CN108486240A - Primer, probe, fluorescent PCR kit and method for detecting people's HLA-B*5801 genes - Google Patents
Primer, probe, fluorescent PCR kit and method for detecting people's HLA-B*5801 genes Download PDFInfo
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Abstract
The present invention is provided to primer, probe, fluorescent PCR kit and the detection methods of the detection of people's HLA B*5801 allele, according to " allele specific pcr " principle, human leucocyte antigen (HLA) HLA B*5801 allele is detected on Real-Time Fluorescent Quantitative PCR Technique platform.
Description
Technical field
The invention belongs to biomedical clinical Molecular Detection fields, and in particular to be detected for HLA-B*5801 allele
Primer, probe, fluorescent PCR kit and detection method.
Background technology
Human leukocyte antigen（HLA）It is one group of gene being located on No. 6 chromosome, is that hitherto known gene is medium
The position highest gene complex of gene pleiomorphism, the whole world has found kind of the allele more than 2700, although wherein how many etc.
Position gene is rare.HLA genes are the genetic polymorphism sexual systems of most complex human, and there are apparent group spies for polymorphism distribution
Sign.So far, the Hans' studies have shown that HLA-B*5801 allele and medicine from China's Mainland, Hong Kong, Taiwan and Thailand
There is very strong correlations for serious skin adverse reaction caused by object Allopurinol.And in Europe and Japanese ethnic group, it is related
Property is then relatively low.
Allopurinol is hypoxanthine oxidase inhibitor, can reduce uric acid and synthesize and reduce uric acid concentration in blood, be to control
Treat the choice drug of gout.But serious drug rash clinic caused by the medicine is appeared in the newspapers repeatly, be drug the most easily caused serious drug eruption it
One.There is result of study to show that the adverse reaction of Allopurinol is mainly shown as skin, mucosa lesions, account for the 88.28% of adverse reaction,
Rest part 57.81% is fever, and about 31.64% damages for liver, and 20.31% is kidney damage, and 9.77% is damaged for hematological system
Evil.And allopurinol causes skin allergic reaction lethality up to 40%
The pharmacological Mechanism how HLA-B*5801 allele causes skin adverse reaction caused by Allopurinol is also unclear at present
Chu, but since the risk for carrying high level HLA-B5801 allele persons generation Allopurinol severe allergic reaction significantly increases, still
It so can be using HLA-B*5801 as the marker of adverse drug reaction.Therefore, before starting Allopurinol in Treatment, high-risk patient is best
The allele can be detected and carry frequency, especially in the Hans.HLA-B*5801 is up to ~ 9% left side in the carrying rate of the Hans
The right side, prevalence of gout rise year by year in China, and to reducing adverse drug reaction, improve therapeutic effect has detection HLA-B*5801
Significance.
HLA allele has more than 2700 kinds, is divided into the gene clusters such as A, B, C.In the Chinese Hans, HLA-B allele is general
There are 150 kinds or so, the carrying rate of HLA-B*5801 reaches ~ 9%, and is in Territorial Difference.Difference between allele is multiple volumes
The SNP site composition of code amino acid.
Currently, analyzing both at home and abroad HLA-B*5801 allele, PCR-SSCP, PCR sequencing PCR are mostly used greatly（SBT）
With fluorescent PCR method.PCR sequencing PCR prepares the most, but needs to analyze sequencing result by professional software, tests and analyzes the period
It is long.PCR-SSCP methods are a kind of methods of classics, to HLA-B*5801 primer amplifieds DNA and pass through electrophoresis knot by several
Fruit is analyzed, and pollution risk is big, the period is long, is not suitable for clinical application.Existing fluorescent PCR method, using dyestuff to the spy of amplification
Heteroleptic generates signal, and specificity is weaker, and can not carry out PCR reaction internal controls.
Invention content
The present invention for the prior art detection HLA-B*5801 allele when it is expensive, the period is long, inefficient, data
The deficiencies of cumbersome is analyzed, a kind of HLA-B*5801 equipotential bases based on Taqman Real-Time Fluorescent Quantitative PCR Technique platforms are provided
Because of the primer and fluorescence probe of detection, kit and detection method, for detecting people's HLA-B*5801 allele.This method needle
Requirement to clinical detection introduces PCR and reacts internal control, ensures the authenticity and accuracy of detection reaction.The present invention quickly, cost
It is low, have and is widely popularized value.
In order to achieve the above objectives, the technical solution adopted by the present invention is：
A kind of detection primer and its correspondent probe for detecting people's HLA-B*5801 allele, it is special according to HLA-B*5801
Polymorphic allele design, sequence is as follows：
Forward primer：
HLA-B*5801 F: 5’-CACGGAACATGAAGGCCTCC -3’ （ SEQ ID No.1）；
Reverse primer：
HLA-B*5801 R: 5’-CGGAGGAGGCGCCCGTAG-3’ （ SEQ ID No.2）；
Fluorescence probe：
TM-HLA-B*5801:5 ' the fluorophor quenching groups of-CCCAGGCGCGTTTACCCGGTTT -3 '（ SEQ ID
No.3）.
The present invention also provides a kind of for detecting the internal control primer pair of people's HLA-B*5801 allele and corresponding fluorescence
Probe, sequence are as follows：
Internal control primer pair：
Forward primer：GAPDH F: 5’-GCTGCTTTTAACTCTGGTAAAGTG-3’ （ SEQ ID No.4）；
Reverse primer：GAPDH R: 5’-TAGCACTCACCATGTAGTTGAG-3’ （ SEQ ID No.5）；
Internal control fluorescence probe：
TM-GAPDH:5 ' fluorophor-TGATGCATCTATGAACGCTTC-3 ' quenching groups（ SEQ ID No.6）.
The fluorescence that the corresponding fluorescence probe 5 ' of probe and internal control primer of above-mentioned detection people HLA-B*5801 allele is held
Group is conventional use of fluorescent reporter group suitable for fluorescent PCR quantitative analysis, preferably FAM, TET, VIC, HEX or ROX,
The quenching group at 3 ' ends is the conventional use of fluorescent quenching group suitable for fluorescent PCR quantitative analysis, preferably BHQ -1, BHQ
- 2, Dabcyl, Eclipse or TAMRA, preferred scheme are that the fluorophor that 5 ' ends of detection fluorescence probe are connected with is FAM,
The quenching group that 3 ' ends are connected with is Dabcyl；The fluorophor that 5 ' ends of internal control fluorescence probe are connected with is ROX, and what 3 ' ends were connected with quenches
The group that goes out is BHQ-2.
The present invention also provides a kind of fluorescent PCR kits for detecting people's HLA-B*5801 allele, including this
Invent the detection primer for detecting people's HLA-B*5801 allele and corresponding fluorescence probe and operation instructions.Specifically
It says, include the PCR reaction solution of detection people's HLA-B*5801 allele, it is necessary slow to contain PCR reactions for wherein PCR reaction solution
Outside the substances such as fliud flushing, magnesium ion, dNTP, also corresponding includes above-mentioned detection primer and probe.The PCR of above-mentioned each specific detection
The sequence of primer and probe that reaction solution separately includes is as follows：
（1）HLA-B*5801 PCR reaction solutions
HLA -B*5801 F：5’- CACGGAACATGAAGGCCTCC -3’；
HLA- B*5801 R：5’- CGGAGGAGGCGCCCGTAG -3’；
TM-HLA- B*5801：5 ' the fluorophor quenching groups of-CCCAGGCGCGTTTACCCGGTTT -3 '；
In the PCR reaction solution preferred embodiment of mentioned reagent box, in addition to the detection including detecting people's HLA B*5801 allele is drawn
Further include a pair of of internal control primer and corresponding fluorescence probe, the sequence of internal control primer and probe is such as outside object and corresponding fluorescence probe
Under：
Internal control primer：
GAPDH F：5’- GCTGCTTTTAACTCTGGTAAAGTG -3’；
GAPDH R：5’- TAGCACTCACCATGTAGTTGAG -3’；
Internal control fluorescence probe：
TM-GAPDH：5 ' the fluorophor quenching groups of-TGATGCATCTATGAACGCTTC -3 '.
Mentioned reagent box operation instructions include the description to PCR amplification condition, and preferred PCR amplification condition is：It is pre- to become
The condition of property is：Temperature is 95 DEG C, and the time is 3 minutes；
PCR reactions are made of first stage and second stage：
First stage is made of 10 amplification cycles, and condition is：
Denaturation：Temperature is 95 DEG C, and the time is 20 seconds；
Annealing extends：Initial temperature is 62 DEG C, and the time is 45 seconds；
Second stage is made of 30 amplification cycles, and condition is：
Denaturation：Temperature is 95 DEG C, and the time is 20 seconds；
Annealing extends：Initial temperature is 62 DEG C, and the time is 45 seconds（Fluorescence signal acquisition is set）.
The present invention also provides a kind of using above-mentioned detection primer and fluorescence probe or it is above-mentioned containing detection HLA-B*
The method that the fluorescent PCR kit of 5801 allele carries out HLA-B*5801 allele detections, step include：
（1）Sample to be tested processing and template extraction：
A. blood sample is extracted from person to be detected；
B. DNA is obtained from blood sample, it is recommended to use commercialized kit extracts peripheral blood genomic DNA；
C. DNA concentration and purity are measured, it is desirable that concentration is more than 10ng/ μ l；OD260nm/OD280nm=1.6~2.0.
（2）Fluorescent PCR expands：
Template DNA to be detected and a certain amount of Taq archaeal dna polymerases are added and contain detection people HLA-B*5801 of the present invention
In the detection primer of allele and corresponding fluorescent probe PCR reaction solution, after being uniformly mixed centrifugation in fluorescent PCR pipe, put
Enter quantitative fluorescent PCR instrument and carry out PCR reactions according to specific temperature cycles and signal acquisition program, preferably by template to be detected
The mentioned reagent box preferred embodiment containing internal control primer and corresponding fluorescence probe is added to a certain amount of Taq archaeal dna polymerases in DNA
PCR reaction solution in, be put into quantitative fluorescent PCR instrument after being uniformly mixed centrifugation in fluorescent PCR pipe and followed according to specific temperature
Ring and signal acquisition program carry out PCR reactions, to monitor response availability.
Above-mentioned fluorescent PCR amplification scheme preferably uses following temperature cycle and signal acquisition program to carry out PCR reactions：
The condition of pre-degeneration is：Temperature is 95 DEG C, and the time is 3 minutes；
PCR reactions are made of first stage and second stage：
First stage is made of 10 amplification cycles, and condition is：
Denaturation：Temperature is 95 DEG C, and the time is 20 seconds；
Annealing extends：Initial temperature is 62 DEG C, and the time is 45 seconds；
Second stage is made of 30 amplification cycles, and condition is：
Denaturation：Temperature is 95 DEG C, and the time is 20 seconds；
Annealing extends：Initial temperature is 62 DEG C, and the time is 45 seconds；（Fluorescence signal acquisition is set）
（3）Analysis result
Under the conditions of above-mentioned PCR reaction systems and temperature cycling program, observes purpose in specific PCR reaction system and detect fluorescence
Whether signal forms logarithmic amplification " S " type curve, and the DNA sample to be checked is the spy if forming logarithmic amplification " S " type curve
HLA-B*5801 allele representated by anisotropic reaction system is the positive.
The present invention also provides a kind of detection primer of detection people's HLA-B*5801 allele and corresponding fluorescence probes
And internal control primer and correspondent probe are in the application for detecting people's HLA-B*5801 allele.
The present invention also provides a kind of detection primer containing detection people's HLA-B*5801 allele and corresponding fluorescence
Probe and the kit of internal control primer and correspondent probe are in the application for detecting people's HLA-B*5801 allele.
Related content in technical solution of the present invention is explained as follows.
1. operation principle of the present invention is：Allele specific pcr (Allele Specific PCR, ASPCR), also known as
For allele specific amplification method (Allele Specific Amplification, ASA) or amplification Refracting Mutation system
It unites (Amplification Refractory Mutation System ARMS-PCR), basic principle is due to Taq DNA
Polymerase lacks 3 ' → 5 ' 5 prime excision enzyme activities, and when carrying out PCR reactions, if the end of primer 3 ' forms mispairing, chain extension reaction will
It is obstructed because of the obstacle that 3 ', 5 '-phosphodiester bonds are formed, PCR product amount is caused drastically to reduce, in certain amplification cycles,
Special amplified production is not will detect that；Conversely, 3 ' end pairings are then capable of detecting when amplified production.Then, for known more
State property site, 3 ' that its polymorphic base is designed at detection primer are held, and after amplified reaction, are passed through gel electrophoresis and are observed product
Whether there is or not the base types that can determine whether polymorphic site.
It is 1996 by Applied Biosystems companies of the U.S. that quantitative fluorescent PCR, which is (Real-time PCR),
A kind of new quantitative experiment technology released is added a specific fluorescence and visits while pair of primers is added in PCR amplification
Needle, the probe are an oligonucleotides by title " TaqMan spies ", both ends respectively one reporter fluorescence group of label and one be quenched it is glimmering
Light group.When probe is complete, the fluorescence signal of reporter group transmitting is quenched group absorptions；When just starting, probe is incorporated in
On any one of DNA is single-stranded；When PCR amplification, 5 ' → 3 ' 5 prime excision enzyme activities of Taq enzyme degrade probe digestion, make reporter fluorescence
Group and the separation of quenching fluorescence group, to which fluorescence monitoring system can receive fluorescence signal, as soon as a DNA chain is often expanded,
Formed there are one fluorescent molecular, realize the accumulation of fluorescence signal and PCR product formed it is fully synchronized.
The product of " allele specific pcr " reaction system is detected by the real-time fluorescent PCR technology based on fluorescence probe,
According to formed amplification curve fluorescence signal in specific threshold recurring number（Ct values）Size can determine whether in corresponding detection architecture
The type of the corresponding allele of template.
2. the design of the primer and probe in technical solution of the present invention：According in US National Biotechnology Information
The reference sequences of HLA-B genes disclosed in the GenBank GeneBank of heart NCBI（NG_023187）And Britain
The information of HLA-B*5801 (HLA00386) disclosed in IMG/HLA databases, using the Primer Express of ABI companies
3.0 softwares separately design the primer and probe that detection HLA-B*5801 etc. is gene.In order to monitor the validity of reaction system,
Internal control primer and probe are added in detection architecture, the present invention chooses one section of sequence of mankind's conservative gene GAPDH（Its GeneBank
Reference sequences are numbered：NG_007073.2）, using ABI companies 3.0 Software for Design detection primers of Primer Express and
Probe.
In technical solution of the present invention, the primer（primer）Refer to the few nucleosides being made of a certain number of dNTP
Acid sequence, it is usually artificial synthesized by DNA synthesizer, it is purified through polyacrylamide gel electrophoresis or other proper methods after synthesis.
In PCR, primer can be combined with region complementary therewith on purpose nucleic acid chain to be amplified, and function is conduct
The starting point of nucleotide polymerization effect, on 3 '-OH of primer, nucleotide is synthesized in the form of diester linkage, therefore primer
3 '-OH must be free.Archaeal dna polymerase can proceed by extension by its 3 ' end, synthesize new nucleic acid chains.
In technical solution of the present invention, the fluorescence probe is a kind of oligonucleotide probe, and fluorophor is connected to
5 ' ends of probe, and quenching group, then in 3 ' ends, generally use DNA synthesizer is artificial synthesized, through polyacrylamide after synthesis
Amine gel electrophoresis or the purifying of other proper methods.Currently used fluorophor has FAM, TET, VIC, HEX, ROX etc..Base is quenched
There are BHQ -1, BHQ -2, Dabcyl, Eclipse, TAMRA etc. in group.
3. in technical solution of the present invention, the internal control primer and its corresponding probe can be used for monitoring reaction
The index of validity, to judge whether that the factors such as template quality, mechanical disorder, reagent stability influence test result
Situation.Under normal circumstances, PCR normally expands the exponential amplification curve that can be formed, and vivid is described as " S " type amplification curve,
Show that system normally expands.When purpose detection fluorescence signal forms logarithmic amplification " S " type curve in specific PCR reaction system
When, it is the positive findings of polymorphism type, also illustrates that PCR system amplification is normal, without internal control primer and its corresponding spy
Needle carries out the verification of result.However, working as purpose detection fluorescence signal in specific PCR reaction system does not form logarithmic amplification " S "
Type curve, testing result are the negative findings of the polymorphism type representated by the specific reaction system, but it could also be possible that mould
The factors such as plate quality, mechanical disorder, reagent stability influence test result, in this case, may be used internal control primer and its
Corresponding probe is excluded.When purpose detection fluorescence signal does not form logarithmic amplification " S " type song in specific PCR reaction system
Line, and when internal control signal forms normal logarithmic amplification " S " type curve, the accurate of the negative findings of polymorphism type can be verified
Property.And works as purpose detection fluorescence signal in specific PCR reaction system and do not form logarithmic amplification " S " type curve, and internal control signal
When also not forming normal logarithmic amplification " S " type curve, then illustrating to go wrong on experiment condition or instrument influences test result, and
The negative findings of non-polymorphism type.
Since above-mentioned technical proposal is used, the present invention has following advantages and effect compared with prior art：
1. in technical solution of the present invention detection people's HLA-B*5801 allele detection primer and corresponding fluorescence probe with
And internal control primer and correspondent probe, PCR detection specificity is very high, and uses real-time fluorescent PCR technology, and testing result is sentenced
It reads to be easy, is more suitable for clinical detection.Technical solution of the present invention uses Taqman probes, and detection signal is than fluorescent dye according to spy
It is different.
2, the detection primer in technical solution of the present invention and probe are cheap, and are not required to be sequenced during the experiment,
While saving testing cost, detection cycle is substantially reduced, improves the efficiency of detection.
3, the detection primer and probe in technical solution of the present invention, using real-time fluorescent PCR technology platform, it can be achieved that high pass
Amount detection.
Description of the drawings
Attached drawing 1 is certain HLA-B*5801 allele carrier's sample reagent box detection figure；
Attached drawing 2 is certain sample HLA-B*5801 allele carrier's sample sequencer maps.
Specific implementation mode
Term as used in the present invention generally there are those of ordinary skill in the art usually to manage unless otherwise indicated
The meaning of solution.
The present invention is described in further detail with reference to specific embodiment and with reference to data.It should be understood that these embodiments are only
It is in order to demonstrate the invention, rather than to limit the scope of the invention in any way.
In the examples below, the various processes and method not being described in detail are conventional methods as known in the art, are led to
Frequently with normal condition such as Sambrook et al., molecular cloning：Laboratory manual（NewYork：Cold Spring Harbor
Laboratory Press, 1989）Described in condition, or according to the method proposed by manufacturer.It is used in the present invention
Fluorescence quantitative PCR instrument model ABI 7500 or BioRad CFX96.
The design of 1. primer and probe of embodiment：
According to HLA-B genes disclosed in the GenBank GeneBank of US National Biotechnology Information center NCBI
Reference sequences（NG_023187）And the information of HLA-B*5801 (HLA00386) disclosed in Britain's IMG/HLA databases,
3.0 softwares of Primer Express of ABI companies are used to separately design detection HLA-B*5801 etc. as the primer of gene and spy
Needle.Primer and probe, it is specific as follows：
Forward primer：
HLA-B*5801 F: 5’-CACGGAACATGAAGGCCTCC -3’；
Reverse primer：
HLA-B*5801 R: 5’-CGGAGGAGGCGCCCGTAG-3’；
Fluorescence probe：
TM-HLA-B*5801: 5’Fam- CCCAGGCGCGTTTACCCGGTTT -3’BHQ1。
In order to monitor the validity of reaction system, internal control primer and probe are added in detection architecture, the present invention chooses people
One section of sequence of class conservative gene GAPDH（Its reference sequences is numbered：NG_007073.2）, using the Primer of ABI companies
3.0 Software for Design detection primers of Express and probe, particular sequence are as follows：
Internal control primer pair：
Forward primer：GAPDH F: 5’- GCTGCTTTTAACTCTGGTAAAGTG -3’；
Reverse primer：GAPDH R: 5’- TAGCACTCACCATGTAGTTGAG -3’；
Internal control fluorescence probe：
TM-GAPDH: 5’ Rox- TGATGCATCTATGAACGCTTC -3’BHQ2。
The preparation of 2. primer of embodiment
It transfers to Synesis Company to synthesize designed primer and probe sequence, is generally synthesized using automation equipment, need to provide
Synthesize qualification test report.
Embodiment 3：Fluorescent PCR detects the preparation of the kit of people's HLA-B*5801 allele
Detection people's HLA-B*5801 allele reaction solutions are prepared, wherein PCR reaction solution contains the necessary buffering of PCR reactions
Outside the substances such as liquid, magnesium ion, dNTP, detection primer and probe and internal control primer and probe are further comprised.Above-mentioned each specificity inspection
The PCR reaction solution concentration of component of survey and comprising primer and probe sequence information it is as follows：
1. PCR reaction solution component of table
| Reaction solution component | Concentration |
| Buffer solution | 10× |
| Magnesium ion | 15mM |
| dNTP（A/T/G/C） | Each 20mM |
| Upstream detection primer | 2µM |
| Downstream detector primer | 2µM |
| Detect signal probe | 2µM |
| Upstream internal control primer | 1µM |
| Downstream internal control primer | 1µM |
| Internal control signal probe | 1µM |
HLA-B*5801 allele
HLA-B*5801 F: 5’-CACGGAACATGAAGGCCTCC -3’
HLA-B*5801 R: 5’-CGGAGGAGGCGCCCGTAG-3’
TM-HLA-B*5801: 5’Fam- CCCAGGCGCGTTTACCCGGTTT -3’BHQ1
GAPDH F: 5’- GCTGCTTTTAACTCTGGTAAAGTG -3’；
GAPDH R: 5’- TAGCACTCACCATGTAGTTGAG -3’；
TM-GAPDH: 5’ Rox- TGATGCATCTATGAACGCTTC -3’BHQ2。
Embodiment 4：The method that fluorescent PCR detects people's HLA-B*5801 allele
The first step：Prepare DNA
（1）Blood sample is extracted from person to be detected；
（2）DNA is obtained from blood sample, it is recommended to use commercialized kit extracts peripheral blood genomic DNA；
（3）DNA concentration and purity are measured, it is desirable that concentration is more than 10ng/ μ l；OD260nm/OD280nm=1.7~2.0。
Second step：PCR reaction systems are prepared
In the dedicated pipe of quantitative fluorescent PCR PCR reaction systems are prepared according to following table
Table 2.PCR reaction systems form
| ddH2O | Add to 25 μ l |
| PCR reaction solution | 2.5μl |
| Archaeal dna polymerase（5.0U/μl） | 0.5μl |
| Genomic DNA | About 80ng |
| Paraffin oil | 20μl |
HLA-B*5801 allele reaction solutions in the kit that above-mentioned PCR reaction solution is prepared using embodiment 3, can also be by
The detection primer containing detection people's HLA-B*5801 allele and probe are prepared according to the preparation method that 3 table 1 of embodiment provides simultaneously
PCR reaction solution containing internal control primer and probe；
Third walks：Upper machine testing
According to following table, temperature cycles and signal acquisition program are set：
3. PCR response procedures of table
Note：Fam and Rox double channels acquisition fluorescence signals are set at " * ".
4th step：Analysis result
Under the conditions of above-mentioned PCR reaction systems and temperature cycling program, normal logarithmic amplification " S " type curve is formed in internal control signal
Precondition under, observe specific PCR reaction system in purpose detection fluorescence signal whether formed logarithmic amplification " S " type song
Line, the DNA sample to be checked is the polymorphism class representated by the specific reaction system if forming logarithmic amplification " S " type curve
The positive of type.Such as：Certain sample to be checked detects fluorescence signal in reaction system（The channels FAM）Logarithmic amplification " S " type curve is formed,
It is that HLA-B*5801 allele is positive then to prompt the sample.
Attached drawing 1 is certain sample HLA-B*5801 allele kits detection figure, detection figure HLA-B*5801 allele
Internal control fluorescence signal in reaction system is qualified, then judges that the sample is positive for HLA-B*5801 allele.
Attached drawing 2 is 1 sample HLA-B*5801 allele sequencer maps of attached drawing, and sequencing result shows sample HLA-B*5801
Allele is positive.Kit testing result is consistent with sequencing result.
Pay attention to：
The above embodiments merely illustrate the technical concept and features of the present invention, and its object is to allow person skilled in the art's energy
Solution present disclosure much of that is simultaneously implemented according to this, and it is not intended to limit the scope of the present invention.It is all according to spirit of that invention
Equivalent change or modification made by essence, should be covered by the protection scope of the present invention.
Sequence table
<110> OrganizationName :Suzhou Kuangyuan Molecular Biotechnology Co., Ltd.
Application Project
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<120> Title :It is a kind of for detect the primer of people's HLA-B*5801 allele, probe, fluorescent PCR kit and
Method
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000
<210> SEQ ID No.6
<213> OrganismName :The mankind (Homo sapiens)
<400> SEQ ID No.6
reequencet ring 14
<212> Type : DNA
000
Claims (8)
1. a kind of detection primer and its correspondent probe for detecting people's HLA-B*5801 allele, it is characterised in that the inspection
It surveys primer and probe to be designed according to HLA-B*5801 allele specifics site, sequence is as follows：
Forward primer：
HLA-B*5801 F: 5’-CACGGAACATGAAGGCCTCC -3’；
Reverse primer：
HLA-B*5801 R: 5’-CGGAGGAGGCGCCCGTAG-3’；
Fluorescence probe：
TM-HLA-B*5801:5 ' the fluorophor quenching groups of-CCCAGGCGCGTTTACCCGGTTT -3 '.
2. a kind of for detecting the internal control primer pair of people's HLA-B*5801 allele and corresponding fluorescence probe, it is characterised in that institute
The sequence for stating internal control primer pair and corresponding fluorescence probe is as follows：
Internal control primer pair：
Forward primer：GAPDH F: 5’-GCTGCTTTTAACTCTGGTAAAGTG-3’；
Reverse primer：GAPDH R:5 '-TAGCACTCACCATGTAGTTGAG-3 ' internal control fluorescence probes；
Fluorescence probe：TM-GAPDH:5 ' fluorophor-TGATGCATCTATGAACGCTTC-3 ' quenching groups.
3. the primer pair as described in claim 1-2 and corresponding fluorescence probe, it is characterised in that the fluorescence probe 5 ' is held glimmering
Light group be FAM, TET, VIC, HEX or ROX, 3 ' end quenching groups be BHQ -1, BHQ -2, Dabcyl, Eclipse or
TAMRA。
4. a kind of kit for detecting people's HLA-B*5801 allele, it is characterised in that the kit includes that right is wanted
It asks detection primer and its correspondent probe described in 1 and/or includes the detection primer and its correspondent probe described in claim 2.
5. the kit of detection people's HLA-B*5801 allele as claimed in claim 4, it is characterised in that the kit
Further include internal control primer pair as claimed in claim 2 and corresponding fluorescence probe.
6. a kind of detection method of people HLA-B*5801 allele, the method includes using claim 1 and/or right to want
Seek the primer and probe described in 2.
7. a kind of detection method of people HLA-B*5801 allele, the method includes using the examination described in claim 4-5
Agent box.
8. the application of primer and probe in detecting people's HLA-B*5801 allele described in claim 1-2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810180207.XA CN108486240A (en) | 2018-03-05 | 2018-03-05 | Primer, probe, fluorescent PCR kit and method for detecting people's HLA-B*5801 genes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810180207.XA CN108486240A (en) | 2018-03-05 | 2018-03-05 | Primer, probe, fluorescent PCR kit and method for detecting people's HLA-B*5801 genes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108486240A true CN108486240A (en) | 2018-09-04 |
Family
ID=63341366
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810180207.XA Pending CN108486240A (en) | 2018-03-05 | 2018-03-05 | Primer, probe, fluorescent PCR kit and method for detecting people's HLA-B*5801 genes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108486240A (en) |
-
2018
- 2018-03-05 CN CN201810180207.XA patent/CN108486240A/en active Pending
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Application publication date: 20180904 |