CN109355358A - A kind of kit and method rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism - Google Patents
A kind of kit and method rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism Download PDFInfo
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Abstract
The invention discloses a kind of kits and method for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism, belong to technical field of gene detection, related gene polymorphism includes HLA-B*1502 and HLA-B*5801 gene pleiomorphism, kit includes sample processing reagent for handling for people's whole blood sample, using gene detection reagent, positive reference substance and the negative controls of the single part premix packing of PCR8 union, positive reference substance is gene plasmid DNA and internal reference Plasmid DNA, and negative controls are the ultrapure water without target gene and reference gene.The present invention is to promote the specificity and sensitivity of detection, using the specificity amplification primer designed according to target-gene sequence to and MGB-NFQ fluorescence probe, specific amplified and real-time detection analysis are carried out, makes detection can accurate from HLA-B up to thousands of kinds of polymorphic alleles, special, delicately detection target gene variation.
Description
Technical field
The present invention relates to a kind of kit of gene pleiomorphism and methods, rapidly and efficiently detect medicine source more particularly to one kind
The kit and method of property skin adverse reaction related gene polymorphism, belong to technical field of gene detection.
Background technique
Investigation shows the drugs such as carbamazepine, phenytoinum naticum for HLA-B*1502 allele positive asian ancestry patient
Treat the first of the serious skin reaction for Glenn Stevens one adherence syndrome sJs and toxic epidermal necrolysis TEN occur
Step data, HLA-B*1502 are a kind of human leucocyte antigen allele, are almost existed only among the ethnic group of Asia, packet
Include Chinese han population, Filipino, Malaysian, South Asia Indian and Thailander.
10%-15% is estimated to be very in China, Thailand, Malaysia, Indonesia, Philippine and Taiwan
HLA-B*1502 allele is carried to more patients, people from South Asia including Indian carries in the ratio of the gene
Deng, average out to 2%-4%, but ratio is higher in some crowds, and the ratio that Japanese and Korean carry this gene is lower, no
To l%.
Currently, the package insert suggestion of the anti-epileptics such as carbamazepine, phenytoinum naticum, may carry HLA-B*1502 equipotential
The patient of gene should carry out HLA-B*1502 equipotential base before receiving the treatment of the anti-epileptics carbamazepine such as carbamazepine, phenytoinum naticum
Because of screening, the patient of the screening results positive should not use the anti-epileptics such as carbamazepine, phenytoinum naticum, unless its benefit is evident is greater than wind
Danger.
HLA-B*58:01 allele is located at No. 6 chromosome of the mankind, is the genotype of human leukocyte antigen, HLA-
The strong correlation of SJS or TEN that B*5801 allele causes with allopurinol are sent out in Chinese Han Population by Taiwan's scholars first
It is existing, and confirmed in Chinese Han Population by Thailand, Singapore, the multiple research groups in Hong Kong and China, in other ethnic groups, Japanese and
Korean is also proved to that very strong correlation is presented, and SJS and HLA-B*5801 present relatively weak related in European patient
Property.
American Society of Rheumatism has updated gout administration guide within 2012, wherein suggesting before using allopurinol, to serious
Anaphylactoid people at highest risk, it is proposed that carry out the detection of HLA-B*5801 allele, which includes South Korea descendants, Chinese descendants
Han nationality and Thailand descendants, therefore, the strong correlation of SJS or TEN that HLA-B*5801 and allopurinol cause have the ethnic difference opposite sex,
HLA-B*5801 gene frequency is 6-15% in the Hans, has areal variation.
Summary of the invention
The main object of the present invention is to provide for a kind of rapidly and efficiently detection drug induccd skin adverse reaction related gene
The kit and method of polymorphism, solve in the prior art detect HLA-B*1502 and HLA-B*5801 gene pleiomorphism when pair
Sample process requires the problems such as high, detection cycle is long, complicated for operation, at high cost and specific not strong.
The purpose of the present invention can reach by using following technical solution:
A kind of kit rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism, related gene are polymorphic
Property includes HLA-B*1502 and HLA-B*5801 gene pleiomorphism, and kit includes sample processing reagent, gene detection reagent, sun
Property reference substance and negative controls.
Further, gene detection reagent includes:
Detect HLA-B*1502 primer pair SEQ ID NO.1 and SEQ ID NO.2；
Detect HLA-B*1502 specific probe SEQ ID NO.3；
Detect HLA-B*5801 primer pair SEQ ID NO.4 and SEQ ID NO.5；
Detect HLA-B*5801 specific probe SEQ ID NO.6；
Probe nucleic acid sequence 5 ' is used using one of FAM, TET, VIC, HEX and ROX modification, probe nucleic acid sequence 3 '
MGB-NFQ base group modification.
Further, detection HLA-B*1502 primer SEQ ID NO.1 is 1502 upstream primers: 5 '-
GCCGCGAGTCGAGGAT-3'；
Detection HLA-B*1502 primer SEQ ID NO.2 is 1502 downstream primers: 5 '-GTTGTAGTACCGCGCAGGT-
3'；
Detection HLA-B*1502 specific probe SEQ ID NO.3 is 1502 fluorescence probes: 5 '-fluorophors-
ACACACAGATCTCC-MGB-NFQ-3'；
Detection HLA-B*5801 primer SEQ ID NO.4 is 5801 upstream primers: 5 '-GACCCAGTTCGTGAGGTTCC-
3'；
Detection HLA-B*5801 primer SEQ ID NO.5 is 5801 downstream primers: 5 '-AGTTCCGTGTCTCCCCGTG-
3'；
Detection HLA-B*5801 specific probe SEQ ID NO.6 is 5801 fluorescence probes: 5 '-fluorophors-
CTCCGTCCTCTGACTC-MGB-NFQ-3’。
Further, gene detection reagent further include for internal reference primer pair SEQ ID NO.7, SEQ ID NO.8 and
Internal reference probe SEQ ID NO.9, probe use TaqMan probe；
SEQ ID NO.7 is internal reference upstream primer:
5'-CCTTCCTGCCACCGCTGAT-3'；
SEQ ID NO.8 is internal reference downstream primer:
5'-TGCCTTCGCAACCATTCCCTTA-3'；
Internal reference probe SEQ ID NO.9:
5 '-fluorophor-CGCTATGCTCCGCGCGCATCGTCTGCTC-BHQ-3 '.
Further, gene detection reagent further includes the PCR premixed liquid TaqPath ProAmp with anti-PCR enzyme inhibitor
Master Mix, premixed liquid include Hotstart-Taq polymerase after modifying, UNG enzyme, ROX fluorescent calibration dyestuff,
dNTP、MgCl2、PCR buffer。
Further, sample processing reagent for people's whole blood sample processing, gene detection reagent using PCR8 union one
Part premix packing.
Further, positive reference substance is mixing HLA-B*15:02, HLA-B*58:01 gene plasmid DNA and internal reference matter
Grain DNA；Negative controls are the ultrapure water without target gene and reference gene.
A method of drug induccd skin adverse reaction related gene polymorphism is rapidly and efficiently detected, is included the following steps:
Step 1: sample is quickly handled, and takes EDTA anticoagulated whole blood sample, takes 2 μ L, 20 μ L sample processing reagents is added, 37
5min is incubated under the conditions of DEG C；
Step 2: by 2 μ L of step 1 gained sample, being added in the gene detection reagent of 18 μ L premix packing, mix laggard
Row PCR amplification；
Step 3: setting instrument reaction process and response parameter；
Step 4: determining sample Genotyping, if testing result has the rise of S type amplification curve, and Ct. value is sentenced less than the positive
Definite value is then positive test symbol, if being greater than positive decision content without curve rise or Ct. value, interpretation is without the genotype.
Further, in step 3, the instrument response parameter of setting are as follows:
1st stage: 95 DEG C, 10min, 1 circulation；
2nd stage: 95 DEG C, 10S, 40 circulations；
3rd stage: 60 DEG C, 30S, 40 circulations open fluorescence channel in the 3rd stage and collect fluorescent value.
Further, positive test symbol decision content is as follows:
Internal reference Ct value :≤32；
Sense channel Ct. value :≤32；
Gene type: containing tested HLA-B5801 or HLA-B1502 gene.
Advantageous effects of the invention:
1, it is provided by the invention rapidly and efficiently detect drug induccd skin adverse reaction related gene polymorphism kit and
Method, for promoted detection specificity and sensitivity, using the specificity amplification primer designed according to target-gene sequence to
MGB-NFQ fluorescence probe carries out specific amplified and real-time detection analysis, makes detection can be from HLA-B up to thousands of kinds of allele
Accurate in polymorphism, special, delicately detection target gene variation.
2, it is provided by the invention rapidly and efficiently detect drug induccd skin adverse reaction related gene polymorphism kit and
Method, detection allele include HLA-B*15:02, HLA-B*58:01, and kit forms of the present invention include fast for sample
The positive reference substance of gene detection reagent, the tested target gene Plasmid DNA of mixing that reagent, the single tube premix of speed processing are completed, no
Negative controls containing target gene.
3, it is provided by the invention rapidly and efficiently detect drug induccd skin adverse reaction related gene polymorphism kit and
Method, gene detection reagent have the function of stronger anti-PCR mortifier ability and multiplex PCR, and when detection extracts pure without gDNA
Change, only the PCR reaction solution that single tube premixes, different tested target bases need to will can be added after the processing of whole blood sample cell rapid cleavage
Gene magnification is carried out because different primer pairs is respectively adopted, and real-time using the probe progress that different fluorescent dye groups mark
It tests and analyzes, realizes detection of the reaction system simultaneously to polygenic variation.
4, it is provided by the invention rapidly and efficiently detect drug induccd skin adverse reaction related gene polymorphism kit and
Method is guaranteeing on the specific and sensitive basis of detection that realization is convenient, fast and efficiently detects, and reduces detection operation step
Suddenly, production cost and testing cost are reduced while the reduction detection reaction time, is conducive to clinical detection assays application.
Detailed description of the invention
Fig. 1 is that testing result of the present invention contains HLA-B*1502, is free of HLA-B*5801；
Fig. 2 is that testing result of the present invention contains HLA-B*5801, is free of HLA-B*1502；
Fig. 3 is that testing result of the present invention contains HLA-B*1502/*5801 simultaneously；
Fig. 4 is that testing result of the present invention is free of HLA-B*1502/*5801 simultaneously.
Specific embodiment
To make the more clear and clear technical solution of the present invention of those skilled in the art, below with reference to examples and drawings
The present invention is described in further detail, and embodiments of the present invention are not limited thereto.
In the present embodiment, according to graphic result interpretation genotype, HLA-B*1502 fluorescence probe is marked to have according to Fig. 1
Amplification curve, label HLA-B*5801 fluorescence probe mark internal reference fluorescence probe to have amplification curve, indicate sample without amplification curve
Containing only the testing result of HLA-B*1502 sample.
In the present embodiment, according to graphic result interpretation genotype, mark according to fig. 2 HLA-B*1502 fluorescence probe without
Amplification curve, label HLA-B*5801 fluorescence probe have amplification curve, and label internal reference fluorescence probe has amplification curve, indicates sample
Containing only the testing result of HLA-B*5801 sample.
In the present embodiment, according to graphic result interpretation genotype, HLA-B*1502 fluorescence probe is marked to have according to Fig. 3
Amplification curve, label HLA-B*5801 fluorescence probe have amplification curve, and label internal reference fluorescence probe has amplification curve, indicates sample
Contain HLA-B*5801/ HLA-B*1502 a pair of alleles.
In the present embodiment, according to graphic result interpretation genotype, internal reference fluorescence probe is marked to have amplification bent according to Fig. 4
Line indicates that the sample had both been free of HLA-B*1502 allele without other signal probe fluorescence curves, is also free of HLA-B*
5801 allele.
The kit provided in this embodiment for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism, phase
Correlation gene polymorphism includes HLA-B*1502 and HLA-B*5801 gene pleiomorphism, and kit includes for being used for people's whole blood
The sample processing reagent of present treatment, gene detection reagent, positive reference substance and the yin dispensed using the single part premix of PCR8 union
Property reference substance, positive reference substance is mixing HLA-B*15:02, HLA-B*58:01 gene plasmid DNA and internal reference Plasmid DNA, negative
Reference substance is the ultrapure water without target gene and reference gene.
In the present embodiment, gene detection reagent includes:
Detection HLA-B*1502 primer pair SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.1 is that 1502 upstreams are drawn
Object: 5 '-GCCGCGAGTCGAGGAT-3 ', SEQ ID NO.2 are 1502 downstream primers: 5 '-GTTGTAGTACCGCGCAGGT-
3'；
Detection HLA-B*1502 specific probe SEQ ID NO.3, SEQ ID NO.3 is 1502 fluorescence probes: 5 '-is glimmering
Light group-ACACACAGATCTCC-MGB-NFQ-3 '；
Detection HLA-B*5801 primer pair SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.4 is that 5801 upstreams are drawn
Object: 5 '-GACCCAGTTCGTGAGGTTCC-3 ', SEQ ID NO.5 are 5801 downstream primers: 5 '-
AGTTCCGTGTCTCCCCGTG-3'；
Detection HLA-B*5801 specific probe SEQ ID NO.6, SEQ ID NO.6 is 5801 fluorescence probes: 5 '-is glimmering
Light group-CTCCGTCCTCTGACTC-MGB-NFQ-3 '.
Probe nucleic acid sequence 5 ' is used using one of FAM, JOE, VIC, NED and ROX modification, probe nucleic acid sequence 3 '
MGB-NFQ base group modification.
In the present embodiment, gene detection reagent further includes primer pair SEQ ID NO.7, the SEQ ID for internal reference
NO.8 and internal reference probe SEQ ID NO.9, probe use TaqMan probe, and 5 ' modification CY5 fluorophors, 3 ' are modified using BHQ；
SEQ ID NO.7 is internal reference upstream primer:
5'-CCTTCCTGCCACCGCTGAT-3'；
SEQ ID NO.8 is internal reference downstream primer:
5'-TGCCTTCGCAACCATTCCCTTA-3'；
Internal reference probe SEQ ID NO.9:
5 '-fluorophor-CGCTATGCTCCGCGCGCATCGTCTGCTC-BHQ-3 '.
In the present embodiment, gene detection reagent further includes the PCR premixed liquid TaqPath with anti-PCR enzyme inhibitor
ProAmp Master Mix, premixed liquid include Hotstart-Taq polymerase, the UNG enzyme, ROX fluorescent calibration dye after modifying
Material, dNTP, MgCl2, PCR buffer etc..
The present embodiment additionally provides the method for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism, packet
Include following steps:
Step 1: sample is quickly handled, and takes EDTA anticoagulated whole blood sample, takes 2 μ L, 20 μ L sample processing reagents is added, 37
5min is incubated under the conditions of DEG C；
Step 2: by 2 μ L of step 1 gained sample, being added in the gene detection reagent of 18 μ L premix packing, mix laggard
Row PCR amplification；
Step 3: setting instrument reaction process and response parameter, the instrument response parameter of setting are as follows:
1st stage: 95 DEG C, 10min, 1 circulation；
2nd stage: 95 DEG C, 10S, 40 circulations；
3rd stage: 60 DEG C, 30S, 40 circulations open fluorescence channel in the 3rd stage and collect fluorescent value；
Step 4: determining sample Genotyping, if testing result has the rise of S type amplification curve, and Ct. value is sentenced less than the positive
Definite value is then positive test symbol, if being greater than positive decision content without curve rise or Ct. value, interpretation is without the genotype.
In the present embodiment, positive test symbol decision content is as follows:
Internal reference Ct value :≤32；
Sense channel Ct. value :≤32；
Gene type: containing tested HLA-B5801 or HLA-B1502 gene.
In the present embodiment, rapidly and efficiently detection drug induccd skin adverse reaction related gene provided in this embodiment is polymorphic
Property kit and method, for promoted detection specificity and sensitivity, using according to target-gene sequence design specificity expand
Increase primer pair and MGB-NFQ fluorescence probe, carries out specific amplified and real-time detection analysis, make detection can be up to thousands of from HLA-B
Accurate, special in kind polymorphic allele, delicately detection target gene variation.
In the present embodiment, rapidly and efficiently detection drug induccd skin adverse reaction related gene provided in this embodiment is polymorphic
Property kit and method, detection allele include HLA-B*15:02, HLA-B*58:01, kit forms packet of the present invention
Include the sun that reagent, the single tube quickly handled for sample premixes the gene detection reagent completed, mixes tested target gene Plasmid DNA
Property reference substance, the negative controls without target gene.
In the present embodiment, rapidly and efficiently detection drug induccd skin adverse reaction related gene provided in this embodiment is polymorphic
The kit and method of property, gene detection reagent have the function of stronger anti-PCR mortifier ability and multiplex PCR, when detection without
The PCR reaction solution for needing gDNA extraction purification, single tube premix need to being only added after the processing of whole blood sample cell rapid cleavage, no
Same tested target gene is respectively adopted different primer pairs and carries out gene magnification, and marked using different fluorescent dye groups
Probe is measured in real time analysis, realizes detection of the reaction system simultaneously to polygenic variation.
In the present embodiment, rapidly and efficiently detection drug induccd skin adverse reaction related gene provided in this embodiment is polymorphic
Property kit and method, guarantee detection it is specific and it is sensitive basis on, realization it is convenient, fast and efficiently detect, subtract
Production cost and testing cost are reduced while detecting operating procedure less, reduce the detection reaction time, is conducive to clinical detection
Analysis application.
In the present embodiment, the present embodiment detects HLA-B*1502 and HLA-B*5801 gene polymorphic for the prior art
Property when the deficiencies of high, detection cycle is long, complicated for operation, at high cost and specific not strong is required to sample process, provide one kind
Detection kit and method based on RT-PCR technology platform.
In the present embodiment, the present embodiment is the specificity and sensitivity for promoting detection, is set using according to target-gene sequence
The specificity amplification primer of meter to and MGB-NFQ fluorescence probe, carry out specific amplified and real-time detection analysis, make detection can be from
Accurate in HLA-B up to thousands of kinds of polymorphic alleles, special, delicately detection target gene variation.
Primer and probe design is as follows:
1502 upstream primers:
5’-GCCGCGAGTCGAGGAT-3’(SEQ ID NO.1)
1502 downstream primers:
5’-GTTGTAGTACCGCGCAGGT-3’(SEQ ID NO.2)
1502 fluorescence probes:
5 '-fluorophor-ACACACAGATCTCC-MGB-NFQ-3 ' (SEQ ID NO.3)
5801 upstream primers:
5’-GACCCAGTTCGTGAGGTTCC-3’(SEQ ID NO.4)
5801 downstream primers:
5’-AGTTCCGTGTCTCCCCGTG-3’(SEQ ID NO.5)
5801 fluorescence probes:
5 '-fluorophor-CTCCGTCCTCTGACTC-MGB-NFQ-3 ' (SEQ ID NO.6)
Internal reference upstream primer:
5’-CCTTCCTGCCACCGCTGAT-3’(SEQ ID NO.7)
Internal reference downstream primer:
5’-TGCCTTCGCAACCATTCCCTTA-3’(SEQ ID NO.8)
Internal reference probe:
5 '-fluorophor-CGCTATGCTCCGCGCGCATCGTCTGCTC-BHQ-3 ' (SEQ ID NO.9)
In the present embodiment, the present embodiment for sample need to carry out the time-consumings such as gDNA extraction purification, effort, consuming behaviour
Make, the mating quick reagent treatment of sample significantly reduces operating procedure and operating time.Specifically processing method are as follows: take 2
μ LEDTA anticoagulated whole blood sample is added to 20 μ L sample processing reagents, and 5min, that is, completion processing is incubated under the conditions of 37 DEG C.
In the present embodiment, the present embodiment is directed to the change of sample process mode and the requirement of multiplex PCR reaction system,
Using the PCR premixed liquid-TaqPath ProAmp containing modified Hotstart-Taq polymerase and the buffer system of optimization
Master Mix greatly promotes detection architecture for the anti-rejection ability of sample, gene detection reagent is made to have high-efficiency multiple
PCR respond, premixed liquid ingredient containing stable reagent, the reagent be conducive under admixture save steadily in the long term, enhance examination
The stability of agent.
In the present embodiment, the present embodiment can only carry out substance PCR reaction for detection, each detection architecture every time, often
The secondary deficiency that can only detect a gene loci polymorphism, gene detection reagent is by multipair primer and a plurality of specificity fluorescent probe
A reaction system is blended together in advance with PCR mix, using TaqPath ProAmp Master Mix premixed liquid multiple reaction ability,
Different target genes high-efficiency multiple PCR under the combination of multipair specific primer is expanded, and utilizes the different fluorescent dye groups of label
MGB-NFQ and TaqMan-BHQ fluorescence probe analysis is measured in real time to target product.All detection primers and specificity
Probe is configured to a detection reagent, and premix single tube packaging facilitates detection, realizes the polygenic variation point of a reaction system
Analysis.Detection efficiency is improved while lowering production and testing cost.Detection reagent constituent is as shown in table 1:
1 detection reagent constituent of table
In the present embodiment, the present embodiment is provided with positive reference substance and negative controls for detection Quality Control demand.
Positive reference substance is the mixing nucleic acid solution for mixing HLA-B*1502, HLA-B*5801 gene and reference gene plasmid, concentration
0.01-1pg/ μ l, blank control are free from the ultrapure water of gene nucleic acid.Reference substance is regarded when detection as sample to be tested, and gene is added
Detection reagent, positive reference substance respectively detects signal and internal reference signal curve answers S-type rise, and corresponding Ct. value is sentenced less than the positive
Definite value.Blank control product should rise without curve, and corresponding Ct. value is greater than positive decision content, and otherwise detection failure, as a result can not
Letter.
In the present embodiment, the testing process of the present embodiment:
Step 1: sample is quickly handled, and takes EDTA anticoagulated whole blood sample, takes 2 μ L, 20 μ L sample processing reagents is added, 37
5min is incubated under the conditions of DEG C.
Step 2: it by 2 μ L of sample obtained by the first step, is added in the gene detection reagent of 18 μ L premix packing, mixes laggard
Row PCR amplification；
Step 3: setting instrument reaction process and parameter are used to detect the response parameter reacted are as follows:
1st stage: 95 DEG C, 10min, 1 circulation；
2nd stage: 95 DEG C, 10S, 40 circulations；
3rd stage: 60 DEG C, 30S, 40 circulations；
Fluorescence channel, which is opened, in the 3rd stage collects fluorescent value.
Step 4: situation is risen according to amplification curve and each fluorescence channel Ct. value determines sample Genotyping, detection sun
Property result should there is typical S type amplification curve to rise, and Ct. value is less than positive decision content.If no curve rises or Ct. value is greater than sun
Sex determination value, interpretation are without the genotype.
Instrument response parameter is set when detection, opens the corresponding phosphor collection channel of instrument.
Fluorescence channel is opened in the 3rd stage and collects fluorescent value, and instrument expands according to channel fluorescence signal value situation of change, production
Increase curve and obtains amplification Ct. value.
Positive value determines:
The Ct. positive decision content of each sense channel is 32, be greater than 32 for feminine gender, less than 30 prompt to sample contain by
Survey target gene.
By goldstandard sanger PCR sequencing PCR be accredited as cdna sample containing HLA-B*1502, cdna sample containing HLA-B*5801,
The sample of the gene containing HLA-B*1502/5801 and while the sample without HLA-B*1502/5801 gene simultaneously.Use this reagent
Box synchronizes Blind Test, finally takes off blind, compares with sequencing result, the result that as a result this kit and detection method measure is and PCR sequencing PCR
As a result it is consistent.
In conclusion in the present embodiment, it is provided in this embodiment rapidly and efficiently to detect drug induccd skin adverse reaction phase
The kit and method of correlation gene polymorphism detect HLA-B*1502 and HLA-B*5801 gene pleiomorphism for the prior art
When the deficiencies of high, detection cycle is long, complicated for operation, at high cost and specific not strong is required to sample process, detected to be promoted
Specificity and sensitivity, using the specificity amplification primer designed according to target-gene sequence to and MGB-NFQ fluorescence probe, into
Row specific amplified and real-time detection analysis, make detection can it is accurate from HLA-B up to thousands of kinds of polymorphic alleles, special,
Delicately detection target gene variation.
The above, further embodiment only of the present invention, but scope of protection of the present invention is not limited thereto, and it is any
Within the scope of the present disclosure, according to the technique and scheme of the present invention and its design adds those familiar with the art
With equivalent substitution or change, protection scope of the present invention is belonged to.
Claims (10)
1. a kind of kit for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism, which is characterized in that phase
Correlation gene polymorphism includes HLA-B*1502 and HLA-B*5801 gene pleiomorphism, and kit includes sample processing reagent, gene
Detection reagent, positive reference substance and negative controls.
2. a kind of reagent for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism as described in claim 1
Box, which is characterized in that gene detection reagent includes:
Detect HLA-B*1502 primer pair SEQ ID NO.1 and SEQ ID NO.2；
Detect HLA-B*1502 specific probe SEQ ID NO.3；
Detect HLA-B*5801 primer pair SEQ ID NO.4 and SEQ ID NO.5；
Detect HLA-B*5801 specific probe SEQ ID NO.6；
Probe nucleic acid sequence 5 ' uses MGB- using one of FAM, JOE, VIC, NED and ROX modification, probe nucleic acid sequence 3 '
NFQ base group modification.
3. a kind of reagent for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism as claimed in claim 2
Box, which is characterized in that detection HLA-B*1502 primer SEQ ID NO.1 is 1502 upstream primers: 5 '-
GCCGCGAGTCGAGGAT-3'；
Detection HLA-B*1502 primer SEQ ID NO.2 is 1502 downstream primers: 5 '-GTTGTAGTACCGCGCAGGT-3 '；
Detection HLA-B*1502 specific probe SEQ ID NO.3 is 1502 fluorescence probes: 5 '-fluorophors-
ACACACAGATCTCC-MGB-NFQ-3'；
Detection HLA-B*5801 primer SEQ ID NO.4 is 5801 upstream primers: 5 '-GACCCAGTTCGTGAGGTTCC-3 '；
Detection HLA-B*5801 primer SEQ ID NO.5 is 5801 downstream primers: 5 '-AGTTCCGTGTCTCCCCGTG-3 '；
Detection HLA-B*5801 specific probe SEQ ID NO.6 is 5801 fluorescence probes: 5 '-fluorophors-
CTCCGTCCTCTGACTC-MGB-NFQ-3’。
4. a kind of reagent for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism as described in claim 1
Box, which is characterized in that gene detection reagent further includes primer pair SEQ ID NO.7, SEQ ID NO.8 and the internal reference for internal reference
Probe SEQ ID NO.9, probe use TaqMan probe；
SEQ ID NO.7 is internal reference upstream primer:
5'-CCTTCCTGCCACCGCTGAT-3'；
SEQ ID NO.8 is internal reference downstream primer:
5'-TGCCTTCGCAACCATTCCCTTA-3'；
Internal reference probe SEQ ID NO.9:
5 '-fluorophor-CGCTATGCTCCGCGCGCATCGTCTGCTC-BHQ3 '.
5. a kind of reagent for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism as described in claim 1
Box, which is characterized in that gene detection reagent further includes the PCR premixed liquid-TaqPath ProAmp with anti-PCR enzyme inhibitor
Master Mix, premixed liquid include Hotstart-Taq polymerase after modifying, UNG enzyme, ROX fluorescent calibration dyestuff, dNTP,
MgCl2、PCR buffer。
6. a kind of reagent for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism as described in claim 1
Box, which is characterized in that for sample processing reagent for the processing of people's whole blood sample, gene detection reagent is pre- using the single part of PCR8 union
Mixed packing.
7. a kind of reagent for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism as described in claim 1
Box, which is characterized in that positive reference substance is mixing HLA-B*15:02, HLA-B*58:01 gene plasmid DNA and internal reference plasmid
DNA；Negative controls are the ultrapure water without target gene and reference gene.
8. a kind of method for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism, which is characterized in that including
Following steps:
Step 1: sample is quickly handled, and takes EDTA anticoagulated whole blood sample, takes 2 μ L, 20 μ L sample processing reagents is added, in 37 DEG C of items
5min is incubated under part；
Step 2: by 2 μ L of step 1 gained sample, being added in the gene detection reagent of 18 μ L premix packing, PCR is carried out after mixing
Amplification；
Step 3: setting instrument reaction process and response parameter；
Step 4: determine sample Genotyping, if testing result has the rise of S type amplification curve, and Ct. value is less than positive decision content,
It is then positive test symbol, if being greater than positive decision content without curve rise or Ct. value, interpretation is without the genotype.
9. a kind of side for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism as claimed in claim 8
Method, which is characterized in that in step 3, the instrument response parameter of setting are as follows:
1st stage: 95 DEG C, 10min, 1 circulation；
2nd stage: 95 DEG C, 10S, 40 circulations；
3rd stage: 60 DEG C, 30S, 40 circulations open fluorescence channel in the 3rd stage and collect fluorescent value.
10. a kind of side for rapidly and efficiently detecting drug induccd skin adverse reaction related gene polymorphism as claimed in claim 8
Method, which is characterized in that positive test symbol decision content is as follows:
Internal reference Ct value :≤32
Sense channel Ct. value :≤32
Gene type: containing tested HLA-B5801 or HLA-B1502 gene.
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| WO2020182189A1 (en) * | 2019-03-12 | 2020-09-17 | Chang Gung Memorial Hospital, Linkou | Methods for detecting or reducing the incidence of adverse drug reactions |
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