CN110106236A - Utilize the kit and its method of high-resolution melting curve method detection Levetiracetam medication related gene SCN1A polymorphism - Google Patents

Utilize the kit and its method of high-resolution melting curve method detection Levetiracetam medication related gene SCN1A polymorphism Download PDF

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Publication number
CN110106236A
CN110106236A CN201910371645.9A CN201910371645A CN110106236A CN 110106236 A CN110106236 A CN 110106236A CN 201910371645 A CN201910371645 A CN 201910371645A CN 110106236 A CN110106236 A CN 110106236A
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scn1a
levetiracetam
melting curve
detection
primer
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何小明
钱怡
孙子奎
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SHANGHAI PERSONAL BIOTECHNOLOGY CO Ltd
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

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Abstract

The invention discloses a kind of kits using high-resolution melting curve method detection Levetiracetam medication related gene SCN1A polymorphism, it is characterized in that, the nucleotide sequence of detection primer including SCN1A gene polynorphisms site rs2298771, the primer is as follows: SCN1A upstream primer: 5 '-CAA GAA AGA CAG TTG TAT GTC CAA TC-3 '；SCN1A downstream primer: 5 '-ACA CTG CTG CCA GTT CCT ATA CC-3 '；Other parts are the conventional reagent of HRM assay kit.The invention also discloses a kind of detection methods of kit using high-resolution melting curve method detection Levetiracetam medication related gene SCN1A polymorphism.Kit of the invention is suitable for being used for quickly detecting Levetiracetam personalized medicine gene, can be widely applied to the genetic test of clinically Levetiracetam personalized medicine solution formulation.

Description

Levetiracetam medication related gene is detected using high-resolution melting curve method The kit and its method of SCN1A polymorphism
Technical field
The invention belongs to genetic test fields, and in particular to a kind of to be used using high-resolution melting curve method detection Levetiracetam The kit and its detection method of medicine related gene SCN1A polymorphism.
Background technique
Levetiracetam (Levetiracetam, Lev, trade name: Keppra, Levetiracetam) is to ratify to use through U.S. FDA for 1999 In adult epilepsy partial seizures.Its oral tablet in 2005 and solution are approved for 4 years old and the partial hair of the above children The adjuvant treatment of work.Research finds that the prominent vesicle protein SV2A of intracerebral is antiepileptic LEV action site.That is LEV combines modification The function of SV2 albumen is to provide anticonvulsant action.
In extensive clinical test prove with LEV adjuvant treatment control Adult Refractory partial seizure in not only effectively but also It is resistant to well.SCN1A gene rs2298771 site mutation leads to LEV Different therapeutical effect.G allele carrier has broken out Full control rate is substantially less than AA type individual, and G allele is the risks and assumptions to affect the treatment.
It can be seen that carrying out parting to the site gene rs2298771 SCN1A detects predictable reaction of the patient to Levetiracetam Property, formulation therapeutic scheme is treated for the first time to epileptic for clinician, and foundation is provided.
Therefore, the site SCN1A gene rs2298771 carries out parting and detects predictable patient to the reactivity of Levetiracetam, is Clinician treats for the first time to epileptic formulates therapeutic scheme offer foundation.Develop quick, efficient, accurate, convenient, warp The kit of the detection SCN1A gene pleiomorphism of Ji will play positive promotion for the individualized clinical treatment of Levetiracetam and make With.
High-resolution melting curve (high-resolution melting, HRM) is that one kind is based on mononucleotide melting temperature not With and form the genetic analysis new technology of different shape melting curve, possess hypersensitivity, it is at low cost, flux is high, speed is fast, ties The features such as limitation in accurate, the not examined site of fruit, stopped pipe operates, meet Big Clinical Samples testing requirements.
Summary of the invention
In order to overcome the drawbacks described above of the prior art, song is melted using high-resolution one of the objects of the present invention is to provide a kind of The kit of collimation method detection Levetiracetam medication related gene SCN1A polymorphism, quick with realization, easy, accurate, efficient, Practical, economic detection Levetiracetam personalized medicine related gene SNP.
The second object of the present invention is that a kind of utilization high-resolution melting curve method detects Levetiracetam medication related gene The detection method of the kit of SCN1A polymorphism.
One of in order to achieve the object of the present invention, used technical solution is:
A kind of kit using high-resolution melting curve method detection Levetiracetam medication related gene SCN1A polymorphism, Detection primer including SCN1A gene polynorphisms site rs2298771, the following institute of the nucleotide sequence of the primer Show:
SCN1A upstream primer: 5 '-CAA GAA AGA CAG TTG TAT GTC CAA TC-3 '；
SCN1A downstream primer: 5 '-ACA CTG CTG CCA GTT CCT ATA CC-3 '；Other parts are HRM analytical reagent The conventional reagent of box.
In order to achieve the object of the present invention two, used technical solution is:
A kind of kit using high-resolution melting curve method detection Levetiracetam medication related gene SCN1A polymorphism Detection method includes the following steps:
DNA extraction step: step 1 saves stand-by to refrigerator after the DNA in sample is extracted
Step 2, high-resolution melting curve reaction step:
20 μ l PCR amplification systems are prepared, include: mix 10 μ l, mg2+1.2 μ l, 0.4 μ l of upstream primer, 0.4 μ l of downstream primer, 7.0 μ l of water, 1.0 μ l of template；
According to following loop parameter, amplification instrument is set:
95 DEG C of 10min, 4.4 DEG C/s initial denaturation；Then successively in 95 DEG C of 10S, 4.4 DEG C/s, 60 DEG C of 15S 2.2 DEG C/s, 72 DEG C of 20S 4.4 DEG C/s, carry out 40 circulations；Then successively in 95 DEG C of 1min, 4.4 DEG C/s, 40 DEG C of 1min 2.2 DEG C/s, 65 DEG C of 1S 4.4 DEG C/s, 95 DEG C of 0.02 DEG C/s continued downs acquire signals, are eventually held in 40 DEG C of holding 30S, obtain melting curve；
Step 3, high-resolution melting curve interpretation of result step: according to the solubility curve obtained in step 2 to SCN1A Rs2298771 single nucleotide polymorphisms carry out detection and interpretation, and the genotype in judgement sample belongs to AA or GG or AG genotype In any one.
The beneficial effects of the present invention are:
Kit of the invention is suitable for being used for quickly detecting Levetiracetam personalized medicine gene, can be widely applied to face The genetic test of Levetiracetam personalized medicine solution formulation on bed.Compared with prior art, it is melted using high-resolution Curve Technique can quickly and accurately carry out short dna sequence analysis, convenient for building normalizing operation process；With high-throughput, low The features such as cost；One step of HRM response procedures is completed, and is not required to carry out the secondary treatments such as product purification sequencing, operation is extremely easy, institute Need sample size small.
Detailed description of the invention
Fig. 1 is the site SCN1A rs2298771 of the present invention wild type, saltant type, heterozygous high-resolution melting curve result.
Specific embodiment
Mentioned reagent box and detection method are described in detail below with reference to embodiment.
The nucleotide sequence in the site SCN1A rs2298771 is as follows
caagaaagacagttgtatgtccaatcatacagcagaaattgggaaagatcttgactatcttaaagatgtaaa tggaactacaagtggtataggaactggcagcagtgt。
Embodiment 1:
Upstream primer: 5 '-CAA GAA AGA CAG TTG TAT GTC CAA TC-3 ' (SEQ ID NO.2)；
Downstream primer: 5 '-ACA CTG CTG CCA GTT CCT ATA CC-3 ' (SEQ ID NO.3)；
1.DNA is extracted
1.1, which test preceding reagent material, prepares and checks that work is as follows:
(1) it checks the kit shelf-life and ensures to have added ethyl alcohol, and the respective identification on bottle in Wash Buffer 1 and 2 Place ticks √；(2) isopropanol (such as nothing, can be substituted with dehydrated alcohol) and 75% ethyl alcohol；(3) in high pressure sterilization validity period 1.5mL Eppendorf pipe and all kinds of liquid transfer gun heads.
1.2 take out the EDTA anticoagulant tube that whole blood is housed from 4 DEG C of refrigerators, turn upside down and mix for several times；
1.3, which correspond to sample unique identification in 1.5mL Eppendorf pipe, marks；
1.4 pipette the 1.5mL Eppendorf pipe that 900 μ l Cell Lysis Solution add to sterilizing respectively；
1.5, which carefully pipette 300 μ l whole bloods, is transferred to the above-mentioned 1.5mL EP pipe added with Cell Lysis Solution；
1.6 cover Eppendorf pipe lid, are incubated at room temperature 10min；
1.7 13,000rpm room temperatures are centrifuged 20 seconds；
1.8 take out Eppendorf pipe, observe white precipitate；
1.9 open Eppendorf pipe lid, hold bottom of the tube, and inclination EP nozzle discards part red supernatant, as far as possible inhales red supernatant To the greatest extent；
1.10 cover Eppendorf pipe, with finger attack Eppendorf bottom of the tube, white precipitate are resuspended；
1.11, which pipette 300 μ l Nuclei Lysis Solution, enters in above-mentioned Eppendorf pipe, covers pipe, turn upside down number Secondary mixing；
1.12 open Eppendorf pipe, pipette 100 μ l Protein Precipitation Solution enter it is above-mentioned In Eppendorf pipe, pipe pipe is covered, is acutely vibrated on oscillator 20 seconds；13,000rpm room temperature is centrifuged 3min；
1.13, which pipette supernatant, is transferred to the new 1.5mL of sterilizing Eppendorf pipe；
1.14, which pipette 300 μ l isopropanols, enters Eppendorf pipe, and lid upper tube cap turns upside down and mixes for several times, it is seen that white flock GDNA is precipitated；
1.15 13,000rpm room temperatures are centrifuged 1min；
1.16 open Eppendorf pipe, and hand pinches bottom of the tube, and inclination nozzle discards supernatant；
1.17, which pipette 300 μ l, 75% ethyl alcohol, is added Eppendorf pipe, and lid upper tube cap, softly turn upside down washing precipitating；
1.18 13,000rpm room temperatures are centrifuged 1min；
1.19 open Eppendorf pipe, hold bottom of the tube, and inclination nozzle discards supernatant；
1.20 place new filter paper on experimental bench, and back-off Eppendorf pipe blots liquid, and Eppendorf pipe side of uncapping is leaked informaton It is dry；
1.21 range estimation precipitating sizes, are added 50~100 μ l DNA Rehydration Solution to precipitating；
Nucleic acid concentration measurement is carried out with NanoDrop ultraviolet specrophotometer after 1.22 dissolutions overnight, nucleic acid concentration is more than or equal to 20ng/ μ l and OD260/OD280 are 1.9 ± 0.2 to be considered as qualification, if concentration is inadequate, ethyl alcohol are added and precipitates DNA again, then weighs Newly plus suitable DNA Rehydration Solution dissolving DNA.
1.23 cover SD sample exclusive number again in tube wall and pipe, and are wound and protected with adhesive tape；
1.24 save nucleic acid sample to 4 DEG C of refrigerators；
2. high-resolution melting curve reacts
2.1 prepare 20 μ l PCR amplification systems (except template addition) in reagent area in preparation, each component and additive amount such as the following table 1:
Table 1
mix 10
F 0.4
R 0.4
mg2+ 1.2
ddH2O 7
2.2 in sample preparation area to adding 1.0 μ l into amplification system after filling the of short duration centrifugation of gDNA template, on PCR pipe wall Sample unique identification is marked, pipe covers label detection project code name.PCR pipe concussion mixes, of short duration centrifugation on tabletop centrifuge；
2.3 carry out HRM reaction in amplification region, amplification instrument are arranged according to following loop parameter, parameter is shown in Table 2:
Table 2
PCR pipe is being put into adapter after 2.4 setting programs, and is being installed in amplification instrument；
2.5, which click " start ", starts instrument operation.
3. pyrosequencing interpretation of result
The double click in " experiment " file opens above-mentioned operating file, selects " gene scaning ", clicks " Calculate " key carries out genotyping to all detection samples.To pattern detection SCN1A rs2298771 locus gene Polymorphism, high-resolution melting curve result such as Fig. 1.
Referring to Fig. 1, it can be seen that kit and method of the invention are used, it can be simple and direct, intuitive and accurate to SCN1A Rs2298771 single nucleotide polymorphisms carry out detection and interpretation.
It is respectively wild type, saltant type, heterozygous that Fig. 1, which prompts the site SCN1A rs2298771,.Clinician can be according to SCN1A Rs2298771 single nucleotide polymorphisms judge that curative effect when different genotype patient is treated using Levetiracetam is reacted.
Screen the comparative example of primer
Primer in the application is got and selecting, other comparison primer detections based on the same specific position It the results are shown in Table 3:
Table 3
Experimental comparison's example
The HRM detection of 50 samples is compared into experiment with generation sequencing.
50 sample HRM are detected 2.5 hours+interpretation of result 0.5 hour, are added up to 3 hours.And generation sequencing 8 hours+result of detection 1h is analyzed, is added up to 9 hours.And HRM is detected as stopped pipe operation, is not required to carry out the secondary treatments such as product purification sequencing, thus also not There are the risks of amplified production pollution.
The comparison of 50 sample HRM and generation sequencing assay result, such as the following table 4:
Table 4
Therefore, main innovation of the invention point is:
Target sequence selected by the present invention, and using kit of the invention can be realized it is quick, easy, accurate, efficient, real With, economic detection SCN1A rs2298771 single nucleotide polymorphisms, the requirement of clinical examination real work can satisfy, benefit It is used in the individuation of Levetiracetam.
Sequence table
<110>biotech inc Shanghai's style Sen Nuo on
<120>kit of high-resolution melting curve method detection Levetiracetam medication related gene SCN1A polymorphism is utilized And its method
<130> 20190321
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 108
<212> DNA
<213> Homo sapiens
<400> 1
caagaaagac agttgtatgt ccaatcatac agcagaaatt gggaaagatc ttgactatct 60
taaagatgta aatggaacta caagtggtat aggaactggc agcagtgt 108
<210> 2
<211> 26
<212> DNA
<213> Artificial sequence
<400> 2
caagaaagac agttgtatgt ccaatc 26
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence
<400> 3
acactgctgc cagttcctat acc 23
<210> 4
<211> 26
<212> DNA
<213> Artificial sequence
<400> 4
caagaaagac agttgtatgt ccaatc 26
<210> 5
<211> 26
<212> DNA
<213> Artificial sequence
<400> 5
cagttcctat accacttgta gttcca 26
<210> 6
<211> 26
<212> DNA
<213> Artificial sequence
<400> 6
aatttattca acagtccttc attagg 26
<210> 7
<211> 28
<212> DNA
<213> Artificial sequence
<400> 7
gttccattta catctttaag atagtcaa 28
<210> 8
<211> 26
<212> DNA
<213> Artificial sequence
<400> 8
gaatttattc aacagtcctt cattag 26
<210> 9
<211> 26
<212> DNA
<213> Artificial sequence
<400> 9
taccacttgt agttccattt acatct 26

Claims (2)

1. a kind of reagent using high-resolution melting curve method detection Levetiracetam medication related gene SCN1A polymorphism Box, which is characterized in that the detection primer including SCN1A gene polynorphisms site rs2298771, the nucleosides of the primer Acid sequence is as follows:
SCN1A upstream primer: 5 '-CAA GAA AGA CAG TTG TAT GTC CAA TC-3 '；
SCN1A downstream primer: 5 '-ACA CTG CTG CCA GTT CCT ATA CC-3 '；Other parts are HRM analytical reagent The conventional reagent of box.
2. a kind of as described in claim 1 detect Levetiracetam medication related gene using high-resolution melting curve method The detection method of the kit of SCN1A polymorphism, which comprises the steps of:
DNA extraction step: step 1 saves stand-by to refrigerator after the DNA in sample is extracted；
Step 2, high-resolution melting curve reaction step:
20 μ l PCR amplification systems are prepared, include: mix10 μ l, mg2+1.2 μ l, 0.4 μ l of upstream primer, 0.4 μ l of downstream primer, water 7.0 μ l, 1.0 μ l of template；
According to following loop parameter, amplification instrument is set:
95 DEG C 10min4.4 DEG C/s initial denaturation；Then successively in 95 DEG C 10S4.4 DEG C/s, 60 DEG C 15S2.2 DEG C/s, 72 DEG C of 20S4.4 DEG C/s, carry out 40 circulations；Then successively in 95 DEG C 1min4.4 DEG C/s, 40 DEG C 1min2.2 DEG C/s, 65 DEG C 1S4.4 DEG C/s, 95 DEG C 0.02 DEG C/s continued down acquires signal, is eventually held in 40 DEG C of holding 30S, obtains melting curve；
Step 3, high-resolution melting curve interpretation of result step: according to the solubility curve obtained in step 2 to SCN1A Rs2298771 single nucleotide polymorphisms carry out detection and interpretation, and the genotype in judgement sample belongs to AA or GG or AG genotype In any one.
CN201910371645.9A 2019-05-06 2019-05-06 Utilize the kit and its method of high-resolution melting curve method detection Levetiracetam medication related gene SCN1A polymorphism Pending CN110106236A (en)

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CN105316401A (en) * 2015-01-23 2016-02-10 复旦大学附属华山医院 Method for measuring ABCC2 gene polymorphism
CN106191299A (en) * 2016-09-20 2016-12-07 广州医科大学附属第二医院 For detecting primer, test kit and the method for SCN1A gene 5 ' end non-coding mutation
CN106701947A (en) * 2016-12-29 2017-05-24 苏州百源基因技术有限公司 Kit for detecting genotype of gene CYP2C19 at SNP site rs4986893
CN107460235A (en) * 2017-01-20 2017-12-12 上海科医联创生物科技有限公司 A kind of mankind's ApoE genetic polymorphism detection kits based on ARMS PCR melting curve methods
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CN105274190A (en) * 2014-07-22 2016-01-27 复旦大学附属华山医院 HRM method for detecting genetic polymorphism of CYP3A4*1G and MDR1C1236T
CN105316401A (en) * 2015-01-23 2016-02-10 复旦大学附属华山医院 Method for measuring ABCC2 gene polymorphism
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CN106701947A (en) * 2016-12-29 2017-05-24 苏州百源基因技术有限公司 Kit for detecting genotype of gene CYP2C19 at SNP site rs4986893
CN107460235A (en) * 2017-01-20 2017-12-12 上海科医联创生物科技有限公司 A kind of mankind's ApoE genetic polymorphism detection kits based on ARMS PCR melting curve methods
CN109504764A (en) * 2018-12-17 2019-03-22 精治基因技术(北京)有限公司 A kind of detection system and application for being used to detect epilepsy medication related gene SNP site based on flight mass spectrum platform
CN109576363A (en) * 2019-02-11 2019-04-05 中日友好医院 A kind of primer design method and kit of people ABCC2 genetic polymorphism detection

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Title
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