CN107190080A - CDA genetic polymorphism detections system and its kit - Google Patents
CDA genetic polymorphism detections system and its kit Download PDFInfo
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- CN107190080A CN107190080A CN201710540256.5A CN201710540256A CN107190080A CN 107190080 A CN107190080 A CN 107190080A CN 201710540256 A CN201710540256 A CN 201710540256A CN 107190080 A CN107190080 A CN 107190080A
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- 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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Abstract
The present invention relates to a kind of CDA 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 CDA Gene A 79C pleomorphism sites, detect the inner side upstream and downstream primer on the inside of CDA Gene A 79C pleomorphism sites, detection probe includes wild-type probe and homozygote probe, peptide nucleic acid includes the CDA 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, CDA Gene A 79C 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.
CDA (Cytidinedeaminase, cytidine deaminase) is a kind of enzyme in pyrimidine rescue approach, the drop of CDA activity
The function of detoxification of the low internal gemcitabine of influence, causes the increase of poisonous side effect of medicine.Neutropenia is Ji Xi
The most common adverse reaction in his shore.SNP in multinomial clinical studies show, CDA gene orders can influence Ji Xi
The blood concentration of his shore, and then influence the adverse reaction after its medication.Clinically by detection CDA gene pleiomorphism, instruct
Medication and personalized treatment.It is main at present to survey using generation sequencing, FLP, the oligonucleotide hybridization of allele specific, equipotential base
Because of technologies such as special PCR and genetic chip, but operating process is cumbersome, it is necessary to change reaction tube and easily pollute, and needs more
Want 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 CDA genetic polymorphism detection kits of the genotype of sample gene C DA A79C polymorphic sites, and used in the kit
Detection architecture.
The present invention is that a kind of technical scheme for solving above-mentioned technical problem proposition is：A kind of CDA genetic polymorphism detections body
System, including detection primer is 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 CDA Gene A 79C pleomorphism sites
The nucleotide sequence such as SEQ ID No.2 on the outside of outside sense primer, detection CDA Gene A 79C pleomorphism sites shown in No.1
Nucleotide sequence on the inside of shown outside anti-sense primer, detection CDA Gene A 79C pleomorphism sites is as shown in SEQ ID No.3
Inner side sense primer, detection CDA Gene A 79C pleomorphism sites on the inside of nucleotide sequence it is interior as shown in SEQ ID No.4
Side anti-sense primer；The outside upstream and downstream primer introduces deoxyinosine；
The detection probe includes the nucleotide sequence such as SEQ ID that detection CDA Gene A 79C pleomorphism sites are wild type
Wild-type probe and detection CDA Gene A 79C pleomorphism sites shown in No.7 are homozygous nucleotide sequence such as SEQ ID
Homozygote probe shown in No.8；The wild-type probe and homozygote probe introduce deoxyinosine and base mismatch；Institute
State nucleotide sequence of the peptide nucleic acid including the inner side sense primer partial complementarity with detecting CDA Gene A 79C pleomorphism sites such as
Peptide nucleic acid shown in SEQ ID No.5 and the inner side anti-sense primer partial complementarity with detecting CDA Gene A 79C pleomorphism sites
Peptide nucleic acid of the nucleotide sequence as shown in SEQ ID No.6.
Above-mentioned CDA genetic polymorphism detections system also include internal control primer to, internal reference probe, Quality Control primer pair and Quality Control visit
Pin；
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.
The volume that above-mentioned CDA genetic polymorphism detections system also includes adding formamide in formamide, reaction system is anti-
Answer the 12% of system cumulative volume.
The present invention is that another technical scheme for solving above-mentioned technical problem proposition is：A kind of above-mentioned detection architecture of use
CDA genetic polymorphism detection products.
The present invention is that another technical scheme for solving above-mentioned technical problem proposition is：A kind of CDA 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 ID on the outside of detection CDA Gene A 79C pleomorphism sites
The nucleotide sequence such as SEQ ID No.2 on the outside of outside sense primer, detection CDA Gene A 79C pleomorphism sites shown in No.1
Nucleotide sequence on the inside of shown outside anti-sense primer, detection CDA Gene A 79C pleomorphism sites is as shown in SEQ ID No.3
Inner side sense primer, detection CDA Gene A 79C pleomorphism sites on the inside of nucleotide sequence it is interior as shown in SEQ ID No.4
Side anti-sense primer；The outside upstream and downstream primer introduces deoxyinosine；
The detection probe includes the nucleotide sequence such as SEQ ID that detection CDA Gene A 79C pleomorphism sites are wild type
Wild-type probe and detection CDA Gene A 79C pleomorphism sites shown in No.7 are homozygous nucleotide sequence such as SEQ ID
Homozygote probe shown in 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 CDA Gene A 79C pleomorphism sites
Peptide nucleic acid of the acid sequence as shown in SEQ ID No.5 and the inner side anti-sense primer portion with detecting CDA Gene A 79C pleomorphism sites
Divide peptide nucleic acid of the complementary nucleotide sequence 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 CDA 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 CDA genetic polymorphism detections kit 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 has positive effect：
(1) CDA genetic polymorphism detections system of the invention and its kit, using special primer probe combinations, are used
Chao Shi PCR after improvement, add the sensitiveness and reliability of detection, stopped pipe operation reduction pollution, with high sensitivity, spirit
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 CDA (A79C) site, can provide guidance for the clinical application of gemcitabine 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 >=100%, 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.
CDA (A79C) genetic polymorphism detection kit of the present embodiment includes：PCR detections liquid, Quality Control detection liquid, the 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
Including detection CDA (A79C) outside sense primer, detection CDA (A79C) outside anti-sense primer, detection CDA (A79C) it is interior
Side sense primer, detection CDA (A79C) inner side anti-sense primer, for CDA (A79C) inner side sense primer peptide nucleic acid, pin
Peptide nucleic acid to CDA (A79C) inner side anti-sense primer, detection CDA (A79C) wild-type probe, detection CDA (A79C) it is pure
Zygote probe, sequence is shown in Table 2, is synthesized by the Li Ge Bioisystech Co., Ltd of Shanghai hundred.Outside primer is in reaction system
Ultimate density is 0.05 μm/l, and ultimate density of the inner primer in reaction system is 0.19 μm/l, and peptide nucleic acid is in reaction system
Final concentration is 1nm/l, and detection CDA (A79C) wild-type probes and detection CDA (A79C) homozygote probe are in end reaction
Ultimate density in system is 0.19 μm/l.In order to reduce non-specific amplification, specificity and the sensitivity of detection are improved, at this
12% carboxamide agents are added in the detection reaction system of 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 free of CDA genes wild type, heterozygote and homozygous plasmid solution for 10ng/ μ l.
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 CDA | 1 | AGCTCCTGTTTCCCGCTGCT |
| Anti-sense primer on the outside of CDA | 2 | AGCGGAGACTGGGGGAAGAAG |
| Sense primer on the inside of CDA | 3 | CACCCTGAAGCCTGAGTGT |
| Anti-sense primer on the inside of CDA | 4 | ACCTTTGAAGATTCTCCCCTC |
| Upstream peptide nucleic acid on the inside of CDA | 5 | CTGAAGCCTGAGT |
| Downstream peptide nucleic acid on the inside of CDA | 6 | TTGAAGATTCTC |
| CDA wild-type probes | 7 | GCTCCCAGGAGGCGAACA |
| CDA homozygote probes | 8 | TGCTCCCAGGAGGCTAATC |
| Internal reference upstream primer | 9 | TGTAGGAGGGACTTAGAGAAG |
| Internal reference downstream primer | 10 | ACCCTTTAGGGAGAAAACGCT |
| Internal reference probe | 11 | CCTGCCCTTTGAGTTTGATGAT |
| Quality Control sense primer | 12 | TCACAGAGCTGGGATTGGAA |
| Quality Control anti-sense primer | 13 | CAGCCACCAAAACAGGTCTGA |
| Quality Control probe | 14 | TTGCTCTGTGGGGAGTGA |
CDA Outside primers are drawing for the lateral extent product length about 300-500bp of CDA (A79C) pleomorphism site two
Thing, CDA inner primers are the primers for the lateral extent about 90-150bp of CDA (A79C) pleomorphism site two, in order that outside is drawn
Thing is combined prior to inner primer with DNA profiling, and it is to be directed to CDA that Outside primer, which introduces peptide nucleic acid on the inside of deoxyinosine, CDA,
(A79C) the DNA analogs of sense primer on the inside of pleomorphism site and the design of inner side anti-sense primer, peptide nucleic acid partial sequence with
Inner primer is overlapped, and can be combined prior to inner side sense primer with DNA profiling, so as to inhibit inner side to draw in the reaction starting stage
The amplification of thing, CDA wild-type probes and CDA homozygote probes are the specificity spies designed for CDA (A79C) pleomorphism site
Pin, 5 ' ends of probe are marked with fluorescence signal group, and fluorescence signal group has FAM, HEX, ROX, CY5 etc., 3 ' end marks of probe
Note has fluorescent quenching group, and fluorescent quenching group is including BHQ1, BHQ2, TAMRA etc., due to wild-type probe and homozygote probe
Sequence only differs a base, and specificity is relatively low, and deoxyinosine is incorporated into detection polymorphic detection specificity by the present invention
In probe, and 1 base mismatch is introduced at 3 ' ends of probe, the specificity of probe is improved to a certain extent.
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 CDA, by detecting the amplification of reference gene, can analyze experiment
As a result validity.The design of primers of Quality Control gene is to exclude the CDA gene other positions conserved region beyond CDA gene Outside primers
Domain, Quality Control primed probe goes without abrupt climatic change site primer and primer binding site, draws in the different section designs of conservative region
Physical prospecting pin, Quality Control sequence amplification is acted on except PCR polymerase inhibitors in recoverable such as DNA content, sample in the present invention,
The difference of amplification efficiency between differential responses pipe, is additionally operable to determine sample gene by the amplification situation of icp gene type and Quality Control
Type.
2nd, the application method of kit.
The specific detecting step of CDA (A79C) 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 CDA (A79C) the genetic polymorphism detections kit examination of the present embodiment, the volume of reaction system
For 10 μ l, the concentration of kit each component and the final concentration in reaction system refer to table 1.(volume of reaction system is also
Can be 20 μ l, 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 CDA HEX signalling channel amplification curves are S-type, and Ct ＜ 20, then sample genome adds excessive, it is proposed that
Detected again after dilution；
Quality Control CDA HEX signalling channel amplification curves are S-type, and 20≤Ct≤30, sample addition is moderate, is appropriate for
Result judgement；
Quality Control CDA HEX signalling channel amplification curves are S-type, Ct ＞ 30, then sample genome addition is low, it is impossible to steady
Fixed 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>CDA genetic polymorphism detections system and its kit
<130>Nothing
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 1
agctcctgtt tcccgctgct 20
<210> 2
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 2
agcggagact gggggaagaa g 21
<210> 3
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 3
caccctgaag cctgagtgt 19
<210> 4
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 4
acctttgaag attctcccct c 21
<210> 5
<211> 13
<212> DNA
<213>It is artificial synthesized
<400> 5
ctgaagcctg agt 13
<210> 6
<211> 12
<212> DNA
<213>It is artificial synthesized
<400> 6
ttgaagattc tc 12
<210> 7
<211> 18
<212> DNA
<213>It is artificial synthesized
<400> 7
gctcccagga ggcgaaca 18
<210> 8
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 8
tgctcccagg aggctaatc 19
<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> 20
<212> DNA
<213>It is artificial synthesized
<400> 12
tcacagagct gggattggaa 20
<210> 13
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 13
cagccaccaa aacaggtctg a 21
<210> 14
<211> 18
<212> DNA
<213>It is artificial synthesized
<400> 14
ttgctctgtg gggagtga 18
Claims (10)
1. a kind of CDA 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 No.1 on the outside of detection CDA Gene A 79C pleomorphism sites
Nucleotide sequence on the outside of shown outside sense primer, detection CDA Gene A 79C pleomorphism sites is as shown in SEQ ID No.2
Outside anti-sense primer, detection CDA Gene A 79C pleomorphism sites on the inside of nucleotide sequence it is interior as shown in SEQ ID No.3
Under inner side of the nucleotide sequence as shown in SEQ ID No.4 on the inside of side sense primer, detection CDA Gene A 79C pleomorphism sites
Swim primer；The outside upstream and downstream primer introduces deoxyinosine；
The detection probe includes the nucleotide sequence such as SEQ ID that detection CDA Gene A 79C pleomorphism sites are wild type
Wild-type probe and detection CDA Gene A 79C pleomorphism sites shown in No.7 are homozygous nucleotide sequence such as SEQ ID
Homozygote probe shown in No.8；The wild-type probe and homozygote probe introduce deoxyinosine and base mismatch；Institute
State nucleotide sequence of the peptide nucleic acid including the inner side sense primer partial complementarity with detecting CDA Gene A 79C pleomorphism sites such as
Peptide nucleic acid shown in SEQ ID No.5 and the inner side anti-sense primer partial complementarity with detecting CDA Gene A 79C pleomorphism sites
Peptide nucleic acid of the nucleotide sequence as shown in SEQ ID No.6.
2. CDA genetic polymorphism detections system according to claim 1, it is characterised in that：Also include internal control primer to, it is interior
Join 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. CDA genetic polymorphism detections system according to claim 2, it is characterised in that：5 ' ends of each probe
Provided with fluorescent reporter group, 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. CDA genetic polymorphism detections system according to claim 2, it is characterised in that：The outside upstream and downstream primer
0.05 μm/l of ultimate density in reaction system, ultimate density 0.19 μ of the inner side upstream and downstream primer in reaction system
M/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. CDA genetic polymorphism detections system according to claim 1 or 2, it is characterised in that：Also include formamide, instead
The volume for answering addition formamide in system is the 12% of reaction system cumulative volume.
6. a kind of CDA genetic polymorphism detection products using detection architecture as claimed in claim 1.
7. a kind of CDA genetic polymorphism detections kit, it is characterised in that：It is right including PCR detections liquid, Quality Control detection liquid, the positive
According to 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 No.1 on the outside of detection CDA Gene A 79C pleomorphism sites
Nucleotide sequence on the outside of shown outside sense primer, detection CDA Gene A 79C pleomorphism sites is as shown in SEQ ID No.2
Outside anti-sense primer, detection CDA Gene A 79C pleomorphism sites on the inside of nucleotide sequence it is interior as shown in SEQ ID No.3
Under inner side of the nucleotide sequence as shown in SEQ ID No.4 on the inside of side sense primer, detection CDA Gene A 79C pleomorphism sites
Swim primer；The outside upstream and downstream primer introduces deoxyinosine；
The detection probe includes the nucleotide sequence such as SEQ ID that detection CDA Gene A 79C pleomorphism sites are wild type
Wild-type probe and detection CDA Gene A 79C pleomorphism sites shown in No.7 are homozygous nucleotide sequence such as SEQ ID
Homozygote probe shown in 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 CDA Gene A 79C pleomorphism sites
Arrange the peptide nucleic acid as shown in SEQ ID No.5 and with detecting that the inner side anti-sense primer part of CDA Gene A 79C pleomorphism sites is mutual
Peptide nucleic acid of the nucleotide sequence of benefit 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 CDA genes wild type, heterozygote and homozygous plasmid solution.
8. CDA genetic polymorphism detections kit according to claim 7, it is characterised in that：The PCR detection liquid and matter
Also include PCR buffer solutions, 2.5mM dNTPs and 5U/ μ l Taq archaeal dna polymerases in control detection liquid；The PCR buffer solutions bag
Include 100mM Tris-HCl, 15mM KCl and 15mM MgCl2, the Tris-HCl bufferings for configuring the PCR buffer solutions
The pH value of liquid is 8.3；The dNTPs includes dATP, dGTP, dCTP and dTTP.
9. CDA genetic polymorphism detections kit according to claim 7, 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.
10. CDA genetic polymorphism detections kit according to claim 7, it is characterised in that：Also include formamide, instead
The volume for answering addition formamide in system is the 12% of reaction system cumulative volume.
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Application publication date: 20170922 |