CN108823301A - It is a kind of for detecting the multiple PCR detection kit of people's drug gene polymorphism - Google Patents

It is a kind of for detecting the multiple PCR detection kit of people's drug gene polymorphism Download PDF

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CN108823301A
CN108823301A CN201810660244.0A CN201810660244A CN108823301A CN 108823301 A CN108823301 A CN 108823301A CN 201810660244 A CN201810660244 A CN 201810660244A CN 108823301 A CN108823301 A CN 108823301A
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dna
homo sapiens
detection kit
pcr amplification
pcr detection
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陈琰
康灿昆
孔礼祥
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Xiamen Spacegen Biotechnology Co Ltd
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Xiamen Spacegen Biotechnology Co Ltd
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

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Abstract

The invention discloses a kind of for detecting the multiple PCR detection kit of people's drug gene polymorphism, the 66 PCR amplification primer pairs reacted in same reaction system including being designed according to VKORC1, UGT1A1, HLA-A, HLA-B, TPMT, NAT1, NAT2, G6PD, F5, F2, IFNL3, APOE, COMT, CYP1A2, CYP2B6, CYP2C19, CYP2C9, CYP2D6, CYP3A5, MTFHR, SLCO1B1, DPYD gene polynorphisms site and the 66 Single base extension primers reacted in same reaction system.Multiple PCR detection kit of the invention can quickly, specifically, efficiently detect the polymorphism of above-mentioned 85 SNP sites of 22 genes, so as to predict cardiovascular and cerebrovascular disease standard scheme curative effect, therapeutic scheme is targetedly formulated convenient for clinician, the other patient of different shaped is taken and treats with a certain discrimination and treats, to improve the treatment level of entire cardiovascular and cerebrovascular disease.

Description

It is a kind of for detecting the multiple PCR detection kit of people's drug gene polymorphism
Technical field
The invention belongs to biomedical field of molecular detection, and in particular to one kind is polymorphic for detecting people's drug gene The multiple PCR detection kit of property.
Background technique
Drug metabolism in vivo, transhipment and the hereditary variation of drug target point gene and its variation of expression can pass through The bulk concentration and sensibility for influencing drug, lead to drug responsiveness individual difference.Recently as the hair of human activities environment Exhibition, pharmacogenomics field are grown rapidly, more and more Drug Discovery biomarkers and its detection method It emerges in large numbers in succession.Pharmacogenomics, which have become, to be instructed clinical individual medication, assessment severe drug adverse reaction occurrence risk, refers to It leads new drug development and evaluates the important tool of new drug, the new drug of part listing is only limitted to the eligible patients of specific genotype.Beauty State's FDa approved increases drug gene group information in the medicine label of more than 140 drugs, the Drug Discovery biology mark being related to Note object 42.In addition, part industry guide is also by the biomarker and its characteristic (such as MgMt gene first of the non-FDa approval in part Base) detection be included in the treatment guidelines of disease.The Molecular Detection of drug response related gene and its expression product is implementation The premise of body drug therapy.
Key link of the pharmacology in conjunction with science of heredity include pharmacokinetics (pharmacokinetics, PK) and Two aspect of pharmacodynamics (pharmacodynamics, PD).Pharmacokinetics is mainly that quantitative study drug exists Biological body absorption, distribution, metabolism and total quantity control lay particular emphasis on the physiological disposition for illustrating drug；Pharmacodynamics are main Drug is studied to the effect of body, action rule and mechanism of action, content includes interacting between drug and target site Caused biochemistry, physiology and morphological change lays particular emphasis on and explains how drug has an effect with action target spot.To drug generation It thanks to enzyme and drug target gene carries out detection and clinic can be instructed to select suitable drug and dosage for specific patient, it is real Existing personalized medicine prevents the generation of severe drug adverse reaction to improve the validity and safety of drug therapy.
Instructing personalized medicine according to Drug Discovery biological marker analyte detection mainly includes two types：First is that according to a The hereditary information adjustment dosage of body reduces the generation of adverse drug reaction to increase curative effect of medication；Second is that according to individual Hereditary information determines the type of medication, avoids using invalid for specific genotype individual or that there may be severe drugs is bad anti- The drug answered.The adjustment of drug dose often needs the result according to Randomized controlled clinical study；The current random controls that lack are faced The hereditary variation of bed research, the magnitude estimation medicament that drug pharmacokinetics area under the curve can be influenced according to genotype Amount；It, can be according to domestic state when interaction is influenced the reactivity of a drug between by multiple genes or gene and environmental factor It is that border clinical trial is derived, incorporate idiotype and the dosage calculation formula of other factors determines medication Dosage.Common drugs metabolic enzyme and drug target gene genetic variation testing result are to the guiding opinion of clinical application.
Summary of the invention
The purpose of the present invention is to provide a kind of for detecting the multiple PCR detection kit of people's drug gene polymorphism.
Testing principle of the invention is as follows：
Multiple PCR detection kit of the invention separately designs the PCR amplification specific primer that synthesis is directed to 66 kinds of sites To and Single base extension primer, then carry out PCR amplification, SAP reaction and iPLEX single base extension.Sample single base is prolonged Product is stretched by resin desalination, then passes through MaLDI-tOF (substance assistant laser desorpted ionized-flight time) mass spectrograph point Analysis, detects the molecular weight difference between different extension products, determines the base type at this 85 SNP sites, so that base will be carried out Because of parting, genotype can be directly judged, to use different therapeutic modalities according to the patient of different genes parting.
Technical scheme is as follows：
It is a kind of for detecting the multiple PCR detection kit of people's drug gene polymorphism, including according to VKORC1, UGT1A1、HLA-A、HLA-B、TPMT、NAT1、NAT2、G6PD、F5、F2、IFNL3、APOE、COMT、CYP1A2、CYP2B6、 The design of CYP2C19, CYP2C9, CYP2D6, CYP3A5, MTFHR, SLCO1B1, DPYD gene polynorphisms site same anti- The 66 PCR amplification primer pairs reacted in system and 66 Single base extensions reacted in same reaction system are answered to draw Object, wherein
Above-mentioned 66 PCR amplification primer pair includes the first to the 66th amplimer pair, and first to the 66th expands Increase the forward primer of primer pair successively as shown in SEQ ID NO 01~066, reverse primer successively such as SEQ ID NO 067~ Shown in 132；Above-mentioned 66 Single base extension primer includes the first to the 66th Single base extension primer, successively such as SEQ ID Shown in NO 133~198；
Its application method includes the following steps：
(1) using genomic DNA to be detected as template, contained using the 66 PCR amplification primer pair one Pcr amplification reaction is carried out in the PCR reaction system of RingCap-Taq enzyme and RingCap buffer, obtains pcr amplification reaction liquid；
(2) above-mentioned pcr amplification reaction liquid is carried out SAP with SAP enzyme to react, obtains SAP reaction solution；
(3) single base extension is carried out to above-mentioned SAP reaction solution with above-mentioned 66 Single base extension primer pair；
(4) by step (3) resulting material after resin desalination, in SpectrocHIP chip loading, mass spectrum inspection is carried out It surveys,.
In a preferred embodiment of the invention, the condition of the pcr amplification reaction includes：95 DEG C of 3min, 1 is followed Ring；95 DEG C of 20s, 60 DEG C of 30s, 72 DEG C of 20s, 45 circulations；72 DEG C of 3min, 1 circulation.
It is further preferred that the condition of the SAP reaction includes：37 DEG C of 40min, 85 DEG C of 5min, 4 DEG C of ∞.
Still more preferably, the condition of the single base extension includes：
First stage recycles for 40 totally：Each circulation includes：
94 DEG C of 30s, 1 circulation, 94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, 5 circulations；
Second stage recycles for 1 totally：72℃ 3min.
In a preferred embodiment of the invention, the RingCap-Taq enzyme is by Taq enzyme, DNA ligase and DNA End modified enzyme composition, ratio 0.8~1.2: 0.8~1.2: 0.8~1.2.
It is further preferred that the RingCap-Taq enzyme is made of Taq enzyme, DNA ligase and the end modified enzyme of DNA, than Example 1: 1: 1.
The beneficial effects of the invention are as follows：
1, multiple PCR detection kit of the invention can quickly, specifically, efficiently detect above-mentioned 85 SNP of 22 genes The polymorphism in site, so as to predict cardiovascular and cerebrovascular disease standard scheme curative effect, targetedly convenient for clinician Therapeutic scheme is formulated, the other patient of different shaped is taken and treats with a certain discrimination and treats, to improve examining for entire cardiovascular and cerebrovascular disease It treats horizontal；
2, PCR amplification primer, the Single base extension provided by the present invention for detecting people's 22 gene, 85 polymorphisms draws Object, detection kit and its detection method have very high clinical value, are particularly suitable for clinical expansion.
Detailed description of the invention
Fig. 1 is the site RS9923231 mass spectral results figure in 22 genes of the detection of the embodiment of the present invention 2.
Fig. 2 is the site RS776746 mass spectral results figure in 22 genes of the detection of the embodiment of the present invention 2.
Specific embodiment
Technical solution of the present invention is further explained and described below by way of specific embodiment combination attached drawing.
Embodiment 1
It is a kind of for detecting the multiple PCR detection kit of people's drug gene polymorphism, including according to VKORC1, UGT1A1、HLA-A、HLA-B、TPMT、NAT1、NAT2、G6PD、F5、F2、IFNL3、APOE、COMT、CYP1A2、CYP2B6、 The design of CYP2C19, CYP2C9, CYP2D6, CYP3A5, MTFHR, SLCO1B1, DPYD gene polynorphisms site same anti- The 66 PCR amplification primer pairs reacted in system and 66 Single base extensions reacted in same reaction system are answered to draw Object；
Above-mentioned 66 PCR amplification primer pair successively includes the first to the 66th amplimer pair, and first to the 60th The forward primer of six amplimers pair is successively as shown in SEQ ID NO 01~066, and reverse primer is successively such as SEQ D NO 067 Shown in~132, shown in table specific as follows：
Above-mentioned 66 Single base extension primer successively includes the first to the 66th Single base extension primer, successively such as Shown in SEQ ID NO 133~198, shown in table specific as follows：
Its application method includes the following steps：
(1) using genomic DNA to be detected as template, contained using the 66 PCR amplification primer pair one Pcr amplification reaction is carried out in the PCR reaction system of RingCap-Taq enzyme and RingCap buffer, obtains pcr amplification reaction liquid； The condition of the pcr amplification reaction includes：95 DEG C of 3min, 1 circulation；95 DEG C of 20s, 60 DEG C of 30s, 72 DEG C of 20s, 45 are followed Ring；72 DEG C of 3min, 1 circulation；The RingCap-Taq enzyme is made of Taq enzyme, DNA ligase and the end modified enzyme of DNA, than Example 0.8~1.2: 0.8~1.2: 0.8~1.2, preferred proportion 1: 1: 1；
(2) above-mentioned pcr amplification reaction liquid is carried out SAP with SAP enzyme to react, obtains SAP reaction solution；The item of the SAP reaction Part includes：37 DEG C of 40min, 85 DEG C of 5min, 4 DEG C of ∞；
(3) single base extension is carried out to above-mentioned SAP reaction solution with above-mentioned 66 Single base extension primer pair；It is described The condition of single base extension includes：
First stage recycles for 40 totally：Each circulation includes：
94 DEG C of 30s, 1 circulation, 94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, 5 circulations；
Second stage recycles for 1 totally：72℃ 3min
(4) by step (3) resulting material after resin desalination, in SpectrocHIP chip loading, mass spectrum inspection is carried out It surveys,.
Specifically, the main agents in kit according to the present invention are as shown in the table：
The detection example of the multiple PCR detection kit of 2 embodiment 1 of embodiment
Detect sample：EDta anticoagulant blood sample comes from Fujian Cancer Hospital's Physical Examination Dept., acquires from routine physical examination Health adult.
Key instrument equipment：
1, DR MassaRRaY mass spectrum gene alaysis system (Guangzhou Da Rui Biotechnology Ltd.)；
2, aB-7500 fluorescence quantitative PCR instrument (Applied biosystems (aBI))；
3, centrifuge (Epponderf)；
4, rotary oscillator (Shanghai).
Detect program：
1, blood sample Genomic DNA Purification：It is extracted using the poba gene group extracts kit of Tiangeng biochemistry.
2, PCR amplification：Using blood sample DNA after purification as template, to be directed to 85 sites in the provided kit of embodiment 1 The PCR amplification primer pair of polymorphism carries out PCR amplification, and reaction system is as follows,
；
It is arranged one group using water as the negative control of sample.
Reaction condition is as follows：
。
3, SAP reacts：It takes above-mentioned 5 μ l pcr amplification reaction liquid that 2 μ l SAP reaction solutions are added respectively and carries out SAP reaction, In, it is as follows that SAP reacts formula of liquid：
；
Reaction condition is as follows：
4, single base extension：It will be separately added into the extension liquid of 2 μ l in above-mentioned SAP digestive juice, carry out single base Extension, wherein extension formula of liquid：
；Wherein, Single base extension primer mix is provided polymorphic for detecting 85 sites in kit by embodiment 1 The mixture of the Single base extension primer of property.
Reaction condition is as follows：
5, resin desalination：41ul water is added in the extension fluid apertures that each has sample to be then centrifuged for.15mg is added Clean resin (Resin)：It gently by sample plane reversion high up in the air, is placed on the dimple plate for having put resin, then will Dimple plate inverts (two allegros are not horizontally moveable in the process) together with sample plane, and resin is allowed to drop in hole.With film handle Plate is sealed, and is placed on oscillation on rotator and is shaken up 15min.Plate is centrifuged 5min with 3200g (4000rpm of on-gauge plate centrifuge).
6, SpectrocHIP chip loading：Use MassaRRaYTM1000 auto sample applicator of RS carries out volume test (Volume check) is to find out suitable point sample speed (dispensing speed).The suitable volumes of point sample be 8-12nL ± 25%.Volume check is carried out with the sample on plate, then uses MassaRRaYTMAfter 1000 auto sample applicator of RS will react Sample point to 96 point SpectrocHIP (chip) on.
7, Mass Spectrometer Method.
Testing result is as depicted in figs. 1 and 2, by the sequencing knot of itself and traditional gene sequencing method two sites detected Fruit is compared, it was demonstrated that the testing result of two kinds of detection methods fits like a glove, this demonstrate that the reliability of the application detection method.
The foregoing is only a preferred embodiment of the present invention, the range that the present invention that therefore, it cannot be limited according to is implemented, i.e., Equivalent changes and modifications made in accordance with the scope of the invention and the contents of the specification should still be within the scope of the present invention.
Sequence table
<110>Xiamen Fei Shuo Bioisystech Co., Ltd
<120>It is a kind of for detecting the multiple PCR detection kit of people's drug gene polymorphism
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<213> Homo sapiens
<400> 57
agtcctctca agtctaatag caactg 26
<210> 58
<211> 25
<212> DNA
<213> Homo sapiens
<400> 58
catttttgct aaggcttcac ctctt 25
<210> 59
<211> 24
<212> DNA
<213> Homo sapiens
<400> 59
atcactcact ttgtgaccat tccg 24
<210> 60
<211> 24
<212> DNA
<213> Homo sapiens
<400> 60
ttcacaaagc ggaagaatgt gtca 24
<210> 61
<211> 28
<212> DNA
<213> Homo sapiens
<400> 61
ataggttgtt taaaggaatc tgggtcat 28
<210> 62
<211> 23
<212> DNA
<213> Homo sapiens
<400> 62
ttcacagata agggtacgcc tct 23
<210> 63
<211> 28
<212> DNA
<213> Homo sapiens
<400> 63
gggataaaac agtttctctt aagtggtg 28
<210> 64
<211> 25
<212> DNA
<213> Homo sapiens
<400> 64
catctttctg cttctgcctg atgta 25
<210> 65
<211> 26
<212> DNA
<213> Homo sapiens
<400> 65
aaaatcttgc cttacaatgt gtggag 26
<210> 66
<211> 28
<212> DNA
<213> Homo sapiens
<400> 66
cttccatctt acatggcacc catttata 28
<210> 67
<211> 22
<212> DNA
<213> Homo sapiens
<400> 67
aacagtaagg gatccctctg gg 22
<210> 68
<211> 25
<212> DNA
<213> Homo sapiens
<400> 68
gctgcaggaa agaatcattc tcaaa 25
<210> 69
<211> 19
<212> DNA
<213> Homo sapiens
<400> 69
acaggtcagc gtgggaaga 19
<210> 70
<211> 27
<212> DNA
<213> Homo sapiens
<400> 70
aataagtcag ctattgcagt agcctag 27
<210> 71
<211> 26
<212> DNA
<213> Homo sapiens
<400> 71
gagtgcccat tgaactacac attaca 26
<210> 72
<211> 28
<212> DNA
<213> Homo sapiens
<400> 72
atgttctttg aaaccctatg aacctgaa 28
<210> 73
<211> 31
<212> DNA
<213> Homo sapiens
<400> 73
gttgatgctt ttgaagaacg acataaaagt t 31
<210> 74
<211> 28
<212> DNA
<213> Homo sapiens
<400> 74
tctttctggt aggacaaata ttggcaaa 28
<210> 75
<211> 30
<212> DNA
<213> Homo sapiens
<400> 75
cacccagcca attttgagta tttttaaaag 30
<210> 76
<211> 27
<212> DNA
<213> Homo sapiens
<400> 76
tcccaggtag gttgaatact acatctg 27
<210> 77
<211> 29
<212> DNA
<213> Homo sapiens
<400> 77
tcaaaatctt caattgttcg aggcttaag 29
<210> 78
<211> 26
<212> DNA
<213> Homo sapiens
<400> 78
gtgaatcatg ccagtgctgt attttt 26
<210> 79
<211> 30
<212> DNA
<213> Homo sapiens
<400> 79
ggtaagggta cagaagatac atgataggtc 30
<210> 80
<211> 24
<212> DNA
<213> Homo sapiens
<400> 80
ttctgtcaag cagaaaatgc aagg 24
<210> 81
<211> 24
<212> DNA
<213> Homo sapiens
<400> 81
atgaagccca ccaaacagta aacc 24
<210> 82
<211> 30
<212> DNA
<213> Homo sapiens
<400> 82
attttcttca aaataacgtg agggtagaga 30
<210> 83
<211> 22
<212> DNA
<213> Homo sapiens
<400> 83
tggtctgaaa accgattgtg gt 22
<210> 84
<211> 22
<212> DNA
<213> Homo sapiens
<400> 84
gtgacctggc caagaagaag at 22
<210> 85
<211> 23
<212> DNA
<213> Homo sapiens
<400> 85
gctcaagctg gaggacttct ttg 23
<210> 86
<211> 23
<212> DNA
<213> Homo sapiens
<400> 86
tgcagatttg ccaacaggat ctt 23
<210> 87
<211> 22
<212> DNA
<213> Homo sapiens
<400> 87
ctcaacaccc aaggagccca tt 22
<210> 88
<211> 24
<212> DNA
<213> Homo sapiens
<400> 88
cgtctgaatg atgcagctct gatc 24
<210> 89
<211> 28
<212> DNA
<213> Homo sapiens
<400> 89
tctgggctaa taggactact tctaatct 28
<210> 90
<211> 23
<212> DNA
<213> Homo sapiens
<400> 90
caccaggtgg tggattctta agt 23
<210> 91
<211> 21
<212> DNA
<213> Homo sapiens
<400> 91
caggctcagg gtcaatcaca g 21
<210> 92
<211> 17
<212> DNA
<213> Homo sapiens
<400> 92
cgcggatggc gctgagg 17
<210> 93
<211> 17
<212> DNA
<213> Homo sapiens
<400> 93
cacctcgccg cggtact 17
<210> 94
<211> 25
<212> DNA
<213> Homo sapiens
<400> 94
ggtgatagtg ggttttcagt gaacg 25
<210> 95
<211> 30
<212> DNA
<213> Homo sapiens
<400> 95
aaattggatg tgaaaaggaa gtttgctatc 30
<210> 96
<211> 23
<212> DNA
<213> Homo sapiens
<400> 96
cgtgatggtg cacacctgta att 23
<210> 97
<211> 18
<212> DNA
<213> Homo sapiens
<400> 97
gggaagccct ccccatcg 18
<210> 98
<211> 27
<212> DNA
<213> Homo sapiens
<400> 98
ctggatacca gaaagactaa gctccat 27
<210> 99
<211> 28
<212> DNA
<213> Homo sapiens
<400> 99
aatctcatag atgactgcct ctgtgtat 28
<210> 100
<211> 23
<212> DNA
<213> Homo sapiens
<400> 100
ccaactgggc cttttatcct gac 23
<210> 101
<211> 19
<212> DNA
<213> Homo sapiens
<400> 101
ggtctctcag aggcaggaa 19
<210> 102
<211> 30
<212> DNA
<213> Homo sapiens
<400> 102
tccaaaatat cactttccat aaaagcaagg 30
<210> 103
<211> 25
<212> DNA
<213> Homo sapiens
<400> 103
atcgtggcgc attatctctt acatc 25
<210> 104
<211> 28
<212> DNA
<213> Homo sapiens
<400> 104
gtattcaaaa atgtacttca gggcttgg 28
<210> 105
<211> 23
<212> DNA
<213> Homo sapiens
<400> 105
atttccaatc actgggagag gag 23
<210> 106
<211> 23
<212> DNA
<213> Homo sapiens
<400> 106
gtcaaggtcc tttgggtcaa tca 23
<210> 107
<211> 21
<212> DNA
<213> Homo sapiens
<400> 107
acccaccctt ggtttttctc a 21
<210> 108
<211> 28
<212> DNA
<213> Homo sapiens
<400> 108
cattactcct tgacctgtta aacatccg 28
<210> 109
<211> 32
<212> DNA
<213> Homo sapiens
<400> 109
cttgggaatg agatagtttc tgaatttaat gt 32
<210> 110
<211> 21
<212> DNA
<213> Homo sapiens
<400> 110
acccaccctt ggtttttctc a 21
<210> 111
<211> 19
<212> DNA
<213> Homo sapiens
<400> 111
gggctacccc gttctgtcc 19
<210> 112
<211> 20
<212> DNA
<213> Homo sapiens
<400> 112
gctgcagcac ttcagcttct 20
<210> 113
<211> 19
<212> DNA
<213> Homo sapiens
<400> 113
tgtccagagg agcccattt 19
<210> 114
<211> 17
<212> DNA
<213> Homo sapiens
<400> 114
gcctgggcaa gaagtcg 17
<210> 115
<211> 20
<212> DNA
<213> Homo sapiens
<400> 115
ggcttgacaa gaggccctga 20
<210> 116
<211> 17
<212> DNA
<213> Homo sapiens
<400> 116
gtccaggccg tgtccaa 17
<210> 117
<211> 20
<212> DNA
<213> Homo sapiens
<400> 117
cccagctgga tgagctgcta 20
<210> 118
<211> 20
<212> DNA
<213> Homo sapiens
<400> 118
ccatctggga aacagtgcag 20
<210> 119
<211> 30
<212> DNA
<213> Homo sapiens
<400> 119
tccaaaatat cactttccat aaaagcaagg 30
<210> 120
<211> 30
<212> DNA
<213> Homo sapiens
<400> 120
acgaatgctc tactgtcatt tctaaccata 30
<210> 121
<211> 28
<212> DNA
<213> Homo sapiens
<400> 121
taactcacca gccctctgat ctataaag 28
<210> 122
<211> 25
<212> DNA
<213> Homo sapiens
<400> 122
gcctacagca tggatgtgat tactg 25
<210> 123
<211> 28
<212> DNA
<213> Homo sapiens
<400> 123
gcatggactc agttgagagt taattcaa 28
<210> 124
<211> 28
<212> DNA
<213> Homo sapiens
<400> 124
ccagcagtgt tctttccttc actttata 28
<210> 125
<211> 27
<212> DNA
<213> Homo sapiens
<400> 125
ctgaaggact actacctctt ctacctg 27
<210> 126
<211> 24
<212> DNA
<213> Homo sapiens
<400> 126
tctcctgact gtcatcccta ttgg 24
<210> 127
<211> 32
<212> DNA
<213> Homo sapiens
<400> 127
tgtgtacatt acctaaatac aaagaagaat gt 32
<210> 128
<211> 28
<212> DNA
<213> Homo sapiens
<400> 128
tctatttctg tttgcaggct atacagtt 28
<210> 129
<211> 28
<212> DNA
<213> Homo sapiens
<400> 129
ccatttttct cttctctgag ctaacatg 28
<210> 130
<211> 27
<212> DNA
<213> Homo sapiens
<400> 130
gtagatgtcc tcatgcatat cttgtgt 27
<210> 131
<211> 25
<212> DNA
<213> Homo sapiens
<400> 131
cctggcttta aatcctcgaa cacaa 25
<210> 132
<211> 26
<212> DNA
<213> Homo sapiens
<400> 132
ttggtgtcaa agtgtcactg aactaa 26
<210> 133
<211> 20
<212> DNA
<213> Homo sapiens
<400> 133
ctgaaaaaca accattggcc 20
<210> 134
<211> 25
<212> DNA
<213> Homo sapiens
<400> 134
tacgtcttca aggtgtaaaa tgctc 25
<210> 135
<211> 21
<212> DNA
<213> Homo sapiens
<400> 135
ccttcccttt gtgacttgaa g 21
<210> 136
<211> 26
<212> DNA
<213> Homo sapiens
<400> 136
gtatttatac tctagaaggg ggcaca 26
<210> 137
<211> 22
<212> DNA
<213> Homo sapiens
<400> 137
cacatactgt ccaattcccc tg 22
<210> 138
<211> 28
<212> DNA
<213> Homo sapiens
<400> 138
gtgtaaatgt atgattttat gcaggttt 28
<210> 139
<211> 28
<212> DNA
<213> Homo sapiens
<400> 139
ggaattgact gtctttttga aaagttat 28
<210> 140
<211> 23
<212> DNA
<213> Homo sapiens
<400> 140
ttgacatgat ttgggataga gga 23
<210> 141
<211> 30
<212> DNA
<213> Homo sapiens
<400> 141
tattttaaca tgttactctt tcttgtttca 30
<210> 142
<211> 22
<212> DNA
<213> Homo sapiens
<400> 142
tgctttgtgg atgttacaca gg 22
<210> 143
<211> 26
<212> DNA
<213> Homo sapiens
<400> 143
tgatctccta gaagacagca aatacc 26
<210> 144
<211> 27
<212> DNA
<213> Homo sapiens
<400> 144
atttttgatc aagttgtgag aagaaat 27
<210> 145
<211> 30
<212> DNA
<213> Homo sapiens
<400> 145
ataggagatt caattataag gacaatacag 30
<210> 146
<211> 19
<212> DNA
<213> Homo sapiens
<400> 146
cttctcctgc aggtgacca 19
<210> 147
<211> 30
<212> DNA
<213> Homo sapiens
<400> 147
aaaaaatata cttatttacg cttgaacctc 30
<210> 148
<211> 18
<212> DNA
<213> Homo sapiens
<400> 148
tgcccaaacc tggtgatg 18
<210> 149
<211> 26
<212> DNA
<213> Homo sapiens
<400> 149
ttttgatcac attgtaagaa gaaacc 26
<210> 150
<211> 19
<212> DNA
<213> Homo sapiens
<400> 150
gcccgaaaac accttcatc 19
<210> 151
<211> 18
<212> DNA
<213> Homo sapiens
<400> 151
cgcctcaaca gccacatg 18
<210> 152
<211> 18
<212> DNA
<213> Homo sapiens
<400> 152
acgcaggcga tgttgtcc 18
<210> 153
<211> 18
<212> DNA
<213> Homo sapiens
<400> 153
aggccaccaa agggtacc 18
<210> 154
<211> 19
<212> DNA
<213> Homo sapiens
<400> 154
agggacctgc agagctctg 19
<210> 155
<211> 18
<212> DNA
<213> Homo sapiens
<400> 155
gcagatccct ggacaggc 18
<210> 156
<211> 23
<212> DNA
<213> Homo sapiens
<400> 156
tcccaataaa agtgactctc agc 23
<210> 157
<211> 20
<212> DNA
<213> Homo sapiens
<400> 157
gtgcaattca accctggttc 20
<210> 158
<211> 18
<212> DNA
<213> Homo sapiens
<400> 158
gccgatgacc tgcagaag 18
<210> 159
<211> 18
<212> DNA
<213> Homo sapiens
<400> 159
gcggacatgg aggacgtg 18
<210> 160
<211> 18
<212> DNA
<213> Homo sapiens
<400> 160
atggtggatt tcgctggc 18
<210> 161
<211> 18
<212> DNA
<213> Homo sapiens
<400> 161
agccatgatt gtggcaca 18
<210> 162
<211> 21
<212> DNA
<213> Homo sapiens
<400> 162
ggcatgacaa ttgcttgaat c 21
<210> 163
<211> 19
<212> DNA
<213> Homo sapiens
<400> 163
cctgggctag gtgtagggg 19
<210> 164
<211> 18
<212> DNA
<213> Homo sapiens
<400> 164
agggtgagct ctgtgggc 18
<210> 165
<211> 26
<212> DNA
<213> Homo sapiens
<400> 165
ctgtacagag agagtctaca gggaga 26
<210> 166
<211> 20
<212> DNA
<213> Homo sapiens
<400> 166
gtggggaatg gatgaaattt 20
<210> 167
<211> 23
<212> DNA
<213> Homo sapiens
<400> 167
aaataccccc aacataccag atc 23
<210> 168
<211> 26
<212> DNA
<213> Homo sapiens
<400> 168
ttttcccact atcattgatt atttcc 26
<210> 169
<211> 26
<212> DNA
<213> Homo sapiens
<400> 169
caaatttgtg tcttctgttc tcaaag 26
<210> 170
<211> 19
<212> DNA
<213> Homo sapiens
<400> 170
ggattgtaag caccccctg 19
<210> 171
<211> 20
<212> DNA
<213> Homo sapiens
<400> 171
aaggaccaca aaaggatcca 20
<210> 172
<211> 26
<212> DNA
<213> Homo sapiens
<400> 172
ccctatgttt gttattttca ggaaaa 26
<210> 173
<211> 18
<212> DNA
<213> Homo sapiens
<400> 173
tctccctcat gacgctgc 18
<210> 174
<211> 22
<212> DNA
<213> Homo sapiens
<400> 174
ttcctgatca aaatggagaa gg 22
<210> 175
<211> 21
<212> DNA
<213> Homo sapiens
<400> 175
gaacgtgtga ttggcagaaa c 21
<210> 176
<211> 20
<212> DNA
<213> Homo sapiens
<400> 176
ggaagaggag cattgaggac 20
<210> 177
<211> 20
<212> DNA
<213> Homo sapiens
<400> 177
caggtcagcc accactatgc 20
<210> 178
<211> 21
<212> DNA
<213> Homo sapiens
<400> 178
gtgtctttgc tttcctggtg a 21
<210> 179
<211> 18
<212> DNA
<213> Homo sapiens
<400> 179
gcagcaggtt gcccagcc 18
<210> 180
<211> 18
<212> DNA
<213> Homo sapiens
<400> 180
gccttcgcca accactcc 18
<210> 181
<211> 18
<212> DNA
<213> Homo sapiens
<400> 181
ccgaaaccca ggatctgg 18
<210> 182
<211> 20
<212> DNA
<213> Homo sapiens
<400> 182
tccaacagga gatcgacgac 20
<210> 183
<211> 22
<212> DNA
<213> Homo sapiens
<400> 183
ggatgagctg ctaactgagc ac 22
<210> 184
<211> 18
<212> DNA
<213> Homo sapiens
<400> 184
gcccccgcct gtaccctt 18
<210> 185
<211> 24
<212> DNA
<213> Homo sapiens
<400> 185
tcccactatc attgattatt tccc 24
<210> 186
<211> 27
<212> DNA
<213> Homo sapiens
<400> 186
ctctttaaag agctcttttg tctttca 27
<210> 187
<211> 23
<212> DNA
<213> Homo sapiens
<400> 187
tttgtagata tgggacccgt aca 23
<210> 188
<211> 28
<212> DNA
<213> Homo sapiens
<400> 188
gatctaagaa accaaatttt aggaactt 28
<210> 189
<211> 18
<212> DNA
<213> Homo sapiens
<400> 189
tccttccagg caccacct 18
<210> 190
<211> 24
<212> DNA
<213> Homo sapiens
<400> 190
cagaaactgc aaaaggagat tgat 24
<210> 191
<211> 20
<212> DNA
<213> Homo sapiens
<400> 191
ggaggagctg accagtgaag 20
<210> 192
<211> 19
<212> DNA
<213> Homo sapiens
<400> 192
gagaaggtgt ctgcgggag 19
<210> 193
<211> 25
<212> DNA
<213> Homo sapiens
<400> 193
tctgggtcat acatgtggat atatg 25
<210> 194
<211> 20
<212> DNA
<213> Homo sapiens
<400> 194
gcagtcgaca atagggcaaa 20
<210> 195
<211> 22
<212> DNA
<213> Homo sapiens
<400> 195
tttggacctt atctggaaca gc 22
<210> 196
<211> 20
<212> DNA
<213> Homo sapiens
<400> 196
cactacatca tacggcagcc 20
<210> 197
<211> 23
<212> DNA
<213> Homo sapiens
<400> 197
gaacacaaac tcatgcaact ctg 23
<210> 198
<211> 23
<212> DNA
<213> Homo sapiens
<400> 198
taaaggctga ctttccagac aac 23

Claims (6)

1. a kind of for detecting the multiple PCR detection kit of people's drug gene polymorphism, it is characterised in that：Including basis VKORC1、UGT1A1、HLA-A、HLA-B、TPMT、NAT1、NAT2、G6PD、F5、F2、IFNL3、APOE、COMT、CYP1A2、 The design of CYP2B6, CYP2C19, CYP2C9, CYP2D6, CYP3A5, MTFHR, SLCO1B1, DPYD gene polynorphisms site The 66 PCR amplification primer pairs reacted in same reaction system and 66 single bases reacted in same reaction system Extension primer；Wherein,
Above-mentioned 66 PCR amplification primer pair includes the first to the 66th amplimer pair, and the first to the 66th amplification is drawn The forward primer of object pair is successively as shown in SEQ D NO 01~066, and reverse primer is successively such as 067~132 institute of SEQ ID NO Show；Above-mentioned 66 Single base extension primer includes the first to the 66th Single base extension primer, successively such as SEQ ID NO Shown in 133~198；
Its application method includes the following steps：
(1) using genomic DNA to be detected as template, contain RingCap-Taq one using the 66 PCR amplification primer pair Pcr amplification reaction is carried out in the PCR reaction system of enzyme and RingCap buffer, obtains pcr amplification reaction liquid；
(2) above-mentioned pcr amplification reaction liquid is carried out SAP with SAP enzyme to react, obtains SAP reaction solution；
(3) single base extension is carried out to above-mentioned SAP reaction solution with above-mentioned 66 Single base extension primer pair；
(4) by step (3) resulting material after resin desalination, in SpectrocHIP chip loading, Mass Spectrometer Method is carried out, i.e., At.
2. multiple PCR detection kit as described in claim 1, it is characterised in that：The condition packet of the pcr amplification reaction It includes：95 DEG C of 3min, 1 circulation；95 DEG C of 20s, 60 DEG C of 30s, 72 DEG C of 20s, 45 circulations；72 DEG C of 3min, 1 circulation.
3. multiple PCR detection kit as claimed in claim 2, it is characterised in that：The condition of SAP reaction includes：37 DEG C 40min, 85 DEG C of 5min, 4 DEG C of ∞.
4. multiple PCR detection kit as claimed in claim 3, it is characterised in that：The condition of the single base extension Including：
First stage recycles for 40 totally：Each circulation includes：
94 DEG C of 30s, 1 circulation, 94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, 5 circulations；
Second stage recycles for 1 totally：72℃ 3min.
5. the multiple PCR detection kit as described in any claim in Claims 1-4, it is characterised in that：It is described RingCap-Taq enzyme is made of Taq enzyme, DNA ligase and the end modified enzyme of DNA, and ratio 0.8~1.2: 0.8~1.2: 0.8~ 1.2。
6. multiple PCR detection kit as claimed in claim 5, it is characterised in that：The RingCap-Taq enzyme by Taq enzyme, DNA ligase and the end modified enzyme composition of DNA, ratio 1: 1: 1.
CN201810660244.0A 2018-06-25 2018-06-25 It is a kind of for detecting the multiple PCR detection kit of people's drug gene polymorphism Pending CN108823301A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN109825572A (en) * 2019-03-13 2019-05-31 陈向东 A kind of kit and its detection method of detection and the susceptibility related gene polymorphism of propofol
CN111057755A (en) * 2019-06-14 2020-04-24 陕西九州医学检验有限公司 Cardiovascular and cerebrovascular disease genetic risk assessment detection panel and application thereof
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Publication number Priority date Publication date Assignee Title
CN109536605A (en) * 2019-01-31 2019-03-29 深圳市第二人民医院 The PCR primer combination and application of statins adverse reaction genotype polymorphism
CN109825572A (en) * 2019-03-13 2019-05-31 陈向东 A kind of kit and its detection method of detection and the susceptibility related gene polymorphism of propofol
CN111057755A (en) * 2019-06-14 2020-04-24 陕西九州医学检验有限公司 Cardiovascular and cerebrovascular disease genetic risk assessment detection panel and application thereof
CN111057755B (en) * 2019-06-14 2022-07-26 陕西九州医学检验有限公司 Cardiovascular and cerebrovascular disease genetic risk assessment detection panel and application thereof
CN112522376A (en) * 2020-12-21 2021-03-19 济南和合医学检验有限公司 Primer group, kit and method for detecting gene polymorphism
CN112522376B (en) * 2020-12-21 2022-07-19 济南和合医学检验有限公司 Primer group, kit and method for detecting gene polymorphism

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