CN107022619A - KRAS gene mutation detection primer probe and its kit - Google Patents
KRAS gene mutation detection primer probe and its kit Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention relates to a kind of KRAS gene mutation detection primer probe and its kit, kit includes digital pcr premixed liquid, droplets stable agent and the primed probe mixed liquor for being used to prepare digital pcr reaction solution；Primed probe mixed liquor includes the upstream and downstream primer A for being used to detect the 12nd codon, the probe A of 12nd codon G12R site mutations, the probe B of 12nd codon G12V site mutations, the probe C of 12nd codon G12D site mutations, the probe D of 12nd codon G12A site mutations, the probe E of the 12nd codon G12S site mutations and probe F of the 12nd codon G12C site mutations, for detecting the upstream and downstream primer B of the 13rd codon and the probe G of the 13rd codon G13D site mutations, and for detecting the upstream and downstream primer C of the 61st codon and the probe H of the 61st codon Q61L site mutations；The KRAS gene mutation detection kit sensitivity and specificity of the present invention are high, and sample requirements are few, can carry out multiplex PCR detection exactly with rapid and convenient.
Description
Technical field
The present invention relates to a kind of primer for detecting KRAS gene mutation, probe and detection product, belong to biological technical field.
Background technology
RAS gene mutations are considered as closely related with human tumor generation.KRAS genes 12,13 passwords in RAS families
The mutation of son has higher discovery rate in colorectal cancer and cancer of pancreas, is considered as generation with tumour, recurrence and patient pre-
It is directly related afterwards.KRAS gene is carried, it is considered as and pancreas with CDKN2A, SMAD4 and TP53 in the change of gene level
The occurrence and development of gland cancer are directly related, and the change of catastrophe helps to understand gene alteration and the state of an illness monitoring KRAS in vivo
Contact between development.
Exploring patient KRAS resistance mechanisms and prediction prognosis turns into feasible approach, now clear and definite tumour cell
Meeting released dna enters the circulatory system so that by way of " liquid biopsy ", can be specific to early stage and late tumor patient
Nucleotide sequence is analyzed, and is a kind of detection oncogene of Noninvasive and a kind of method of clinical therapeutic efficacy, and suffer from
Person is higher to the acceptance of Noninvasive sample collection mode.
But it is due to that cfDNA contents are low in most people serum/plasma, in tumour early stage, ctDNA only accounts for total free
The 0.01% of DNA, and the reason such as cfDNA fragments are relatively small, at present the clinical detection to tumor-related gene be mainly PCR sequencing PCR
With ARMS methods, and do not apply to.Therefore, ctDNA detection is accomplished by a kind of more special technology sensitiveer than conventional method.Use
The relatively low conventional method detection of sensitivity can only find tumour variation of the subset of patients before particular treatment is received, using Gao Ling
It can be found that the carrying saltant type patient of higher proportion if sensitivity method (such as dPCR) is detected.In terms of digital pcr is due to sensitivity
Advantage, by unimolecule amplification low-abundance gene signal is distinguished from complex background, available for disease or virus
Early diagnosis or curative effect monitoring.Digital pcr technology unimolecule amplification after to sample DNA accurate quantification, with than other PCR
Higher measurement accuracy.
The sensitivity of digital pcr depends primarily on 2 main aspects：Efficiency and specific detection that nucleic acids in samples is extracted
The sensitivity of system, both determine the detection sensitivity of target sequence together.Droplet type digital pcr detects extremely low copy number sample
Its testing result of this when is unstable, it is necessary to repeatedly parallel laboratory test.Due to the limitation of hardware device, it is difficult to be difficult to realize multiple sites
While detect, and dPCR Multiple detection systems are limited to the length of pcr amplification product, and the design to TaqMan probe has
Very high requirement.The present invention is improved from digital pcr detection architecture, improves stability and the sensitivity of digital pcr detection.
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 KRAS gene mutation detection primer probe and its kit of multiplex PCR detection.
The present invention is that a kind of technical scheme for solving above-mentioned technical problem proposition is：A kind of KRAS gene mutation detection primer
Probe, including for detecting the upstream and downstream primer A of the 12nd codon, it is the probe A of the 12nd codon G12R site mutations, the 12nd close
The probe B of numeral G12V site mutations, the probe C of the 12nd codon G12D site mutations, the 12nd codon G12A site mutations
Probe D, the probe E of the 12nd codon G12S site mutations and the 12nd codon G12C site mutations probe F, for detecting
The upstream and downstream primer B of 13rd codon and the 13rd codon G13D site mutations probe G, and for detecting the 61st codon
Upstream and downstream primer C and the 61st codon Q61L site mutations probe H；
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 probe A is as shown in SEQ ID No.3, the nucleotides sequence of the probe B
Row are as shown in SEQ ID No.4, and the nucleotide sequence of the probe C is as shown in SEQ ID No.5, the nucleotides of the probe D
Sequence is as shown in SEQ ID No.6, and the nucleotide sequence of the probe E is as shown in SEQ ID No.7, the nucleosides of the probe F
Acid sequence as shown in SEQ ID No.8,
The nucleotide sequence of the sense primer B is as shown in SEQ ID No.9, the nucleotide sequence of the anti-sense primer B
As shown in SEQ ID No.10, the nucleotide sequence of the probe G as shown in SEQ ID No.11,
The nucleotide sequence of the sense primer C is as shown in SEQ ID No.12, the nucleotide sequence of the anti-sense primer C
As shown in SEQ ID No.13, the nucleotide sequence of the probe H is as shown in SEQ ID No.14.
Above-mentioned probe A, B, C, D, E, F, G, H have carried out LNA modifications.
Ultimate densities of the above-mentioned upstream and downstream primer A in reaction system is 0.5~0.8 μM/L, and upstream and downstream primer B is in reaction
Ultimate density in system is 0.1~0.3 μM/L, ultimate densities of the upstream and downstream primer C in reaction system be 0.1~0.3 μM/
The ultimate density of L, probe A, B, C, D, E, F, G, H in reaction system is 0.15~0.30 μM/L.
Ultimate densities of the above-mentioned upstream and downstream primer A in reaction system is 0.55 μM/L, and the upstream and downstream primer B is in reaction
Ultimate density in system is 0.2 μM/L, and ultimate densities of the upstream and downstream primer C in reaction system is 0.2 μM/L, described
The ultimate density of probe A, B, C, D, E, F, G, H in reaction system be respectively 0.21 μM/L, 0.18 μM/L, 0.22 μM/L,
0.22μM/L、0.19μM/L、0.21μM/L、0.19μM/L、0.18μM/L。
Above-mentioned probe A, B, C, D, E, F, G, H 5 ' ends are provided with reporter fluorescence group, and 3 ' ends are provided with quenching fluorescence group.
The present invention is that a kind of technical scheme for solving above-mentioned technical problem proposition is：A kind of above-mentioned primed probe of use
KRAS gene mutation detects product.
The present invention is that a kind of technical scheme for solving above-mentioned technical problem proposition is：A kind of KRAS gene mutation detection reagent
Box, including for preparing digital pcr premixed liquid, droplets stable agent, detection liquid A and the detection liquid B of digital pcr reaction solution；
The detection liquid A includes upstream and downstream primer A, the 12nd codon G12R site mutations for being used to detect the 12nd codon
Probe A, the probe B of the 12nd codon G12V site mutations and the 12nd codon G12D site mutations probe C, and be used for
Detect the upstream and downstream primer B of the 13rd codon and the probe G of the 13rd codon G13D site mutations；
The detection liquid B includes upstream and downstream primer A, the 12nd codon G12A site mutations for being used to detect the 12nd codon
Probe D, the probe E of the 12nd codon G12S site mutations and the 12nd codon G12C site mutations probe F, and be used for
Detect the upstream and downstream primer C of the 61st codon and the probe H of the 61st codon Q61L site mutations；
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 probe A is as shown in SEQ ID No.3, the nucleotides sequence of the probe B
Row are as shown in SEQ ID No.4, and the nucleotide sequence of the probe C is as shown in SEQ ID No.5, the nucleotides of the probe D
Sequence is as shown in SEQ ID No.6, and the nucleotide sequence of the probe E is as shown in SEQ ID No.7, the nucleosides of the probe F
Acid sequence as shown in SEQ ID No.8,
The nucleotide sequence of the sense primer B is as shown in SEQ ID No.9, the nucleotide sequence of the anti-sense primer B
As shown in SEQ ID No.10, the nucleotide sequence of the probe G as shown in SEQ ID No.11,
The nucleotide sequence of the sense primer C is as shown in SEQ ID No.12, the nucleotide sequence of the anti-sense primer C
As shown in SEQ ID No.13, the nucleotide sequence of the probe H is as shown in SEQ ID No.14.
Above-mentioned probe A, B, C, D, E, F, G, H have carried out LNA modifications；The upstream and downstream primer A in reaction system most
The ultimate density of final concentration of 0.5~0.8 μM/L, upstream and downstream primer B in reaction system is 0.1~0.3 μM/L, and upstream and downstream is drawn
Ultimate densities of the thing C in reaction system is 0.1~0.3 μM/L, and probe A, B, C, D, E, F, G, H are final in reaction system
Concentration is 0.15~0.30 μM/L.4th, KRAS gene mutation detection primer probe according to claim 3, its feature exists
In：Ultimate densities of the upstream and downstream primer A in reaction system is 0.55 μM/L, and the upstream and downstream primer B is in reaction system
In ultimate density be 0.2 μM/L, ultimate densities of the upstream and downstream primer C in reaction system be 0.2 μM/L, the probe
The ultimate density of A, B, C, D, E, F, G, H in reaction system be respectively 0.21 μM/L, 0.18 μM/L, 0.22 μM/L, 0.22 μM/
L、0.19μM/L、0.21μM/L、0.19μM/L、0.18μM/L；Described probe A, B, C, D, E, F, G, H 5 ' ends are glimmering provided with reporting
Light group, 3 ' ends are provided with quenching fluorescence group.
Above-mentioned KRAS gene mutation detection kit also includes histone deacetylase solution, the digital pcr premix
Liquid includes archaeal dna polymerase, ultra-pure water, dNTPs mixed liquors, reference fluorescent ROX and buffer solution；The droplets stable agent includes
Mineral oil.
During above-mentioned KRAS gene detecting kits reaction, reaction system includes the volume of digital pcr premixed liquid 10, detects liquid
A or B2 volumes, the volume of droplets stable agent 2, the volume of template 1, sterilize the volume of ultra-pure water 5, and the concentration of the template is
0.1ng/ul~1ng/ul.
The present invention has positive effect：
1) the KRAS gene mutation detection kit of the invention production different, of the invention from conventional multi-primerses design principle
Thing length and primer length are shorter, and special modification is carried out to primer and probe, improve for mutational site and wild type
The specificity of the respective probe in site, reaches the purpose of probe and target sequence specific binding, detection architecture is optimized so that this hair
Bright sensitivity is 0.01%, and specificity is 100%, and the sample of lower mutation abundance can be detected.
2) KRAS gene mutation detection kit of the invention can also carry out multiplex PCR point except being outside one's consideration with ultra-high sensitive
Analysis.4 re-detections are reached by the fluorescence signal and regulation concentration and probe concentration that design probe, 1 KRAS is detected by 1 original chip
Sudden change sample is changed into 1 chip and detects 4 KRAS samples, and testing cost saves 50-100 members, greatly reduces cost.
Quick, economic, sensitive detection is realized to mutation in the case of high flux sample.
3) sample extracting, probe modification mode and the journey of KRAS gene mutation detection kit of the invention to reaction system
Sequence is optimized, and sample process process optimization avoids harmful effect of the pre-treatment step to testing result accuracy, and probe is excellent
Change is prevented effectively from the suppression between multiplex PCR probe and causes amplification efficiency unstable, it is ensured that enter in same reaction system
The stability of the re-detection of row 4.The combination of ddPCR and Multiple techniques improves the flux of genetic analysis so that can be from each patient
More information is obtained in sample.Patient is higher to the acceptance of Noninvasive sample collection mode, definitely fixed by the kit
Amount has been carried out time and spatial analysis to tumor patient, changes the sampling mode in Tumor mutations phase analysis, can be effectively
CtDNA levels (copy number of mutation allele in per ml blood plasma) performance of tumor patient is monitored, can not only be from KRAS
" having " or "None" are mutated the difference of matter, and to " many " or the amount difference of " few " mutation, treatment is reacted in time, so as to influence treatment
The selection and decision of scheme, more preferably clinical application provide theoretical foundation.
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 1
First, the composition of kit.
The KRAS gene mutation detection kit of the present embodiment, including digital pcr premixed liquid, droplets stable agent, primer spy
Pin mixed liquor, histone deacetylase solution and positive control.Each composition of kit is as shown in table 1.
The Kit components table of table 1
| Composition | Packing | Reagent Company |
| Digital pcr premixed liquid | 1 pipe | Life technologies |
| Droplets stable agent | 1 pipe | Raindance technologies |
| Primed probe mixed liquor | 1 pipe | Hundred power lattice |
| Positive control | 1 pipe | —— |
| Histone deacetylase | 1 pipe | Beijing Suo Laibao Science and Technology Ltd |
Reagent constituents is described as follows in above-mentioned table 1：
1) main component of digital pcr premixed liquid includes archaeal dna polymerase, ultra-pure water, dNTPs mixed liquors, reference fluorescent
ROX, buffer solution etc. (are provided, article No. by Life technologies companies：1508099).
2) main component of droplets stable agent is mineral oil, (is provided by Raindance technologies companies, goods
Number：30-00826), main function is that reaction system is formed Water-In-Oil droplet by droplet processing procedure.
3) primed probe mixed liquor includes being used to detect KRAS the 12nd, 13, the primer and probe of 61 codons, is to use respectively
Probe A, the 12nd codon G12V of upstream and downstream primer A, the 12nd codon G12R site mutations in the 12nd codon of detection
The probe B of point mutation, the probe C of the 12nd codon G12D site mutations, the probe D of the 12nd codon G12A site mutations,
The probe E of the 12 codon G12S site mutations and probe F of the 12nd codon G12C site mutations, for detecting the 13rd codon
Upstream and downstream primer B and the 13rd codon G13D site mutations probe G, and for detect the 61st codon upstream and downstream draw
Thing C and the 61st codon Q61L site mutations probe H.
The present embodiment has carried out LNA modifications to probe A, B, C, D, E, F, G, H.One is introduced i.e. in oligonucleotide probe
LNA bases, and adjust by adjusting the position of LNA bases within the probe the specificity of optimal fusing point (Tm) and hybridization.By
Probe after modification has higher Tm values, more preferably can be combined with mutagenesis template, the real-time quantitative PCR probe after modification, long
Degree is shorter, can increase the flexibility of design, crosses the mutant probe of LNA modifications and has more preferable saltant type to have more preferably to saltant type
Sub-clustering effect.
According to the requirement of instrument and Multiple experiments, certain specific mutant form is needed with two different fluorescence labelings
Taqman probes, are marked to the end of Taqman probes 5 ' using two kinds of fluorescence of VIC and FAM, use MGB quenching groups pair
The end of Taqman probes 3 ' is marked.If the design of primer and probe is improper, the selection to crucial target sequence is directly affected,
The sensitivity and specificity of PCR detections are reduced, or even are fallen flat.Thus, it is necessary to there is sufficient understanding to extension increasing sequence, and advise
Model PCR Lab operating technology, uses identical sense primer and anti-sense primer for wild and mutation, prevents due to primer mistake
Interfere more, reduce the sensitivity of detection.
That fluorescence labelings are held in probe A, probe B, probe D, the 5 ' of probe E is FAM, probe C, probe F, probe G, probe H
5 ' end fluorescence labelings be VIC, when detection template (DNA profiling) is undergone mutation, corresponding detection probe has VIC fluorescence
Signal or FAM fluorescence signals.Primed probe mixed liquor is divided into two pipes in the KRAS gene mutation detection kit of the present embodiment, point
Be not detection liquid A and detection liquid B, detection liquid A include upstream and downstream primer A, probe A, probe B, probe C, upstream and downstream primer B and
Probe G, detection liquid B includes upstream and downstream primer A, probe D, probe E, probe F, upstream and downstream primer C and probe H.
4) histone deacetylase is provided by Beijing Suo Laibao Science and Technology Ltd, according to product description that enzyme is dilute
Release to 10ug ml, every milliliter of serum or blood plasma add 10ul dilutions.
5) positive control is from positive cell strain (including the password of the 12nd, the 13rd and the 61st for carrying KRAS gene mutation
Saltant type including son), the DNA of saltant type and wild type KRAS genes is proportionally 1:100.
2nd, primed probe is designed.
Because the length of tumour dissociative DNA is very short, length≤180bp, the cracking of normal cell can discharge largely in blood
DNA, further reduction ctDNA abundance.Nuclease can also influence the stability of ctDNA in blood so that partial target piece
Duan Xulie may be shorter, if the amplified production of design primer is long, leading mutagenic sequence can not detect, the present invention
Product length is detected according to changing, it is found that the longer product mutation recall rate of shorter product mutation recall rate has bigger advantage,
It is more beneficial for realizing the detection of KRAS.The primed probe length that the present invention is designed be 14~18bp, product length 50~
80bp, prevents from causing Positive rate to reduce because amplified production amplification is long, as shown in table 2.
The primer probe sequence table of table 2
| Position | Detect classification | Seq No. | Sequence(5’—3’) |
| 12 codons | Sense primer A | 1 | CATGTTCTAATATAGTC |
| 12 codons | Anti-sense primer A | 2 | TGATCGTCAAGGCACTC |
| G12R | Probe A | 3 | TAGTTGGAGCTGGTCG |
| G12V | Probe B | 4 | AGCTGGTGTCGTA |
| G12D | Probe C | 5 | TGGAGCTGGTGACGT |
| G12A | Probe D | 6 | TGGAGCTGGTGCCGTA |
| G12S | Probe E | 7 | GAGCTGGTAGCGTA |
| G12C | Probe F | 8 | TTGGAGCTGGTTGC |
| 13 codons | Sense primer B | 9 | CCTGCTGAAAATGACTG |
| 13 codons | Anti-sense primer B | 10 | TGATTCTGAATTAGCTC |
| G13D | Probe G | 11 | AGCTGGTGGCGTAGG |
| 16 codons | Sense primer C | 12 | GATTCCTACAGGAAGCA |
| 16 codons | Anti-sense primer C | 13 | ACTGGTCCCTCATTGCAC |
| Q61L | Probe H | 14 | CACAGCAGGTCATGA |
Primed probe has the synthesis of the Li Ge Bioisystech Co., Ltd of Shanghai hundred, and work is diluted to by dry powder according to primer specification
Make mother liquor, then detection working solution is configured to by mother liquor, primer and probe mixed liquor is by mother liquor addition sterilizing ultra-pure water dilution
Into.
Too high concentration and probe concentration can improve the probability with wild-type template mispairing, to meet during multiple digital pcr design
Need, according to different fluorescence labelings, and by adjusting concentration and probe concentration so that the drop of different mutation types is able to put down in two dimension
Distinguished in face.For detecting that ultimate densities of the upstream and downstream primer A of the 12nd codon in reaction system is 0.5~0.8 μM/L,
It is preferred that end reaction concentration be 0.55 μM/L；For detect the 12nd codon upstream and downstream primer B in reaction system most
Final concentration of 0.1~0.3 μM/L, end reaction concentration preferably is 0.2 μM/L；For detecting that the upstream and downstream of the 61st codon is drawn
Ultimate densities of the thing C in reaction system is 0.1~0.3 μM/L, and end reaction concentration preferably is 0.2 μM/L；Probe A, B,
The ultimate density of C, D, E, F, G, H in reaction system is 0.15~0.30 μM/L, and end reaction concentration preferably is respectively
0.21μM/L、0.18μM/L、0.22μM/L、0.22μM/L、0.19μM/L、0.21μM/L、0.19μM/L、0.18μM/L。
The application method of three kits.
1st, sample DNA is extracted
DNA sources can be serum/plasma, blood plasma, peripheral blood, mucous membrane of mouth etc..The serum of 50 tumor patients of collection/
Plasma sample 5ml, high speed centrifugation separates supernatant, obtains serum/plasma.The serum/plasma provided with German Qiagen companies is total
DNA extraction kit (article No.：55114), the dissociative DNA in patients serum/blood plasma is extracted according to kit operational manual.Obtain
Obtain after serum/plasma sample dissociative DNA, pass through Thermo-Fisher companies3.0 nucleic acid-protein fluorescent quantitation instruments, are surveyed
Determine serum/plasma sample dissociative DNA concentration and purity, -20 DEG C of preservations.
Because normal cell etc. can be cracked in the blood after blood sampling in blood, substantial amounts of DNA is discharged, reduction ctDNA's is rich
Degree, nuclease can also influence the stability meeting of ctDNA in blood, cause both sides to influence on ctDNA detection, increase
The difficulty of ctDNA detections.The present embodiment, due to that can not carry out subsequent detection to sample in time, is dividing for a large amount of detection samples
Histone deacetylase (SIR2 enzymes) is added in the DNA sample after blood plasma or extracting from after, ensure that ctDNA's is steady
It is qualitative, and enzyme inactivates at high temperature, has no effect on follow-up amplification experiment.
2nd, prepared by digital pcr reaction solution
PCR reaction systems in table 3, take 20 μ l digital pcr premixed liquid in kit, 4 μ l primed probe to mix
Liquid is closed, 2ul template, 4 μ l droplets stable agent is added, sterilizing ultra-pure water is added and mends to 40 μ l, digital pcr reaction response is made
Liquid, prepares quantitative reaction system, and vibration is mixed, and centrifuges bubble removing.Template refers to the sample DNA after serum/plasma Sample Dilution
And negative control, negative control is autoclaving water.
Table 3PCR reaction system tables
| Reacted constituent | Add concentration | Add volume |
| Digital pcr premixed liquid | 2× | 20ul |
| Detect liquid A/B | --- | 4ul |
| Droplets stable agent | 10× | 4ul |
| Template | 0.1ng/ul~10ng/ul | 2ul |
| ddH2O | to 40ul |
3rd, PCR reacts droplet and prepared
Digital pcr mixed liquor is fabricated to the micro- reaction drops of PCR, 500~8,000,000 Water-In-Oil drops are formed.This experiment
Reaction system by drop generator formation picoliters level size Water-In-Oil droplet, DNA molecular mixture is distributed to
In the droplet of million, each reative cell averagely contains one or zero target molecule.Droplet generation plate is put into 8 passage droplets
In maker, the drop generators RainDrop Source produced with Raindance companies carry out droplet processing to sample.
4th, PCR is expanded
Performing PCR amplification is entered to the droplet of generation using the PCR amplification instrument of Bo companies.Response procedures are as follows：Pre-degeneration rank
94 DEG C of the condition of section, 8min；1 circulation；94 DEG C of 10s, 52 DEG C of 10s, 36 circulations；94 DEG C, 8min, 1 circulation；12℃
20min, 1 circulation；4 DEG C, insulation.
5th, droplet is detected
After PCR reactions terminate, 8 unions are placed in droplet analyzer, the droplet analyzer produced with Raindance companies
RainDrop Sense are analyzed the fluorescence signal of each droplet the droplet containing fluorescence signal labeled as 1, without fluorescence
Signal is labeled as 0, then droplet is counted, and software is automatically analyzed.Because digital pcr is a kind of terminal analysis method, if target molecule
Without discretization well, such as some droplets include multiple target nucleic acid molecules, then the result obtained in theory will be inaccurate,
Therefore introduce Poisson probability distribution function and be used to analyze data.
Poisson distribution formula is as follows：
According to the coefficient of dilution of droplet sum, the droplet number containing fluorescence signal and sample, the ctDNA water of sample can obtain
The abundance of flat and corresponding KRAS gene mutation.The ctDNA levels of sample represent the copy number of every milliliter of blood plasma allelic,
Gene mutation abundance represents the mutant copies number of KRAS genes in peripheral blood genome, and mutation includes the codon of the 12nd, 13 and 61
Mutation.
Comparative example 1
In order to improve the sensitivity of detection, those skilled in the art have carried out contrast experiment, the detection examination of this comparative example
Agent box, remainder is same as Example 1, and difference is：
1) primed probe design is using conventional primed probe design principle, and design primer length is 18~25bp, probe
Length is 20~30bp, and product length is 80~150bp, and probe does not carry out LNA modifications, primer probe sequence such as table 4
It is shown.
The custom primer probe sequence table of table 4
2) histone deacetylase dilution is not added in sample extraction step.
3) PCR amplification programs are：95 DEG C of the condition in pre-degeneration stage, 10min；1 circulation；94 DEG C of 10s, 52 DEG C of 15s, 60
DEG C 45s, 36 circulations；98 DEG C, 10min, 1 circulation；12 DEG C of 30min, 1 circulation；4 DEG C, insulation.
Take 50 samples that embodiment 1 is respectively adopted and the kit of comparative example 1 is detected, the KRAS genes of 50 samples
The abundance of mutation is as shown in table 5 and table 6.
The comparative example 1 of table 5 detects the KRAS gene mutation abundance table (%) of 50
The embodiment 1 of table 6 detects the KRAS gene mutation abundance table (%) of 50
As a result show, use the positive rate of kit of comparative example 1 for 20%, the abundance of KRAS gene mutation it is flat
Average is 1.8%；The positive rate of kit of embodiment 1 is used for 28%, the average value of the abundance of KRAS gene mutation
For 8.9%.
Comparative example 2
In order to optimize the detection kit of this comparative example, remainder is same as Example 1, and difference is：
1) probe does not carry out LNA modification.
2) histone deacetylase (SIR2 is added in the blood plasma after being separated when using or the DNA sample after extracting
Enzyme).
The positive sample of 20 KRAS gene mutations is taken, takes 20 samples that the reagent of embodiment 1 and comparative example 2 is respectively adopted
Box is detected that the abundance of the KRAS gene mutation of 20 samples is as shown in table 7 and table 8.
The comparative example 2 of table 7 detects the abundance table (%) of the KRAS gene mutation of 20
The embodiment 1 of table 8 detects the abundance table (%) of the KRAS gene mutation of 20
Histone deacetylase is added in blood plasma after being separated during due to using or the DNA sample after extracting
High 6~10ng/ul of ctDNA concentration ratios comparative example 2 in (SIR2 enzymes), the sample serum of embodiment 1.The KRAS of embodiment 1
The average value of the KRAS gene mutation abundance of the abundance ratio comparative example 2 of gene mutation is high by 7.0%.
Above-described embodiment is only intended to clearly illustrate example of the present invention, and is not the embodiment party to the present invention
The restriction of formula.For those of ordinary skill in the field, other differences can also be made on the basis of the above description
The change or variation of form.There is no necessity and possibility to exhaust all the enbodiments.And these belong to the essence of the present invention
Among the obvious changes or variations that god extends out is still in protection scope of the present invention.
SEQUENCE LISTING
<110>Shanghai Saian Biological Medical Technology Co., Ltd.
<120>KRAS gene mutation detection primer probe and its kit
<130>Nothing
<160> 14
<170> PatentIn version 3.3
<210> 1
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<212> DNA
<213>It is artificial synthesized
<400> 1
catgttctaa tatagtc 17
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<212> DNA
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<400> 2
tgatcgtcaa ggcactc 17
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<400> 3
tagttggagc tggtcg 16
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<211> 13
<212> DNA
<213>It is artificial synthesized
<400> 4
agctggtgtc gta 13
<210> 5
<211> 15
<212> DNA
<213>It is artificial synthesized
<400> 5
tggagctggt gacgt 15
<210> 6
<211> 16
<212> DNA
<213>It is artificial synthesized
<400> 6
tggagctggt gccgta 16
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gagctggtag cgta 14
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ttggagctgg ttgc 14
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<400> 9
cctgctgaaa atgactg 17
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tgattctgaa ttagctc 17
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<400> 11
agctggtggc gtagg 15
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<400> 12
gattcctaca ggaagca 17
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<212> DNA
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actggtccct cattgcac 18
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cacagcaggt catga 15
Claims (10)
1. a kind of KRAS gene mutation detection primer probe, it is characterised in that：Including the upstream and downstream for detecting the 12nd codon
Primer A, the probe A of the 12nd codon G12R site mutations, probe B, the 12nd codon of the 12nd codon G12V site mutations
The probe C of G12D site mutations, the probe D of the 12nd codon G12A site mutations, the spy of the 12nd codon G12S site mutations
Pin E and the 12nd codon G12C site mutations probe F, upstream and downstream primer B and the 13rd password for detecting the 13rd codon
The probe G of sub- G13D site mutations, and for detecting upstream and downstream primer C and the 61st codon Q61L sites of the 61st codon
The probe H of mutation；
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 probe A is as shown in SEQ ID No.3, the nucleotide sequence such as SEQ of the probe B
Shown in ID No.4, the nucleotide sequence of the probe C is as shown in SEQ ID No.5, the nucleotide sequence such as SEQ of the probe D
Shown in ID No.6, the nucleotide sequence of the probe E is as shown in SEQ ID No.7, the nucleotide sequence such as SEQ of the probe F
Shown in ID No.8,
The nucleotide sequence of the sense primer B is as shown in SEQ ID No.9, the nucleotide sequence such as SEQ of the anti-sense primer B
Shown in ID No.10, the nucleotide sequence of the probe G as shown in SEQ ID No.11,
The nucleotide sequence of the sense primer C is as shown in SEQ ID No.12, and the nucleotide sequence of the anti-sense primer C is such as
Shown in SEQ ID No.13, the nucleotide sequence of the probe H is as shown in SEQ ID No.14.
2. KRAS gene mutation detection primer probe according to claim 1, it is characterised in that：The probe A, B, C, D,
E, F, G, H have carried out LNA modifications.
3. KRAS gene mutation detection primer probe according to claim 1, it is characterised in that：The upstream and downstream primer A
Ultimate density in reaction system is 0.5~0.8 μM/L, ultimate densities of the upstream and downstream primer B in reaction system be 0.1~
0.3 μM/L, ultimate densities of the upstream and downstream primer C in reaction system is 0.1~0.3 μM/L, and probe A, B, C, D, E, F, G, H exist
Ultimate density in reaction system is 0.15~0.30 μM/L.
4. KRAS gene mutation detection primer probe according to claim 3, it is characterised in that：The upstream and downstream primer A
Ultimate density in reaction system is 0.55 μM/L, and ultimate densities of the upstream and downstream primer B in reaction system is 0.2 μ
The ultimate density of M/L, the upstream and downstream primer C in reaction system is 0.2 μM/L, and described probe A, B, C, D, E, F, G, H exist
Ultimate density in reaction system is respectively 0.21 μM/L, 0.18 μM/L, 0.22 μM/L, 0.22 μM/L, 0.19 μM/L, 0.21 μ
M/L、0.19μM/L、0.18μM/L。
5. KRAS gene mutation detection primer probe according to claim 1, it is characterised in that：The probe A, B, C, D,
E, F, G, H 5 ' ends are provided with reporter fluorescence group, and 3 ' ends are provided with quenching fluorescence group.
6. a kind of KRAS gene mutation detection product using primed probe as claimed in claim 1.
7. a kind of KRAS gene mutation detection kit, it is characterised in that：Including the numeral for preparing digital pcr reaction solution
PCR premixed liquids, droplets stable agent, detection liquid A and detection liquid B；
The detection liquid A includes upstream and downstream primer A, the spy of the 12nd codon G12R site mutations for being used to detect the 12nd codon
Pin A, the probe B of the 12nd codon G12V site mutations and the 12nd codon G12D site mutations probe C, and for detecting
The upstream and downstream primer B of 13rd codon and the 13rd codon G13D site mutations probe G；
The detection liquid B includes upstream and downstream primer A, the spy of the 12nd codon G12A site mutations for being used to detect the 12nd codon
Pin D, the probe E of the 12nd codon G12S site mutations and the 12nd codon G12C site mutations probe F, and for detecting
The upstream and downstream primer C of 61st codon and the 61st codon Q61L site mutations probe H；
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 probe A is as shown in SEQ ID No.3, the nucleotide sequence such as SEQ of the probe B
Shown in ID No.4, the nucleotide sequence of the probe C is as shown in SEQ ID No.5, the nucleotide sequence such as SEQ of the probe D
Shown in ID No.6, the nucleotide sequence of the probe E is as shown in SEQ ID No.7, the nucleotide sequence such as SEQ of the probe F
Shown in ID No.8,
The nucleotide sequence of the sense primer B is as shown in SEQ ID No.9, the nucleotide sequence such as SEQ of the anti-sense primer B
Shown in ID No.10, the nucleotide sequence of the probe G as shown in SEQ ID No.11,
The nucleotide sequence of the sense primer C is as shown in SEQ ID No.12, and the nucleotide sequence of the anti-sense primer C is such as
Shown in SEQ ID No.13, the nucleotide sequence of the probe H is as shown in SEQ ID No.14.
8. KRAS gene mutation detection kit according to claim 6, it is characterised in that：The probe A, B, C, D, E,
F, G, H have carried out LNA modifications；Ultimate densities of the upstream and downstream primer A in reaction system is 0.5~0.8 μM/L, upstream and downstream
Ultimate densities of the primer B in reaction system is 0.1~0.3 μM/L, and ultimate densities of the upstream and downstream primer C in reaction system is
0.1~0.3 μM/L, the ultimate density of probe A, B, C, D, E, F, G, H in reaction system is 0.15~0.30 μM/L；It is described to visit
Pin A, B, C, D, E, F, G, H 5 ' ends are provided with reporter fluorescence group, and 3 ' ends are provided with quenching fluorescence group.
9. KRAS gene mutation detection kit according to claim 6, it is characterised in that：Also include histone deacetylase
Change enzyme solutions, the digital pcr premixed liquid includes archaeal dna polymerase, ultra-pure water, dNTPs mixed liquors, reference fluorescent ROX and eased up
Fliud flushing；The droplets stable agent includes mineral oil.
10. KRAS gene mutation detection kit according to claim 6, it is characterised in that：During reaction, in reaction system
Including the volume of digital pcr premixed liquid 10, liquid A or B2 volume, the volume of droplets stable agent 2, the volume of template 1, sterilizing ultra-pure water 5 are detected
Volume, the concentration of the template is 0.1ng/ul~1ng/ul.
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| CN107557469A (en) * | 2017-09-27 | 2018-01-09 | 广州漫瑞生物信息技术有限公司 | A kind of specific primer in detection KRAS gene G12C sites is to, probe and kit |
| CN110373453A (en) * | 2019-07-24 | 2019-10-25 | 湖南大地同年生物科技有限公司 | A kind of detection primer, probe and the kit in KRAS gene mutation site |
| CN110452983A (en) * | 2019-07-05 | 2019-11-15 | 凯威康达(厦门)生物医药科技有限公司 | A kind of primer, probe, detection architecture and kit and method detecting 12 6 kinds of hot spot mutations of codon of KRAS gene |
| CN112342275A (en) * | 2020-11-26 | 2021-02-09 | 厦门大学 | Method and kit for detecting whether target nucleic acid contains mutation |
| CN114196757A (en) * | 2021-12-28 | 2022-03-18 | 普瑞斯新(上海)生物医疗科技有限公司 | KRAS gene mutation multiple detection primer probe and kit thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557469A (en) * | 2017-09-27 | 2018-01-09 | 广州漫瑞生物信息技术有限公司 | A kind of specific primer in detection KRAS gene G12C sites is to, probe and kit |
| CN110452983A (en) * | 2019-07-05 | 2019-11-15 | 凯威康达(厦门)生物医药科技有限公司 | A kind of primer, probe, detection architecture and kit and method detecting 12 6 kinds of hot spot mutations of codon of KRAS gene |
| CN110452983B (en) * | 2019-07-05 | 2022-09-20 | 凯威康达(厦门)生物医药科技有限公司 | Primer, probe, detection system, kit and method for detecting 6 hot spot mutations of KRAS gene 12 codons |
| CN110373453A (en) * | 2019-07-24 | 2019-10-25 | 湖南大地同年生物科技有限公司 | A kind of detection primer, probe and the kit in KRAS gene mutation site |
| CN112342275A (en) * | 2020-11-26 | 2021-02-09 | 厦门大学 | Method and kit for detecting whether target nucleic acid contains mutation |
| CN114196757A (en) * | 2021-12-28 | 2022-03-18 | 普瑞斯新(上海)生物医疗科技有限公司 | KRAS gene mutation multiple detection primer probe and kit thereof |
| CN114196757B (en) * | 2021-12-28 | 2024-02-23 | 普瑞斯新(上海)生物医疗科技有限公司 | KRAS gene mutation multiple detection primer probe and kit thereof |
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