A kind of kit and method for predicting the SNP site of leflunomide curative effect of medicationTechnical field
The present invention relates to molecular biology and medical domain, and more specifically, the present invention relates to one group of leflunomide drugs to treatThe system and kit of effect and adverse reaction risk SNP site, by detecting leflunomide curative effect of medication and adverse reaction simultaneouslyMononucleotide polymorphism site (SNP) genotype of the closely related DHODH genes of risk and CYP1A2 genes carrys out fluorine to assessCurative effects and adverse reaction risk of the meter Te in rheumatoid arthritis and other diseases use.
Background technology
Leflunomide is a kind of slow effect antirheumatic object (DMARD) being directed to rheumatoid arthrosis (RA).Previous facesBed experiment confirm leflunomide treatment patient in have 30-50% reach disease activity decline 20% (ACR20), in addition with firstCurative effect is also very notable when aminopterin is combined.But still have quite a few patient reactionless to its, up to 40% patient in additionIt must discontinue medication because of toxicity；There is gastrointestinal toxicity (diarrhea, nausea and vomiting) in the patient of about 20-30%, this is oftenCome across medication early stage and may spontaneous remission after long-term use.Patient invalid traditional DMARDs would generally be consideredBiological DMARDs, but biology DMARDs may increase the risk of certain types of cancer and infection and expensive.So using compared withIt is still a kind of preferable selection for economy and drug (such as leflunomide) that recurrence rate can be reduced.Therefore, prediction patient is to comingHow quite important fluorine rice spy reaction is, but the effect of usual clinical judgment leflunomide needs 3 months, and and drug responseRelevant hereditary feature contributes to early stage to be prejudged before the treatment starts, to help clinician to determine therapeutic choice rapidly.
The mechanism of action of leflunomide relates generally to the inhibition to enzyme DHODH, and DHODH extremely closes the de novo formation of pyrimidineIt is important.By inhibiting the synthesis of pyrimidine, the proliferation of T cell is suppressed, and the gene of encoding D HODH is located at No. 16 chromosomeOn long-armed, length about 16kbp.Pawlik et al. analyzes the non-synonymous SNP in first exon of DHODH genes(rs3213422), as a result, it has been found that C allele is related to the enhancing of leflunomide therapeutic response, at the same C allele also with treatmentRisk of toxicity decline it is related.
Leflunomide is a kind of pro-drug, in vivo rapidly by conversion teriflunomide (active metabolite).Cell colorPlain P-450 enzymes CYP1A2 is responsible for leflunomide being metabolized as teriflunomide, research shows that the gene pleiomorphism of CYP1A2 may be withThe toxicity of leflunomide increases and the variation of plasma concentration is related.There is research to confirm that the A allele toxicity of CYP1A2C734A is anti-The risk answered increases by 9.7 times (P=0.002, OR=9.708,95%CI=2.276,41.403).
Invention content
It is mentioned above in order to solve the problems, such as, the present invention provides one kind can predict patient to leflunomide reaction howSystem, kit and method.
A kind of system for predicting the SNP site of leflunomide curative effect of medication and risk, the kit include：
Following distinguished sequence primer：
Following Sequence-specific probe：
Following sequence reporter probe：
A kind of kit for predicting the genotype of leflunomide curative effect of medication and risk, the kit includeAbove-mentioned distinguished sequence primer, probe and reporter probe.
A method of the SNP site for predicting leflunomide curative effect of medication and risk, the method include as followsStep：PCR reactions are carried out first, realize amplification；Multiple OLA reactions, label and connection are carried out again；Then hybridization reaction is carried out,Finally carry out the analysis of genotype.
Preferably, the PCR reaction systems are as follows：2x qiagen Hotstar MM 5ul, primer mix1ul,DNA sample 2ul, aqua sterilisa 2ul；PCR reaction conditions be 95 DEG C, 15min, carry out 30 cycle 94 DEG C, 30 seconds, 60 DEG C, 30Second, 72 DEG C, 30 seconds；72 DEG C, 7 minutes, 4 DEG C of maintenances.
Preferably, OLA reactions are as follows：2xOLA master mix are prepared, OLA master mix and PCR is anti-It answers product to mix, reaction is attached after mixing.
Preferably, preparing 2xOLA master mix includes：10x Taq Ligase buffer 2ul,40000U/mlTaq DNA Ligase 0.25ul, wild-type probe mix 1ul, saltant type probe mix 2ul, deionized water 4.75ul.
Preferably, connection reaction condition is as follows：96 DEG C of 2min, the 94 DEG C of 15s, 37 DEG C of 1min of 30 cycles；4 DEG C of maintenances.
Preferably, the washing procedure process of hybridization reaction is as follows：The magnetic bead of corresponding reporter probe is selected, is resuspended, thenEach magnetic bead is mixed, dilution is up to 100u/ul and magnetic is added after mixing with 2X Tm hybridization bufferIn pearl mixture to every hole, 1-5ul OLAreaction and 25ul dH are added2O carries out PCR reactions to each hole：96℃90s, 37 DEG C of 30min, siphons away supernatant, magnetic bead is resuspended with 1x Tm hybridization buffer, magnetically attractive 30-60s inhales againSupernatant is walked, repeats that magnetic bead is resuspended with 1x Tm hybridization buffer, magnetically attractive 30-60s siphons away supernatant for the third time, usesMagnetic bead is resuspended in 1x Tm hybridization buffer, and 15min is incubated at 37 DEG C, and 50ul reaction products are added to LUMINEXMiddle analysis.
Preferably, the not washing process of hybridization reaction is as follows：1, magnetic bead is selected, and is resuspended；2, each magnetic bead is mixed, andIt is diluted to 100u/ul, with 2X Tm hybridization buffer, oscillation mixing；3, magnetic bead mix is added to every holeIn；4, it adds in sample to every hole；5, PCR reactions are carried out：96 DEG C of 90s, 37 DEG C of 30min；6, prepare 6ug/ml SAPE in 1xhybridization buffer；7,100ul SAPEmix, mixing are added；8,37 DEG C of incubation 15min；9,100ul to 37 is addedDEG C luminex in analyze.
The beneficial effects of the present invention are：The primer, probe and reporter probe for providing distinguished sequence, innovatively to oneGroup leflunomide curative effect of medication and the gene loci of adverse reaction risk are detected, and predict rheumatoid arthritis and other patientsCurative effect and the risk of adverse reaction after leflunomide are taken, the accurate medication of patient is conducive to.
Specific implementation mode
The present invention provides a kind of kit, and kit is directed in human genome DNA leflunomide curative effect of medication and badReact the gene loci of risk, i.e. DHODH (19C>A) i.e. rs3213422 and CYP1A2 (734C>A) i.e. rs762551.DesignSpecific primer and wild type/saltant type probe, the MagPlex-TAG magnetic beads of reporter probe corresponding to coupling hybridize anti-It answers, is detected on 200 instruments of luminex by chromogenic reaction.Pass through each SNP site of reading interpretation to signal valueBase type judged.
Specific primer sequences involved in kit are as follows：
The specific probe sequence being related to is as follows：
The reporter probe sequence being related to is as follows：
Its detection process is as follows：
(1) 2 SNP site carries out PCR reactions in same pipe, and the system of reaction is 10 μ l of total volume, including 2x qiagenHotstar MM 5ul, primer mix 1ul, DNA sample 2ul, aqua sterilisa 2ul.
It is reacted in ABI9700 type PCR amplification instruments, reaction condition is 95 DEG C, 15min, carries out the 94 of 30 cyclesDEG C, 30 seconds, 60 DEG C, 30 seconds, 72 DEG C, 30 seconds；72 DEG C, 7 minutes, 4 DEG C of maintenances.
(2) multiple OLA reactions：Prepare 2xOLA master mix：10x Taq Ligase buffer 2ul,Taq DNALigase (40,000U/ml) 0.25ul, wild-type probe mix (100nM each) 1ul, saltant type probe mix (2.5uMEach) 2ul, deionized water 4.75ul.OLA master mix are mixed with reaction product：2xOLA master mix 10ul,The PCR product 5ul of amplification, sterile deionized water 5ul.Piping and druming mixing up and down covers reaction tube, expand in ABI9700 types PCRIncrease and connects reaction on instrument.96 DEG C of 2min, the 94 DEG C of 15s, 37 DEG C of 1min of 30 cycles；4 DEG C of maintenances.
(3) hybridization reaction：The MagPlex-TAG magnetic beads of corresponding reporter probe are selected, and are resuspended.Each magnetic bead is mixed,And it is diluted to 100u/ul, with 2X Tm hybridization buffer, oscillation mixing 20s.25ul magnetic bead mix is addedInto every hole.(2500 pearls/each reaction should be provided).1-5ul OLA reaction and 25ul dH2O are added to everyA hole.The volume for adjusting H2O, makes total volume close to 50ul.It closes the lid, carries out PCR reactions：96℃90s,37℃30min.It putsThe 30s-60s in magnetic board, makes magnetic bead be sucked.Supernatant carefully is siphoned away, does not siphon away magnetic bead.With 1x Tm hybridizationMagPlex-TAG magnetic beads, magnetically attractive 30-60s is resuspended in buffer 75ul.Supernatant carefully is siphoned away, does not siphon away magnetic bead.Repetition 1xMagPlex-TAG magnetic beads, magnetically attractive 30-60s is resuspended in Tm hybridization buffer 75ul.Carefully siphon away supernatant.With75ul 1x Tm hybridization buffer (including 2-8ug/ml SAPE), are resuspended magnetic bead, 15min are incubated at 37 DEG C.In 37 DEG C, it is added in 50ul reaction products to LUMINEX and analyzes.
(4) interpretation of result
It is compareed by plasmid and water, obtains background signal, when detecting sample results, after subtracting background, numerical value is more than 200For positive reaction.
2 SNP types can be provided in examining report as a result, reference gene is wild type gene.Detect DHODH (19C>A)The corresponding result of gene rs3213422 saltant types is shown as AA, and the corresponding result of heterozygous is shown as CA, the corresponding knot of wild typeFruit is shown as CC.CYP1A2(734C>A) the corresponding result of gene rs762551 saltant types is shown as AA, the corresponding knot of heterozygousFruit is shown as AC, and the corresponding result of wild type is shown as CC.
Sequence table
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