CN109112188A - A kind of detection method and detection kit of HLA-B*1502 gene - Google Patents
A kind of detection method and detection kit of HLA-B*1502 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*1502 gene.The detection method includes the following steps: that (1) designs specific primer for HLA-B*1502 gene；(2) target fragment of the specific PCR amplification comprising HLA-B*1502 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*1502 is tested and analyzed by nucleic acid mass spectrograph.The detection method of above-mentioned HLA-B*1502 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*1502 gene, including the specific primer pair for expanding HLA-B*1502 gene.The detection kit of above-mentioned HLA-B*1502 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*1502 gene
Test agent box.
Background technique
Carbamazepine (carbamazepine) alias carbamazepine, carbamazepine, carbamoyl nitrogen etc. are a kind of
For treating the drug of epilepsy, peripheral neuralgia, neurogenic diabetes insipidus, prevention or treatment manic-depressive psychosis.Carbamazepine is normal
The side effect seen mainly has: nervous system damage, hemopoietic system damage, hepatorenal damage, gastrointestinal lesions and skin lesion.In beauty
State, carbamazepine is for treating epilepsy, mania/depression and neurogenic pain.U.S. Food and Drug Administration
(FDA) safety information of the publication about carbamazepine, information claim, and carbamazepine can cause dangerous even fatal skin anti-
(Stevens-Johnson syndrome and toxic epidermal necrolysis disease) is answered, especially in allele containing human leucocyte antigen (HLA)
It is easier to occur in the patient of HLA- B*1502.Stevens-Johnson syndrome (Stevens Johnson syndr
Ome, SJS) and toxic epidermal necrolysis disease (toxic epidermal necrolysis, TEN) be serious skin and
It is even fatal to can lead to permanent disability for mucosa reaction.Carbamazepine causes the probability of SJS/TEN very low, in white people country
What is carried out causes the total evaluation of SJS/TEN to show for carbamazepine, and S JS/TEN incidence only has a ten thousandth to very much
Six.But the marketed products adverse events report display received according to the World Health Organization (WHO) and carbamazepine pharmaceutical manufacturer, one
The probability that SJS/TEN occur in a little Asian countries will be about being higher by 10 times.TaiWan, China, Europe and the studies have shown that of Hong-Kong
The increase of SJS/TEN risk is related with HLA-B*1502.Almost only Asian ancestry patient carries HLA-B*1502 allele, packet
Include the Indian in South Asia.HLA-B*1502 can pass through heredity test detection.The patient for carrying HLA-B*1502 gene is starting
Before being treated using carbamazepine, HLA-B*1502 allele detection should be carried out, is such as positive through testing result, then should not be made
With carbamazepine, except the prospective earnings of non-drug are significantly greater than the increase of serious skin reaction risk.The study found that in HLA-B
Nearby there is the SNP chain with certain hypotype height in locus, i.e., " Tag SNP ", the Tag SNP of HLA-B*1502 is
The C allele of rs10484555, the specificity of the two is up to 99.27%.Therefore, sample is detected by detection Tag SNP
The genotype of H LA-B be a kind of new research direction.The present invention is directed to which the screening of rs10484555 polymorphism is unfolded to patient,
Carry out personalized precisely diagnosis and treatment for such patient and establishes clinical and scientific research basis.
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*1502 gene.
A kind of detection method of HLA-B*1502 gene, the detection method include the following steps:
(1) specific primer is designed for HLA-B*1502 gene；
(2) target fragment of the specific PCR amplification comprising HLA-B*1502 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*1502 is tested and analyzed by nucleic acid mass spectrograph.
The detection method of above-mentioned HLA-B*1502 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 H LA-B*
The specific primer pair of 1502 genes, the specific primer is to anti-comprising a forward primer SEQ I D Nos:1 and one
To primer SEQ ID Nos:2, the sequence of forward primer SEQ ID Nos:1 are as follows: TCAAGCTAGGAAAGTTGCCAA reversely draws
The sequence of object SEQ ID Nos:2 are as follows: A GAACATTTTCATCATCCCAAGA.
The response procedures that specific PCR expands in the step (2) in one of the embodiments, are as follows: 98 DEG C of heat open
It is dynamic, 10 minutes；95 DEG C of denaturation, 30 seconds；58 DEG C of annealing, 30 seconds；72 DEG C of extensions, 30 seconds；Expand 40 circulations；72 DEG C extend eventually, and 5
Minute.
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: CTCAAAATTTATGGAT TTACTTCATTG.
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；55 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 instantaneously from
The heart；
(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*1502 gene, including for expanding HLA- B*1502
The specific primer pair of gene, the specific primer reversely draw to comprising a forward primer SEQ ID N os:1 and one
The sequence of object SEQ ID Nos:2, forward primer SEQ ID Nos:1 are as follows: TCAAGCTAGGAAAGTTGCCAA, reverse primer
The sequence of SEQ ID Nos:2 are as follows: AGA ACATTTTCATCATCCCAAGA.
The detection kit of above-mentioned HLA-B*1502 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: CTCAAAATTTATG GATTTACTTCATTG.
Compared with prior art, the present invention has following beneficial effect；
(1) detection method of HLA-B*1502 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*1502 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*1502 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*1502 gene, the detection method include the following steps:
(1) specific primer is designed for HLA-B*1502 gene；
(2) target fragment of the specific PCR amplification comprising HLA-B*1502 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*1502 is tested and analyzed by nucleic acid mass spectrograph.
The detection method of HLA-B*1502 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*1502 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: TCAAGCT AGGAAAGTTGCCAA, reverse primer SEQ ID Nos:
2 sequence are as follows: AGAACATTTTC ATCATCCCAAGA.
Compared to the technological means of other traditional detection HLA-B*1502 genes, the present invention is through detecting HLA- B*1502
The SNP site of gene region, this point are just more different from traditional quantitative fluorescent PCR or Sanger PCR sequencing PCR.As above-mentioned
The mass spectrographic principle of nucleic acid only extends the alkali with polymorphism in destination region SNP site using nucleic acid Mass Spectrometry detection method
Base does not need the genetic fragment for detecting entire specific amplification.The flux of whole detection, experimental period, reaction cost, result into
Degree has greatly improved and is optimized compared with conventional method, and 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；58 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 N os:3 in the step (4), is drawn
The sequence of object SEQ ID Nos:3 are as follows: CTCAAAATTTATGGATTTACTTCATT G.
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；55 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 instantaneously from
The heart；
(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 30pp m 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 equal
It is 100%.
A kind of detection kit of HLA-B*1502 gene, including the specific primer for expanding HLA-B*1502 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 TCAAGCTAGG AAAGTTGCCAA, reverse primer SEQ ID Nos:2
Are as follows: AGAACATTTTCATC ATCCCAAGA.
Preferably, the kit further includes the Single base extension primer SEQ I D Nos for single base extension:
The sequence of 3, primer SEQ ID Nos:3 are as follows: CTCAAAATTTATGGATTTACTTC ATTG.
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*1502 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*1502 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*1502 gene is amplified using specific PCR technology, according to the reactant of table 2
Amplified reaction is prepared by system, and 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 dNT P in removal system.Each
The alkali formula phosphatase of 0.5U is added in reaction system, adjusts buffer concentration, each reaction with matched 10*Tris-Buffer
10*Tris-Buffer 0.5ul is added.Then 37 DEG C are carried out in the water-bath of preheating digestion process 45 minutes, then 85 DEG C
Heat denatured 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
| 5ul/ul | |
| 10*iPLEX buffer | 0.5 |
| iPLEX ddNTP | 0.2 |
| IPLEX extension primer HLA-B*1502E | 0.5 |
| IPLEX single base extension enzyme | 0.2 |
| 10*Tris Buffer | 3.6 |
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 25ulddH2O, 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, falls into air-dried resin in the reacting hole of sample plane, sealed with sealed membrane, 3000 revs/min instantaneously from
The heart；
(d) sample plane is placed on rotator and is rotated at a slow speed 20 minutes, is centrifuged 5 minutes at 3000 revs/min, 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*1502 gene, which is characterized in that the detection method includes the following steps:
(1) specific primer is designed for HLA-B*1502 gene；
(2) target fragment of the specific PCR amplification comprising HLA-B*1502 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*1502 is tested and analyzed by nucleic acid mass spectrograph.
2. the detection method of HLA-B*1502 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*1502 gene, and the specific primer is to including one
A forward primer SEQ ID Nos:1 and reverse primer SEQID Nos:2.
3. the detection method of HLA-B*1502 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；58 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*1502 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*1502 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*1502 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*1502 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；55 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*1502 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*1502 gene, which is characterized in that including for expanding HLA-B*1502 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*1502 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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