CN108624676A - Kit and its detection method for detecting HLA-B*5801 allele and application - Google Patents
Kit and its detection method for detecting HLA-B*5801 allele and application Download PDFInfo
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Abstract
The invention discloses for detecting HLA B*5801 allele kit and its detection method and application, belong to technical field of biological.The kit includes the primer for determining sample parting to be detected as HLA B*58, and for determining that the HLA B*58 sample partings of the detection are the primer of HLA B*5801；It is first enriched with by designing the primer of target fragment to be detected by the present invention by PCR amplification, then the target fragment to be detected by amplification detection primer, PCR amplification detection can reduce false positive rate；And testing result can be made more accurate by two pairs of special primers of design, two pairs of detection primers corresponding with its, wherein first pair of special primer and the first detection primer can determine that sample to be tested be parting is HLA B*58；Second pair of special primer and the second detection primer can determine that sample parting to be detected is HLA B*5801；The interference performance for excluding non-specific amplification is strong.
Description
Technical field
The present invention relates to for detecting HLA-B*5801 allele kit and its detection method and application, belong to raw
Analyte detection technical field.
Background technology
The common drug for the treatment of gout includes that colchicin, benzene smell Ma Long and do not purine alcohol (Allopurinol) etc. at present；
Colchicin is the first-line drug of current treatment gout, can inhibit apocyte and migrates to joint and inhibit its phagocytic function, together
When inhibit oneself to enter the cell release lactic acid and lysosomal enzyme of articular cavity again, prevent the mitosis of cell, can relieve pain rapidly.But
It is to have direct repression to marrow due to colchicin, agranulocytosis, alpastic anemia etc. can be caused serious bad
Reaction, so being only limitted to use as analgesic drug product in acute gout arthritis.Benzene smells Ma Long and do not purine alcohol is usually used in gout
Symptom remission, but the two mechanism of action is completely different, wherein and benzene smells the grand category uricosureic agent of horse, to kidney proximal tubule
One anion exchange system of lithate has strong and reversible inhibiting effect, to reduce the reabsorption of uric acid, reaches reduction blood urine
The effect of acid；As the drug for resisting uric acid synthesis, do not purine alcohol and its fast cry of certain animals alcohol of metabolite oxygen can inhibit do not purine alcohol
Yellow fast cry of certain animals oxidizing ferment, prevents hypoxanthine and xanthine from being metabolized as uric acid, to reduce the generation of uric acid, makes the urine in blood and urine
Acid content reduces to solubility or less level, prevents uric acid from forming crystallization deposition in its hetero-organization.As uric acid building-up process
The inhibitor of final step, allopurinol are that a few can inhibit one of drug of uric acid generation at present；It is serious in gout
When smell Ma Long with uricosuric benzene and share, reinforce curative effect.
Therefore, for uric acid generate it is excessive, to uricosuric allergy or invalid, and uricotelic drugs should not be used
The primary of (if any renal insufficiency) and secondary gout patient, allopurinol are suitble to the most.Nearly 5% allopurinol is taken
Patient will appear serious skin adverse reaction, including Steven-Johnson syndromes (SJS) and toxic epidermal necrosis pine
Solve disease (TEN).Steven-Johnson syndromes show as serious erythema multiforme, can involve skin and mucous membrane, including mouth,
Nose, eye, vagina, urethra, gastrointestinal tract and lower respiratory tract mucous membrane, patient even have the anxiety of blindness, and further development then forms poisoning
Property epidermal necrolysis, whole body eats film and festers, and slackness bulla or epidermis stripping occur in erythema；If meet slight touching or
Drawing can cause epidermis large area to be removed, it was reported that the lethality of SJS/TEN is up to 26%.
Therefore, the use of allopurinol clinically at present is extremely restricted, but due to the uniqueness of its mechanism of action,
The inhibiting effect generated to uric acid can be substituted without other drugs, eligible patients can only emit will appear at any time it is serious bad
The risk of reaction takes the medicine.Meanwhile as a kind of delayed hypersensitivity, usually several weeks are just after the tablet has been ingested by the SJS/TEN
It will appear symptom, and patient occurs general medical to dept. of dermatology first after dermatitis, once medication history narration is unclear, easily causes to leak
It examines, mistaken diagnosis, to be delayed the opportunity of drug withdrawal and treatment.
HLA (human leukocyte antigen, human leukocyte antigen) system, by No. 6 the short arm of a chromosome of the mankind
The closely chain multiple allele coding of a group is Gene Density highest and polymorphism in current known human chromosomal
Region the abundantest, be mainly responsible in immune system it is intercellular be mutually distinguishable and induce immune response, adjust immune answers
It answers.Include mainly HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ and HLA-DP.HLA gene polynorphisms determine individual
Between the HLA protein moleculars expressed it is different, also determine Different Individual processing and present same antigen ability it is different, this is to exempt from
The most basic point that epidemic disease response is induced and adjusted, to make Different Individual can express out individual difference to the immune response of same antigen
It is different.This individual difference exclusive or generates protective immunity, or forms immune tolerance, or autoimmunity tendency occurs, or shows as HLA
Relevant disease.Such as：HLA-B*5801, HLA-B*1502 allele are related to the allergic reaction of drug；HLA-B*5801 etc.
Position gene and Stevens-Johnson syndromes/toxic epidermal necrolysis (Stevens- caused by allopurinol
Johnson syndrome/toxic epidermal necrolysis, SJS/TEN) it is related.Occur after taking allopurinol
100% exists in the patient of serious adverse reaction, and in the patient (tolerance crowd) for not occurring adverse reaction and Normal group
In, carrying rate is only that high (6-8% is arrived HLA-B*5801 positive ratio white men in 15% and 20% Chinese Han nationality, Thailander
2%) the risk bigger of hypersensitivity, occurs.
High degree of polymorphism and complexity and numerous studies in view of HLA systems prove that allopurinol is relevant serious super quick anti-
Should be closely related with the anti-HLA-B*5801 of leucocyte, determine that the detection method of HLA allele is different from common polymorphic position
The detection of point.Existing detection method mainly has electrophoretic techniques, fluorescent probe technique and sequencing technologies etc..
But due to the sequence polymorphism of HLA gene families, relative to the detection of HLA allele, the technology is so far very
It is applied to the detection of HLA-B*5801 allele less, basic reason is that multiple HLA-B allele have with HLA-B*5801
There is high sequence homology (90% or more), thus designs a set of multicolor fluorescence PCR reaction high specifics that are suitable for and expand
The primer combination of probe of HLA-B*5801 allele is very difficult.
Invention content
The purpose of the present invention is to provide a kind of kit and its detection sides for detecting HLA-B*5801 allele
Method and application, so as to by the method for fluorescent PCR, be detected to HLA-B*5801 allele, and specificity is high,
Testing result is accurate.
Unless specifically stated otherwise, " R " herein refers both to " fluorophor ", and " Q " is referred both to " quenching group ".
Unless specifically stated otherwise, " HLA-B*5801-P1 " herein, " HLA-B*5801-P2 " is respectively two check bits
Point, corresponding primer are respectively first pair of special primer and second pair of special primer.
In order to achieve the above objectives, the present invention provides the following technical solutions：
The first purpose of the invention is to provide a kind of primers for detecting HLA-B*5801 allele, including are used for
Determine that sample parting to be detected is the primer of HLA-B*58, and for detecting determining HLA-B*58 sample partings as HLA-B*
5801 primer.
In one embodiment of the invention, described for determining that sample parting to be detected is the primer packet of HLA-B*58
First pair of special primer and the first detection primer are included, wherein：
Sense primer in first pair of special primer includes such as SEQ ID NO:Nucleotide sequence shown in 1 and and its
5 ends ＇ be connected 0~5 arbitrary base,
SEQ ID NO:1：GTCTGCGCGGAGGCCTTCAT；
Downstream primer in first pair of special primer includes such as SEQ ID NO:Nucleotide sequence shown in 2 and and its
5 ends ＇ be connected 0~5 arbitrary base,
SEQ ID NO:2：CGCGAGTCCGAGGACGGA；
First detection primer is such as SEQ ID NO:Nucleotide sequence shown in 3,
GCCTTCATGTTCCGTGTCTCCCC(SEQ ID NO:3)；
It is described specifically to draw including second Dui for detecting the primer that determining HLA-B*58 sample partings are HLA-B*5801
Object and the second detection primer, wherein
Sense primer in second pair of special primer includes such as SEQ ID NO:Nucleotide sequence shown in 4 and and its
5 ends ＇ be connected 0~5 arbitrary base,
SEQ ID NO:4：CCGTGTGGCGGAGCAGCT；
Downstream primer in second pair of special primer includes such as SEQ ID NO:Nucleotide sequence shown in 5 and and its
5 ends ＇ be connected 0~5 arbitrary base,
SEQ ID NO:5：CCGCGCGCTGCAGCGTCT；
Second detection primer is such as SEQ ID NO:Nucleotide sequence shown in 6,
SEQ ID NO:6：GAGCCTACCTGGAGGGCCT.
In one embodiment of the invention, first detection primer is in 5 ends ＇ jointing sequences 1, the second detection
Primer is in 5 ends ＇ jointing sequences 2；Or first detection primer is in 5 ends ＇ jointing sequences 2, the second detection primer
In 5 ends ＇ jointing sequences 1；
The joint sequence 1 is such as SEQ ID NO:Nucleotide sequence shown in 17：
SEQ ID NO:17：TTCATTAGCGGCGCAATTT；
The joint sequence 2 includes such as SEQ ID NO:Nucleotide sequence shown in 18 and be connected at 5 ends ＇ with it 0~5
A arbitrary base：
SEQ ID NO:18：CTAGCCCGAACGAATACTCA.
Second object of the present invention is to provide application of the primer in detecting HLA-B*5801 allele.
Third object of the present invention is to provide a kind of kits for detecting HLA-B*5801 allele, including with
The upper primer.
In one embodiment of the invention, further include internal control primer, under the sense primer of the internal control primer includes
State SEQ ID NO:Nucleotide sequence shown in 7,
SEQ ID NO:7：R-TCGCTCCGTCAGGCTTTCGGTGACGTGGACATCCGC AAA, wherein R indicate fluorescence
Group is also associated with 0-5 arbitrary bases between the R and 5 ends ＇；
The downstream primer of the internal control primer includes following SEQ ID NO:Nucleotide sequence shown in 8,
SEQ ID NO:8：GGAAAGACACCCACCTTGATCT-Q, wherein Q expression are quenched gene, the Q and 5 ends ＇ it
Between be also associated with 0-5 arbitrary bases.
In one embodiment of the invention, further include universal fluorescent probe, the fluorescence probe includes normal chain probe
With minus strand probe, the normal chain probe includes oligonucleotide positive strand sequence and joint sequence II compositions, the minus strand probe packet
Include the oligonucleotide negative strand sequence with oligonucleotide positive strand sequence complementation；The oligonucleotide positive strand sequence is using such as
SEQ ID NO：Nucleotide sequence shown in any one of 9-12；The oligonucleotide negative strand sequence uses such as SEQ ID NO：
Nucleotide sequence shown in any one of 13-16；The joint sequence II uses such as SEQ ID NO：Shown in any one of 19-22
Nucleotide sequence；5 ends ＇ of the normal chain probe are connected with fluorophor, and 3 ends ＇ of the minus strand probe, which are connected with, is quenched base
Group.
In one embodiment of the invention, the fluorophor includes FAM, TET, Texas Red, VIC or Cy5,
The quenching group includes BHQ1 and/or BHQ2.
Fourth object of the present invention is to provide a kind of method of detection HLA-B*5801 allele, including following step
Suddenly：
1) upstream and downstream primer of the upstream and downstream primer of first pair of special primer and second pair of special primer is mixed into
Mix primer 1；
2) by the universal fluorescent probe, the first detection primer, the second detection primer, first pair of special primer downstream draw
The downstream primer of object and second pair of special primer is mixed into mix primer 2；
3) genomic DNA of sample is extracted；
4) mix primer 1 is configured to PCR reaction systems, the genomic DNA obtained to step 3) extraction carries out PCR
Amplification, obtains target fragment to be measured；
5) mix primer 2 is configured to PCR reaction systems, the target fragment to be measured that step 4) amplification obtains is carried out real-time
Fluorescent PCR detects
6) interpretation of result is carried out according to the fluorescence signal intensity detected in real time.
In one embodiment of the invention, further include by the sense primer of internal control primer and downstream in step 5)
Primer mixes, and obtains mix primer 3, then during carrying out real-time fluorescence PCR detection to target fragment to be measured, and meanwhile it is right
Reference gene carries out real-time fluorescence PCR detection.
Fifth object of the present invention is to provide application of the kit in detecting HLA-B*5801 allele.
The beneficial effects of the present invention are：By designing the primer of two target fragments to be detected, first it is expanded by PCR
Increase enrichment, then the detection primer of two target fragments to be detected by amplification, carries out PCR amplification detection and determine sample to be detected
Parting is HLA-B*5801, can reduce false positive rate；And pass through two pairs of special primers of design, two pairs of inspections corresponding with its
Surveying primer can make testing result more accurate, wherein first pair of special primer and the first detection primer can determine sample to be tested
It is HLA-B*58 for parting；Second pair of special primer and the second detection primer can determine that sample parting to be detected is HLA-B*
5801；In addition by internal control primer, the accuracy of detection can be further provided for；And by compared with sequencing result, examining
The accuracy of survey reaches 100%；Single tube detects distinguished sequence and characteristic sequence simultaneously, and testing result is easy to interpretation, and this method is same
Single probe Comparison between detecting methods exclude the interference of non-specific amplification, such as HLA-B*5802；Compare with electrophoresis, the method is real
Existing two site of single tube is detected simultaneously, pollution-free, and accuracy is high；Without synthesizing expensive TaqMan probe, R＆D costs and
Testing cost is extremely low.
Above description is only the general introduction of technical solution of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention and after coordinating attached drawing to be described in detail such as.
Description of the drawings
Fig. 1 is the design schematic diagram of first pair of special primer in the embodiment of the present invention；
Fig. 2 is the design schematic diagram of second pair of special primer in the embodiment of the present invention；
Fig. 3 is the amplification figure of the target fragment in the embodiment of the present invention；
Fig. 4 is the amplification figure of the internal control primer in the embodiment of the present invention；
Fig. 5 is the testing result figure of the detected sample in the embodiment of the present invention；
Fig. 6 is the sequencing result after the positive HLA-B*5801-P1 special primers amplification in the embodiment of the present invention
Scheme HLA-B*5801-P1 and HLA-B*5801-P2；
Fig. 7 is the sequencing result after the positive HLA-B*5801-P2 special primers amplification in the embodiment of the present invention
Figure；
Fig. 8 is the testing result figure being detected to 101 example clinical samples in the embodiment of the present invention.
Specific implementation mode
With reference to the accompanying drawings and examples, the specific implementation mode of the present invention is described in further detail.Implement below
Example is not limited to the scope of the present invention for illustrating the present invention.
Unless specifically stated otherwise, the reagent in following embodiment is that analysis level is pure, and can be commercially available from regular channel.
Embodiment 1：The design of primer
In HLAAlleles, specific network address is the data source for all HLA allele that PCR primer design is wanted：
http://hla.alleles.org/alleles/text_index.html.Design of primers uses manual method, design to draw
Object is compared in IMGT databases, confirms that the primer can specifically bind HLA-B*5801 allele.Choose two positions
Point design primer, a site are denoted as HLA-B*5801-P1, and corresponding primer is first pair of special primer, a site note
For HLA-B*5801-P2, corresponding primer is second pair of special primer, wherein the design principle of first pair of special primer is as schemed
Shown in 1, the design principle of second pair of special primer is as shown in Figure 2.
The design of internal control primer and universal fluorescent probe is referring in particular to patent CN105861706A.Universal fluorescent probe includes
Normal chain probe and minus strand probe, normal chain probe include the oligonucleotide positive strand sequence and connector being sequentially connected to 3 ends ＇ by 5 ends ＇
Sequence II compositions, minus strand probe includes the oligonucleotide negative strand sequence with oligonucleotide positive strand sequence complementation；
Oligonucleotide positive strand sequence uses such as SEQ ID NO：Nucleotide sequence shown in any one of 9-12；
R-CCCGCCGCGTAGATCGAATA(SEQ ID NO：9)
R-CCAAGGCCGATCGCATGTTC(SEQ ID NO：10)
R-ACCTTCGGATCGCGGGTCT(SEQ ID NO：11)
R-CCTCCGAATCCGCCGTCGTACAAG(SEQ ID NO：12)
Oligonucleotide negative strand sequence uses such as SEQ ID NO：Nucleotide sequence shown in any one of 13-16；
TATTCGATCTACGCGGCGGG-Q(SEQ ID NO：13)
GAACATGCGATCGGCCTTGG-Q(SEQ ID NO：14)
AGACCCGCGATCCGAAGGT-Q(SEQ ID NO：15)
CTTGTACGACGGCGGATTCGGAGG-Q(SEQ ID NO：16)
5 ends ＇ of normal chain probe are connected with fluorophor, and 3 ends ＇ of the minus strand probe are connected with quenching group.
Fluorophor (R) includes FAM, TET, Texas Red, VIC or Cy5, quenching group (Q) include BHQ1 and/or
BHQ2。
Joint sequence II uses such as SEQ ID NO：Nucleotide sequence shown in any one of 19-22：
TTCATTAGCGGCGCAATTT(SEQ ID NO：19)
CTAGCCCGAACGAATACTCA(SEQ ID NO：20)
TGATACAACCCGTAACCGT(SEQ ID NO：21)
ACTCATCGATCCCGCATAC(SEQ ID NO：22)
The present invention uses primer and universal fluorescent probe, is synthesized by the Suzhou bio tech ltd Jin Weizhi.
Specific design of primers result is as described in Table 1：
The list of primers that table 1 designs
Embodiment 2：The amplification of target fragment in sample to be tested
S1, the genomic DNA for extracting sample (samples sources include saliva, blood and the tissue etc. of people)；
S2, the sense primer of HLA-B*5801-P1 and HLA-B*5801-P2 and downstream primer are mixed into composition mix primer
1 (i.e. the sense primer and downstream primer of first pair of specific primer and second pair of specific primer), it is spare；
S3, mix primer 1 is configured to PCR reaction systems, target fragment to be measured is enriched with, PCR results are enrichment
Product；Wherein, reaction system is 12 μ L, including 2X TaKaRa TaqTMHS Perfect Mix 6u1, and mix primer 1 is 1 μ
L, DNA5 μ L.
Amplification obtains HLA-B*5801-P1 and HLA-B*5801- as shown in figure 3, as can be seen from the figure expanding
P2 target fragments.
Embodiment 3：The detection of sample to be tested
S1, by universal fluorescent probe, the first detection primer as sense primer and the second detection primer, first pair it is special
The downstream primer of property primer and the downstream primer of second pair of specific primer are mixed in a certain ratio as mix primer 2, spare；
S2, the sense primer of internal control primer and downstream primer are mixed in a certain ratio as mix primer 3, it is spare；
S3, mix primer 2 is set to PCR reaction systems, real-time fluorescence PCR detection is carried out to target fragment to be measured；Wherein,
Reaction system is 15 μ L, including 6 μ L of 2X TaKaRa TaqTMHS Perfect Mix, and mix primer 2 is 4 μ L, enriched product
For 5 μ L；
S4, S3 steps to target fragment to be measured carry out real-time fluorescence PCR detection while, carry out reference gene ACTB inspections
It surveys；Wherein, reaction system is 15 μ L, including 6 μ L of 2X TaKaRa TaqTMHS Perfect Mix, and mix primer 3 is 4 μ L,
Enriched product is 5 μ L；
S5, interpretation of result, the wherein testing result of reference gene ACTB are carried out according to the fluorescence signal intensity detected in real time
As shown in figure 4, Successful amplification reference gene as can be seen from Figure 4；Target fragment testing result to be measured is as shown in figure 5, from Fig. 5
In it can be seen that the square method of the present invention can well separate target gene to be measured and other sites, suitable for multiple genes
The Genotyping in site detects.Specific testing result interpretation method, i.e., when HLA-B*5801-P1 and HLA-B*5801-P2 are special
When primer has detection fluorescence signal, judge the sample for allele containing HLA-B*5801.
Embodiment 4：The inspection and verification of sample to be tested detection
101 clinical samples (being derived from The Third Xiangya Hospital of Central South University, and patient has signed informed consent form) are taken, are pressed
The method of embodiment 2-3 is detected it, the results showed that wherein 12 testing results are the positive, recall rate 11.9%, tool
Body result referring to Fig. 8, as can be seen from the figure only and meanwhile with HLA-B*5801-P1 and HLA-B*5801-P2 detection sites into
When row detection, 11.9% sample detected is just positive, i.e. sample parting is HLA-B*5801.
Then reselection positive sample, and sequence verification is carried out to the positive sample of selection, as a result as shown in fig. 6-7, from
The strictly HLA-B*5801-P1 and HLA-B*5801-P2 that it can be seen from the figure that is amplified by two pairs of special primers of design,
Show that the accurate match degree of the detection method of the present invention is 100%, illustrates that the detection method of the present invention is accurately high, arrange interference performance
By force.
In addition, applicant also increases 4-5 base by 5 ends of the primer with asterisk in listed primer in table 1,
Specific detection is carried out, the results showed that it is HLA-B*5801, inspection that these primers, which can effectively determine sample parting to be detected,
It is 100% to survey accurate match degree, and the detection method further illustrated the present invention is accurately high, and row's interference performance is strong.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
It encloses without being limited thereto.Those skilled in the art on the basis of the present invention made by equivalent substitute or transformation, in the present invention
Protection domain within.Protection scope of the present invention is subject to claims.
Claims (10)
1. a kind of primer for detecting HLA-B*5801 allele, which is characterized in that including being used to determine sample to be detected
Parting is the primer of HLA-B*58, and for detecting the primer that determining HLA-B*58 sample partings are HLA-B*5801.
2. the primer according to claim 1 for detecting HLA-B*5801 allele, which is characterized in that described to be used for
Determine that the primer that sample parting to be detected is HLA-B*58 includes first pair of special primer and the first detection primer, wherein：
Sense primer in first pair of special primer includes such as SEQ ID NO:Nucleotide sequence shown in 1 and with it in 5 ＇
0~5 connected arbitrary base of end,
Downstream primer in first pair of special primer includes such as SEQ ID NO:Nucleotide sequence shown in 2 and with it in 5 ＇
0~5 connected arbitrary base of end,
First detection primer includes such as SEQ ID NO:Nucleotide sequence shown in 3；
It is described for detect the primer that determining HLA-B*58 sample partings are HLA-B*5801 include second pair of special primer and
Second detection primer, wherein
Sense primer in second pair of special primer includes such as SEQ ID NO:Nucleotide sequence shown in 4 and with it in 5 ＇
0~5 connected arbitrary base of end,
Downstream primer in second pair of special primer includes such as SEQ ID NO:Nucleotide sequence shown in 5 and with it in 5 ＇
0~5 connected arbitrary base of end,
Second detection primer includes such as SEQ ID NO:Nucleotide sequence shown in 6.
3. the primer according to claim 2 for detecting HLA-B*5801 allele, which is characterized in that described first
Detection primer is connected with joint sequence 1 at 5 ends ＇, and the second detection primer is in 5 ends ＇ jointing sequences 2；Or first inspection
Primer is surveyed in 5 ends ＇ jointing sequences 2, the second detection primer is in 5 ends ＇ jointing sequences 1；The joint sequence 1 is such as
SEQ ID NO:Nucleotide sequence shown in 17；The joint sequence 2 includes such as SEQ ID NO:Nucleotide sequence shown in 18
With 0~5 arbitrary base being connected at 5 ends ＇ with it.
4. application of the primer in detecting HLA-B*5801 allele described in a kind of any one of claims 1 to 3.
5. a kind of kit for detecting HLA-B*5801 allele, which is characterized in that draw including described in claim 1
Object.
6. kit according to claim 5, which is characterized in that further include internal control primer, the upstream of the internal control primer
Primer includes such as SEQ ID NO:Nucleotide sequence shown in 7 and 0~5 arbitrary base being connected at 5 ends ＇ with it；Described
5 ends ＇ of the sense primer of internal control primer are also associated with fluorophor；
The downstream primer of the internal control primer includes such as SEQ ID NO:Nucleotide sequence shown in 8 and it is connected at 5 ends ＇ with it
0~5 arbitrary base；It is also associated with fluorophor at 5 ends ＇ of the sense primer of the internal control primer and gene is quenched.
7. kit according to claim 5, which is characterized in that further include universal fluorescent probe, the universal fluorescent is visited
Needle includes normal chain probe and minus strand probe, and the normal chain probe includes oligonucleotide positive strand sequence and joint sequence II, described
Minus strand probe includes the oligonucleotide negative strand sequence with the oligonucleotide positive strand sequence complementation；The oligonucleotide is just
Chain-ordering is SEQ ID NO：Nucleotide sequence shown in any one of 9-12；The oligonucleotide negative strand sequence is SEQ ID
NO：Nucleotide sequence shown in any one of 13-16；The joint sequence II is SEQ ID NO：Shown in any one of 19-22
Nucleotide sequence；5 ends ＇ of the normal chain probe are connected with fluorophor, and 3 ends ＇ of the minus strand probe, which are connected with, is quenched base
Group.
8. a kind of method detecting HLA-B*5801 allele using claim 5 to 7 any one of them kit, special
Sign is, includes the following steps：
1) upstream and downstream primer of the upstream and downstream primer of first pair of special primer and second pair of special primer is mixed into mixing
Primer 1；
2) by the universal fluorescent probe, the first detection primer, the second detection primer, the downstream primer of first pair of special primer and
The downstream primer of second pair of special primer is mixed into mix primer 2；
3) genomic DNA of sample is extracted；
4) genomic DNA obtained to step 3) extraction using the mix primer 1 carries out PCR amplification, obtains purpose piece to be measured
Section；
5) target fragment to be measured obtained to step 4) amplification using mix primer 2 carries out real-time fluorescence PCR detection.
Further include drawing the upstream of internal control primer 9. according to the method described in claim 8, it is characterized in that, in step 5)
Object and downstream primer mixing, obtain mix primer 3, while carrying out real-time fluorescence PCR detection to target fragment to be measured, carry out
The step of mix primer 3 carries out real-time fluorescence PCR detection to reference gene.
10. application of the kit in detecting HLA-B*5801 allele described in any one of claim 5~7.
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