CN107841541A - IDH1/2 detection in gene mutation system and its kit - Google Patents
IDH1/2 detection in gene mutation system and its kit Download PDFInfo
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Abstract
The present invention relates to a kind of detection architecture and its kit of IDH1/2 gene mutations, kit includes PCR detection liquid and SYBR GreenI mixed liquors；PCR detection liquid A includes being used for the upstream and downstream primer A and peptide nucleic acid A for detecting the codon C394T sites of IDH1/2 genes the 394th and the 395th codon G395A sites, PCR detection liquid B include being used for the upstream and downstream primer C and peptide nucleic acid C for detecting the codon G515A sites of IDH1/2 genes the 515th including the upstream and downstream primer B for being used to detect the codon G419A sites of IDH1/2 genes the 419th and peptide nucleic acid B, PCR detection liquid C.The IDH1/2 detection in gene mutation system and its kit sensitivity and specificity of the present invention is high, and sample requirements are few, can carry out IDH1/2 detection in Gene Mutation exactly with rapid and convenient.
Description
Technical field
The present invention relates to a kind of method of detection IDH1/2 gene mutations and detection product, belong to biological technical field.
Background technology
2008, Parsons etc. carried out high flux expression analysis research to 20661 protein coding genes, sends out first
There are about in present glioblastoma 12% there occurs isocitric dehydrogenase 1 (isocitrate dehydrogenase 1,
IDH1 gene mutation), then, more scholars are further investigation revealed that IDH1/2 mutation are generally deposited as prognosis bona's mark
It is II grade and III grade of glioma and Secondary cases glioblastoma, and is sent out in the generation, development and evolution process of tumour
Wave important function.IDH1/2 gene mutations are most common metabolic enzyme gene mutation types, in glioma and acute myeloid
It is fairly common in some hypotypes of leukaemia.Chondrosarcoma, cholangiocarcinoma, Chromaffionoma, colorectal cancer, prostate cancer, lung cancer etc.
Solid tumor also has been reported that.IDH mutation status and cancer pathology parting are closely related, and IDH mutation are that glioma is in progress
Earliest events, therefore, IDH abrupt climatic changes can not only in diagnosis more it is timely comprehensively, and for further further investigation
Tumorigenesis and biological property are significant.
IDH1/2 detection in Gene Mutation contributes to the exploitation of the target therapeutic agent for the mutational site and cancer metabolin,
So that targeted therapy has more validity, specificity, and reduce adverse reaction.Therefore the foundation of IDH1/2 detection method of gene mutation
Prevention and treatment to tumour have important directive significance.
The clinical detection to tumor-related gene is mainly PCR sequencing PCR and ARMS methods at present, and is not applied to.Use sensitivity
Relatively low conventional method detection can only find tumour variation of the subset of patients before particular treatment is received, using high sensitivity method
It can be found that the carrying saltant type patient of higher proportion if (such as dPCR) detection.The side of IDH1/2 detection in Gene Mutation is carried out at present
Method mainly has chip technology, probe primer technology, digestion, pyrosequencing techniques and HRM analytical technologies etc., price, cumbersome
Or accuracy is not high.Therefore, develop a kind of product of detection IDH1/2 gene mutations, with realization more high sensitivity, it is more inexpensive,
The IDH1/2 detection in Gene Mutation of more convenient and quicker is necessary.
The content of the invention
High the technical problem to be solved in the present invention is to provide a kind of sensitivity and specificity, sample requirements are few, can be fast
Speed easily and accurately carries out the IDH1/2 detection in gene mutation system and its kit of IDH1/2 detection in Gene Mutation.
The present invention is to solve a kind of technical scheme that above-mentioned technical problem proposes to be：A kind of IDH1/2 detection in Gene Mutation body
System, including the upstream and downstream for detecting the codon C394T sites of IDH1/2 genes the 394th and the 395th codon G395A sites are drawn
Thing A, for detecting the upstream and downstream primer B in the codon G419A sites of IDH1/2 genes the 419th, for detecting IDH1/2 genes
The upstream and downstream primer C in 515 codon G515A sites, fluorescent dye SYBR GreenI, and peptide nucleic acid；
The nucleotide sequence of the sense primer A is as shown in SEQ ID No.1, the nucleotide sequence of the anti-sense primer A
As shown in SEQ ID No.2；
The nucleotide sequence of the sense primer B is as shown in SEQ ID No.3, the nucleotide sequence of the anti-sense primer B
As shown in SEQ ID No.4；
The nucleotide sequence of the sense primer C is as shown in SEQ ID No.5, the nucleotide sequence of the anti-sense primer C
As shown in SEQ ID No.6；
The peptide nucleic acid includes nucleotide sequence and the codon C394T sites of IDH1/2 wild type genes the 394th and wild
The peptide nucleic acid A of the codon G395A site complete complementaries of type the 395th and nucleotide sequence as shown in SEQ ID No.7, nucleotides sequence
Row are with the codon G419A site complete complementaries of IDH1/2 wild type genes the 419th and nucleotide sequence is as shown in SEQ ID No.8
Peptide nucleic acid B, nucleotide sequence and the codon G515A site complete complementaries of IDH1/2 wild type genes the 515th and nucleotides sequence
Arrange the peptide nucleic acid C as shown in SEQ ID No.9.
Above-mentioned upstream and downstream primer has carried out LNA modifications.
Ultimate density of the above-mentioned upstream and downstream primer in reaction system is 0.1~0.3 μM/L.
Ultimate densities of the above-mentioned upstream and downstream primer A in reaction system is 0.19 μM/L, and the upstream and downstream primer B is reacting
Ultimate density in system is 0.2 μM/L, and ultimate densities of the upstream and downstream primer C in reaction system is 0.15 μM/L.
Ultimate density of the above-mentioned peptide nucleic acid in reaction system is 1~1.5 μM/L.
The ultimate density of above-mentioned peptide nucleic acid A, B, C in reaction system is 1.25 μM/L.
The present invention is to solve a kind of technical scheme that above-mentioned technical problem proposes to be：It is a kind of using above-mentioned detection architecture
IDH1/2 detection in Gene Mutation products.
The present invention is to solve a kind of technical scheme that above-mentioned technical problem proposes to be：A kind of IDH1/2 detection in Gene Mutation examination
Agent box, including PCR detection liquid A, PCR detection liquid B, PCR detection liquid C and SYBR GreenI mixed liquors；
The PCR detection liquid A includes being used to detect the codon C394T sites of IDH1/2 genes the 394th and wild type the 395th
The upstream and downstream primer A and peptide nucleic acid A, PCR detection liquid B in codon G395A sites include close for detecting IDH1/2 genes the 419th
The upstream and downstream primer B and peptide nucleic acid B, PCR detection liquid C in numeral G419A sites include being used to detect the password of IDH1/2 genes the 515th
The upstream and downstream primer C and peptide nucleic acid C in sub- G515A sites；
The nucleotide sequence of the sense primer A is as shown in SEQ ID No.1, the nucleotide sequence of the anti-sense primer A
As shown in SEQ ID No.2；
The nucleotide sequence of the sense primer B is as shown in SEQ ID No.3, the nucleotide sequence of the anti-sense primer B
As shown in SEQ ID No.4.
The nucleotide sequence of the sense primer C is as shown in SEQ ID No.5, the nucleotide sequence of the anti-sense primer C
As shown in SEQ ID No.6；
The peptide nucleic acid includes nucleotide sequence and the codon C394T sites of IDH1/2 wild type genes the 394th and wild
The peptide nucleic acid A of the codon G395A site complete complementaries of type the 395th and nucleotide sequence as shown in SEQ ID No.7, nucleotides sequence
Row are with the codon G419A site complete complementaries of IDH1/2 wild type genes the 419th and nucleotide sequence is as shown in SEQ ID No.8
Peptide nucleic acid B, nucleotide sequence and the codon G515A site complete complementaries of IDH1/2 wild type genes the 515th and nucleotides sequence
Arrange the peptide nucleic acid C as shown in SEQ ID No.9.
Above-mentioned upstream and downstream primer has carried out LNA modifications；Ultimate density of the upstream and downstream primer in reaction system be
0.1~0.3 μM/L；Ultimate density of the peptide nucleic acid in reaction system is 1~1.5 μM/L.
Above-mentioned IDH1/2 gene mutation detection kits also include positive control P1, positive control P2 and negative control；
The positive control solution P1 be concentration be 100ng/ μ L comprising IDH1/2 gene Cs 394T, G395A, G419A,
The wild plasmid solution in G515A sites；
The positive control solution P2 be concentration be 100ng/ μ L comprising IDH1/2 gene Cs 394T, G395A, G419A,
The mutant plasmids solution in G515A sites；
The negative controls are free from the Genomic DNA solution of IDH1/2 genes.
The present invention has positive effect：
(1) present invention clamps down on real-time quantitative PCR detection method using PNA, it is only necessary to passes through melting curve analysis, you can will
IDH1/2 genic mutation types distinguish with wild type.By single step reaction, can solve PCR amplifications, mutation enrichment and quantify whole
Process, without subsequent steps such as electrophoresis, hybridization, digestions, experimentation is enormously simplify, whole course of reaction is in seal pipe
Interior completion, reduce the possibility of pollution to greatest extent, reduce false positive rate.
(2) of the invention IDH1/2 detection method of gene mutation and its kit, can detect the 394/395th simultaneously, 419,
Any one mutation of 515 codons, high sensitivity.Sensitivity can reach 0.1%~0.5%, compared with the concentration of Standard PCR,
The present invention easily detects the genotype in the IDH1/2 sites of very low concentration sample, and overcoming TaqMan probe method needs to make
The shortcomings that being detected respectively with a plurality of specific probe, meet the requirement of large sample size Clinical screening.
(3) present invention selection SYBR GreenI are cheap as fluorescent dye, substantially reduce testing cost.
Brief description of the drawings
Fig. 1 detects positive control P1 C394T and G395A sites wild type specific product using the kit of embodiment
Peak Tm values.
Fig. 2 is the G419A sites wild type specific product peak Tm using the kit detection positive control P1 of embodiment
Value.
Fig. 3 detects positive control P1 G515A sites wild type specific product peak Tm values using the kit of embodiment.
Fig. 4 is C394T the and G395A site mutations type specificity production using the kit detection positive control P2 of embodiment
Thing peak Tm values.
Fig. 5 is the G419A site mutation type specificity products peak Tm using the kit detection positive control P2 of embodiment
Value.
Fig. 6 is the G515A site mutation type specificity products peak Tm using the kit detection positive control P2 of embodiment
Value.
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
So that some nonessential modifications and adaptations are made 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 obtain 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.
First, the composition of kit.
The IDH1/2 gene mutation detection kits of the present embodiment, including PCR detection liquid A, PCR detection liquid B, PCR detections
Liquid C, SYBR GreenI mixed liquors, negative control, positive control P1 and positive control P2.Each composition of kit is as shown in table 1.
The Kit components table of table 1
| Composition | Packing | Composition |
| PCR detection liquid A | 1 pipe | C394T and G395A sites primer, peptide nucleic acid |
| PCR detection liquid B | 1 pipe | G419A sites primer, peptide nucleic acid |
| PCR detection liquid C | 1 pipe | G515A sites primer, peptide nucleic acid |
| SYBR GreenI mixed liquors | 1 pipe | —— |
| Positive control P1 | 1 pipe | The plasmid of the genes of IDH1/2 containing wild type |
| Positive control P2 | 1 pipe | The plasmid of the genes of IDH1/2 containing saltant type |
| Negative control | 1 pipe | Genomic DNA solution without IDH1/2 genes |
Reagent constituents is described as follows in above-mentioned table 1：
1) main component of SYBR GreenI mixed liquors includes archaeal dna polymerase, ultra-pure water, dNTPs mixed liquors, SYBR
GreenI, buffer solution etc. (are provided, article No. by Bioer companies：B255l1).
2) PCR detects liquid A and included:For detecting the upstream and downstream primer A in IDH1/2 gene C 394T sites and G395A sites
With the peptide nucleic acid A with wild type C394T sites and G395A sites complete complementary.PCR detections liquid B includes:For detecting G419A
The upstream and downstream primer B in the site and peptide nucleic acid B with wild type G419A sites complete complementary.PCR detections liquid C includes:For detecting
The upstream and downstream primer C in the G515A sites and peptide nucleic acid C with wild type G515A sites complete complementary.
The present embodiment has carried out LNA modifications to upstream and downstream primer.A LNA base is introduced i.e. in oligonucleotide chain, and
The specificity of optimal fusing point (Tm) and hybridization is adjusted by adjusting position of the LNA bases in primer.Drawing after modification
Thing has higher Tm values, more preferably can be combined with mutagenesis template, and the real-time quantitative PCR primer after modification, length is shorter, can be with
Increase the flexibility of design.
The present embodiment adds a small amount of peptide nucleic acid (PNA) in PCR detects liquid, is not enough to completely inhibit wild type gene expansion
Increase, now the Tm values of wild type are unchanged, and amplification efficiency substantially reduces when not adding PNA, and expand nothing to mutated genes
Influence, but its Tm declines substantially, the Tm differences increase of the two, can now be analyzed by solubility curve by two kinds of gene regions
Separate.
3) positive control solution P1 is the solution for the wild plasmid of gene containing IDH1/2 that concentration is 100ng/ μ l.
4) positive control solution P2 is the solution for the plasmid of genic mutation type containing IDH1/2 that concentration is 100ng/ μ l.
5) negative controls are free from the Genomic DNA solution of IDH1/2 genes.
Wild plasmid is retrieved as wild plasmid routine construction step：Design is located at the primer of mutational site both sides,
The wild type product containing corresponding site is expanded, is built into plasmid, selects simultaneously sequence verification.
Mutant plasmids are retrieved as homozygous mutation plasmid routine construction step：The corresponding polymorphic position of one covering of design
Point simultaneously will wait bit base to be incorporated into special primer in primer sequence, and corresponding upstream or the pairing of downstream wild primers,
Purpose fragment product of the amplification containing 4 kinds of mutational sites, is built into plasmid, selects simultaneously sequence verification.
2nd, primer, peptide nucleic acid design.
The primer length that the present invention designs is 15~22bp, product length 200~220bp. specific peptide nucleic acid PNA fragments
With wild type polymorphic position point sequence complete complementary, length is 15~20bp.As shown in table 2.
The primer peptide nucleic acid sequence table of table 2
| Position | Detect classification | Seq No. | Sequence(5’—3’) |
| C394T/G395A | Sense primer A | 1 | TATCCCCCGGCTTGT |
| C394T/G395A | Anti-sense primer A | 2 | GCAAAATCACATTA |
| C394T/G395A | Peptide nucleic acid A | 7 | TATCATCATAGGTT |
| G419A | Sense primer B | 3 | AGTTCAAGCTGAAGA |
| G419A | Anti-sense primer B | 4 | CCAGGGACTAGGCGT |
| G419A | Peptide nucleic acid B | 8 | AATGGAACTATCCA |
| G515A | Sense primer C | 5 | ACGCCTAGTCCCT |
| G515A | Anti-sense primer C | 6 | CAGGTCAGTGGAT |
| G515A | Peptide nucleic acid C | 9 | ATCACCATTGGCAA |
Primer and peptide nucleic acid are synthesized by the Li Ge Bioisystech Co., Ltd of Shanghai hundred, are diluted according to primer specification by dry powder
It is configured to detect working solution, primer and peptide nucleic acid mixed liquor by mother liquor addition sterilizing ultra-pure water into working stocks, then by mother liquor
Dilution forms.
Concentration of the peptide nucleic acid PNA in reaction system is most important in the present invention, 0.25~8.0 μm of ol/L concentration range
It is interior, when PNA concentration is less than 3.75 μm of ol/L, there is wild specific amplification peak on the melting curve of wild-type template, show
Wild type gene background does not completely inhibit, can not now judge whether mutator；When PNA concentration is in 3.75~4.0 μ
During mol/L, melting curve of the saltant type template after PCR is expanded can be observed to be mutated peak, and wild type only has primer dimer
Peak, i.e. wild-type template are suppressed can not expand completely；With the increase of PNA concentration, it can be observed either that wild type is still
The amplification efficiency of saltant type template gradually reduces, when PNA concentration is higher than 40 times of primer, during up to 8.0 μm of ol/L, and all templates
It is suppressed and without any amplified peak exist.
The application method of three kits.
1st, sample DNA extracts
Sample blood sample is extracted using kit (Axygen Multisource Genomic DNA Miniprep Kit)
This DNA, or use buccal swab genome DNA extracting reagent kit (article No. of the health for 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.
After obtaining sample DNA, concentration is determined by Thermo-Fisher nucleic acid-proteins quantitative instrument (NanoDrop 2000)
And purity, sample quality is controlled, ultimately joins the sample in reaction system.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 the IDH1/2 gene mutation detection kits examination of the present embodiment, the volume of reaction system is 10 μ
2 μ l, SYBR GreenI mixed liquors of l, wherein PCR detection liquid 5 μ l, the μ l of template DNA 1, the μ l of ultra-pure water 2.(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).Primer is corresponded to using mutational site
PCR reaction systems A, B and C detect same sample respectively.
1) 10 μ l detection PCR reaction systems are prepared：
Detect PCR reaction systems：Take respectively 2 μ l PCR detect liquid, 5 μ l SYBR GreenI mixed liquors, 2 μ l ultra-pure waters, 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 respectively.
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.
The PCR response procedures tables of table 3
Preferably, the condition of PCR amplifications is：95 DEG C of the condition in pre-degeneration stage, 1min；Circulate 0 circulation of Isosorbide-5-Nitrae, condition
For 60 DEG C of 95 DEG C of 15s, 70 DEG C of PNA combination 10s, primer annealing and elongating temperature, time 1min；；2 conditions are circulated as 4 DEG C of guarantors
Temperature.
Reaction carries out solubility curve analysis immediately after terminating：95 DEG C of lasting 15s, 65 DEG C of holding 1min are cooled to, and with 0.3
DEG C/s speed is gradually heating to 95 DEG C of lasting 15s.Instrument software automatically analyzes out Ct values, i.e. S YBR Green I detections are glimmering
Optical signal is significantly higher than period during background signal.
4th, PCR result judgements.
1) judgement of kit validity.
Positive control P1：All amplification curves of positive control FAM signalling channels have obvious Exponential growth stage, and Ct values
Positive≤30；Solubility curve is analyzed, and the Tm values of each site PCR primer are as shown in table 4.
Positive control P2:All amplification curves of positive control FAM signalling channels have obvious Exponential growth stage, and Ct values
Positive≤30；Solubility curve is analyzed, and the Tm values of each site PCR primer are as shown in table 4.
Negative control：All amplification curves of negative control FAM signalling channels are bent without obvious Exponential growth stage, or dissolving
Without specific product peak on line.
The Tm values of each site PCR primer of the IDH1/2 genes of table 4
| C394T/G395A | G419A | G515A | |
| Positive control P1 | 89.2±0.6℃ | 80.1±0.6℃ | 81.0±0.5℃ |
| Positive control P2 | 84.1±0.5℃ | 74.9±0.5℃ | 76.9±0.7℃ |
2) judgement of detection in Gene Mutation result.
By above step, under the premise of determining that experimental result is effective, then the result of sample is judged.According to sample
The difference that solubility curve Tm values compare solubility curve Tm values with wild-type positive carries out interpretation to pattern detection result, determines sample
Genotype.The △ Tm values of effective detection sample are calculated according to formula " the Tm values of △ Tm values=positive control P1 Tm values-sample ",
And interpretation is carried out to pattern detection result according to following steps and table 5, determine sample with the presence or absence of mutation.
The result judgement of table 5
If the △ Tm values of pattern detection reaction tube are more than corresponding △ Tm Cut-Off reference values, the sample is the reaction
Mutation is positive corresponding to pipe；It is on the contrary then be wild type sample corresponding to the reaction tube.
Product peak Tm values are similar to shown in Fig. 1, and the IDH1/2 gene C 394T or G395A sites for detecting sample are wild type.
Product peak Tm values are similar to shown in Fig. 2, and the IDH1/2 gene G419A sites for detecting sample are wild type.Product peak Tm values are similar
It is wild type in shown in Fig. 3, detecting the IDH1/2 gene G515A sites of sample.Product peak Tm values are similar to shown in Fig. 4, detection
The IDH1/2 gene C 394T or G395A sites of sample are saltant type.Product peak Tm values are similar to shown in Fig. 5, detect sample
IDH1/2 gene G419A sites are saltant type.Product peak Tm values are similar to the IDH1/2 genes G515A for shown in Fig. 6, detecting sample
Site is saltant type.
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 table
<110>Match peaceful thing medical sci-tech limited company in Shanghai
<120>IDH1/2 detection in gene mutation system and its kit
<130>Nothing
<160> 9
<170> SIPOSequenceListing 1.0
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<211> 15
<212> DNA
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<400> 1
tatcccccgg cttgt 15
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<211> 14
<212> DNA
<213>Artificial sequence (nothing)
<400> 2
gcaaaatcac atta 14
<210> 3
<211> 15
<212> DNA
<213>Artificial sequence (nothing)
<400> 3
agttcaagct gaaga 15
<210> 4
<211> 15
<212> DNA
<213>Artificial sequence (nothing)
<400> 4
ccagggacta ggcgt 15
<210> 5
<211> 13
<212> DNA
<213>Artificial sequence (nothing)
<400> 5
acgcctagtc cct 13
<210> 6
<211> 13
<212> DNA
<213>Artificial sequence (nothing)
<400> 6
caggtcagtg gat 13
<210> 7
<211> 14
<212> DNA
<213>Artificial sequence (nothing)
<400> 7
tatcatcata ggtt 14
<210> 8
<211> 14
<212> DNA
<213>Artificial sequence (nothing)
<400> 8
aatggaacta tcca 14
<210> 9
<211> 14
<212> DNA
<213>Artificial sequence (nothing)
<400> 9
atcaccattg gcaa 14
Claims (10)
- A kind of 1. IDH1/2 detection in gene mutation system, it is characterised in that：Including for detecting the codon of IDH1/2 genes the 394th C394T sites and the upstream and downstream primer A in the 395th codon G395A sites, for detecting the codon of IDH1/2 genes the 419th The upstream and downstream primer B in G419A sites, it is glimmering for detecting the upstream and downstream primer C in the codon G515A sites of IDH1/2 genes the 515th Photoinitiator dye SYBR Green, and peptide nucleic acid；The nucleotide sequence of the sense primer A is as shown in SEQ ID No.1, the nucleotide sequence such as SEQ of the anti-sense primer A Shown in ID No.2；The nucleotide sequence of the sense primer B is as shown in SEQ ID No.3, the nucleotide sequence such as SEQ of the anti-sense primer B Shown in ID No.4；The nucleotide sequence of the sense primer C is as shown in SEQ ID No.5, the nucleotide sequence such as SEQ of the anti-sense primer C Shown in ID No.6；The peptide nucleic acid includes nucleotide sequence and the codon C394T sites of IDH1/2 wild type genes the 394th and wild type the The peptide nucleic acid A of 395 codon G395A site complete complementaries and nucleotide sequence as shown in SEQ ID No.7, nucleotide sequence with The peptide of the codon G419A site complete complementaries of IDH1/2 wild type genes the 419th and nucleotide sequence as shown in SEQ ID No.8 Nucleic acid B, nucleotide sequence and the codon G515A site complete complementaries of IDH1/2 wild type genes the 515th and nucleotide sequence are such as Peptide nucleic acid C shown in SEQ ID No.9.
- 2. IDH1/2 detection in gene mutation system according to claim 1, it is characterised in that：The upstream and downstream primer is carried out LNA modifications.
- 3. IDH1/2 detection in gene mutation system according to claim 1, it is characterised in that：The upstream and downstream primer is anti- It is 0.1~0.3 μM/L to answer the ultimate density in system.
- 4. IDH1/2 detection in gene mutation system according to claim 3, it is characterised in that：The upstream and downstream primer A exists Ultimate density in reaction system is 0.19 μM/L, ultimate densities of the upstream and downstream primer B in reaction system be 0.2 μM/ The ultimate density of L, the upstream and downstream primer C in reaction system is 0.15 μM/L.
- 5. IDH1/2 detection in gene mutation system according to claim 1, it is characterised in that：The peptide nucleic acid is in reactant Ultimate density in system is 1~1.5 μM/L.
- 6. IDH1/2 detection in gene mutation system according to claim 5, it is characterised in that：Described peptide nucleic acid A, B, C exist Ultimate density in reaction system is 1.25 μM/L.
- A kind of 7. IDH1/2 detection in Gene Mutation products using detection architecture as claimed in claim 1.
- A kind of 8. IDH1/2 gene mutation detection kits, it is characterised in that：Including PCR detection liquid A, PCR detection liquid B, PCR inspections Survey liquid C and SYBR GreenMixed liquor；The PCR detection liquid A includes being used to detect the codon C394T sites of IDH1/2 genes the 394th and the password of wild type the 395th The upstream and downstream primer A and peptide nucleic acid A, PCR detection liquid B in sub- G395A sites include being used to detect the codon of IDH1/2 genes the 419th The upstream and downstream primer B and peptide nucleic acid B, PCR detection liquid C in G419A sites include being used to detect the codon of IDH1/2 genes the 515th The upstream and downstream primer C and peptide nucleic acid C in G515A sites；The nucleotide sequence of the sense primer A is as shown in SEQ ID No.1, the nucleotide sequence such as SEQ of the anti-sense primer A Shown in ID No.2；The nucleotide sequence of the sense primer B is as shown in SEQ ID No.3, the nucleotide sequence such as SEQ of the anti-sense primer B Shown in ID No.4；The nucleotide sequence of the sense primer C is as shown in SEQ ID No.5, the nucleotide sequence such as SEQ of the anti-sense primer C Shown in ID No.6；The peptide nucleic acid includes nucleotide sequence and the codon C394T sites of IDH1/2 wild type genes the 394th and wild type the The peptide nucleic acid A of 395 codon G395A site complete complementaries and nucleotide sequence as shown in SEQ ID No.7, nucleotide sequence with The peptide of the codon G419A site complete complementaries of IDH1/2 wild type genes the 419th and nucleotide sequence as shown in SEQ ID No.8 Nucleic acid B, nucleotide sequence and the codon G515A site complete complementaries of IDH1/2 wild type genes the 515th and nucleotide sequence are such as Peptide nucleic acid C shown in SEQ ID No.9.
- 9. IDH1/2 gene mutation detection kits according to claim 8, it is characterised in that：The upstream and downstream primer is entered LNA modifications are gone；Ultimate density of the upstream and downstream primer in reaction system is 0.1~0.3 μM/L；The peptide nucleic acid exists Ultimate density in reaction system is 1~1.5 μM/L.
- 10. IDH1/2 gene mutation detection kits according to claim 8, it is characterised in that：Also include positive control P1, positive control P2 and negative control；The positive control solution P1 is that concentration includes IDH1/2 gene C 394T, G395A, G419A, G515A positions for 100ng/ μ L The wild plasmid solution of point；The positive control solution P2 is that concentration includes IDH1/2 gene C 394T, G395A, G419A, G515A positions for 100ng/ μ L The mutant plasmids solution of point；The negative controls are free from the Genomic DNA solution of IDH1/2 genes.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114752677A (en) * | 2022-06-13 | 2022-07-15 | 北京思诺普斯生物科技有限公司 | Primer group and kit for rapidly detecting IDH1 mutation, amplification method and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436613A (en) * | 2013-08-21 | 2013-12-11 | 中国人民解放军第二军医大学 | Fluorescent PCR (Polymerase Chain Reaction) detection kit for IDH1/IDH2 (isocitrate dehydrogenase 1/isocitrate dehydrogenase 2) gene mutation and application thereof |
| CN105368924A (en) * | 2014-11-29 | 2016-03-02 | 上海赛安生物医药科技有限公司 | IDH1/2 gene mutation detection system and kit thereof |
-
2017
- 2017-12-18 CN CN201711361069.7A patent/CN107841541A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436613A (en) * | 2013-08-21 | 2013-12-11 | 中国人民解放军第二军医大学 | Fluorescent PCR (Polymerase Chain Reaction) detection kit for IDH1/IDH2 (isocitrate dehydrogenase 1/isocitrate dehydrogenase 2) gene mutation and application thereof |
| CN105368924A (en) * | 2014-11-29 | 2016-03-02 | 上海赛安生物医药科技有限公司 | IDH1/2 gene mutation detection system and kit thereof |
Non-Patent Citations (1)
| Title |
|---|
| 林寒等: ""应用肽核酸钳制 PCR技术检测胰腺癌 K-ras基因突变"", 第二军医大学学报, vol. 30, no. 7, pages 762 - 764 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114752677A (en) * | 2022-06-13 | 2022-07-15 | 北京思诺普斯生物科技有限公司 | Primer group and kit for rapidly detecting IDH1 mutation, amplification method and application |
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