CN106929591A - A kind of human HLA B*5801 genetic polymorphism detection kits - Google Patents
A kind of human HLA B*5801 genetic polymorphism detection kits Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention relates to a kind of human HLA B5801 genetic polymorphism detection kits, the kit includes PCR buffer solutions, specific primer, specific probe, interior mark system, Taq enzyme, UNG enzymes, weakly positive control and blank.This kit also includes blood treatment agent, and blood sample just can directly enter performing PCR amplification through simple process, eliminate DNA extraction process, save the operating time.The present invention realizes two kinds of different genotype of detection in a pipe using the technology of SNP probe combination ARMS primers.Mark system is used for monitor sample quality design simultaneously in, and the control of design weakly positive and blank are used for monitoring reagent box quality.The present invention is for detecting the HLA B*5801 allele advantage such as have high specificity, sensitivity high, quick, safety simple to operate, result interpretation objective.
Description
Technical field
The invention belongs to biotechnology and medical domain, in particular to a kind of inspection of human HLA-B*5801 genes
Test agent box.
Background technology
Allopurinol is the first-line drug for treating gout, is currently the only xanthine oxidase inhibitor, is mainly used in
Treatment gout and the auxiliary treatment for preventing gouty nephropathy, secondary hyperuricemia and severe epilepsy.Its metabolite oxygen
By suppressing the activity of xanthine oxidase, (the latter can make hypoxanthine switch to xanthine to purine alcohol, then be transformed into xanthine
Uric acid), generate uric acid and reduce, the uric acid content in blood and in urine is reduced to the level below solubility, so as to prevent uric acid
The deposition of calculus, contributes to the dissolving again of tophus and uric acid crystal.But allopurinol can cause serious side effect, bag
Include Stevens-John syndromes (SJS) and toxic epidermal's necrosis (TEN).
Scientific research finds the serious skin adverse reaction (SCARs) that HLA-B*5801 allele causes with allopurinol
There is very strong relevance, and the frequency that this allele is carried in Chinese population is higher, therefore U.S.'s rheumatism management in 2012 years
Association have updated gout administration guide, wherein suggestion is using before allopurinol, to people at highest risk (the Chinese descendants of severe allergic reaction
Han nationality, South Korea descendants, Thailand descendants), it should carry out HLA-B*5801 allele detections.
HLA (HLA) can be divided three classes, wherein the HLA-B of the HLA-B series antigens in coding I quasi-molecules
Gene has thousands of kinds of allele, and HLA-B*5801 allele is one kind therein.
Japanese scholars research finds the SJS/ caused by the SNP-rs9263726 and allopurinol of PSORS1C1 gene locis
TEN is related, and the SNP and HLA-B*5801 complete linkages, and this is found to be detection HLA-B*5801 allele and have found letter
Single quickly method.
Detection method currently for gene pleiomorphism is a lot, such as direct sequencing, and pyrosequencing method, high-resolution is molten
Solution curve detection method (High Resolution Melting Analysis, HRM), fluorescence quantitative PCR method etc..It is wherein most common
Method is PCR sequencing PCR, and the method expense is relatively low, but operation time-consuming and sensitivity is low；High-resolution solubility curve method will to equipment
Ask comparing special, there is certain difficulty in clinical expansion；Conventional fluorescent quantitative PCR method clinically applies relatively broad, but
The method detection gene pleiomorphism is typically detected that the method is to sample type, sample quality using Genotyping methods
And polymorphism distribution there are certain requirements, whole blood sample and low concentration genomic DNA can not be detected accurately.Therefore, need
Setting up one kind fast and effectively can be with direct detection whole blood, while the detection method of compatible detection low concentration genomic DNA.
In view of this, it is special to propose the present invention.
The content of the invention
It is an object of the invention to provide a kind of detection kit of HLA-B*5801 gene pleiomorphisms, solved with this existing
Whole blood sample cannot be detected directly in genetic polymorphism detection technology, and low concentration genomic DNA Detection results are paid no attention to
The problem thought.This kit can be used for high flux, inexpensive quick detection HLA-B*5801 gene pleiomorphisms, and its sensitivity is high,
High specificity, is applicable to EDTA, citrate anticoagulation fresh whole blood or blood plasma and is directly detected, it is also possible to suitable for from
The detection of the human genome DNA extracted in EDTA, citrate anticoagulation fresh whole blood or blood plasma.In order to realize on of the invention
Purpose is stated, spy uses following technical scheme：
A kind of human HLA-B*5801 genetic polymorphism detection kits, including：
Nucleotides sequence is classified as SEQ ID NO:Positive outer primer shown in 1；
Nucleotides sequence is classified as SEQ ID NO:Reverse outer primer shown in 2；
Nucleotides sequence is classified as SEQ ID NO:The ARMS primers in the mutational site for testing goal gene shown in 3；
Nucleotides sequence is classified as SEQ ID NO:The Taqman probes for detecting wild type site shown in 4；
Nucleotides sequence is classified as SEQ ID NO:The Taqman probes for detecting mutational site shown in 5.
Wherein SEQ ID NO:1、SEQ ID NO:2 is common PCR primers, and amplification includes HLA-B*5801 gene pleiomorphisms
DNA fragmentation；SEQ ID NO:3 is ARMS primers, and the DNA pieces of HLA-B*5801 codons CAT are contained for specific amplification
Section.The end of ARMS primers 3 ' is matched with mutant bases, SEQ ID NO:3 ARMS primers 3 ' ends, 3rd increase reciprocal
Base mismatch.
The kit that the present invention is provided is used for the SNP of the Serial No. rs9263726 for detecting HLA-B*5801 allele
Site：
Table 1：The type of HLA-B*5801 allele
Wherein, rs9263726G is wild type site, and rs9263726A is saltant type site.
Compared with prior art, beneficial effects of the present invention are：
1) sensitivity is high, can accurately detect the genotype of 0.2ng genomic DNAs；
2) specificity is good, the technology being combined using ARMS specific primers and SNP typing probes, can be expanded with specificity
Increase the corresponding genotype of identification；
3) real results reliability, detection process is stopped pipe reaction, significantly reduces pollution, and add UNG enzymes antifouling
Dye.Humanized's internal standard monitor sample quality and operating accuracy are used in addition, and examination is monitored using weakly positive control and blank
Agent box quality, it is to avoid the generation of false positive and false negative result；
4) the clear easy analysis of results, wild-type probe, saltant type probe and internal standard probe are respectively with different fluorescence
Mark, three chrominance channels detection, with reference to result interpretation method, you can clearly it is concluded that；
5) is simple to operate, takes short, and this kit detects two kinds of polymorphisms using a pipe, it is to avoid be in charge of；Again will simultaneously
Enzyme is directly mixed in PCR system, greatly simplifies operating process；
6) can directly carry out whole blood test, be not required to extract genomic DNA again.
Brief description of the drawings
In order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art, below will be to specific
The accompanying drawing to be used needed for implementation method or description of the prior art is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is the qPCR experimental results of the embodiment of the present invention 2；
Fig. 2 is the qPCR experimental results of the embodiment of the present invention 2；
Fig. 3 is the qPCR experimental results of the embodiment of the present invention 2；
Fig. 4 is the qPCR experimental results of the embodiment of the present invention 3；
Fig. 5 is the qPCR experimental results of the embodiment of the present invention 3；
Fig. 6 is the qPCR experimental results of the embodiment of the present invention 3.
Specific embodiment
The present invention relates to a kind of human HLA-B*5801 genetic polymorphism detection kits, including：
Nucleotides sequence is classified as SEQ ID NO:Positive outer primer shown in 1；
Nucleotides sequence is classified as SEQ ID NO:Reverse outer primer shown in 2；
Nucleotides sequence is classified as SEQ ID NO:The ARMS primers in the mutational site for testing goal gene shown in 3；
Nucleotides sequence is classified as SEQ ID NO:The Taqman probes for detecting wild type site shown in 4；
Nucleotides sequence is classified as SEQ ID NO:The Taqman probes for detecting mutational site shown in 5.
Wherein SEQ ID NO:1、SEQ ID NO:2 is common PCR primers, and amplification includes HLA-B*5801 gene pleiomorphisms
DNA fragmentation；SEQ ID NO:3 is ARMS primers, and the DNA pieces of HLA-B*5801 codons CAT are contained for specific amplification
Section.The end of ARMS primers 3 ' is matched with mutant bases, SEQ ID NO:3 ARMS primers 3 ' ends, 3rd increase reciprocal
Base mismatch.
Preferably, it is SNP probes the invention provides two Taqman probes, can be with specific recognition aim sequence.
Preferably, kit as described above, the end of Taqman probes 5 ' is connected with fluorescent reporter group, in 3 ' end connections of nucleic acid
There is fluorescent quenching group, and two fluorescent reporter groups of Taqman probes are different；
The fluorescent reporter group is selected from any one in FAM, VIC, ROX, CY3 and CY5；The fluorescent quenching group choosing
Any one from TAMRA, BHQ1, BHQ2 and NFQ.
In certain embodiments of the present invention, saltant type is detected in 5 ' ends of the probe of detection wild type using FAM marks
The end of probe 5 ' using VIC marks, 3 ' end fluorescent quenching groups of two probes are NFQ (Non-Fluorescent
Quencher), the group does not produce fluorescence in itself, therefore can substantially reduce the intensity of background signal.It is further preferred that two
Bar probe in a reaction tube, establishes same two kinds of multi-fluorescences of different polymorphisms of reaction tube detection same gene simultaneously
Quantitative PCR detection
In certain embodiments of the present invention, all it is connected with MGB on the Taqman probes of wild type and saltant type
(Minor Groove Binder) modification group；
Preferably, the MGB modification groups are connected with the fluorescent quenching group；Such as MGB-NFQ；
MGB modification groups do not produce fluorescence in itself, therefore can substantially reduce the intensity of background signal, while the specificity
MGB modification groups are also associated with probe, the Tm values of the probe 10 DEG C or so, therefore same Tm values can be improved, MGB is visited
Pin can be more shorter than what general T aqman probes were designed so that probe is special when identification has Single nuclear polymorphism site
The opposite sex is stronger.
In certain embodiments of the present invention, the kit additionally provides interior mark system, and the interior mark system includes
Interior label primer and internal standard probe；
Preferably, the interior label primer and internal standard probe are humanized's internal standard；
It is highly preferred that the end of internal standard probe 5 ' is connected with fluorescent reporter group, being connected with fluorescence at 3 ' ends of nucleic acid quenches
Go out group；The fluorescent reporter group is selected from any one in FAM, VIC, ROX, CY3 and CY5；The fluorescent quenching group choosing
Any one from TAMRA, BHQ1, BHQ2 and NFQ；And the fluorescence report base on the internal standard probe and the Taqman probes
Group is different；
It is highly preferred that the interior label primer is classified as SEQ ID NO for nucleotides sequence:Positive interior label primer shown in 6, nucleosides
Acid sequence is SEQ ID NO:Reverse interior label primer shown in 7；The nucleotides sequence of the internal standard probe is classified as SEQ ID NO:8 institutes
Show；
Using internal standard system monitoring, the quantitative fluorescent PCR is reacted with the presence or absence of suppressing, due to certain in sample to be detected
A little compositions may contain causes PCR to occur partially or completely suppressing；Additionally, between amplification instrument there may be the hole higher than allowed band
Difference, causes amplification efficiency difference between different pipes, and artificial sample-adding mistake is likely to cause the appearance of false negative result, therefore this in addition
Above-mentioned hidden danger is eliminated in inventive embodiments using interior mark system, it is ensured that the accuracy of testing result；SEQ ID NO:6~8
The sequence selection of the internal standard compound that shown primer is detected be human genome DNA one section of GAPDH sequence；Described
GAPDH sequences are present in human genome simultaneously with target sequence, and with target gene to be checked without homology.Therefore ensure that interior
Index thing only expands GAPDH sequences, without influence target sequence amplification；
Preferably, the internal standard probe 5 ' is terminal modified a ROX, and 3 ' terminal modified have BHQ2.
In certain embodiments of the present invention, the kit preferably also includes weakly positive comparison liquid and/or blank pair
According to liquid；
The weakly positive comparison liquid is that, containing 2 kinds of mixed liquors of plasmid, 2 kinds of plasmids contain 2 kinds of different bands respectively
There is the HLA-B*5801 allele in mutational site to be detected (comprising rs9263726G or rs9263726A mutation position i.e. in plasmid
The DNA fragmentation of point)；The plasmid can be plasmid well known to those skilled in the art, and this 2 kinds of plasmid concentrations can be with identical, it is preferable that
The concentration of the plasmid is 2000~3000copies/ μ l；
The blank liquid is Tris-HCl buffer solutions；Preferably, the concentration of the Tris-HCl buffer solutions be 7~
13mM, pH are 7.5~8.5.
Preferably, kit as described above, the kit is also included in blood treatment agent, PCR buffer solutions, enzyme and water
One or more；
Preferably, kit as described above, the blood treatment agent is the 0.9% NaCl aqueous solution；
Blood sample can directly enter performing PCR and expand after 10~20 times of dilutions of blood inorganic agent, eliminate DNA and extract step
Suddenly.
Preferably, kit as described above, the PCR buffer solutions include PCR additives, MgCl2In dNTPs
One or more；
Preferably, MgCl in PCR buffer solutions2Concentration be 1.0~5.0mM；
Preferably, the PCR additives include one or more in DMSO, glycerine, BSA；
Preferably, the dNTPs includes dATP, dGTP, dCTP, dUTP；It is highly preferred that dATP, dUTP, dGTP and dCTP
Each 0.1~0.5mM of concentration.
Preferably, kit as described above, the enzyme includes Taq enzyme, UNG enzymes；
Preferably, the Taq enzyme and UNG enzymes are individually dispensed or are mixed and made into solution；
Preferably, the Taq enzyme and UNG enzymes are mixed into PCR working solutions；Direct point sample can enter performing PCR amplification, greatly
Save the operating time, whole reaction only needs to complete for 90 minutes.
It is highly preferred that when the Taq enzyme and UNG enzymes are mixed and made into solution, the total amount ratio of the Taq enzyme and UNG enzymes is：
(0.5U~1.0U)：(0.1U~0.5U).
Contain Taq enzyme, preferably HotStart Taq enzymes in the enzyme solutions that the present invention is provided, it is that PCR reactions are necessary,
And the enzyme solutions also contain UNG enzymes.Wherein UNG (uracil-N-glycosylase) enzyme is uracil-N-glycosylase, and it is special
Point is that optimum activity temperature is 50 DEG C, 95 DEG C of inactivations, and its action principle is the selective hydrolysis fracture double-strand containing dU or single-stranded
Uracil glycosidic bond in DNA, is formed with the DNA of missing base.In PCR reactions, non-specificity can be prevented using UNG enzymes
PCR is expanded and polluted.
The invention further relates to a kind of method that human HLA-B*5801 genetic tests are carried out using kit as described above, bag
Include
(1) reagent in the kit and the sample containing DNA are carried out mixing composition PCR reaction systems, and is carried out
PCR reacts；(2) fluorescence signal intensity in analysing amplified product.
Preferably, it is positive outer primer, reverse outer primer, ARMS primers, wild for detecting in the PCR reaction systems
The Taqman probes in type site, the concentration ratio of Taqman probes for detecting mutational site are 0.2 μM -1.0 μM：0.2μM-
1.0μM：0.5μM-1.5μM：0.1μM-0.5μM：0.2μM-1.0μM；
It is highly preferred that also including positive interior label primer, reverse interior label primer and internal standard probe in working as the PCR reaction systems
When, SEQ ID NO:The concentration ratio of 1~8 shown primer is：0.2μM-1.0μM：0.2μM-1.0μM：0.5μM-1.5μM：0.1
μM-0.5μM：0.2μM-1.0μM：0.1μM-0.5μM：0.1μM-0.5μM：0.1μM-0.5μM.
Preferably, method as described above, the sample containing DNA is EDTA and/or citrate anticoagulation is fresh complete
Blood or blood plasma；
Or the human genome DNA extracted from EDTA and/or citrate anticoagulation fresh whole blood or blood plasma.
The detection method specifically may include following steps：
(1) design and screen specific amplification and combined and probe groups with the primer of detection HLA-B*5801 gene pleiomorphisms
Close；Prepare human HLA-B*5801 gene detecting kits of the invention.
(2) blood sample treatment；
(3) fluorescent quantitative PCR is carried out：Often added in pipe specific primer group and probe groups, PCR reaction buffers,
Taq enzyme, UNG enzymes, interior mark system.Reaction solution prepare after the completion of be dispensed into PCR reacting holes, addition treatment after blood sample or
The genomic DNA for having extracted, performing PCR of going forward side by side reaction.
Specifically, by taking 25 μ l reaction systems as an example, to each in the mixture obtained after addition detected sample in reaction tube
Component final concentration and content are as follows：
Above-mentioned system is only illustrative, can proportionally expand or shrink in actual applications the volume of mixture and its
Middle each component content.
Specifically, in above-mentioned 25 μ l reaction systems, each pair of primer includes 1 pair of specificity in above-mentioned steps (1)
Primer, 1 ARMS screens primer and interior label primer, and each bar probe includes specific probe in above-mentioned steps (1) and interior
Mark probe, the sample is whole blood sample or genomic DNA.
Specifically, the quantitative fluorescent PCR reaction condition in step (3) is：
37 DEG C are processed 10 minutes, 95 DEG C of predegenerations 5 minutes,
40 circulations：95 DEG C 15 seconds, 60 DEG C 60 seconds and are collected fluorescence signal.
After above-mentioned PCR reactions, acquired results carry out result judgement by table 1：
The result judgement of table 2
Detection method of the invention has sensitivity high, high specificity, the believable advantage of real result, while the detection side
Method is simple to operate quick, and detection can be completed in 90 minutes, and result interpretation is simply objective, is easy to analysis.
The present invention is expanded on further below by way of specific embodiment.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, are
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
Human HLA-B*5801 gene detecting kits of the invention are prepared, is comprised the following steps：
1. primer and probe synthesize：
Design and synthesize specific primer SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3, specific probe SEQ
ID NO:4 and SEQ ID NO:5, and in SEQ ID NO:45 ' end flag F AM fluorophors, 3 ' end mark NFQ-MGB do not send out
Optical quenching group, in SEQ ID NO:55 ' end mark VIC fluorophors, 3 ' the end not luminous quenching groups of mark NFQ-MGB.
Primer, probe are prepared into 100 μM of mother liquor storage respectively.
2. mark system in preparing：Design and synthesize 1 pair of interior label primer for humanized's gene GAPDH, the primer pair sequence
It is classified as SEQ ID NO:6 and SEQ ID NO:7；Internal standard probe is designed and synthesized, the probe is SEQ ID NO:8.In SEQ ID
NO:85 ' end mark ROX fluorophors, 3 ' the end not luminous quenching groups of mark BHQ2.The interior label primer, internal standard are matched somebody with somebody respectively
It is made 100 μM of mother liquor storage.
3. other reagents are prepared：PCR buffer solutions are prepared, wherein the MgCl containing 3.0mM2, dATP, dUTP, dGTP and
DCTP each 0.2mM, DMSO 2ul, BSA 0.1mg/ml；Enzyme mixation is prepared, wherein containing Taq enzyme 0.5U, UNG enzymes 0.25U.
4. weakly positive comparison liquid and blank liquid are prepared, 2 kinds of DNAs, these matter are contained in the weakly positive comparison liquid
Different many of 2 kinds of the HLA-B*5801G plasmids being related to containing kit of the present invention respectively in grain DNA and HLA-B*5801A plasmids
State property plasmid, the selection of the plasmid and be designed as those skilled in the art know, be 2500copies/ μ l；The blank
Liquid is Tris-HCl (10mM) buffer solution.
5.PCR reaction solutions are prepared：It is carried out according to the following table the preparation of PCR reaction solutions：
Each composition usage amount of 20 person-portions is calculated, each component is once added, fully mixed.
6. blood treatment agent is prepared, and blood treatment agent is 0.9% NaCl.
7. kit is assembled：Kit each component is subsequently assembled into finished product kit, No. 1 pipe HLA-B* is specifically included
5801PCR reaction solutions, No. 2 pipe blood treatment agent, No. 3 pipe weakly positive controls, No. 4 pipe blanks.
Embodiment 2
Human HLA-B*5801 the gene detecting kits prepared with embodiment 1 are detected to testing sample.
10 EDTA anticoagulant whole blood samples are collected in the present embodiment, is processed with blood treatment agent, then with embodiment 1
Human HLA-B*5801 the gene detecting kits of preparation are detected.
1. blood sample treatment
The μ l of whole blood 10 are taken, adds the blood treatment agent of 90 μ l, concussion to mix 1min.
2. the fluorescence quantitative PCR detection of sample
By sample in step 1 take 2 μ l be added separately to 23 μ l embodiment 1 kit HLA-B*5801 detect reaction
In system, make reaction system cumulative volume for 25 μ l, and be put into quantitative real time PCR Instrument, after PCR response procedures are set as described below
Carry out amplified reaction：
37℃10min；
95℃5min；
95 DEG C of 15s, 60 DEG C of 60s, 40 circulations；The fluorescence signal of FAM, VIC and ROX is collected after each circulation.
3. the Analysis of test results of sample:The testing result of 10 samples is as follows：
Wild 6 of HLA-B*5801 sites G/G homozygosis, one of testing result is as shown in Figure 1；HLA-B*5801 sites
1, A/A homozygous mutations sample, testing result is as shown in Figure 2；3, HLA-B*5801 sites G/A heterozygous mutants sample, wherein one
Individual testing result is as shown in Figure 3；
Above-mentioned 10 sample fluorescences quantitative PCR detection result is consistent with sequencing result.Result above shows implementation of the present invention
The detection kit that example is provided is used for human HLA-B*5801 genetic polymorphism detection reliable results, is reached with direct Sequencing concordance rate
To 100%.
Embodiment 3
Testing result is the remaining whole blood sample of heterozygous mutant in Example 2, extracts genomic DNA, determines DNA concentration
Afterwards, DNA is diluted to 10ng/ μ l, 1ng/ μ l and 0.1ng/ μ l, the detection performance of kits for evaluation successively.
1. extracting genome DNA
The μ l of whole blood 300 are taken, the cell pyrolysis liquid CL of 900 μ l is added, is overturned and is mixed, stand 5min；
10,000rpm (11,500 × g) are centrifuged 1min, suck supernatant, cell precipitation are left, to the cell being collected by centrifugation
Add 200 μ l buffer solution GS, vibration to thoroughly mixing in precipitation；
20 μ l Proteinase K solution are added, is mixed；
Plus 200 μ l buffer solution GB, fully reverse to mix, 56 DEG C of placement 10min, overturn mix for several times therebetween, solution strain
It is limpid (such as solution does not become limpid thoroughly, please extend pyrolysis time to solution it is limpid)；
Plus 200 μ l absolute ethyl alcohols, it is fully reverse to mix, now it is possible that flocculent deposit；
Previous step resulting solution and flocculent deposit all add in an adsorption column CB3 (adsorption column CB3 is put into collecting pipe
In), 12,000rpm (13,400 × g) centrifugation 30sec outwell the waste liquid in collecting pipe, and adsorption column CB3 is put into collecting pipe；
To adding 500 μ l buffer solutions GD (please first checked whether before and added absolute ethyl alcohol) in adsorption column CB3,12,
000rpm (13,400 × g) is centrifuged 30sec, outwells the waste liquid in collecting pipe, and adsorption column CB3 is put into collecting pipe；
To adding 600 μ l rinsing liquids PW (please first checked whether before and added absolute ethyl alcohol) in adsorption column CB3,12,
000rpm (13,400 × g) is centrifuged 30sec, outwells the waste liquid in collecting pipe, and adsorption column CB3 is put into collecting pipe；
To 600 μ l rinsing liquids PW, 12,000rpm (13,400 × g) centrifugation 2min are added in adsorption column CB3, waste liquid is outwelled；
Adsorption column CB3 is placed in room temperature and places several minutes, thoroughly to dry the rinsing liquid of remnants in sorbing material；
Adsorption column CB3 is transferred in 1.5ml centrifuge tubes, 100 μ l elution buffers are vacantly added dropwise to adsorbed film centre position
TB, room temperature places 2-5min, 12,000rpm (13,400 × g) centrifugation 2min, and solution is collected into centrifuge tube；
Take 2 μ l resulting solutions survey OD values to determine DNA concentration, then by sample DNA be diluted to 10ng/ μ l, 1ng/ μ l and
0.1ng/μl。
2. the fluorescence quantitative PCR detection of sample
DNA sample after being diluted in step 1 takes the kit that 2 μ l are added separately to the embodiment 1 of 23 μ l successively
In HLA-B*5801 detection reaction systems, make reaction system cumulative volume for 25 μ l, and be put into quantitative real time PCR Instrument, by following institute
Amplified reaction is carried out after showing setting PCR response procedures：
37℃10min；
95℃5min；
95 DEG C of 15s, 60 DEG C of 60s, 40 circulations；The fluorescence signal of FAM, VIC and ROX is collected after each circulation.
3. the Analysis of test results of sample：
3 groups of pattern detection results are as shown in accompanying drawing 4~6.
A group DNA total amounts are 20ng (10ng/ μ l, μ l of loading 2)；
B group DNA total amounts are 2ng (1ng/ μ l, μ l of loading 2)；
C group DNA total amounts are 0.2ng (0.1ng/ μ l, μ l of loading 2)；
As can be seen from Figure, during heterozygous sample loading total amount as little as 0.2ng, system FAM (inspection wild type),
VIC (inspection saltant type) and ROX (internal standard) triple channel are still normally expanded, therefore kit detection sensitivity of the invention can reach
Arrive：The as little as genomic DNA of 0.2ng accurately detects genotype.Detection method sensitivity is higher than traditional sequencing methods, behaviour
Make simple and quick, beneficial to large-scale promotion.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
Pipe has been described in detail with reference to foregoing embodiments to the present invention, but it will be understood by those within the art that：Its
The technical scheme described in foregoing embodiments can still be modified, or to which part or all technical characteristic
Carry out equivalent；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill
The scope of art scheme.
SEQUENCE LISTING
<110>Wuhan You Zhiyou medical science and technologies limited company
<120>A kind of human HLA-B5801 genetic polymorphism detection kits
<160> 8
<170> PatentIn version 3.3
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cttaacacag atcccagctc 20
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gctggctcca tgtggcaa 18
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cccagctccg aggaaacsca 20
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aggaaactcg tccccccca 19
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aggaaactca tccccccca 19
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gggccactag gcgctca 17
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ctctccctcc gcgcagccg 19
Claims (10)
1. a kind of human HLA-B*5801 genetic polymorphism detection kits, it is characterised in that including：
Nucleotides sequence is classified as SEQ ID NO:Positive outer primer shown in 1；
Nucleotides sequence is classified as SEQ ID NO:Reverse outer primer shown in 2；
Nucleotides sequence is classified as SEQ ID NO:The ARMS primers in the mutational site for testing goal gene shown in 3；
Nucleotides sequence is classified as SEQ ID NO:The Taqman probes for detecting wild type site shown in 4；
Nucleotides sequence is classified as SEQ ID NO:The Taqman probes for detecting mutational site shown in 5.
2. kit according to claim 1, it is characterised in that the end of Taqman probes 5 ' is connected with fluorescence report base
Group, fluorescent quenching group is connected with 3 ' ends of nucleic acid, and two fluorescent reporter groups of Taqman probes are different；
The fluorescent reporter group is selected from any one in FAM, VIC, ROX, CY3 and CY5；The fluorescent quenching group is selected from
Any one in TAMRA, BHQ1, BHQ2 and NFQ.
3. kit according to claim 2, it is characterised in that be connected with MGB modification groups on the Taqman probes；
Preferably, the MGB modification groups are connected with the fluorescent quenching group.
4. the kit according to Claims 2 or 3, it is characterised in that the kit also includes interior label primer and internal standard
Probe；
Preferably, the interior label primer and internal standard probe are humanized's internal standard；
It is highly preferred that the interior label primer is classified as SEQ ID NO for nucleotides sequence:Positive interior label primer shown in 6, nucleotides sequence
It is classified as SEQ ID NO:Reverse interior label primer shown in 7；The nucleotides sequence of the internal standard probe is classified as SEQ ID NO:Shown in 8.
5. kit according to claim 4, it is characterised in that the end of internal standard probe 5 ' is connected with fluorescence report base
Group, fluorescent quenching group is connected with 3 ' ends of nucleic acid；The fluorescent reporter group is selected from FAM, VIC, ROX, CY3 and CY5
Any one；The fluorescent quenching group is selected from any one in TAMRA, BHQ1, BHQ2 and NFQ；And the internal standard probe with
Fluorescent reporter group on the Taqman probes is different.
6. the kit according to any one of claims 1 to 3, it is characterised in that the kit also includes weakly positive pair
According to liquid and/or blank liquid；
The weakly positive comparison liquid is that, containing 2 kinds of mixed liquors of plasmid, 2 kinds of plasmids need containing 2 kinds of different bands respectively
Detect the HLA-B*5801 allele in mutational site；Preferably, the concentration of the plasmid is 2000~3000copies/ μ l；
The blank liquid is Tris-HCl buffer solutions；Preferably, the concentration of the Tris-HCl buffer solutions is 7~13mM,
PH is 7.5~8.5.
7. the kit according to any one of claims 1 to 3, it is characterised in that the kit also includes blood treatment
One or more in agent, PCR buffer solutions, enzyme and water.
8. kit according to claim 7, it is characterised in that the blood treatment agent is the 0.9% NaCl aqueous solution.
9. kit according to claim 7, it is characterised in that the PCR buffer solutions include PCR additives, MgCl2
With one or more in dNTPs；
Preferably, the PCR additives include one or more in DMSO, glycerine, BSA；
Preferably, the dNTPs includes dATP, dGTP, dCTP, dUTP.
10. kit according to claim 7, it is characterised in that the enzyme includes Taq enzyme, UNG enzymes；
Preferably, the Taq enzyme and UNG enzymes are individually dispensed or are mixed and made into solution；
It is highly preferred that when the Taq enzyme and UNG enzymes are mixed and made into solution, the total amount ratio of the Taq enzyme and UNG enzymes is：
(0.5U~1.0U)：(0.1U~0.5U).
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| CN201710267541.4A CN106929591A (en) | 2017-04-21 | 2017-04-21 | A kind of human HLA B*5801 genetic polymorphism detection kits |
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| CN201710267541.4A CN106929591A (en) | 2017-04-21 | 2017-04-21 | A kind of human HLA B*5801 genetic polymorphism detection kits |
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| CN107988353A (en) * | 2017-12-08 | 2018-05-04 | 益善生物技术股份有限公司 | A kind of mankind MTHFR and/or MTRR genetic polymorphism detection kit |
| CN107988334A (en) * | 2017-12-22 | 2018-05-04 | 美因健康科技（北京）有限公司 | The method that buccal swab Direct PCR carries out SNP partings |
| CN108048547A (en) * | 2017-12-22 | 2018-05-18 | 美因健康科技（北京）有限公司 | The method that blood Direct PCR carries out Taqman partings |
| CN108179183A (en) * | 2018-02-09 | 2018-06-19 | 苏州百源基因技术有限公司 | A kind of method for detecting LRP2 gene SNP site rs2544390 genotype |
| CN108977524A (en) * | 2018-06-26 | 2018-12-11 | 苏州道尔盾基因科技有限公司 | A kind of detection method and detection kit of HLA-B*5801 gene |
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| CN110923314A (en) * | 2019-12-30 | 2020-03-27 | 广州白云山拜迪生物医药有限公司 | Primer group for detecting SNP locus rs9263726, crRNA sequence and application thereof |
| CN111073964A (en) * | 2019-12-20 | 2020-04-28 | 江苏伟禾生物科技有限公司 | Kit for detecting human leukocyte antigen HLA-ABCCDRDQ genotyping |
| CN112813151A (en) * | 2021-03-05 | 2021-05-18 | 为朔医学数据科技（北京）有限公司 | Whole blood direct fluorescence PCR detection method and application of whole blood sample treatment fluid in preparation of gene detection kit |
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| CN107988353B (en) * | 2017-12-08 | 2019-02-12 | 益善生物技术股份有限公司 | A kind of mankind MTHFR and/or MTRR genetic polymorphism detection kit |
| CN107988353A (en) * | 2017-12-08 | 2018-05-04 | 益善生物技术股份有限公司 | A kind of mankind MTHFR and/or MTRR genetic polymorphism detection kit |
| CN107988334A (en) * | 2017-12-22 | 2018-05-04 | 美因健康科技（北京）有限公司 | The method that buccal swab Direct PCR carries out SNP partings |
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| CN108179183A (en) * | 2018-02-09 | 2018-06-19 | 苏州百源基因技术有限公司 | A kind of method for detecting LRP2 gene SNP site rs2544390 genotype |
| CN108977524A (en) * | 2018-06-26 | 2018-12-11 | 苏州道尔盾基因科技有限公司 | A kind of detection method and detection kit of HLA-B*5801 gene |
| CN108998517A (en) * | 2018-09-06 | 2018-12-14 | 武汉康录生物技术股份有限公司 | A kind of mankind SLCO1B1 and ApoE genetic polymorphism detection kit and its preparation method and application |
| CN108998517B (en) * | 2018-09-06 | 2019-11-15 | 武汉康录生物技术股份有限公司 | A kind of mankind SLCO1B1 and ApoE genetic polymorphism detection kit and its preparation method and application |
| CN111073964A (en) * | 2019-12-20 | 2020-04-28 | 江苏伟禾生物科技有限公司 | Kit for detecting human leukocyte antigen HLA-ABCCDRDQ genotyping |
| CN111073964B (en) * | 2019-12-20 | 2022-10-04 | 江苏伟禾生物科技有限公司 | Kit for detecting human leukocyte antigen HLA-ABCCDRDQ genotyping |
| CN110923314A (en) * | 2019-12-30 | 2020-03-27 | 广州白云山拜迪生物医药有限公司 | Primer group for detecting SNP locus rs9263726, crRNA sequence and application thereof |
| CN110923314B (en) * | 2019-12-30 | 2023-04-28 | 广州白云山拜迪生物医药有限公司 | Primer for detecting SNP locus rs9263726, crRNA sequence and application thereof |
| CN112813151A (en) * | 2021-03-05 | 2021-05-18 | 为朔医学数据科技（北京）有限公司 | Whole blood direct fluorescence PCR detection method and application of whole blood sample treatment fluid in preparation of gene detection kit |
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Application publication date: 20170707 |