CN108998517B - A kind of mankind SLCO1B1 and ApoE genetic polymorphism detection kit and its preparation method and application - Google Patents
A kind of mankind SLCO1B1 and ApoE genetic polymorphism detection kit and its preparation method and application Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of mankind SLCO1B1 and ApoE genetic polymorphism detection kit and its preparation method and application.The kit by: be respectively used to the detection site SLCO1B1 gene rs2306283, the site SLCO1B1 gene rs4149056, the site gene rs429358 ApoE and the site ApoE gene rs7412 PCR premix reaction solution, positive reference substance and negative controls form, the PCR premix reaction solution includes specific primer sequence group, probe groups and the PCR reaction solution for expanding each site, and the ARMs primer of specificity of the specific primer group by common outer primer and with fluorescence labels forms.Kit of the present invention has many advantages, such as that high sensitivity, specificity is high, easy to operate, result is reliable for detecting mankind's SLCO1B1 and ApoE gene pleiomorphism, and detection can be completed in 1 hour, and result interpretation is simply objective.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of mankind SLCO1B1 and the examination of ApoE genetic polymorphism detection
Agent box and its preparation method and application.
Background technique
The mankind SLCO1B1 assignment of genes gene mapping encodes organic anion and transports polypeptide OATP1B1 in No. 12 chromosomes.OATP1B1
For transmembrane transporter, it is distributed mainly on liver, is had and is mediated liver plasma membrane to transport inside and outside source property substance and it is metabolized
With the physiological function of elimination, important regulating and controlling effect is played in transhipment statins.Research shows that SLCO1B1 gene has
Genetic polymorphism, wherein 388A > G, 521T > C are two kinds of common single nucleotide polymorphism, can form 4 kinds of haplotypes
SLCO1B1*1a (388A-521T), SLCO1B1*1b (388G-521T), SLCO1B1*5 (388A-521C) and SLCO1B1*15
(388G-521C).Saltant type SLCO1B1 gene causes the OATP1B1 transport protein vigor of coding to weaken, and shows as liver intake
Drug ability reduces, and blood concentration is caused to rise.The ApoE assignment of genes gene mapping is in the 19th pair of chromosome, guidance synthesis apo E
(apolipoprotein E, ApoE), ApoE are one of apolipoproteins important in blood plasma, mainly the table in liver and brain tissue
It reaches, identifies ldl receptor and ApoE receptor, mediate the metabolism of VLDL and IDL.There are mainly two types of mononucleotide polymorphics for ApoE gene
Property, rs7412 (526C > T) and rs429358 (388T > C), can be formed 3 kinds of haplotypes be respectively ApoE3 (388T-526C),
ApoE2(388T-526T),ApoE4(388C-526C).Document report, drug of the ApoE4 carrier compared to ApoE2 carrier
Metabolic capability is weaker.Fig. 1 is that SLCO1B1 gene and ApoE gene polymorphism sites and genotype frequency summarize.Statins
It is the choice drug of the diseases such as current prevention and treatment of coronary heart disease, headstroke, hyperlipidemia.When SLCO1B1 gene mutates, transhipment
Function reduction, the statins concentration that will lead in blood rise, and increase adverse reaction (such as rhabdomyolysis of drug
Deng)；When ApoE gene generates variation, it can lead to TC, LDL-C level and generate difference, so that the curative effect to statins generates
It influences.
Clinically following four mode is mainly taken to detect SLCO1B1 and ApoE gene polynorphisms at present:
1.PCR- PCR sequencing PCR
2. high-resolution solubility curve method
3. chip hybridization methods
4.Taqman-qPCR method
PCR- PCR sequencing PCR sensitivity is low, and time-consuming for experiment, is not suitable for clinical expansion；Chip hybridization methods, detection sensitivity are low
And poor specificity is easy to appear false positive results；High-resolution solubility curve method is special to equipment requirement, and unsuitable clinic pushes away
Extensively, and detection sensitivity is not high.Taqman-qPCR method, detection sensitivity is high, but often due to site sequence reason, causes to examine
It is not high to survey specificity, and it carries out parting by Genotyping method, is distributed with and has to sample size and polymorphism
It asks, is not suitable for the too low site of the detection frequency of mutation.
Summary of the invention
The present invention is in view of the deficiencies of the prior art, and it is an object of the present invention to provide a kind of mankind SLCO1B1 and ApoE gene pleiomorphism
Detection kit and its preparation method and application.This kit has high sensitivity, high specific, low cost high throughput, operation
It easy, the features such as experimental period is short, can be with fast accurate to SLCO1B1 and ApoE gene pleiomorphism in human genome DNA
It is detected.
For achieving the above object, the technical solution adopted by the present invention are as follows:
A kind of mankind SLCO1B1 and ApoE genetic polymorphism detection primer sets, the primer sets by common outer primer and
The ARMs primer composition of specificity with fluorescence labels, particular sequence are as follows:
A kind of mankind SLCO1B1 and ApoE genetic polymorphism detection probe groups, the probe groups are by specific taqman-
MGB probe and the lock nucleic acid probe composition for carrying quenching group, particular sequence are as follows:
Internal control-P:5 '-ROX-ACCACCACGGCCGAGC-MGB-BHQ-3 ' SEQ ID NO.19
Wild type quenching group lock nucleic acid: 5 '-BHQ-ACGATGACAAGGGAT-3 ' SEQ ID NO.20
Saltant type quenching group lock nucleic acid: 5 '-BHQ-TCATCAATGTATCTT-3 ' SEQ ID NO.21.
A kind of mankind SLCO1B1 and ApoE genetic polymorphism detection kit, the kit by: be respectively used for amplifying
SLCO1B1 gene rs2306283, SLCO1B1 gene rs4149056, ApoE gene rs429358 and ApoE gene rs7412 tetra-
PCR premix reaction solution, positive reference substance and the negative controls composition in a site；
The concrete component of the PCR premix reaction solution is as follows:
(1) for expanding SLCO1B1 gene rs2306283 site PCR premix reaction solution: being used for as described in claim 1
4 primers in the site SLCO1B1 gene rs2306283, primer sequence are expanded as shown in SEQ ID NO.1~SEQ ID NO.4,
Internal control primer, primer sequence are as shown in SEQ ID NO.17~SEQ ID NO.18, probe groups described in claim 2, nucleotide
Sequence is as shown in SEQ ID NO.19~SEQ ID NO.21, PCR reaction solution composition；
(2) for expanding SLCO1B1 gene rs4149056 site PCR premix reaction solution: being used for as described in claim 1
4 primers in the site SLCO1B1 gene rs4149056, sequence are expanded as shown in SEQ ID NO.5~SEQ ID NO.8, internal control
Primer, sequence are as shown in SEQ ID NO.17~SEQ ID NO.18, such as SEQ ID of probe groups, sequence described in claim 2
Shown in NO.19~SEQ ID NO.21, PCR reaction solution composition；
(3) for expanding ApoE gene rs429358 site PCR premix reaction solution: as benefit require 1 described in for expanding
4 primers in the site ApoE gene rs429358, sequence are as shown in SEQ ID NO.9~SEQ ID NO.12；Internal control primer, sequence
It arranges as shown in SEQ ID NO.17~SEQ ID NO.18, such as SEQ ID NO.19 of probe groups, sequence described in claim 2~
Shown in SEQ ID NO.21, PCR reaction solution composition；
(4) for expanding ApoE gene rs7412 site PCR premix reaction solution: as benefit require 1 described in for expanding ApoE
4 primers in the site gene rs7412, sequence are as shown in SEQ ID NO.13~SEQ ID NO.16, and internal control primer, sequence are such as
Shown in SEQ ID NO.17~SEQ ID NO.18, such as SEQ ID NO.19~SEQ ID of probe groups, sequence described in claim 2
Shown in NO.21, PCR reaction solution composition；
The positive reference substance includes: the site SLCO1B1 gene rs2306283 wild plasmid, SLCO1B1 gene
Rs2306283 site mutation type plasmid, the site SLCO1B1 gene rs4149056 wild plasmid, SLCO1B1 gene
Rs4149056 site mutation type plasmid, the site ApoE gene rs429358 wild plasmid, the site gene rs429358 ApoE are prominent
Modification plasmid, the site ApoE gene rs7412 wild plasmid, ApoE gene rs7412 site mutation type plasmid and reference gene
Plasmid DNA；The negative controls include the recombinant plasmid not comprising internal standard gene and target gene site.
In above scheme, in PCR premix reaction solution, in probe groups the concentration of taqman-MGB probe be 50~
100nM, the concentration for carrying the lock nucleic acid probe of quenching group is 100~400nM；For expand each site without fluorescent base
Each primer concentration of group is 100~400nM, and each primer concentration with fluorophor is 50~100nM；For drawing for internal control
The concentration of object is 100~400nM.
In above scheme, the PCR reaction solution includes Premix qPCRmix and TE buffer.
In above scheme, the negative controls are made of PUC-19 plasmid empty carrier and TE buffer.
Above-mentioned mankind SLCO1B1 and ApoE genetic polymorphism detection kit preparation for detect mankind SLCO1B1 and
Application in ApoE gene pleiomorphism product.
It is more using mankind's SLCO1B1 and ApoE genetic polymorphism detection kit detection mankind's SLCO1B1 and ApoE gene
The detection process of state property, specifically comprises the following steps:
(1) genomic DNA of human peripheral to be measured is extracted as sample to be detected using genome DNA extracting reagent kit；
(2) PCR premix reaction solution and sample to be tested DNA fluorogenic quantitative detection: are added into reaction tube；The positive is set simultaneously
Control group and negative control group, then by reaction tube be placed in fluorescent PCR instrument carry out pcr amplification reaction, and acquire FAM, VIC and
ROX fluorescence signal；
(3) after pcr amplification reaction, result judgement is carried out according to fluorescence signal initial line situation collected.
In above scheme, it is 0.1~100ng/ μ L that the sample DNA to be detected, which is diluted to concentration, in step (1), then into
Row pcr amplification reaction.
In above scheme, the condition of pcr amplification reaction described in step (2) are as follows: 95 DEG C of 1 minute initial denaturations；15 circulations:
95 DEG C 5 seconds, 61 DEG C 32 seconds, do not collect fluorescence；30 circulation: 95 DEG C 5 seconds, 61 DEG C 32 seconds, collect fluorescence.
In above scheme, the principle of step (3) described result judgement is (as shown in table 5 below): 1. when positive controls are equal
There are FAM, VIC, ROX fluorescence signal initial line, for negative control group without FAM, VIC, ROX fluorescence initial line, sample to be detected has ROX glimmering
When optical signal initial line, determines Success in Experiment, subsequent parting judgement can be carried out；2. being believed according to FAM, VIC fluorescence of detection sample
Number judge the parting in the site SLCO1B1 gene rs2306283: VIC fluorescence signal initial line, the site SLCO1B1 gene rs2306283
Parting is homozygous wildtype；FAM fluorescence signal initial line, SLCO1B1 gene rs2306283 site parting are homozygous mutant；
The equal initial line of VIC, FAM fluorescence signal, SLCO1B1 gene rs2306283 site parting are heterozygous mutant；3. according to detection sample
FAM, VIC fluorescence signal judge the parting in the site SLCO1B1 gene rs4149056: VIC fluorescence signal initial line, SLCO1B1 base
Because the site rs4149056 parting is homozygous wildtype；FAM fluorescence signal initial line, the site SLCO1B1 gene rs4149056 parting
For homozygous mutant；The equal initial line of VIC, FAM fluorescence signal, SLCO1B1 gene rs4149056 site parting are heterozygous mutant；
4. judging the parting in the site ApoE gene rs429358 according to FAM, VIC fluorescence signal of detection sample: VIC fluorescence signal rises
Line, ApoE gene rs429358 site parting are homozygous wildtype；FAM fluorescence signal initial line, the site ApoE gene rs429358
Parting is homozygous mutant；The equal initial line of VIC, FAM fluorescence signal, ApoE gene rs429358 site parting are heterozygous mutant；
5. the parting in the site ApoE gene rs7412: VIC fluorescence signal initial line is judged according to FAM, VIC fluorescence signal of detection sample,
ApoE gene rs7412 site parting is homozygous wildtype；FAM fluorescence signal initial line, ApoE gene rs7412 site parting are pure
Close saltant type；The equal initial line of VIC, FAM fluorescence signal, ApoE gene rs7412 site parting are heterozygous mutant.
Testing principle such as Fig. 2 and Fig. 3 institute of mankind SLCO1B1 and ApoE genetic polymorphism detection kit of the present invention
Showing, the ARMs primer with sequence label for carrying FAM fluorescence specific recognition and can expand the template of mutated-genotype, and
With the consumption of primer, more FAM fluorescence are released, at the end of circulation, each channel fluorescence is acquired, it is bent to generate fluorescence
Line；The ARMs primer with sequence label for carrying VIC fluorescence specific recognition and can expand the template of wild-type genotype, and
With the consumption of primer, more VIC fluorescence are released, at the end of circulation, each channel fluorescence is acquired, it is bent to generate fluorescence
Line.Internal control probe uses MGB probe, and specific recognition internal control corresponding sequence, by amplification, more and more ROX fluorescence are released
It releases.So final display only has internal control (ROX) and wild (VIC) glimmering when the genomic templates of detection are homozygous wildtypes
Light curve can normal initial line and curve it is S-type, initial line or initial line be not very low and not S-type for mutation (FAM) fluorescence curve.When
The genomic templates of detection are homozygous mutants, and final display only has internal control (ROX) and mutation (FAM) fluorescence curve can be normal
Initial line and curve is S-type, initial line or initial line be not very low and not S-type for wild (VIC) fluorescence curve.When the genome mould of detection
Plate is heterozygote, and finally showing internal control (ROX), mutation (FAM) and wild (VIC) fluorescence curve can normal initial line and song
Line is S-type.
Beneficial effects of the present invention are as follows: mankind SLCO1B1 and ApoE genetic polymorphism detection kit of the present invention
Detection mankind's SLCO1B1 and ApoE typing gene polymorphisms that can be simple and quick, using ARMs primer binding specificity fluorescence
Label carries out PCR reaction and realizes that each gene polynorphisms parting need to only be can be completed by a multi-PRC reaction, each
Contain internal control in PCR reaction, guarantees the accuracy of testing result, prevent the appearance of false negative and false positive results；Each PCR
The tape label ARMs primer that FAM or VIC fluorescence is carried in reaction system can accordingly be had the lock nucleic acid locking of quenching group
So that fluorescence is in cancellation state, in PCR reaction process, carries the tape label ARMs primer and cls gene to be checked of FAM or VIC fluorescence
Release fluorescence generates fluorescence curve and carries out result interpretation after template specific binding；The present invention utilizes carrying fluorophor and label
The specific ARMs primer of sequence combines the lock nucleic acid sequence for carrying quenching group, specific internal control probe and specificity
Internal control primer can greatly improve the specificity and accuracy of Genotyping, meanwhile, two outer primers F1 and R1 can also be carried out
Amplification, is further enriched with genomic templates, can greatly improve parting efficiency, and then can detecte the genome of lower concentration
Sample, kit of the present invention is minimum to carry out polymorphic detection to 0.1ng genomic DNA；In addition, reagent of the present invention
Box is packed by the way of premixing, and when use only needs that genomic DNA is added, while simplifying the response procedures of PCR, can
The detection of aspirin resistance gene pleiomorphism is completed in 1 hour, result interpretation, interpretation directly can be carried out according to fluorescence curve
As a result easy to be objective, convenient for analysis.
Detailed description of the invention
Fig. 1 is clinically SLCO1B1 and ApoE gene loci polymorphism and its occurrence frequency.
Fig. 2 is the detection schematic diagram of kit of the present invention.
Fig. 3 is the detection schematic diagram of kit of the present invention.
Fig. 4 is the site SLCO1B1 gene rs2306283 homozygous wildtype testing result.
Fig. 5 is the site SLCO1B1 gene rs2306283 heterozygous testing result.
Fig. 6 is the site SLCO1B1 gene rs2306283 homozygous mutant testing result.
Fig. 7 is the site SLCO1B1 gene rs4149056 homozygous wildtype testing result.
Fig. 8 is the site SLCO1B1 gene rs4149056 heterozygous testing result.
Fig. 9 is the site SLCO1B1 gene rs4149056 homozygous mutant testing result.
Figure 10 is the site ApoE gene rs429358 homozygous wildtype testing result.
Figure 11 is the site ApoE gene rs429358 heterozygous testing result.
Figure 12 is the site ApoE gene rs429358 homozygous mutant testing result.
Figure 13 is the site ApoE gene rs7412 homozygous wildtype testing result.
Figure 14 is the site ApoE gene rs7412 heterozygous testing result.
Figure 15 is the site ApoE gene rs7412 homozygous mutant testing result.
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but it is of the invention
Content is not limited solely to the following examples.
Embodiment 1 prepares SLCO1B1 and ApoE gene detecting kit of the invention
One, design of primers and synthesis:
SLCO1B1 388, SLCO1B1 521, draw comprising 6 in 526 4 each systems in site of ApoE 388 and ApoE
Object；Two of them positive-sense strand upstream primer F1 and F2, two antisense strand downstream primers R1 and R2, F1 and R1 are general primer, F2
It is the specific ARMs primer with fluorophor and sequence label with R2, is screened by primer and PCR reaction condition, guarantees primer
F1R1 is enriched with template, and F2 and R1, F1 and R2 can normally amplify the specific genotype PCR product with fluorescence；Often
Internal control primer is added in a gene loci.
Particular sequence is as follows:
Specific combination is as follows:
Wherein SEQ ID NO.1 and SEQ ID NO.4 is produced for expanding SLCO1B gene loci 388A DNA fragmentation, amplification
Object is 107bp, and for expanding SLCO1B1 388G DNA fragmentation, amplified production is SEQ ID NO.2 and SEQ ID NO.3
102p, SEQ ID NO.1 and SEQ ID NO.3 expand two kinds of genotype；
Wherein SEQ ID NO.5 and SEQ ID NO.8 is produced for expanding SLCO1B gene loci 521T DNA fragmentation, amplification
Object is 133bp, and SEQ ID NO.6 and SEQ ID NO.7 is for expanding SLCO1B gene loci 521C DNA fragmentation, amplified production
Two kinds of genotype are expanded for 141bp, SEQ ID NO.5 and SEQ ID NO.7；
Wherein SEQ ID NO.9 and SEQ ID NO.12 is produced for expanding ApoE gene loci 388T DNA fragmentation, amplification
Object is 104bp, and SEQ ID NO.10 and SEQ ID NO.11 is for expanding ApoE gene loci 388C DNA fragmentation, amplified production
Two kinds of genotype are expanded for 135bp, SEQ ID NO.9 and SEQ ID NO.11；
Wherein SEQ ID NO.13 and SEQ ID NO.16 is produced for expanding ApoE gene loci 526C DNA fragmentation, amplification
Object is 110bp, and SEQ ID NO.14 and SEQ ID NO.15 is for expanding ApoE gene loci 526T DNA fragmentation, amplified production
Two kinds of genotype are expanded for 99bp, SEQ ID NO.13 and SEQ ID NO.15；
SEQ ID NO 17 and SEQ ID NO18 is for expanding internal control DNA fragmentation, amplified production 186bp；
Two, probe design and synthesis:
It SLCO1B1 388, SLCO1B1 521, include 2 spies in 526 4 each systems in site of ApoE 388 and ApoE
The opposite sex is with the lock nucleic acid probe of quenching group and 1 internal reference specificity MGB probe, the lock nucleic acid of 2 specific band quenching groups
Probe wherein one be used for lock-in detection wild type the specific ARMs primer with VIC fluorophor and sequence label, it is another
Item is used for the specific ARMs primer with FAM fluorophor and sequence label of lock-in detection saltant type；Internal reference specificity MGB is visited
Needle internal reference site for identification.
Particular sequence is as follows:
Internal control-P:5 '-ROX-ACCACCACGGCCGAGC-MGB-BHQ-3 ' SEQ ID NO.19
Wild type quenching group lock nucleic acid: 5 '-BHQ-ACGATGACAAGGGAT-3 ' SEQ ID NO.20
Saltant type quenching group lock nucleic acid: 5 '-BHQ-TCATCAATGTATCTT-3 ' SEQ ID NO.21.
Specific combination is as follows:
Wherein SEQ ID NO.19 specific recognition internal reference site；
Wherein SEQ IDNO.20 specific recognition and lock-in detection wild type with VIC fluorophor and sequence label
Specific ARMs primer；
The wherein specificity with FAM fluorophor and sequence label of SEQ ID NO.21 specific recognition detection saltant type
ARMs primer；
Three, PCR premixes reaction solution configuration
PCR premixes reaction solution 1: by 4 primers for expanding SLCO1B1 gene rs2306283 site A388G, primer
Sequence is as shown in SEQ ID NO.1~SEQ ID NO.4；Internal control primer, primer sequence such as SEQ ID NO.17~SEQ ID
Shown in NO.18；Probe groups, nucleotide sequence is as shown in SEQ ID NO.19~SEQ ID NO.21；PCR reaction solution composition, tool
Body ingredient such as the following table 1:
1 SLCO1B1 gene rs2306283 site PCR reaction solution reagent of table composition
PCR premixes reaction solution 2: by 4 primers for expanding the site the SLCO1B1 gene rs4149056 site T521C,
Primer sequence is as shown in SEQ ID NO.5~SEQ ID NO.8；Internal control primer, primer sequence such as SEQ ID NO.17~SEQ
Shown in ID NO.18；Probe groups, nucleotide sequence is as shown in SEQ ID NO.19~SEQ ID NO.21；PCR reaction solution composition.
Specific ingredient following 2:
2 SLCO1B1 gene rs4149056 site PCR reaction solution reagent of table composition
| Component | Proportion |
| Premix qPCR MIX | 25μL |
| SEQ ID NO.5 | 100~400nM |
| SEQ ID NO.6 | 50~100nM |
| SEQ ID NO.7 | 100~400nM |
| SEQ ID NO.8 | 50~100nM |
| SEQ ID NO.17 | 100~400nM |
| SEQ ID NO.18 | 100~400nM |
| SEQ ID NO.19 | 50~100nM |
| SEQ ID NO.20 | 100~400nM |
| SEQ ID NO.21 | 100~400nM |
| TE buffer | Complement to 30 μ L |
PCR premixes reaction solution 3: by 4 primers for expanding ApoE gene rs429358 site T388C, primer sequence
As shown in SEQ ID NO.9~SEQ ID NO.12；Internal control primer, primer sequence such as SEQ ID NO.17~SEQ ID NO.18
It is shown；Probe groups, nucleotide sequence is as shown in SEQ ID NO.19~SEQ ID NO.21；PCR reaction solution composition.Specific ingredient
Such as the following table 3:
3 ApoE gene rs429358 site PCR reaction solution reagent of table composition
PCR premixes reaction solution 4: by 4 primers for expanding ApoE gene rs7412 site C526T, primer sequence is such as
Shown in NO.13~16 SEQ ID；Internal control primer, primer sequence is as shown in SEQ ID NO.17~SEQ ID NO.18；Probe
Group, nucleotide sequence is as shown in SEQ ID NO.19~SEQ ID NO.21；PCR reaction solution composition.Specific ingredient such as the following table 4:
4 ApoE gene rs7412 site PCR reaction solution reagent of table composition
| Component | Proportion |
| Premix qPCR MIX | 25μL |
| SEQ ID NO.13 | 100~400nM |
| SEQ ID NO.14 | 50~100nM |
| SEQ ID NO.15 | 100~400nM |
| SEQ ID NO.16 | 50~100nM |
| SEQ ID NO.17 | 100~400nM |
| SEQ ID NO.18 | 100~400nM |
| SEQ ID NO.19 | 50~100nM |
| SEQ ID NO.20 | 100~400nM |
| SEQ ID NO.21 | 100~400nM |
| TE buffer | Complement to 30 μ L |
Four, reference substance configures
Positive reference substance: concentration be respectively 10^4copy/ μ l the site SLCO1B1 gene rs2306283 wild plasmid,
SLCO1B1 gene rs2306283 site mutation type plasmid, the site SLCO1B1 gene rs4149056 wild plasmid, SLCO1B1
Gene rs4149056 site mutation type plasmid, the site ApoE gene rs429358 wild plasmid, ApoE gene rs429358
Point mutation type plasmid, the site ApoE gene rs7412 wild plasmid, ApoE gene rs7412 site mutation type plasmid and internal reference
Gene plasmid DNA and TE buffer.The plasmid sequence is that will not enumerate its sequence herein known to those skilled in the art.
Negative controls: PUC-19 plasmid empty carrier and TE buffer.
Five, kit is assembled
Kit includes that 4 pipes detect SLCO1B1 388,526 gene position of SLCO1B1 521, ApoE 388 and ApoE respectively
The PCR of point polymorphism premixes reaction solution, 1 pipe positive reference substance, 1 pipe negative controls.The usage amount for calculating 20 person-portion specifications, matches
Make the ingredient in each pipe and assembling.
Embodiment 2
Using mankind's SLCO1B1 and ApoE the genetic polymorphism detection kit prepared in embodiment 1 to sample to be tested into
Row detection.The present embodiment collects the anticoagulant venous whole sample of 100 EDTA, extracts genomic DNA, using mankind SLCO1B1 and
It is more that ApoE genetic polymorphism detection kit detects SLCO1B1 388, SLCO1B1 521, ApoE 388 and 526 gene of ApoE
State property situation, specific operation process are as follows:
(1) blood sample extracting genome DNA: commodity in use extracts kit carries out extracting genome DNA, has extracted
TE buffer eluted dna is used at rear, and measures DNA concentration；Genomic DNA is diluted to 20ng/ μ l；
(2) 30 μ l PCR premix reaction solution and 20 μ l sample to be tested DNA fluorogenic quantitative detection: are added into reaction tube；PCR
Response procedures are as follows: 95 DEG C of 1 minute initial denaturations；15 circulation: 95 DEG C 5 seconds, 61 DEG C 32 seconds, do not collect fluorescence；30 circulations: 95 DEG C
5 seconds, 61 DEG C 32 seconds, collect fluorescence.
(3) PCR carries out result interpretation after reaction according to table 5.
5 result interpretation of table
Note: "+" represents deta Rn end point fluorescence value > 100,000, and amplification curve is S-type；It is whole that "-" represents deta Rn
Point fluorescent value < 100,000 or the non-S type of curve；
Interpretation result is as follows:
Homozygous wild 8, sample of the site SLCO1B1 gene rs2306283 1075, one of testing result such as Fig. 4 institute
Show；
36,1075 heterozygosis sample, the site SLCO1B1 gene rs2306283, one of testing result is as shown in Figure 5；
56,1075 homozygous mutation sample, the site SLCO1B1 gene rs2306283, one of testing result such as Fig. 6 institute
Show；
Homozygous wild 70, sample of the site SLCO1B1 gene rs4149056, one of testing result is as shown in Figure 7；
28, heterozygosis sample, the site SLCO1B1 gene rs4149056, one of testing result is as shown in Figure 8；
2, homozygous mutation sample, the site SLCO1B1 gene rs4149056, one of testing result is as shown in Figure 9；
Homozygous wild 80, sample of the site ApoE gene rs429358, one of testing result is as shown in Figure 10；
19, heterozygosis sample, the site ApoE gene rs429358, one of testing result is as shown in figure 11；
1, homozygous mutation sample, the site ApoE gene rs4293583, testing result is as shown in figure 12；
Homozygous wild 85, sample of the site ApoE gene rs7412, one of testing result is as shown in figure 13；
14, heterozygosis sample, the site ApoE gene rs7412, one of testing result is as shown in figure 14；
1, homozygous mutation sample, the site ApoE gene rs7412, testing result is as shown in figure 15.
Above-mentioned 100 sample fluorescence quantitative detection results and sequencing result 100% are consistent.This shows the mankind of the present invention
SLCO1B1 and ApoE genetic polymorphism detection kit is used for mankind's SLCO1B1 and ApoE genetic polymorphism detection result very
Reliably, and the detection sensitivity of kit of the present invention is much higher than PCR sequencing PCR.The mankind are detected using kit of the present invention
SLCO1B1 and ApoE gene pleiomorphism has many advantages, such as that high sensitivity, specificity is high, easy to operate, result is reliable, furthermore this hair
The bright response procedures for simplifying PCR by the annealing in routine PCR reaction and extend step (annealing extension in 30 seconds 1 minute) simplification
It is a step (only needing 61 degree to react 32 seconds) so detection can be completed in 1 hour, and the present invention can be directly according to fluorescence
Curve carries out result interpretation, visual result, interpretation simplicity.
Embodiment 3
The lowest detection line of this kit is verified in the kit detection prepared using embodiment 1, specifically includes following operation
Step:
(1) using the sample of known SLCO1B1 and ApoE corresponding site genotype in embodiment 2, each site is selected respectively
Take wild type gene group, mutated genes group and heterozygous genome sample a case each, by above-mentioned Sample Dilution to 10ng/ μ L,
1ng/μL,0.5ng/μL,0.25ng/μL,0.1ng/μL,0.05ng/μL,0.025ng/μL,0.001ng/μL；
(2) 30 μ l PCR premix reaction solution and 20 μ l sample to be tested DNA fluorogenic quantitative detection: are added into reaction tube；PCR
Response procedures are as follows: 95 DEG C of 1 minute initial denaturations；15 circulation: 95 DEG C 5 seconds, 61 DEG C 32 seconds, do not collect fluorescence；30 circulations: 95 DEG C
5 seconds, 61 DEG C 32 seconds, collect fluorescence；Each concentration gradient counterpoise reinspection of each sample is surveyed 20 times；
(3) PCR carries out result interpretation after reaction according to table 5；
(4) detection success rate (the parting as detection consistent with known type of the above-mentioned each concentration gradient of sample is counted
Success detects success rate=testing result consistent results quantity/20 × 100%), it is shown in Table 6~9.
6 SLCO1B1 gene rs2306283 site primer successful rate statistics of table
7 SLCO1B1 gene rs4149056 site primer successful rate statistics of table
8 ApoE gene rs429358 site primer successful rate statistics of table
9 ApoE gene rs7412 site primer successful rate statistics of table
The above results show that the present invention can carry out the mankind SLCO1B1 and ApoE to the DNA sample of concentration down to 0.1ng/ μ L
Genetic polymorphism detection.
Obviously, above-described embodiment is only intended to clearly illustrate made example, and is not the limitation to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And the obvious variation or change therefore amplified
It moves within still in the protection scope of the invention.
Sequence table
<110>Wuhan Kang Lu Biotechnology Ltd.
<120>a kind of mankind SLCO1B1 and ApoE genetic polymorphism detection kit and its preparation method and application
<160> 21
<210> 1
<211> 22bp
<212> DNA
<213>artificial sequence
400 > 1 of <
atatttctct gtatttctag ga 22
<210> 2
<211> 24bp
<212> DNA
<213>artificial sequence
400 > 2 of <
atcccttgtc atcgttgatg aatc 24
<210> 3
<211> 17bp
<212> DNA
<213>artificial sequence
400 > 3 of <
attcttacct tttccca 17
<210> 4
<211>25bp
<212> DNA
<213>artificial sequence
400 > 4 of <
aagatacatt gatgaactaa tatca 25
<210> 5
<211>25bp
<212> DNA
<213>artificial sequence
400 > 5 of <
aagatacatt gatgaactaa tatca 25
<210> 6
<211>24bp
<212> DNA
<213>artificial sequence
400 > 6 of <
atcccttgtc atcgtccatg aacg 24
<210> 7
<211>20bp
<212> DNA
<213>artificial sequence
400 > 7 of <
tgatcataca atttaatatt 20
<210> 8
<211>26bp
<212> DNA
<213>artificial sequence
400 > 8 of <
aagatacatt gatgatggat atatgt 26
<210> 9
<211>18bp
<212> DNA
<213>artificial sequence
400 > 9 of <
actggaggaa caactgac 18
<210> 10
<211>22bp
<212> DNA
<213>artificial sequence
400 > 10 of <
atcccttgtc atcgtggccg cg 22
<210> 11
<211>20bp
<212> DNA
<213>artificial sequence
400 > 11 of <
ctgcaggtca tcggcatcgc 20
<210> 12
<211>24bp
<212> DNA
<213>artificial sequence
400 > 12 of <
aagatacatt gatgaaggac gtgt 24
<210> 13
<211>17bp
<212> DNA
<213>artificial sequence
400 > 13 of <
gtgcaggcca tgctcgg 17
<210> 14
<211>25bp
<212> DNA
<213>artificial sequence
400 > 14 of <
atcccttgtc atcgtctgcc aggca 25
<210> 15
<211>16bp
<212> DNA
<213>artificial sequence
400 > 15 of <
cacgcggccc tgttcc 16
<210> 16
<211>25bp
<212> DNA
<213>artificial sequence
400 > 16 of <
aagatacatt gatgactgca gaagc 25
<210> 17
<211>20bp
<212> DNA
<213>artificial sequence
400 > 17 of <
cgggacctga ctgactacct 20
<210> 18
<211>20bp
<212> DNA
<213>artificial sequence
400 > 18 of <
ggccatctct tgctcgaagt 20
<210> 19
<211>16bp
<212> DNA
<213>artificial sequence
400 > 19 of <
accaccacgg ccgagc 16
<210> 20
<211>15bp
<212> DNA
<213>artificial sequence
400 > 20 of <
acgatgacaa gggat 15
<210> 21
<211>15bp
<212> DNA
<213>artificial sequence
400 > 21 of <
tcatcaatgt atctt 15
Claims (8)
1. a kind of detection kit, which is characterized in that the kit by: be respectively used for amplifying SLCO1B1 gene
Tetra- sites rs2306283, SLCO1B1 gene rs4149056, ApoE gene rs429358 and ApoE gene rs7412
PCR premixes reaction solution, positive reference substance and negative controls composition；
The concrete component of the PCR premix reaction solution is as follows:
(1) described for expanding SLCO1B1 gene rs2306283 site PCR premix reaction solution: by for expanding SLCO1B1 base
Because of 4 primers in the site rs2306283, primer sequence is as shown in SEQ ID NO.1 ~ SEQ ID NO.4, wherein SEQ ID
5 ' ends of primer shown in NO.2 carry FAM fluorophor, and 5 ' ends of primer shown in SEQ ID NO.4 carry VIC fluorophor；It is interior
Primer is controlled, primer sequence is as shown in SEQ ID NO.17 ~ SEQ ID NO.18；Probe groups, by specific taqman-MGB probe
With the lock nucleic acid probe composition for carrying quenching group, sequence is as shown in SEQ ID NO.19 ~ SEQ ID NO.21；PCR reaction solution
Composition；
(2) for expanding SLCO1B1 gene rs4149056 site PCR premix reaction solution: by for expanding SLCO1B1 gene
4 primers in the site rs4149056, sequence is as shown in SEQ ID NO.5 ~ SEQ ID NO.8, wherein shown in SEQ ID NO.6
5 ' ends of primer carry FAM fluorophor, and 5 ' ends of primer shown in SEQ ID NO.8 carry VIC fluorophor；Internal control primer,
Sequence is as shown in SEQ ID NO.17 ~ SEQ ID NO.18；Probe groups are quenched by specific taqman-MGB probe and carrying
The lock nucleic acid probe of group forms, and sequence is as shown in SEQ ID NO.19 ~ SEQ ID NO.21；PCR reaction solution composition；
(3) for expanding ApoE gene rs429358 site PCR premix reaction solution: by for expanding ApoE gene rs429358
4 primers in site, sequence is as shown in SEQ ID NO.9 ~ SEQ ID NO.12, wherein primer shown in SEQ ID NO.10
5 ' ends carry FAM fluorophor, and 5 ' ends of primer shown in SEQ ID NO.12 carry VIC fluorophor；Internal control primer, sequence
Column are as shown in SEQ ID NO.17 ~ SEQ ID NO.18；Base is quenched by specific taqman-MGB probe and carrying in probe groups
The lock nucleic acid probe composition of group, sequence is as shown in SEQ ID NO.19 ~ SEQ ID NO.21；PCR reaction solution composition；
(4) for expanding ApoE gene rs7412 site PCR premix reaction solution: by for expanding the site ApoE gene rs7412
4 primers, sequence is as shown in SEQ ID NO.13 ~ SEQ ID NO.16, and wherein 5 ' ends of primer shown in SEQ ID NO.14 are taken
5 ' ends of band FAM fluorophor, primer shown in SEQ ID NO.16 carry VIC fluorophor；Internal control primer, sequence such as SEQ ID
Shown in NO.17 ~ SEQ ID NO.18；Probe groups are visited by specific taqman-MGB probe and the lock nucleic acid for carrying quenching group
Needle composition, sequence is as shown in SEQ ID NO.19 ~ SEQ ID NO.21；PCR reaction solution composition；
The concrete form of the probe groups is as follows:
Internal control-P:5 '-ROX-ACCACCACGGCCGAGC-MGB-BHQ-3 '
Wild type quenching group lock nucleic acid: 5 '-BHQ-ACGATGACAAGGGAT-3 '
Saltant type quenching group lock nucleic acid: 5 '-BHQ-TCATCAATGTATCTT-3 '；
The positive reference substance includes: the site SLCO1B1 gene rs2306283 wild plasmid, SLCO1B1 gene
Rs2306283 site mutation type plasmid, the site SLCO1B1 gene rs4149056 wild plasmid, SLCO1B1 gene
Rs4149056 site mutation type plasmid, the site ApoE gene rs429358 wild plasmid, the site gene rs429358 ApoE are prominent
Modification plasmid, the site ApoE gene rs7412 wild plasmid, ApoE gene rs7412 site mutation type plasmid and reference gene
Plasmid DNA；
The negative controls include recombinant plasmid, and the recombinant plasmid does not include internal standard gene and target gene site.
2. detection kit according to claim 1, which is characterized in that in the PCR premix reaction solution, in probe groups
The concentration of taqman-MGB probe is 50 ~ 100nM, and the concentration for carrying the lock nucleic acid probe of quenching group is 100 ~ 400nM；With
It is 100 ~ 400nM in each primer concentration without fluorophor for expanding each site, each primer concentration with fluorophor is equal
For 50 ~ 100nM；The concentration of primer for internal control is 100 ~ 400nM.
3. detection kit according to claim 1, which is characterized in that the PCR reaction solution includes Premix qPCRmix
With TE buffer.
4. any detection kit of claim 1 ~ 3 is in preparation for detecting mankind's SLCO1B1 and ApoE gene pleiomorphism
Application in product.
5. applying according to claim 4, which is characterized in that specific detection method includes the following steps:
(1) genomic DNA of human peripheral to be measured is extracted as sample to be detected using genome DNA extracting reagent kit；
(2) PCR premix reaction solution and sample to be tested DNA fluorogenic quantitative detection: are added into reaction tube；Positive control is set simultaneously
Then reaction tube is placed in fluorescent PCR instrument and carries out pcr amplification reaction by group and negative control group, and it is glimmering to acquire FAM, VIC and ROX
Optical signal；
(3) after pcr amplification reaction, result judgement is carried out according to fluorescence signal initial line situation collected.
6. application according to claim 5, which is characterized in that by the sample to be detected in step (1)
It is 0.1 ~ 100ng/ μ L that DNA, which is diluted to concentration, then carries out pcr amplification reaction.
7. application according to claim 5, which is characterized in that the condition of step (2) described pcr amplification reaction are as follows: 95 DEG C
1 minute initial denaturation；15 circulation: 95 DEG C 5 seconds, 61 DEG C 32 seconds, do not collect fluorescence；30 circulation: 95 DEG C 5 seconds, 61 DEG C 32
Second, collect fluorescence.
8. application according to claim 5, which is characterized in that the criterion of step (3) described result judgement are as follows:
1. when positive controls have FAM, VIC, ROX fluorescence signal initial line, negative control group is risen without FAM, VIC, ROX fluorescence
Line when sample to be detected has ROX fluorescence signal initial line, determines Success in Experiment, can carry out subsequent parting judgement；2. according to inspection
FAM, VIC fluorescence signal of test sample sheet judge the parting in the site SLCO1B1 gene rs2306283: VIC fluorescence signal initial line,
SLCO1B1 gene rs2306283 site parting is homozygous wildtype；FAM fluorescence signal initial line, SLCO1B1 gene
The site rs2306283 parting is homozygous mutant；The equal initial line of VIC, FAM fluorescence signal, the site SLCO1B1 gene rs2306283
Parting is heterozygous mutant；3. judging SLCO1B1 gene rs4149056 according to FAM, VIC fluorescence signal of detection sample
The parting of point: VIC fluorescence signal initial line, SLCO1B1 gene rs4149056 site parting are homozygous wildtype；FAM fluorescence letter
Number initial line, SLCO1B1 gene rs4149056 site parting are homozygous mutant；The equal initial line of VIC, FAM fluorescence signal,
SLCO1B1 gene rs4149056 site parting is heterozygous mutant；4. being judged according to FAM, VIC fluorescence signal of detection sample
The parting in the site ApoE gene rs429358: VIC fluorescence signal initial line, ApoE gene rs429358 site parting are homozygous wild
Raw type；FAM fluorescence signal initial line, ApoE gene rs429358 site parting are homozygous mutant；VIC, FAM fluorescence signal are equal
Initial line, ApoE gene rs429358 site parting are heterozygous mutant；5. being sentenced according to FAM, VIC fluorescence signal of detection sample
The parting in the disconnected site ApoE gene rs7412: VIC fluorescence signal initial line, ApoE gene rs7412 site parting are homozygous wild
Type；FAM fluorescence signal initial line, ApoE gene rs7412 site parting are homozygous mutant；The equal initial line of VIC, FAM fluorescence signal,
ApoE gene rs7412 site parting is heterozygous mutant.
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