CN108977524A - A kind of detection method and detection kit of HLA-B*5801 gene - Google Patents
A kind of detection method and detection kit of HLA-B*5801 gene Download PDFInfo
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Abstract
The present invention relates to the detection methods and detection kit of a kind of HLA-B*5801 gene.The detection method includes the following steps: that (1) designs specific primer for HLA-B*5801 gene；(2) target fragment of the specific PCR amplification comprising HLA-B*5801 gene is utilized；(3) digestion with restriction enzyme PCR product segment is utilized；(4) single base extension is carried out；(5) desalting and purifying processing is carried out；(6) gene order of target gene HLA-B*5801 is tested and analyzed by nucleic acid mass spectrograph.The detection method of above-mentioned HLA-B*5801 gene, detection accuracy is high, experiment can principal characteristic be high, flux is big, at low cost.The present invention also provides a kind of detection kits of HLA-B*5801 gene, including the specific primer pair for expanding HLA-B*5801 gene.The detection kit of above-mentioned HLA-B*5801 gene, simplifies experiment flow, and personnel's operating time is short, difficulty is small, and laboratory automation degree is high.
Description
Technical field
The present invention relates to technical field of biological, more particularly to the detection method and inspection of a kind of HLA-B*5801 gene
Test agent box.
Background technique
With high protein, high purine, high heat food in people's diet intake be substantially increased and drinking amount
Increase, activity is reduced etc., it results in fat and metabolic syndrome patient and increases, so that adjoint hyperuricemia and primary
The disease incidence of gout is in rising trend.Studies have shown that gout is a kind of due to purine substance metabolic disorder, uric acid generate it is excessive or
Uric acid in blood is caused to increase because uric acid excretion is bad, urate crystal is deposited in synovium of joint, synovial bursa, cartilage and its hetero-organization
Caused repeated relapsing diseases associated with inflammation shows as patient articular's limitation of activity, heat, pain, swells, with joint deformity, knot
Section property gout calculus even renal lesions, the illness bring considerable distress to patient.
Allopurinol is a kind of xanthine oxidase inhibitor, by inhibiting the activity of the enzyme, reduces hypoxanthine and Huang is fast
Purine synthesizes uric acid, to reduce the generation of uric acid, so that the uric acid content in blood and urine is reduced to solubility or less level, prevents from urinating
Acid formed crystallization deposition in its hetero-organization, be widely used in the treatment of gout, hyperuricemia, can also pre- preventing leukemia,
Lymthoma or other tumours uric acid mineralization, kidney stone in secondary tissue after chemotherapy or radiotherapy, are particularly suitable for uric acid generation
Excessively, to uricosuric allergy or in vain, and the patient of uricotelic drugs should not be used.But the medicine can cause serious secondary anti-
It answers, including Stevens-John syndrome (SJS) and toxic epidermal's necrosis (TEN).It is red that SJS shows as serious pleomorphism
Spot can be involved skin and mucous membrane, including mouth, nose, eye, vagina, urethra, gastrointestinal tract and lower respiratory tract mucous membrane, patient have blindness
Anxiety, further can develop to form TEN, and whole body mucous membrane festers, and slackness bulla or epidermis removing occur in erythema, if meeting slight
Touching or drawing can lead to the removing of epidermis large area.Studies have shown that HLA-B*5801 allele cause with allopurinol it is tight
Very strong correlation is presented in pipe inflammation side reaction, and 100% is up to especially in the Hans, and the patient of nearly all side reaction is
The carrier of HLA-B*5801, and there was only 15% or so HLA-B*5801 carrier in the resistance to receptor of allopurinol, detect HLA-
B*5801 allele can prevent the SJS or TEN of allopurinol initiation.In the gout that American Society of Rheumatism in 2012 updates
Also suggest coping with the people at highest risk of severe allergic reaction, it is proposed that carry out HLA-B*5801 using before allopurinol in administration guide
Allele detection.But in view of the complexity of HLA-B polymorphic site, HLA-B*5801 detection method is cumbersome, interference because
Element is more, thereby increases and it is possible to since the interference of rare or unknown HLA allele causes result is equivocal to be difficult to interpretation.
SJS/ caused by the SNP-rs9263726 and allopurinol of Japanese scholars research discovery PSORS1C1 gene loci
TEN is related, and the SNP and HLA-B*5801 complete linkage, this is found to be detection HLA-B*5801 allele and has found letter
Single quick method.Currently, the method detected both at home and abroad to HLA-B*5801 allele mainly has DNA direct sequencing
With Taqman sonde method etc., DNA direct sequencing is the goldstandard of current allele detection, but there are time-consuming and laborious etc. scarce
It falls into, is a kind of detection method of small throughput, be not suitable for great amount of samples detection；Taqman is the quantitative PCR technique of high special,
It is a kind of allele detection of moderate fluxes its main feature is that being adapted to the site primer of a small amount of allele of large sample, and it is right
Detect the sites of multiple allele in simultaneously, then it is costly, be not appropriate for using.
Summary of the invention
Based on this, it is necessary to detect this research side of the genotype of HLA-B of sample for by detection Tag SNP
To providing the detection method and detection kit of HLA-B*5801 gene.
A kind of detection method of HLA-B*5801 gene, the detection method include the following steps:
(1) specific primer is designed for HLA-B*5801 gene；
(2) target fragment of the specific PCR amplification comprising HLA-B*5801 gene is utilized；
(3) digestion with restriction enzyme PCR product segment is utilized；
(4) single base extension is carried out；
(5) desalting and purifying processing is carried out；
(6) gene order of target gene HLA-B*5801 is tested and analyzed by nucleic acid mass spectrograph.
The detection method of above-mentioned HLA-B*5801 gene, detection accuracy is high, with the result of goldstandard Sanger PCR sequencing PCR
It compares, accuracy rate is more than 99.7%, while can directly detect DNA mass spectrum, can detect three equipotential SNP sites in sample
In the presence of experiment can principal characteristic height.Compared with traditional SNP site, other detection techniques are much bigger on detection flux for this method,
The Multiple experiments of 384 biological samples can be completed on one chip, unit point cost is than other similar detections after cost conversion
The technical method of SNP site is spent less, is suitble to the demand of a variety of users and batch detection, is the ideal tools for detecting SNP site.
The specific primer designed in the step (1) in one of the embodiments, includes for expanding HLA-B*
The specific primer pair of 5801 genes, the specific primer is to reversed comprising a forward primer SEQ ID Nos:1 and one
The sequence of primer SEQ ID Nos:2, forward primer SEQ ID Nos:1 are as follows: CCAGCTTTACAAGGACCCCAG, reverse primer
The sequence of SEQ ID Nos:2 are as follows: TTGGCTCTCACCAGAAATGG.
The response procedures that specific PCR expands in the step (2) in one of the embodiments, are as follows: 98 DEG C of thermal startings,
10 minutes；95 DEG C of denaturation, 30 seconds；55 DEG C of annealing, 30 seconds；72 DEG C of extensions, 30 seconds；Expand 40 circulations；72 DEG C extend eventually, and 5 points
Clock.
The restriction enzyme in the step (3) carries out digestion process condition in one of the embodiments, are as follows: 37 DEG C
Digestion process 45 minutes, 85 DEG C heat denatured 5 minutes, ice bath 10 minutes.
The restriction enzyme in the step (3) is shrimp alkaline phosphotase in one of the embodiments,.
Single base extension includes Single base extension primer SEQ in the step (4) in one of the embodiments,
The sequence of ID Nos:3, primer SEQ ID Nos:3 are as follows: GATCCCAGCTCCGAGGAAACTC.
In one of the embodiments, in the step (4) single base extension response procedures are as follows: 95 DEG C of heat open
It is dynamic, 1 minute；95 DEG C of denaturation, 5 seconds；56 DEG C of annealing, 5 seconds；80 DEG C of extensions, 5 seconds；Expand 40 circulations；72 DEG C extend eventually, and 3 points
Clock.
The specific steps of the nucleic acid Mass Spectrometer Method of the step (6) in one of the embodiments, are as follows:
(1) stand-by resin is completed on resin plate, is air-dried 5-10 minutes；
(2) sample to be measured is added into the reacting hole of sample plane, supplements 25ul ddH2O, is sealed with sealed membrane, 3000
Rev/min brief centrifugation；
(3) sealed membrane is removed, sample plane is tipped upside down on the resin plate of step (1) and is fixed, then by sample plane and resin
Plate is overturn together, is fallen into air-dried resin in the reacting hole of sample plane, is sealed with sealed membrane, 3000 revs/min of brief centrifugations；
(4) sample plane is placed on rotator and is rotated at a slow speed 20 minutes, then 3000 revs/min are centrifuged 5 minutes, then point sample
Upper machine is detected with nucleic acid mass spectrograph.
The present invention also provides a kind of detection kits of HLA-B*5801 gene, including for expanding HLA-B*5801 base
The specific primer pair of cause, the specific primer is to including a forward primer SEQ ID Nos:1 and a reverse primer
The sequence of SEQ ID Nos:2, forward primer SEQ ID Nos:1 are as follows: CCAGCTTTACAAGGACCCCAG, reverse primer SEQ
The sequence of ID Nos:2 are as follows: TTGGCTCTCACCAGAAATGG.
The detection kit of above-mentioned HLA-B*5801 gene, simplifies experiment flow, and personnel's operating time is short, difficulty is small,
Laboratory automation degree is high.
The kit further includes the Single base extension primer for single base extension in one of the embodiments,
The sequence of SEQ ID Nos:3, primer SEQ ID Nos:3 are as follows: GATCCCAGCTCCGAGGAAACTC.
Compared with prior art, the present invention has following beneficial effect；
(1) detection method of HLA-B*5801 gene of the present invention is on detection flux compared with traditional position SNP
Other detection techniques of point are much bigger, and the Multiple experiments of 384 biological samples can be completed on a chip, are detections SNP
The ideal tools of point；
(2) the detection method detection accuracy of HLA-B*5801 gene of the present invention is high, surveys with goldstandard Sanger
The result of sequence method is compared, and accuracy rate is more than 99.7%, while can directly detect DNA mass spectrum, can be detected third in sample
The presence of position SNP site, experiment can principal characteristic height.
(3) detection method low cost, the cost performance of HLA-B*5801 gene of the present invention are high, can be on a chip
The Multiple experiments of 384 biological samples are completed, the technical side of unit point cost detection SNP site more similar than other after cost conversion
Method is spent less, is suitble to the demand of a variety of users and batch detection.
(4) when gene order SNP site nucleic acid Mass Spectrometry detection method experiment flow of the present invention is simple, personnel operate
Between it is short, difficulty is small, laboratory automation degree is high, and data processing software processing is convenient, and report result is clearly understandable.
Detailed description of the invention
Fig. 1 is the nucleic acid mass spectral analysis figure of one embodiment of the invention.
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text
Word can be implemented accordingly.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other elements or combinations thereof.
A kind of detection method of HLA-B*5801 gene, the detection method include the following steps:
(1) specific primer is designed for HLA-B*5801 gene；
(2) target fragment of the specific PCR amplification comprising HLA-B*5801 gene is utilized；
(3) digestion with restriction enzyme PCR product segment is utilized；
(4) single base extension is carried out；
(5) desalting and purifying processing is carried out；
(6) gene order of target gene HLA-B*5801 is tested and analyzed by nucleic acid mass spectrograph.
The detection method of HLA-B*5801 gene of the present invention is based primarily upon PCR and Single base extension technology, former
Reason is to be expanded first by PCR primer to the target fragment containing SNP to be checked, and restriction enzyme is added after PCR and goes
Except the dNTP in reaction solution.Then the related components such as SNP extension primer and ddNTP are added in reaction solution and carry out single base and prolong
Reaction is stretched, SNP extension primer in conjunction with the 5 ' terminal sequences of SNP to be detected and can extend a base in reaction process, according to not
Different extension products can be obtained with SNP template.
Preferably, the specific primer designed in the step (1) includes for expanding the special of HLA-B*5801 gene
Property primer pair, the specific primer is to including a forward primer SEQ ID Nos:1 and a reverse primer SEQ ID
The sequence of Nos:2, forward primer SEQ ID Nos:1 are as follows: CCAGCTTTACAAGGACCCCAG, reverse primer SEQ ID Nos:2
Sequence are as follows: TTGGCTCTCACCAGAAATGG.
Compared to the technological means of other traditional detection HLA-B*5801 genes, the present invention is through detecting HLA-B*5801 base
Because of the SNP site in region, this point is just more different from traditional quantitative fluorescent PCR or Sanger PCR sequencing PCR.As above-mentioned core
The mass spectrographic principle of acid only extends the base with polymorphism in destination region SNP site using nucleic acid Mass Spectrometry detection method,
The genetic fragment of entire specific amplification is not needed to detect.The flux of whole detection, experimental period, reaction cost, result progress
Have greatly improved compared with conventional method and optimize, there is good market should be worth.
Preferably, the response procedures that specific PCR expands in the step (2) are as follows: 98 DEG C of thermal startings, 10 minutes；95℃
Denaturation, 30 seconds；55 DEG C of annealing, 30 seconds；72 DEG C of extensions, 30 seconds；Expand 40 circulations；72 DEG C extend eventually, and 5 minutes.
Preferably, the restriction enzyme in the step (3) carries out digestion process condition are as follows: 37 DEG C of digestion process 45 are divided
Clock, 85 DEG C heat denatured 5 minutes, ice bath 10 minutes.
Preferably, the restriction enzyme in the step (3) is shrimp alkaline phosphotase.
Preferably, single base extension includes Single base extension primer SEQ ID Nos:3, primer in the step (4)
The sequence of SEQ ID Nos:3 are as follows: GATCCCAGCTCCGAGGAAACTC.
Preferably, in the step (4) single base extension response procedures are as follows: 95 DEG C of thermal startings, 1 minute；95℃
Denaturation, 5 seconds；56 DEG C of annealing, 5 seconds；80 DEG C of extensions, 5 seconds；Expand 40 circulations；72 DEG C extend eventually, and 3 minutes.
Preferably, the specific steps of the nucleic acid Mass Spectrometer Method of the step (6) are as follows:
(1) stand-by resin is completed on resin plate, is air-dried 5-10 minutes；
(2) sample to be measured is added into the reacting hole of sample plane, supplements 25ul ddH2O, is sealed with sealed membrane, 3000
Rev/min brief centrifugation；
(3) sealed membrane is removed, sample plane is tipped upside down on the resin plate of step (1) and is fixed, then by sample plane and resin
Plate is overturn together, is fallen into air-dried resin in the reacting hole of sample plane, is sealed with sealed membrane, 3000 revs/min of brief centrifugations；
(4) sample plane is placed on rotator and is rotated at a slow speed 20 minutes, then 3000 revs/min are centrifuged 5 minutes, then point sample
Upper machine is detected with nucleic acid mass spectrograph.
Ion exchange is carried out by mixing reaction solution with resin when nucleic acid Mass Spectrometer Method, is adsorbed in for removing in liquid
K on DNA fragmentation+、Na+、Mg2+Plasma prevents it from interfering Mass Spectrometer Method result.
Reaction solution and matrix crystallize on target plate, carry out Mass Spectrometer Method and obtain map.Due to each extension products molecular weight
Difference, therefore can check whether detection peak occur in respective molecular weight, then judge the SNP parting of the sample.Core
Sour mass spectrometric platforms specificity is good, can reach 30ppm using general international standard product, and lowest detection is limited to 5ng genomic DNA,
The goldstandard verification technique that comparative test selects is Sanger sequencing technologies and the granted testing product of same type, and accordance is
100%.
A kind of detection kit of HLA-B*5801 gene, including the specific primer for expanding HLA-B*5801 gene
Right, the specific primer is to comprising a forward primer SEQ ID Nos:1 and a reverse primer SEQ ID Nos:2, just
To the sequence of primer SEQ ID Nos:1 are as follows: the sequence of CCAGCTTTACAAGGACCCCAG, reverse primer SEQ ID Nos:2
Are as follows: TTGGCTCTCACCAGAAATGG.
Preferably, the kit further includes the Single base extension primer SEQ ID Nos for single base extension:
The sequence of 3, primer SEQ ID Nos:3 are as follows: GATCCCAGCTCCGAGGAAACTC.
Below in conjunction with specific embodiment, the present invention is further elaborated.
Embodiment 1: the detection using nucleic acid Mass Spectrometry detection method to people's HLA-B*5801 gene SNP site
1. human gene group DNA extracts
(1) extracting genome DNA operation is carried out to the blood sample of patient in Biohazard Safety Equipment.Using Blood&Cell
Culture DNA Mini Kit (Qiagen Cat No:13323) extracts experiment.
(2) design of primers
SNP site design of primers is carried out for people's HLA-B*5801 gene SNP site, it is special for this SNP design respectively
Anisotropic PCR primer and Single base extension primer, designed PCR primer sequence is as shown in table 1.
Table 1
(3) specific PCR reacts
The target fragment comprising HLA-B*5801 gene is amplified using specific PCR technology, according to the reaction system of table 2
Amplified reaction is prepared, reaction system is expanded after the completion of preparing according to the response procedures of table 3.
Table 2
Table 3
(4) alkali formula phosphoric acid collagenase treatment
Multi-PRC reaction product is digested using shrimp alkali formula phosphatase, extra dNTP in removal system.It is each anti-
The alkali formula phosphatase that 0.5U is added in system is answered, adjusts buffer concentration with matched 10*Tris-Buffer, each reaction adds
Enter 10*Tris-Buffer 0.5ul.Then 37 DEG C are carried out in the water-bath of preheating digestion process 45 minutes, then add for 85 DEG C
Thermal denaturation 5 minutes, last ice bath 10 minutes.
(5) single base extension is carried out
Single base extension system is prepared, is configured according to the reaction system of table 4.Take the prepared Single base extension of 2ul
Before the reaction solution of reaction is added in the system of step digestion reaction, it is anti-that Single base extension then is carried out according to the response procedures of table 5
It answers.
Table 4
Table 5
(6) machine pre-treatment on product desalting and purifying and nucleic acid mass spectrum
(a) stand-by resin is completed on resin plate, is air-dried 5-10 minutes；
(b) sample to be measured is added into the reacting hole of sample plane, supplements 25ul ddH2O, is sealed with sealed membrane, 3000
Rev/min brief centrifugation；
(c) sealed membrane is removed, sample plane is tipped upside down on the resin plate of step (1) and is fixed, then by sample plane and resin
Plate is overturn together, is fallen into air-dried resin in the reacting hole of sample plane, is sealed with sealed membrane, 3000 revs/min of brief centrifugations；
(d) sample plane is placed on rotator and is rotated at a slow speed 20 minutes, then 3000 revs/min are centrifuged 5 minutes, then point sample
Upper machine is detected with nucleic acid mass spectrograph.
(7) machine and software data analysis on nucleic acid mass spectrograph
Upper machine testing operation is carried out according to the operating instruction of instrument, is finished to sample detection, software is carried out to testing result
Processing analysis, finally obtains testing result, nucleic acid mass spectral analysis figure is specifically shown in Fig. 1.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Claims (10)
1. a kind of detection method of HLA-B*5801 gene, which is characterized in that the detection method includes the following steps:
(1) specific primer is designed for HLA-B*5801 gene；
(2) target fragment of the specific PCR amplification comprising HLA-B*5801 gene is utilized；
(3) digestion with restriction enzyme PCR product segment is utilized；
(4) single base extension is carried out；
(5) desalting and purifying processing is carried out；
(6) gene order of target gene HLA-B*5801 is tested and analyzed by nucleic acid mass spectrograph.
2. the detection method of HLA-B*5801 gene according to claim 1, which is characterized in that set in the step (1)
The specific primer of meter includes the specific primer pair for expanding HLA-B*5801 gene, and the specific primer is to including one
A forward primer SEQ ID Nos:1 and reverse primer SEQ ID Nos:2.
3. the detection method of HLA-B*5801 gene according to claim 1, which is characterized in that special in the step (2)
The response procedures of specific PCR amplification are as follows: 98 DEG C of thermal startings, 10 minutes；95 DEG C of denaturation, 30 seconds；55 DEG C of annealing, 30 seconds；72 DEG C are prolonged
It stretches, 30 seconds；Expand 40 circulations；72 DEG C extend eventually, and 5 minutes.
4. the detection method of HLA-B*5801 gene according to claim 1, which is characterized in that in the step (3)
Restriction enzyme carry out digestion process condition are as follows: 37 DEG C digestion process 45 minutes, 85 DEG C heat denatured 5 minutes, ice bath 10 divides
Clock.
5. the detection method of HLA-B*5801 gene according to claim 1, which is characterized in that in the step (3)
Restriction enzyme is shrimp alkaline phosphotase.
6. the detection method of HLA-B*5801 gene according to claim 1, which is characterized in that single in the step (4)
Base extension includes Single base extension primer SEQ ID Nos:3.
7. the detection method of HLA-B*5801 gene according to claim 1, which is characterized in that single in the step (4)
The response procedures of base extension are as follows: 95 DEG C of thermal startings, 1 minute；95 DEG C of denaturation, 5 seconds；56 DEG C of annealing, 5 seconds；80 DEG C of extensions,
5 seconds；Expand 40 circulations；72 DEG C extend eventually, and 3 minutes.
8. the detection method of HLA-B*5801 gene according to claim 1, which is characterized in that the core of the step (6)
The specific steps of sour Mass Spectrometer Method are as follows:
(1) stand-by resin is completed on resin plate, is air-dried 5-10 minutes；
(2) sample to be measured is added into the reacting hole of sample plane, supplements 25ul ddH2O is sealed with sealed membrane, and 3000 revs/min
Clock brief centrifugation；
(3) sealed membrane is removed, sample plane is tipped upside down on the resin plate of step (1) and is fixed, then by sample plane and resin plate one
Overturning is played, air-dried resin is fallen into the reacting hole of sample plane, is sealed with sealed membrane, 3000 revs/min of brief centrifugations；
(4) sample plane is placed on rotator and is rotated at a slow speed 20 minutes, then 3000 revs/min are centrifuged 5 minutes, then on point sample
Machine is detected with nucleic acid mass spectrograph.
9. a kind of detection kit of HLA-B*5801 gene, which is characterized in that including for expanding HL A-B*5801 gene
Specific primer pair, the specific primer is to including a forward primer SEQ ID Nos:1 and a reverse primer SEQ ID
Nos:2.
10. the detection kit of HLA-B*5801 gene according to claim 9, which is characterized in that the kit is also
Including the Single base extension primer SEQ ID Nos:3 for single base extension.
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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 |
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