase (Luciferase),
Apyrase (Apyrase) and dNTP, and configure the Klenow enzymes (KlenowFragment) of reaction solution.
It is furnished with complete operating method in kit, such as sample to be amplified is untreated whole blood sample, need to use and use
In the buffer solution of whole blood sample PCR, wherein containing 100mmol/L Tris-HCl, 50mmol/L KCl, pH 9.3~9.5.
One, amplimer designs
The SNP segments of HLA-DRB1 are expanded using linearized index PCR in the present invention, are amplified enough single-stranded
DNA profiling is to carry out pyrosequencing.According to the record to the SNP site of HLA-DRB1 in NCBI, No. rs of HLA-DRB1 is
701831, the long 1101bp of sequence, SNP site are located at the 501st, and A/T/G/C is likely to occur mispairing, specific sequence when mispairing occurs
Row such as sequence table SEQ ID NO:Shown in 1, N indicates the SNP site of HLA-DRB1, SEQ ID NO:Lower stroke specific as follows of 1 sequence
Line part is specific primer coupling part：
TTACTACACTCTGTACTTTCCAAGATTTGTAAACAGTTTGACAATGCATGCCGATTTAAA
ACTATGAAGAAACAAACACAATTTTTCACAACACCTCTCAAATCTAATGGGTCCTCACTG
TCAAGATTAAATTCCAGGCTGATGACACTGTAAGGTCACATGGCCAGCTGTGCTATAGGC
CTGGTCAAGGCCAGAGCCTGGGTTTGCAGAGAAGCAGACACACAGCCAAACCAGGAGACT
TACTCTGTCTTCCTGACTCATTCCCTCTACGTTGTTTTTCTCCTAGTCCAACCTAAGGTG
ACTGTATATCCTTCAAAGACCCAGCCCCTGCAGCACCACAACCTCCTGGTCTGCTCTGTG
AGTGGTTTCTATCCAGGCAGCATTGAAGTCAGGTGGTTCCTGAACGGCCAGGAAGAGAAG
GCTGGGATGGTGTCCACAGGCCTGATCCAGAATGGAGACTGGACCTTCCAGACCCTGGTG
ATGCTGGAAACAGTTCCTCG
N
AGTGGAGAGGTTTACACCTGCCAAGTGGAGCACCCAAGCGTGACAAGCCCTCTCACAGTG
GAATGGAGTGAGCAGCTTTCTGACTTCCTAAATTTCTCACCCACTAAGAAGGGGACCGTG
CTAATCCCTGAGTGTCAGGTTCCTCCTCTCCCACATCCTATTTTAATTTGCTCCATGTTC
TCATCTCCATCAGCACAGGTCACTGGGGGTAGCCCTGTAGGTGTTTCTAGAAACACCTGT
ACCTCCTGGAGAAGCAGTCTCGCCTGCCAGGCGGGAGAGACTGTCCCTCTTTTGAACCTC
CCCATGATTTTGCAGGTCAGGGTCACCCACTCTCCCAGGCTCCAGGCCCTGCCTCTGGGT
CTGAGACTGAGTTTCTGGTGCTGTTGCTCTGAGTTATTTTTTGTGATCTGGGAAGAGGAG
AAGTGTAGGGGCCTACCTGACATGAGGGGAGTCCAATCTCAGCCCTGCTTTTTATTAGCT
TTGTCACTCTAGACAAACTA
According to said gene sequence, it was using the amplification of 5 Software for Design of Primer Primer and annealing primer, wherein E
Measure primer, Excess primer sequence such as sequence table SEQ ID NO：Shown in 2, L is restricted primer, restricted primer sequence such as sequence
Table SEQ ID NO：Shown in 3, A is amplified production.TmThe calculating of value is calculated using 3.1 softwares of Oligo Analyzer.Primer sequence
Row and parameter are as shown in table 1 below：
Table 1
Two, linearized index PCR amplification
1. sampling
100 human blood samples are chosen in this embodiment originally to be tested, samples sources in Wuhan downtown blood station, sample into
Row pretreatment is DNA profiling, and the immunoglobulin G in blood, hemoglobin and lactoferrin is avoided to do PCR reaction systems
It disturbs.
2. linearized index expands
The reaction system of linearized index amplification is 25 μ L systems, and specific ingredient is as follows in system：
1×PCR buffer(50mmol KCl、15mmol Tris-HCl、pH 8.0)、3mmol/L MgCl2、100μ
Mol/L dNTPs, 1.5U Taq DNA polymerases, the restricted primer P of 0.1 μm of ol/LL, 1 μm of ol/L Excess primers PE, 13% glycerine
With 4%BSA 2mmol/L；1 μ L DNA profilings, add water to be supplemented to 25 μ L.
Amplification condition is：95 DEG C of 5min, 60 DEG C of amplification cycles (87 DEG C of 10s, 66 DEG C of 10s, 72 DEG C of 20s), 72 DEG C of 10min, 4
DEG C keep.
Three, pyrosequencing
3.1 design of primers
Annealing primer, annealing primer sequence such as sequence table SEQ ID NO are designed according to amplified production：Shown in 4：5'-
GCCAAGTGGAGCACCCAAGCGT-3'。
The preparation of 3.2 reaction solutions
Reagent A：0.1mol/L Tris- acetic acid (pH 7.7), 2mmol/L EDTA, 10mmol/L magnesium acetates, 0.1%
BSA, 1mmol/L dithiothreitol (DTT) (DTT), 2 μm of ol/L APS, 0.4g/L PVP, 200U/L ATPsulfurylase, 18U/
ML Klenow, 0.8mmol/L D- fluoresceins, 6mg/L luciferases；
Reagent B：Reagent A, 3.2U/mL Apyrase.
The preparation of 3.3 single-stranded templates
1~2 μ L LATE-PCR products and 1.2 μ L APS (1mmol/L) are taken, 30 μ L reagent As are added, 10 are placed after mixing
~15min, then 40 μ L reagent B mixings is added to place 3~5min, annealing primer (final concentration reaches 0.3 μm of ol/L), room is then added
10~15min of temperature annealing.
3.4 pyrosequencing
Reaction solution in 3.3 steps is all transferred in pyrosequencing reaction tank, according to designed primer sequence
DNTP is added, the pyrosequencing instrument that declaration is produced using our company is sequenced, dNTP addition sequences and testing result such as Fig. 2
Shown, wherein Fig. 2 show the pyrosequencing figure of HLA-DRB1.
3.5 statistical result
It is for statistical analysis according to the testing result to 20 samples in the present embodiment, the distribution of HLA-DRB1 genotype frequencies
Situation is as shown in table 2 below：
Table 2HLA-DRB1 genotype frequency distribution statistics tables
Detection sample in the present embodiment is smaller, and the site of HLA-DRB1 is more in addition and has the spy of Regional Distribution unevenness
Point, therefore this testing result does not represent the overall distribution situation of the genotype, only indicates the statistical result of this embodiment detection.
Four, testing result is verified
4.1 agarose gel electrophoresis
2% agarose gel electrophoresis detection, electrophoresis result figure such as Fig. 1 will be carried out by the amplified production of linearized index amplification
Shown, non-label band is DNA Marker in figure, and 1~5 is the amplified production randomly selected, and 6 be negative control.In figure
The target fragment size of purpose band position display amplification is correct.
It is it is necessary to described herein finally：Above example is served only for making technical scheme of the present invention further detailed
Ground illustrates, should not be understood as limiting the scope of the invention, those skilled in the art's the above according to the present invention
Some the nonessential modifications and adaptations made all belong to the scope of protection of the present invention.
Sequence table
<110>New increase income is won freely（Wuhan）Bio tech ltd
<120>A kind of the rapid amplifying diagnostic kit and amplification method of HLA high-resolution gene loci
<130> 2017
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1001
<212> DNA
<213> Homo sapiens
<220>
<221> gene
<222> (1)..(1001)
<223>HLA-DRB1 gene orders
<220>
<221> misc_feature
<222> (501)..(501)
<223> n is a, c, g, t or u
<400> 1
ttactacact ctgtactttc caagatttgt aaacagtttg acaatgcatg ccgatttaaa 60
actatgaaga aacaaacaca atttttcaca acacctctca aatctaatgg gtcctcactg 120
tcaagattaa attccaggct gatgacactg taaggtcaca tggccagctg tgctataggc 180
ctggtcaagg ccagagcctg ggtttgcaga gaagcagaca cacagccaaa ccaggagact 240
tactctgtct tcctgactca ttccctctac gttgtttttc tcctagtcca acctaaggtg 300
actgtatatc cttcaaagac ccagcccctg cagcaccaca acctcctggt ctgctctgtg 360
agtggtttct atccaggcag cattgaagtc aggtggttcc tgaacggcca ggaagagaag 420
gctgggatgg tgtccacagg cctgatccag aatggagact ggaccttcca gaccctggtg 480
atgctggaaa cagttcctcg nagtggagag gtttacacct gccaagtgga gcacccaagc 540
gtgacaagcc ctctcacagt ggaatggagt gagcagcttt ctgacttcct aaatttctca 600
cccactaaga aggggaccgt gctaatccct gagtgtcagg ttcctcctct cccacatcct 660
attttaattt gctccatgtt ctcatctcca tcagcacagg tcactggggg tagccctgta 720
ggtgtttcta gaaacacctg tacctcctgg agaagcagtc tcgcctgcca ggcgggagag 780
actgtccctc ttttgaacct ccccatgatt ttgcaggtca gggtcaccca ctctcccagg 840
ctccaggccc tgcctctggg tctgagactg agtttctggt gctgttgctc tgagttattt 900
tttgtgatct gggaagagga gaagtgtagg ggcctacctg acatgagggg agtccaatct 960
cagccctgct ttttattagc tttgtcactc tagacaaact a 1001
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(22)
<223>HLA-DRB1 gene Excess primer sequences
<400> 2
gagtggtttc tatccaggca gc 22
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
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<222> (1)..(22)
<223>The restricted primer sequence of HLA-DRB1 genes
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tgagagggct tgtcacgctt gg 22
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<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(22)
<223>HLA-DRB1 gene annealing primers
<400> 4
gccaagtgga gcacccaagc gt 22
Claims (10)
1. a kind of rapid amplifying diagnostic kit of HLA high-resolution gene loci, which is characterized in that the amplification kit includes
Archaeal dna polymerase and amplimer, HLA high-resolution gene locis are HLA-DRB1, and amplification template sequence is included in such as sequence table
SEQ ID NO：In sequence shown in 1, amplimer is used for linearized index PCR amplification, such as sequence table of amplimer upstream sequence
SEQ ID NO：Shown in 2, downstream sequence such as sequence table SEQ ID NO：Shown in 3.
2. the rapid amplifying diagnostic kit of HLA high-resolution gene loci according to claim 1, which is characterized in that institute
It is Excess primer to state in amplimer one, another is restricted primer.
3. the rapid amplifying diagnostic kit of HLA high-resolution gene loci according to claim 1, it is characterised in that：Institute
The quantity for stating Excess primer is at least 10 times of restricted primer.
4. the rapid amplifying diagnostic kit of HLA high-resolution gene loci according to claim 1, it is characterised in that：Institute
Stating kit further includes：Amplification buffer, dNTPs and Mg2+。
5. the rapid amplifying diagnostic kit of HLA high-resolution gene loci according to claim 1, it is characterised in that：Institute
It states and obtains gene order through pyrosequencing after amplimer is annealed.
6. the rapid amplifying diagnostic kit of HLA high-resolution gene loci according to claim 5, it is characterised in that：It is burnt
The primer sequence such as sequence table SEQ ID NO of phosphoric acid sequencing：Shown in 4.
7. a kind of amplification method using according to claim 1~6 any one of them HLA high-resolution gene locis, feature
It is, the amplification system of the amplification method is：1×PCR buffer、3mmol/L MgCl2、100μmol/L dNTPs、
1.5U Taq DNA polymerases, the restricted primers of 0.1 μm of ol/L, 1 μm of ol/L Excess primer, 13% glycerine and 4%BSA 2mmol/
L；1 μ L DNA profilings, add water to be supplemented to 25 μ L.
8. the amplification method of HLA high-resolution gene loci according to claim 7, it is characterised in that：The amplification condition
For：95 DEG C of pre-degeneration 5min, 60 DEG C of amplification cycles, 72 DEG C of extension 10min, 4 DEG C of holdings.
9. the amplification method of HLA high-resolution gene loci according to claim 7, it is characterised in that：60 DEG C of amplifications
Cycle forms a cycle by 87 DEG C of 10s, 66 DEG C of 10s and 72 DEG C of 20s.
10. the application of a kind of specific primer and amplification system of HLA-DQB1 genes in rapid amplifying diagnostic kit,
It is characterized in that：The kit expands HLA-DQB1 genes in such a way that linearized index PCR is combined with pyrosequencing
Increase diagnosis.
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