CN107267620A - GSTP1 genetic polymorphism detections system and its kit - Google Patents
GSTP1 genetic polymorphism detections system and its kit Download PDFInfo
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- CN107267620A CN107267620A CN201710540244.2A CN201710540244A CN107267620A CN 107267620 A CN107267620 A CN 107267620A CN 201710540244 A CN201710540244 A CN 201710540244A CN 107267620 A CN107267620 A CN 107267620A
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Abstract
The present invention relates to a kind of GSTP1 genetic polymorphism detections system, including detection primer pair, detection probe and peptide nucleic acid, detection primer is to including the outside upstream and downstream primer on the outside of detection GSTP1 Gene A 342G pleomorphism sites, detect the inner side upstream and downstream primer on the inside of GSTP1 Gene A 342G pleomorphism sites, detection probe includes wild-type probe and homozygote probe, peptide nucleic acid includes the GSTP1 genetic polymorphism detections system and its kit with the peptide nucleic acid of inner side sense primer partial complementarity and with the peptide nucleic acid present invention of inner side anti-sense primer partial complementarity, GSTP1 Gene A 342G polymorphic sites can be detected, sensitivity is high, high specificity, it is efficient and convenient.
Description
Technical field
The present invention relates to the primer used in a kind of detection product of gene pleiomorphism and the product, probe, detection body
System, belongs to biological technical field.
Background technology
Genomic DNA changes on a certain specific nucleotide position, transversion, insertion or missing, and it is in group
Frequency >=1% occurred in body, referred to as SNP (SNP), the reparation work(of gene pleiomorphism and some genomic DNAs
Can be relevant, polymorphism can significantly affect sensitiveness of the individual to medicine, and the medical diagnosis on disease of molecular level can be to tumour individuation
Treatment and Index for diagnosis provide guidance.
GSTP1 (glutathione S-transferase pi-1), i.e. Glutathione S-transferases M1, are that a class is responsible for
A member in the enzyme family GSTs of a variety of carcinogen metabolism, because II phase metabolic enzyme GSTs participates in the solution of carcinogenic substance in vivo
Malicious process, while the encoding gene of many members (such as GSTP1) has polymorphism in the enzyme system, the variation of gene can change phase
The enzyme activition or the ability of the heterologous substrate of inactivation answered, may the individual danger for suffering from tumour of increase, the base that research has been found that
A kind of A342G base mutations multiformity of cause, makes the amino acids of coding the 105th change over Val from Ile, have impact on the work(of gene
Can, so as to equivalent to the concentration for adding environmental toxin and carcinogen, improve a series of wind that individual suffers from types of cancer
Danger.The genotype in GSTP1A342G sites is detected, for the early warning of pathogenesis of cancer, the drug screening for the treatment of of cancer and pre-
All there is important effect afterwards.It is main at present to survey using generation sequencing, FLP, the oligonucleotide hybridization of allele specific, equipotential
The technologies such as the PCR and genetic chip of gene specific, but operating process is cumbersome, it is necessary to change reaction tube and easily pollute, it is many
Need electrophoresis and multistep to post-process, influence the accuracy of testing result.Or instrument and equipment is expensive, it is difficult to large-scale promotion application.
Traditional nest-type PRC reaction not only takes, open pipe operation easily causes cross pollution to lead due to there is 2 PCR amplifications
The inaccuracy of false positive and result is caused, and testing result needs electrophoresis and gel imaging system just to see result, so as to drop
The low possibility of the multiple target spots of amplification, comes out from PCR to testing result and takes around 5h, is mainly used in molecular biology neck
In domain and medical science detection research.
The content of the invention
Can accurately it be detected under conditions of sample original template amount is low the technical problem to be solved in the present invention is to provide one kind
Go out the GSTP1 genetic polymorphism detection kits of the genotype of sample gene GSTP1 A342G polymorphic sites, and the reagent
Detection architecture used in box.
The present invention is that a kind of technical scheme for solving above-mentioned technical problem proposition is：A kind of GSTP1 genetic polymorphism detections
System, including detection primer is to, detection probe and peptide nucleic acid,
The detection primer is to including the nucleotide sequence such as SEQ on the outside of detection GSTP1 Gene A 342G pleomorphism sites
The nucleotide sequence such as SEQ on the outside of outside sense primer, detection GSTP1 Gene A 342G pleomorphism sites shown in ID No.1
The nucleotide sequence such as SEQ on the inside of outside anti-sense primer, detection GSTP1 Gene A 342G pleomorphism sites shown in ID No.2
The nucleotide sequence such as SEQ on the inside of inner side sense primer, detection GSTP1 Gene A 342G pleomorphism sites shown in ID No.3
Inner side anti-sense primer shown in ID No.4；The outside upstream and downstream primer introduces deoxyinosine；
The detection probe includes the nucleotide sequence such as SEQ that detection GSTP1 Gene A 342G pleomorphism sites are wild type
Wild-type probe shown in ID No.7 and detection GSTP1 Gene A 342G pleomorphism sites be homozygous nucleotide sequence such as
Homozygote probe shown in SEQ ID No.8；The wild-type probe and homozygote probe introduce deoxyinosine and mispairing
Base；The peptide nucleic acid includes the nucleosides of the inner side sense primer partial complementarity with detecting GSTP1 Gene A 342G pleomorphism sites
Peptide nucleic acid of the acid sequence as shown in SEQ ID No.5 and the inner side anti-sense primer with detecting GSTP1 Gene A 342G pleomorphism sites
Peptide nucleic acid of the nucleotide sequence of partial complementarity as shown in SEQ ID No.6.
Above-mentioned GSTP1 genetic polymorphism detections system also include internal control primer to, internal reference probe, Quality Control primer pair and Quality Control
Probe；
The nucleotide sequence of the internal control primer sense primer as shown in SEQ ID No.9, the internal reference downstream primer
Nucleotide sequence is as shown in SEQ ID No.10, and the nucleotide sequence of the internal reference probe is as shown in SEQ ID No.11；
The nucleotide sequence of the Quality Control sense primer is as shown in SEQ ID No.12, the nucleosides of the Quality Control anti-sense primer
Acid sequence is as shown in SEQ ID No.13, and the nucleotide sequence of the Quality Control probe is as shown in SEQ ID No.14.
5 ' ends of each above-mentioned probe are provided with fluorescent reporter group, and 3 ' ends of each probe are provided with fluorescent quenching base
Group；The fluorescent reporter group is FAM, HEX, ROX or Cy5, and the fluorescent quenching group is BHQ1, BHQ2 or TAMRA.
Ultimate density 0.05 μm/l of the above-mentioned outside upstream and downstream primer in reaction system, the inner side upstream and downstream primer exists
0.19 μm/l of ultimate density in reaction system, ultimate density of the peptide nucleic acid in reaction system is 1nm/l, described wild
Ultimate density of the type probe in reaction system is 0.19 μm/l, and ultimate density of the homozygote probe in reaction system is
0.19μm/l；
Ultimate density of the Quality Control primer pair in reaction system is 0.19 μm/l, and the internal control primer is in reactant
Ultimate density in system is 0.19 μm/l, and ultimate density of the internal reference probe in reaction system is 0.19 μm/l, the matter
It is 0.19 μm/l to control ultimate density of the probe in reaction system.
Above-mentioned GSTP1 genetic polymorphism detections system also includes formamide, and the volume of formamide is added in reaction system and is
The 12% of reaction system cumulative volume.
The present invention is that a kind of technical scheme for solving above-mentioned technical problem proposition is：A kind of above-mentioned detection architecture of use
GSTP1 genetic polymorphism detection products.
The present invention is that a kind of technical scheme for solving above-mentioned technical problem proposition is：A kind of GSTP1 genetic polymorphism detections
Kit, including PCR detections liquid, Quality Control detection liquid, positive control and negative control；
PCR detection liquid includes detection primer to, detection probe and peptide nucleic acid；
The detection primer is to including the nucleotide sequence such as SEQ on the outside of detection GSTP1 Gene A 342G pleomorphism sites
The nucleotide sequence such as SEQ on the outside of outside sense primer, detection GSTP1 Gene A 342G pleomorphism sites shown in ID No.1
The nucleotide sequence such as SEQ on the inside of outside anti-sense primer, detection GSTP1 Gene A 342G pleomorphism sites shown in ID No.2
The nucleotide sequence such as SEQ on the inside of inner side sense primer, detection GSTP1 Gene A 342G pleomorphism sites shown in ID No.3
Inner side anti-sense primer shown in ID No.4；The outside upstream and downstream primer introduces deoxyinosine；
The detection probe includes the nucleotide sequence such as SEQ that detection GSTP1 Gene A 342G pleomorphism sites are wild type
Wild-type probe shown in ID No.7 and detection GSTP1 Gene A 342G pleomorphism sites be homozygous nucleotide sequence such as
Homozygote probe shown in SEQ ID No.8；The wild-type probe and homozygote probe introduce deoxyinosine and mispairing
Base；
The peptide nucleic acid includes the inner side sense primer partial complementarity with detecting GSTP1 Gene A 342G pleomorphism sites
Peptide nucleic acid of the nucleotide sequence as shown in SEQ ID No.5 and the inner side downstream with detecting GSTP1 Gene A 342G pleomorphism sites
Peptide nucleic acid of the complementary nucleotide sequence of primer portion as shown in SEQ ID No.6；
Quality Control detection liquid include internal control primer to, internal reference probe, Quality Control primer pair and Quality Control probe；
The nucleotide sequence of the internal control primer sense primer as shown in SEQ ID No.9, the internal reference downstream primer
Nucleotide sequence is as shown in SEQ ID No.10, and the nucleotide sequence of the internal reference probe is as shown in SEQ ID No.11；
The nucleotide sequence of the Quality Control sense primer is as shown in SEQ ID No.12, the nucleosides of the Quality Control anti-sense primer
Acid sequence is as shown in SEQ ID No.13, and the nucleotide sequence of the Quality Control probe is as shown in SEQ ID No.14；
The positive control solution is that concentration is wild plasmid and the mixed solution of homozygote plasmid；
The negative controls are that concentration is without GSTP1 genes wild type, heterozygote and homozygous plasmid solution.
Also include PCR buffer solutions, 2.5mM dNTPs and 5U/ μ l Taq in above-mentioned PCR detection liquid and Quality Control detection liquid
Archaeal dna polymerase；The PCR buffer solutions include 100mM Tris-HCl, 15mM KCl and 15mM MgCl2, for configuring
The pH value for stating the Tris-HCl buffer solutions of PCR buffer solutions is 8.3；The dNTPs includes dATP, dGTP, dCTP and dTTP.
Ultimate density 0.05 μm/l of the above-mentioned outside upstream and downstream primer in reaction system, the inner side upstream and downstream primer exists
0.19 μm/l of ultimate density in reaction system, ultimate density of the peptide nucleic acid in reaction system is 1nm/l, described wild
Ultimate density of the type probe in reaction system is 0.19 μm/l, and ultimate density of the homozygote probe in reaction system is
0.19μm/l；
Ultimate density of the Quality Control primer pair in reaction system is 0.19 μm/l, and the internal control primer is in reactant
Ultimate density in system is 0.19 μm/l, and ultimate density of the internal reference probe in reaction system is 0.19 μm/l, the matter
It is 0.19 μm/l to control ultimate density of the probe in reaction system.
Above-mentioned GSTP1 genetic polymorphism detections kit also includes the volume that formamide is added in formamide, reaction system
It is the 12% of reaction system cumulative volume.
The present invention has positive effect：
(1) GSTP1 genetic polymorphism detections system of the invention and its kit, using special primer probe combinations, are adopted
With the Chao Shi PCR after improvement, the sensitiveness and reliability of detection are added, stopped pipe operation reduction is polluted, with high sensitivity,
Sensitivity can reach 0.5%~1%, compared with the concentration of Standard PCR, does invention and easily detects unusual low concentration sample
The genotype in GSTP1 (A342G) site, can provide guidance for the clinical application of platinum medicine.
(2) hypersensitivity for remaining original fluorescent quantitation of the invention and it is specific on the basis of, greatly shorten
Experimental period, improves detection efficiency, sample, and the genetic polymorphism detection kit of the present invention is saved, using goldstandard
Control experiment is carried out, genotype coincidence rate >=99%, the molecule parting degree of accuracy is high.
(3) present invention devises internal reference and Quality Control and the whole detection process of detection is monitored, it is ensured that sample can detect result
Accuracy and reliability.
Brief description of the drawings
Fig. 1 detects positive control amplification curve using the kit of embodiment.
Fig. 2 detects wild sample amplification curve using the kit of embodiment.
Fig. 3 detects homozygosis sample amplification curve using the kit of embodiment.
Fig. 4 detects heterozygosis sample amplification curve using the kit of embodiment.
Embodiment
The present invention is specifically described below by embodiment, it is necessary to it is pointed out here that be that following examples are only used
It is further described in the present invention, it is impossible to be interpreted as limiting the scope of the invention, person skilled in art can
To make some nonessential modifications and adaptations to the present invention according to the invention described above content.In following embodiments, if not specially
Show, reagent used is that analysis is pure, and agents useful for same can be obtained from commercial channel.The experiment of unreceipted actual conditions in text
Method, what the Science Press generally write according to normal condition such as J. Pehanorm Brookers etc. published for 2002《Molecular cloning is real
Test guide》Condition described in one book, or according to the condition proposed by manufacturer.Unless otherwise defined, the institute used in text
There is specialty identical with meaning known to one skilled in the art with scientific words.In addition, it is any similar to described content or
Impartial method and material all can be applied in the present invention.
Embodiment
First, the composition of kit.
GSTP1 (A342G) genetic polymorphism detection kit of the present embodiment includes：PCR detection liquid, Quality Control detection liquid,
Positive control, negative control, specific composition are as shown in table 1.
The kit forms table of table 1
Kit each component is described as follows in above-mentioned table 1：
PCR detects liquid by 10 × PCR buffer solutions, dNTPs, thermal starting enzyme, primer pair, probe and peptide nucleic acid Compound mixed solution
Form.10 × PCR buffer solutions include 100mM Tris-HCl, 500mM KCl and 15mM MgCl2, for configuring PCR bufferings
The pH value of the Tris-HCl buffer solutions of liquid is 8.3.DNTPs includes dATP, dGTP, dCTP and dTTP, dense eventually in reaction system
Spend for 0.2mM.Thermal starting enzyme is the Taq archaeal dna polymerases that concentration is 5U/ μ l, the final concentration 0.05U/ μ in reaction system
l.10 × PCR buffer solutions, dNTPs and thermal starting enzyme are all from Takara (article No.s：R007A).Primed probe peptide nucleic acid mixed liquor
Outside sense primer including detection GSTP1 (A342G), detection GSTP1 (A342G) outside anti-sense primer, detection GSTP1
(A342G) inner side sense primer, detection GSTP1 (A342G) inner side anti-sense primer, on GSTP1 (A342G) inner side
Swim primer peptide nucleic acid, for GSTP1 (A342G) inner side anti-sense primer peptide nucleic acid, detection GSTP1 (A342G) it is wild
The homozygote probe of type probe, detection GSTP1 (A342G), sequence is shown in Table 2, by the Li Ge Bioisystech Co., Ltd of Shanghai hundred
Synthesis.Ultimate density of the Outside primer in reaction system is 0.05 μm/l, ultimate density of the inner primer in reaction system
For 0.19 μm/l, peptide nucleic acid is 1nm/l, detection GSTP1 (A342G) wild-type probes and detection in the final concentration of reaction system
Ultimate density of GSTP1 (A342G) the homozygote probes in end reaction system is 0.19 μm/l.In order to reduce non-specific expansion
Increase, improve specificity and the sensitivity of detection, 12% carboxamide agents are added in the detection reaction system of the present invention.
Control liquid is formed by 10 × PCR buffer solutions, dNTPs, thermal starting enzyme, primer pair and probe Compound mixed solution.10×
PCR buffer solutions include 100mM Tris-HCl, 500mM KCl and 15mM MgCl2, the Tris- for configuring PCR buffer solutions
The pH value of HCl buffer solutions is 8.3.DNTPs includes dATP, dGTP, dCTP and dTTP, the final concentration of 0.2mM in reaction system.
Thermal starting enzyme is the Taq archaeal dna polymerases that concentration is 5U/ μ l, the final concentration 0.05U/ μ l in reaction system.10 × PCR delays
Fliud flushing, dNTPs and thermal starting enzyme are all from Takara (article No.s：R007A).Primed probe mixed liquor includes detection internal reference upstream and drawn
Thing, the anti-sense primer for detecting internal reference, the probe for detecting internal reference, detection Quality Control sense primer, detection Quality Control anti-sense primer, detection matter
The probe of control, sequence is shown in Table 2, and primer, probe and peptide nucleic acid are synthesized by Shanghai Sheng Gong bioengineering Co., Ltd.Internal control primer
With probe reaction system ultimate density be 0.19 μm/l, Quality Control primer and probe reaction system ultimate density be 0.19
μm/l.In order to reduce non-specific amplification, specificity and the sensitivity of detection are improved, is added in the Quality Control reaction system of the present invention
Enter 12% carboxamide agents.
Positive control solution is the mixed solution of the wild plasmid that concentration is 10ng/ μ l and 10ng/ μ l homozygote plasmid.
Negative controls are that concentration is molten without GSTP1 genes wild type, heterozygote and homozygous plasmid for 10ng/ μ l
Liquid.
Wild plasmid is retrieved as the conventional construction step of wild plasmid：Design is located at the primer of mutational site both sides,
The wild type product in the site containing correspondence is expanded, plasmid is built into, selected and sequence verification.
Homozygote plasmid is retrieved as the conventional construction step of homozygote plasmid：The corresponding pleomorphism site of one covering of design is simultaneously
Bit base will be waited to be incorporated into the special primer in primer sequence, matched with corresponding upstream or downstream wild primers, amplification
Purpose fragment product containing corresponding site, is built into plasmid, selects and sequence verification.
Sense primer, anti-sense primer, probe and peptide nucleic acid nucleotide sequence it is as shown in table 2.
The primer of table 2, probe, peptide nucleic acid sequence table
| Primed probe title | Seq No. | Nucleotide sequence (5 ' -3 ') |
| Sense primer on the outside of GSTP1 | 1 | CTCATCCTTCCACGCACATCC |
| Anti-sense primer on the outside of GSTP1 | 2 | CCCCCATGACCCGTTA |
| Sense primer on the inside of GSTP1 | 3 | GTGGACATGGTGAATGACG |
| Anti-sense primer on the inside of GSTP1 | 4 | TGGTGCAGATGCTCACATAGT |
| Upstream peptide nucleic acid on the inside of GSTP1 | 5 | CATGGTGAATGACG |
| Downstream peptide nucleic acid on the inside of GSTP1 | 6 | GATGCTCACATAG |
| GSTP1 wild-type probes | 7 | GGACCTCCGCTGCAAGTACA |
| GSTP1 homozygote probes | 8 | AGGACCTCCGCTGCAACTACG |
| Internal reference upstream primer | 9 | TGTAGGAGGGACTTAGAGAAG |
| Internal reference downstream primer | 10 | ACCCTTTAGGGAGAAAACGCT |
| Internal reference probe | 11 | CCTGCCCTTTGAGTTTGATGAT |
| Quality Control sense primer | 12 | TGGGAGGGATGAGAGTAGGAT |
| Quality Control anti-sense primer | 13 | CCCACAATGAAGGTCTTGC |
| Quality Control probe | 14 | GATGACTATGTGAAGGCACT |
GSTP1 Outside primers are drawing for GSTP1 (A342G) pleomorphism site both sides product length about 300-500bp
Thing, GSTP1 inner primers are the primers for GSTP1 (A342G) pleomorphism sites both sides product length about 90-150bp, in order to
Outside primer is set to be combined prior to inner primer with DNA profiling, Outside primer introduces peptide nucleic acid on the inside of deoxyinosine, GSTP1
It is the DNA analogs for the sense primer on the inside of GSTP1 (A342G) pleomorphism site and the design of inner side anti-sense primer, peptide core
Acid moieties sequence is overlapped with inner primer, can be combined prior to inner side sense primer with DNA profiling, so that in the reaction starting stage
The amplification of inner primer is inhibited, GSTP1 wild-type probes and GSTP1 homozygote probes are to be directed to GSTP1 (A342G) polymorphism
The specific probe of site design, 5 ' ends of probe are marked with fluorescence signal group, fluorescence signal group have FAM, HEX, ROX,
CY5 etc., 3 ' ends of probe are marked with fluorescent quenching group, and fluorescent quenching group is including BHQ1, BHQ2, TAMRA etc., due to wild
Type probe and homozygote probe sequence only differ a base, and specificity is relatively low, and deoxyinosine is incorporated into inspection by the present invention
Survey in polymorphic detection specific probe, and 1 base mismatch is introduced at 3 ' ends of probe, probe is improved to a certain extent
Specificity.
The fluorescence signal group of the wild-type probe of the present embodiment is FAM, and the fluorescence signal group of homozygote probe is
HEX。
Internal control primer probe is the GAPDH genes for being different from GSTP1, by detecting the amplification of reference gene, can analyze reality
Test result validity.
The design of primers of Quality Control gene is that the GSTP1 genes other positions excluded beyond GSTP1 gene Outside primers are guarded
Region, Quality Control primed probe goes without abrupt climatic change site primer and primer binding site, in the different section designs of conservative region
Primed probe, Quality Control sequence amplification is acted on except PCR polymerize enzyme level in recoverable such as DNA content, sample in the present invention
The difference of amplification efficiency between thing, differential responses pipe, is additionally operable to determine sample base by the amplification situation of icp gene type and Quality Control
Because of type.
2nd, the application method of kit.
The specific detecting step of GSTP1 (A342G) genetic polymorphism detection kit of the present embodiment is as follows：
1st, sample DNA is extracted.
Sample blood sample is extracted using kit (Axygen Multisource Genomic DNA Miniprep Kit)
This DNA, or health is used for the buccal swab genome DNA extracting reagent kit (article No. of century bio tech ltd：
CW0530S mucous membrane of mouth sample DNA) is extracted, is specifically operated referring to reagent kit product specification.
2nd, sample DNA quality testing.
Obtain after sample DNA, concentration is determined by Thermo-Fisher nucleic acid-proteins quantitative instrument (NanoDrop 2000)
And purity, sample quality is controlled, the sample in reaction system is ultimately joined.OD260/OD280 ratio is between 1.8~2.0
Obtain peak optimization reaction result, concentration dilution is 5~20ng/ μ l.
3rd, PCR reacts.
Detected using GSTP1 (A342G) the genetic polymorphism detections kit examination of the present embodiment, the body of reaction system
Product is 10 μ l, and the concentration of kit each component and the final concentration in reaction system refer to table 1.(the volume of reaction system
20 μ l are can also be, when preparing that the component in 10 μ l reaction systems is double).
1) 10 μ l Quality Control PCR reaction systems and 10 μ l detection PCR reaction systems are prepared：
Quality Control PCR reaction systems：9 μ l Quality Control is taken to detect liquid and 1 μ l sample DNAs.Detect PCR reaction systems：9 μ l are not taken
Detection reagent and 1 μ l templates (if DNA sample, 5~20ng/ of concentration μ l), and repetition is set.
Template in reaction system refers to corresponding sample DNA, positive control, negative control, blank control, blank pair respectively
It is ultra-pure water according to liquid.
2) PCR response procedures.
Each reaction system carries out real-time fluorescence PCR reaction on ABI real-time fluorescence quantitative PCRs amplification instrument (stepone), most
Excellent response procedures are as shown in table 3.
Table 3PCR response procedures tables
Preferably, the condition of PCR amplifications is：95 DEG C of the condition in pre-degeneration stage, 10min；1,10 circulations of circulation, condition
For 95 DEG C of 15s, 55 DEG C of annealing temperature, the time is 10 seconds, 72 DEG C of elongating temperature, 30 seconds time；Circulation 2,30 is circulated, and condition is
94 DEG C of 15s, 60 DEG C of annealing temperature, the time is 10 seconds；It is 4 DEG C of insulations to circulate 3 conditions.The present invention is in order to prevent circulation section 2 longer
Fragment non-specific amplification, be not provided with extension program.
4th, PCR result judgements.
1) judgement of kit validity.
Positive control：All amplification curves of positive control FAM and HEX signalling channel have obvious Exponential growth stage, and
The Ct values positive≤25.
Negative control：All amplification curves of negative control FAM, HEX signalling channel are without obvious Exponential growth stage, or Ct
Value feminine gender >=40.
Blank control：All amplification curves of blank control negative control FAM, HEX signalling channel increase without obvious index
For a long time.
2) judgement of sample availability is detected.
Judged according to the CT values of internal reference FAM passages and Quality Control HEX passages：
Internal reference GAPDH FAM signalling channel amplification curves are S-type, but Ct<18, or Ct>32, testing result is invalid；
Internal reference GAPDH FAM signalling channel amplification curves are not S-type, but and 18≤Ct≤32 between, pattern detection result
It is invalid；
Internal reference GAPDH FAM signalling channel amplification curves are S-type, and between 18≤Ct≤32, sample is effective.
Quality Control GSTP1 HEX signalling channel amplification curves are S-type, and Ct ＜ 20, then sample genome is added excessively, is built
Detected again after view dilution；
Quality Control GSTP1 HEX signalling channel amplification curves are S-type, and 20≤Ct≤30, sample addition is moderate, be adapted into
Row result judgement；
Quality Control GSTP1 HEX signalling channel amplification curves are S-type, Ct ＞ 30, then sample genome addition is low, it is impossible to
Stable carry out mutation result judgement.
3) judgement of polymorphism result.
By above step, it is determined that under the premise of detecting sample and experimental result effectively, then the result of sample is carried out
Judge.Wild-type genotype detection is the amplification situation for detecting FAM signalling channels in reaction solution, and homozygous genotype detection is that detection is anti-
Answer the amplification situation of HEX signalling channels in liquid.Interpretation is carried out to pattern detection result according to following steps, sample gene is determined
Type.
If the amplification curve for A. having FAM signalling channels in detection liquid has obvious Exponential growth stage, the expansion of HEX signalling channels
Increase curve without obvious Exponential growth stage, and 20≤Ct≤35, then it is wild type to detect sample.
If the amplification curve for B. having HEX signalling channels in detection liquid has obvious Exponential growth stage, the expansion of FAM signalling channels
Increase curve without obvious Exponential growth stage, and 20≤Ct≤35, then detect sample to be homozygous.
If the amplification curve for C. having FAM and HEX signalling channels in detection liquid has obvious Exponential growth stage, and |
Ct(detection FAM)-Ct(detection HEX)|≤5, then it is heterozygous to detect sample.
If the amplification curve for D. having FAM and HEX signalling channels in detection liquid has obvious Exponential growth stage, |
Ct(detection FAM)-Ct(detection HEX)|>5, then the less CT values of detection are compared with Quality Control CT, difference between the two is denoted as △ △ CT,
If △ △ CT=| Ct(detection FAM/HEX)-Ct(Quality Control HEX)|≤7, then it is the smaller testing result genotype of CT values, if △ △ CT=|
Ct(detection FAM/HEX)-Ct(Quality Control HEX)|>7, detect again.
Obviously, above-described embodiment is only intended to clearly illustrate example of the present invention, and is not to the present invention
The restriction of embodiment.For those of ordinary skill in the field, it can also be made on the basis of the above description
Its various forms of changes or variation.There is no necessity and possibility to exhaust all the enbodiments.And these belong to this hair
Among the obvious changes or variations that bright spirit is extended out is still in protection scope of the present invention.
SEQUENCE LISTING
<110>Match peaceful thing medical sci-tech limited company in Shanghai
<120>GSTP1 genetic polymorphism detections system and its kit
<130>Nothing
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 1
ctcatccttc cacgcacatc c 21
<210> 2
<211> 16
<212> DNA
<213>It is artificial synthesized
<400> 2
cccccatgac ccgtta 16
<210> 3
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 3
gtggacatgg tgaatgacg 19
<210> 4
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 4
tggtgcagat gctcacatag t 21
<210> 5
<211> 14
<212> DNA
<213>It is artificial synthesized
<400> 5
catggtgaat gacg 14
<210> 6
<211> 13
<212> DNA
<213>It is artificial synthesized
<400> 6
gatgctcaca tag 13
<210> 7
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 7
ggacctccgc tgcaagtaca 20
<210> 8
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 8
aggacctccg ctgcaactac g 21
<210> 9
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 9
tgtaggaggg acttagagaa g 21
<210> 10
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 10
accctttagg gagaaaacgc t 21
<210> 11
<211> 22
<212> DNA
<213>It is artificial synthesized
<400> 11
cctgcccttt gagtttgatg at 22
<210> 12
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 12
tgggagggat gagagtagga t 21
<210> 13
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 13
cccacaatga aggtcttgc 19
<210> 14
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 14
gatgactatg tgaaggcact 20
Claims (10)
1. a kind of GSTP1 genetic polymorphism detections system, it is characterised in that：Including detection primer to, detection probe and peptide nucleic acid,
The detection primer is to including the nucleotide sequence such as SEQ ID on the outside of detection GSTP1 Gene A 342G pleomorphism sites
The nucleotide sequence such as SEQ ID on the outside of outside sense primer, detection GSTP1 Gene A 342G pleomorphism sites shown in No.1
The nucleotide sequence such as SEQ ID on the inside of outside anti-sense primer, detection GSTP1 Gene A 342G pleomorphism sites shown in No.2
The nucleotide sequence such as SEQ ID on the inside of inner side sense primer, detection GSTP1 Gene A 342G pleomorphism sites shown in No.3
Inner side anti-sense primer shown in No.4；The outside upstream and downstream primer introduces deoxyinosine；
The detection probe includes the nucleotide sequence such as SEQ ID that detection GSTP1 Gene A 342G pleomorphism sites are wild type
Wild-type probe and detection GSTP1 Gene A 342G pleomorphism sites shown in No.7 are homozygous nucleotide sequence such as SEQ
Homozygote probe shown in ID No.8；The wild-type probe and homozygote probe introduce deoxyinosine and base mismatch；
The peptide nucleic acid includes the nucleotides sequence of the inner side sense primer partial complementarity with detecting GSTP1 Gene A 342G pleomorphism sites
Arrange the peptide nucleic acid as shown in SEQ ID No.5 and the inner side anti-sense primer part with detecting GSTP1 Gene A 342G pleomorphism sites
Peptide nucleic acid of the complementary nucleotide sequence as shown in SEQ ID No.6.
2. GSTP1 genetic polymorphism detections system according to claim 1, it is characterised in that：Also include internal control primer to,
Internal reference probe, Quality Control primer pair and Quality Control probe；
The nucleotide sequence of the internal control primer sense primer is as shown in SEQ ID No.9, the nucleosides of the internal reference downstream primer
Acid sequence is as shown in SEQ ID No.10, and the nucleotide sequence of the internal reference probe is as shown in SEQ ID No.11；
The nucleotide sequence of the Quality Control sense primer is as shown in SEQ ID No.12, the nucleotides sequence of the Quality Control anti-sense primer
Row are as shown in SEQ ID No.13, and the nucleotide sequence of the Quality Control probe is as shown in SEQ ID No.14.
3. GSTP1 genetic polymorphism detections system according to claim 2, it is characterised in that：The 5 ' of each probe
End is provided with fluorescent reporter group, and 3 ' ends of each probe are provided with fluorescent quenching group；The fluorescent reporter group be FAM,
HEX, ROX or Cy5, the fluorescent quenching group are BHQ1, BHQ2 or TAMRA.
4. GSTP1 genetic polymorphism detections system according to claim 2, it is characterised in that：The outside upstream and downstream is drawn
Ultimate density 0.05 μm/l of the thing in reaction system, ultimate density 0.19 of the inner side upstream and downstream primer in reaction system
μm/l, ultimate density of the peptide nucleic acid in reaction system is 1nm/l, and the wild-type probe is final in reaction system
Concentration is 0.19 μm/l, and ultimate density of the homozygote probe in reaction system is 0.19 μm/l；
Ultimate density of the Quality Control primer pair in reaction system is 0.19 μm/l, and the internal control primer is in reaction system
Ultimate density be 0.19 μm/l, ultimate density of the internal reference probe in reaction system is 0.19 μm/l, and the Quality Control is visited
Ultimate density of the pin in reaction system is 0.19 μm/l.
5. GSTP1 genetic polymorphism detections system according to claim 1 or 2, it is characterised in that：Also include formamide,
The volume that formamide is added in reaction system is the 12% of reaction system cumulative volume.
6. a kind of GSTP1 genetic polymorphism detection products using detection architecture as claimed in claim 1.
7. a kind of GSTP1 genetic polymorphism detections kit, it is characterised in that：Including PCR detections liquid, Quality Control detection liquid, the positive
Control and negative control；
PCR detection liquid includes detection primer to, detection probe and peptide nucleic acid；
The detection primer is to including the nucleotide sequence such as SEQ ID on the outside of detection GSTP1 Gene A 342G pleomorphism sites
The nucleotide sequence such as SEQ ID on the outside of outside sense primer, detection GSTP1 Gene A 342G pleomorphism sites shown in No.1
The nucleotide sequence such as SEQ ID on the inside of outside anti-sense primer, detection GSTP1 Gene A 342G pleomorphism sites shown in No.2
The nucleotide sequence such as SEQ ID on the inside of inner side sense primer, detection GSTP1 Gene A 342G pleomorphism sites shown in No.3
Inner side anti-sense primer shown in No.4；The outside upstream and downstream primer introduces deoxyinosine；
The detection probe includes the nucleotide sequence such as SEQ ID that detection GSTP1 Gene A 342G pleomorphism sites are wild type
Wild-type probe and detection GSTP1 Gene A 342G pleomorphism sites shown in No.7 are homozygous nucleotide sequence such as SEQ
Homozygote probe shown in ID No.8；The wild-type probe and homozygote probe introduce deoxyinosine and base mismatch；
The peptide nucleic acid includes the nucleosides of the inner side sense primer partial complementarity with detecting GSTP1 Gene A 342G pleomorphism sites
Peptide nucleic acid of the acid sequence as shown in SEQ ID No.5 and the inner side anti-sense primer with detecting GSTP1 Gene A 342G pleomorphism sites
Peptide nucleic acid of the nucleotide sequence of partial complementarity as shown in SEQ ID No.6；
Quality Control detection liquid include internal control primer to, internal reference probe, Quality Control primer pair and Quality Control probe；
The nucleotide sequence of the internal control primer sense primer is as shown in SEQ ID No.9, the nucleosides of the internal reference downstream primer
Acid sequence is as shown in SEQ ID No.10, and the nucleotide sequence of the internal reference probe is as shown in SEQ ID No.11；
The nucleotide sequence of the Quality Control sense primer is as shown in SEQ ID No.12, the nucleotides sequence of the Quality Control anti-sense primer
Row are as shown in SEQ ID No.13, and the nucleotide sequence of the Quality Control probe is as shown in SEQ ID No.14；
The positive control solution is that concentration is wild plasmid and the mixed solution of homozygote plasmid；
The negative controls are that concentration is without GSTP1 genes wild type, heterozygote and homozygous plasmid solution.
8. GSTP1 genetic polymorphism detections kit according to claim 7, it is characterised in that：PCR detection liquid and
Also include PCR buffer solutions, 2.5mM dNTPs and 5U/ μ l Taq archaeal dna polymerases in Quality Control detection liquid；The PCR buffer solutions
The KCl and 15mM of Tris-HCl, 15mM including 100mM MgCl2, for configure the PCR buffer solutions Tris-HCl delay
The pH value of fliud flushing is 8.3；The dNTPs includes dATP, dGTP, dCTP and dTTP.
9. GSTP1 genetic polymorphism detections kit according to claim 7, it is characterised in that：The outside upstream and downstream
Ultimate density 0.05 μm/l of the primer in reaction system, ultimate density of the inner side upstream and downstream primer in reaction system
0.19 μm/l, ultimate density of the peptide nucleic acid in reaction system is 1nm/l, and the wild-type probe is in reaction system
Ultimate density is 0.19 μm/l, and ultimate density of the homozygote probe in reaction system is 0.19 μm/l；
Ultimate density of the Quality Control primer pair in reaction system is 0.19 μm/l, and the internal control primer is in reaction system
Ultimate density be 0.19 μm/l, ultimate density of the internal reference probe in reaction system is 0.19 μm/l, and the Quality Control is visited
Ultimate density of the pin in reaction system is 0.19 μm/l.
10. GSTP1 genetic polymorphism detections kit according to claim 7, it is characterised in that：Also include formamide,
The volume that formamide is added in reaction system is the 12% of reaction system cumulative volume.
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Application publication date: 20171020 |