CN108728492A

CN108728492A - A kind of preparation method of gene mutation and fusion positive reference product

Info

Publication number: CN108728492A
Application number: CN201710255423.1A
Authority: CN
Inventors: 高劲松; 张英杰; 魏潇; 魏奇
Original assignee: LIAONING QIRUN BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: LIAONING QIRUN BIOLOGICAL TECHNOLOGY Co Ltd
Priority date: 2017-04-19
Filing date: 2017-04-19
Publication date: 2018-11-02

Abstract

The purpose of the present invention is to provide the preparation methods of a kind of gene mutation and fusion positive reference product, the preparation method is using the slow virus or retrovirus being integrated into alien gene in cellular genome as carrier, prepare the sequence for meeting different genes mutation or fusion, after sequence verification is errorless, it is embedded into cell genomic dna by virus, drug sieve selects the monoclonal cell system with gene mutation or fusion, to obtain endlessly positive reference product.Wherein the cell line is preferably 293T cell lines, the mutation can be the single mutational site of term single gene, the multiple site mutations of term single gene, the multiple site mutations of multiple genes, the fusion can be single fusion, can also be multiple concatenated fusions；This method has the advantages that quick, accurate, simple, at low cost, high sensitivity, can be produced on a large scale, is conducive to using and promoting for market.

Description

A kind of preparation method of gene mutation and fusion positive reference product

Technical field

The invention belongs to genetic test fields, especially provide a kind of preparation of gene mutation and fusion positive reference product Method.

Background technology

It is well known that gene diagnosis is to develop an extremely rapid field in recent years, in the molecule diagnosis of tumour and target The genotype (SNP) treated to treatment, infectious disease pathogens determination and other diseased individualsization is required for using when being checked or analyzed To technique of gene detection, therefore, the clinical demand of genetic test is increasing.In detection method such as two generations generally used at present, survey Sequence technology (Next-generation sequencing technology), amplification refractory mutation system (Amplification Refractory mutation system, ARMS)-PCR, digital pcr (Digital PCR, dPCR) technology etc., according to these Technology is developing corresponding gene detection kit, and has inevitably been required for positive reference in the kit developed Positive control when product are as detection, because the kit based on clinical application must have positive control sample in the process of development The corresponding experiment of this progress kit, other than preventing false negative from occurring, it is also necessary to be made to the property indices of kit Judge and evaluate, being also required for corresponding positive or negative reference material in subsequent kit registers approval process is registered It examines；Called reference product, that is, have it is one or more enough it is uniform excellent characteristics is determined, to calibrating measuring device, Measurement method of grading or a kind of material or substance to material assignment.

Most common positive reference product are the artificial matter with corresponding mutational site in gene mutation detection kit at present Grain, still, Plasmid DNA is not the genomic DNA in histocyte, it is the product manually prepared, and molecular weight is only 3-4 thousand Base-pair, and sample to be detected is all complete genome DNA, molecular weight are 3,000,000,000 base-pairs, exist in the matrix of detection compared with Large deviation, therefore certain distortion can be caused, while in preparation and detection, plasmid is readily formed aerosol and pollutes ring Border, and then there is false positive；Positive plasmid only has the mutational site of cls gene to be checked, cannot detect internal control and external control index, Therefore integrality is short of using plasmid as positive criteria, in addition, its stability cannot be kept when plasmid concentration is less than a certain amount of For 24 hours, i.e., do not have stability.For the detection of fusion, plasmid as many deficiencies existing for positive reference product, because For the target of fusion detection, often mRNA, and plasmid is DNA, and detection mRNA has to by reverse transcription at cDNA's Step, detection DNA does not need this step then, therefore there are prodigious differences for the detecting step of the two, and it is same to make reference product with plasmid The problem of it is easy to appear false negative or false positives, the reliability of detection is had a greatly reduced quality.In addition to positive plasmid is as positive reference Outside product, the genomic DNA of some mutant tissues being easy to get or cell origin can also be used as positive reference product, but tumour base Because of mutation or normal SNP, type is various, is ever-changing, by the rarity and a series of sociology human relations of clinical sample The restriction of factor of science, it is difficult to be collected into the clinical sample of sufficient amount, the heterogeneity of tumor tissues, is sequenced with Sanger in addition Method is usually present the shortcoming of sensitivity and confusion occurs, and therefore, this reference material from tumor tissues or cell is by pole Big limitation, and the plasmid reference material generally used is obviously a short slab in some present molecule diagnosis kit components.

State Food and Drug Administration medical instrument evaluation center clearly requires reference material, i.e. reference material Matrix should use identical matrix, the sample of such as identical matrix to be not easy to obtain with the tested sample of clinic, should use as far as possible With the close sample of the tested sample matrix of clinic, matrix described herein is exactly the basic composition substance in sample to be detected, such as Virus in detection blood, matrix is when being serum or blood plasma, and such as detection is oncogene mutation, and it is to come from group that matrix, which is worked as, It knits or the genomic DNA of cell, such as detection is tumour fusion, matrix reference material and clinical is examined when being cell RNA Measured object in test sample sheet is consistent as far as possible.If the setting of reference material is unreasonable or existing defects, that will result in whole The raw material selection of a product, the composition of the determination of production technology and reaction system there is a problem of larger, lead to product Quality cannot reach safety and the validity of Clinical practice, be easy error result occur during clinical detection and influence to face The diagnosing and treating of bed.It can be seen that a good reference material is how important to the manufacturing enterprise of detection reagent, but country now There are no the reference standard product of gene mutation and fusion coherent detection reagent, this is also that national departments concerned is urgently to be resolved hurrily Problem.

Invention content

In view of the foregoing drawbacks, the technical problem to be solved in the present invention is to provide it is a kind of can meet all gene mutation types and The preparation method of Gene Fusion positive reference product, to avoid because nucleic acid extraction and detection error lead to the false sun of false negative Property and stability difference situation occur.

The present invention provides the preparation method of a kind of gene mutation type and Gene Fusion positive reference product, this method is exactly It, can be whole by slow virus or retrovirus by the virus particle for meeting gene mutation or fusion extracellularly prepared It is incorporated into the characteristic of cellular genome, mutation or fusion site are fitted into cell genomic dna, then uses drug screening Go out the monoclonal cell system with external mutation or fusion, pass through mass propgation to such cell line and storage, so that it may To ensure endlessly positive reference product, and it is this with the genomic DNA being fitted into mutation or fusion site, completely Above-mentioned deficiency is overcome, obtains stable mutant DNA or fusion gene mRNA, and inexhaustible.

The present invention provides the preparation methods of a kind of gene mutation and fusion positive reference product, are as follows：

(1) structure accordingly meets the virus particle of gene mutation and fusion gene sequence

A. the mRNA sequence of the genomic dna sequence and fusion in the corresponding mutational site of gene to be detected is recalled；

B. amplimer is selected in the upstream and downstream of gene mutation site and fusion, and is added respectively at the both ends of primer Digestion point sequence appropriate；

C. PCR amplification is carried out to gene mutation site or fusion, digestion purifying is carried out to PCR product, while to phase The viral vectors answered carries out digestion purifying；

D. digestion PCR product after purification and virus particle are attached reaction, are converted by heat shock bacterium, positive gram Grand screening, plasmid extraction and gene sequencing obtain the recombinant plasmid that sequence complies fully with gene mutation and fusion gene sequence；

(2) stable cell lines of the structure with gene mutation or fusion

A. transfected virus incasing cells prepares virus to the recombinant virus plasmid that prepared by viral packaging plasmid and above-mentioned (1) together Supernatant；

B. drug sieve selects the cell line of embedded recombinant viral genome sequence, is as follows：

The viral supernatants prepared in right amount are added in the fresh medium, and are added final concentration of 6 μ g/ml's Polybrene (Sigma products), is positioned over 37 DEG C of 5%CO₂Incubator in cultivate for 24 hours after, be added final concentration of 2 μ g/ml's The cell not infected is killed in puromycin (Sigma products) processing；

C. it verifies mutational site and fusion obtains gene mutation and fusion positive reference product；

Invention further provides the preparation method of a kind of gene mutation and fusion positive reference product, including Following steps：Take respectively quick 3 μ l of 2XT4DNA connections enzyme reaction buffer solution of Promega companies, 2 μ l of PCR digestions purified product, 1 μ l of digestion purified virus carrier, 1 μ l of T4DNA ligases centrifuge mixing, place room temperature 30min, then take 2 μ l of product after connection, It is added in the DH5 α competent cells (NEB companies of the U.S.) of 20 μ l, after ice bath 0.5h, then 42 DEG C of heat shock 30sec are added 200 μ l SOC culture solutions, 37 DEG C of shake culture 1h, then this culture is uniformly coated on the fine jade containing 100 μ g/ml ampicillins On fat culture plate, places 37 DEG C of incubators and be incubated overnight.

The another step of the present invention provides a kind of preparation method of gene mutation and fusion positive reference product, including Following steps：Viral packaging plasmid and the recombinant virus plasmid with mutational site or fusion are transfected together to being ready to 293T cells, it is specific as follows：1.5ml sterilizing EP pipes are taken, 1.0 μ g packagings mixing plasmids and 1.0 μ g mutated viruses plasmids are added And 250 μ l serum-free Opti MEM (Life companies of the U.S.).Soft mixing is incubated at room temperature 5min, while 1.5ml being taken to sterilize EP is managed, and 6 μ l liposomes Lipofectamine 2000 (U.S.'s Life Products) is taken to be dissolved in 250 μ l serum-free Opti-MEM I In culture medium, soft mixing is incubated at room temperature 5min；By plasmid solution and the soft mixing of liposome solutions, it is incubated at room temperature 20min； In six orifice plates, plasmid liposome complex is added；It is then placed into 37 DEG C of 5%CO₂Incubator in cultivate, for 24 hours afterwards with fresh DMEM culture mediums containing 10%FBS change liquid；Continue harvest after cultivating 48-72h and centrifuge 20min containing viral supernatant, 3000rpm, Removal precipitation, -80 DEG C of storages of viral supernatants or immediately infection cell.

To improve the purity of gene mutation and fusion positive reference product of the present invention, it is added in the fresh medium suitable The viral supernatants prepared are measured, and the polybrene (Sigma products) of final concentration of 6 μ g/ml is added, are positioned over 37 DEG C 5% CO₂Incubator in cultivate for 24 hours after, puromycin (Sigma products) processing that final concentration of 2 μ g/ml are added is killed and is not infected Cell.

As a preferred embodiment of gene mutation of the present invention and fusion positive reference product preparation method, selection carries There are Tumor mutations site fusion or the artificial recombination viral vectors of SNP site, you can alien gene is integrated into cell Viral vectors in genome, including slow virus carrier and retroviral vector.

It is therein as another preferred embodiment of gene mutation of the present invention and fusion positive reference product preparation method Cell line is 293T cell lines, further include various kinds of cell is available, such as other kinds of 293, CHO, Hela and A549 Deng optimal selection 293T cell lines.

Further step provides a kind of preparation method of gene mutation and fusion positive reference product to the present invention, wherein passing through The individual cells that limiting dilution method filters out are in 37 DEG C of 5%CO₂Incubator in cultivated, and to the correct cell of sequence verification System is enlarged culture, obtains monospecific polyclonal cell line.

As another preferred embodiment of gene mutation of the present invention and fusion positive reference product preparation method, wherein institute The cell line stated is the cell line in the single mutational site of term single gene, the cell line in the multiple mutational sites of term single gene and multiple bases Because of the cell line in multiple mutational sites, the cell line of single fusion and a variety of fusions.Prepare gene mutation or fusion The positive template of gene, amplification that can be by PCR rapid mutations method or RT-PCR to patient specimen can also be direct It synthesizes to obtain by gene chemical synthesis company.

To enable gene mutation of the present invention and fusion positive reference product extensive use, the preparation method that can make For the positive reference product of base replacement, frameshit, missing, insertion mutation, fusion, chromosome shift and its rare mutation.

Description of the drawings

The specific implementation mode of the present invention is described in further detail below in conjunction with the accompanying drawings, wherein：

Fig. 1 is the amplification figure in mutational site；

Fig. 2 is wild type site amplification figure corresponding with mutational site；

Fig. 3 is internal reference gene GAPDH amplification figures；

Wherein, curve 1,2,3,4,5 points of tables indicate 10 in figure shown in Fig. 1, Fig. 2 and Fig. 3⁵A mutant cell, 2X10⁴ A mutant cell, 4X10³A mutant cell, 8X10²A mutant cell and 1.6X10²The amplification curve of a mutant cell；

Fig. 4 is that 50ng/ μ l detection of nucleic acids result figures are added in A549 cells and the 293T cells of the mutational site containing Gly12Ser；

Fig. 5 is that A549 cells and the 293T cellular nucleic acids concentration of the mutational site containing Gly12Ser dilute the inspection after 100 times simultaneously Survey result figure；

1 indicates the detection of nucleic acids of A549 cells as a result, 2 indicate the 293T cells of the mutational site containing Gly12Ser in Fig. 4 and Fig. 5 Detection of nucleic acids result.

Specific implementation mode

Case study on implementation is described further the present invention below：

Embodiment 1

BRAF V600E positive gene mutation reference materials are prepared using PCDH slow virus as carrier：

(1) the genomic DNA sequence in the corresponding mutational site of gene to be detected is recalled from the Genbank of NCBI network address first Row, such as：The BRAF gene group DNA sequence dna seen around the mutational sites V600E is looked into BRAF V600E gene mutations, and in mutation position Point upstream and downstream about 500bp replicates its sequence, and sequence is as follows： ATCTATTAGTCCCTTTCAGACCTCTGACCTTGCTCAGTGGTAGTTGAGATATAACTGAAGACTCTAAATTATATAAC AATGAGGTGAGAAAAACATAATATTTCTCTTCCCTAAGTGCAGACTAAGATACTATCTGCAGCATCTTCATTCCAAT GAAGAGCCTTTACTGCTCGCCCAGGAGTGCCAAGAGAATATCTGGGCCTACATTGCTAAAATCTAATGGGAAAGTTT TAGGTTCTCCTATAAACTTAGGAAAGCATCTCACCTCATCCTAACACATTTCAAGCCCCAAAAATCTTAAAAGCAGG TTATATAGGCTAAATAGAACTAATCATTGTTTTAGACATACTTATTGACTCTAAGAGGAAAGATGAAGTACTATGTT TTAAAGAATATTATATTACAGAATTATAGAAATTAGATCTCTTACCTAAACTCTTCATAATGCTTGCTCTGATAGGA AAATGAGATCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGG TGATTTTGGTCTAGCTACAGTGAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGT GGATGGTAAGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCTGAGATGCTGCTGAGTTACTAGAAAGTCA TTGAAGGTCTCAACTATAGTATTTTCATAGTTCCCAGTATTCACAAAAATCAGTGTTCTTATTTTTTATGTAAATAG ATTTTTTAACTTTTTTCTTTACCCTTAAAACGAATATTTTGAAACCAGTTTCAGTGTATTTCAAACAAAAATATATG TCTTATAAACAGTGTTTCATATTTTATTCTTAAATAAATATGAACCCTTAAAACGAATATTTTGAAACCAGTTTCAG TGTATTTCAAACAAAAATATATGTCTTATAAACAGTGTTTCATATTTTATTCTAAATTGTTTAAAGTATTTTGTGTT CAAAATGTTCTGTGTACCCTGTTGAAAAAAAAAACAGGTATGCAATTTAAGGCAGGTGTGATCCACAGCCATTATTA TGGTTTTGCTAAGAGAACTACTCCTTTTAACAGAGAAGCTGT

Wherein upstream primer sequence is：GCTCTAGAGCATCTCACCTCATCCTAAC, downstream primer sequence are： GGAATTCTAGTAACTCAGCAGCATCTCAG, whereinTCTAGAWithGAATTCRespectively XbaI and EcoRI restriction enzyme sites.

(2) it using people's blood genomic DNA as template and the pairs of amplimer of above-mentioned synthesis, is tried with standard PCR amplification containing Taq enzyme Agent carries out PCR amplification according to operation instruction；

Reagent dosage：0.1 μ g/ pipes of DNA；Every primer 10pm/ pipe, 30 μ l/ pipes of reaction volume；

Reaction condition：94 DEG C of 3min pre-degenerations, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 32 amplification cycles, most 72 DEG C of extension 10min afterwards, respectively take 5 μ l of PCR product to be identified into row agarose gel electrophoresis after completion of the reaction.

(3) PCR product electroresis appraisal shows single band and molecular weight is correct, using the QIAquick of Qiagen companies PCR purification kits purify remaining PCR product, and operation by specification carries out.

To PCR product after purification, it is positioned over 37 DEG C using corresponding restriction enzyme and carries out digestion in 1 hour, then Again use Qiagen companies QIAquick PCR purification kits, the product after digestion is purified, operation by specification into Row, PCR product after purification are quantitative determined with ultraviolet specrophotometer 260nm, are adjusted to respective concentration with pure water.

(4) by taking PCDH slow virus carriers as an example, PCDH slow virus carriers are carried out with identical XbaI and EcoRI restriction endonucleases Digestion, the plasmid obtained after purification are quantitative determined in ultraviolet specrophotometer 260nm, are adjusted to 50-100ng/ μ l with pure water；It is right The ratio of PCR product and plasmid by specification after purification is attached, and its step are as follows：

Take quick 3 μ l of 2XT4DNA connections enzyme reaction buffer solution of Promega companies, 2 μ l of PCR product, 1 μ l of viral vectors, 1 μ l of T4DNA ligases centrifuge mixing, place room temperature 30 minutes, then take 2 μ l of product after connection, and the DH5 α competence of 20 μ l is added In cell (NEB companies of the U.S.), after ice bath 0.5h, then 200 μ l SOC culture solutions, 37 DEG C of shakes are added in 42 DEG C of heat shock 30sec Culture 1h is swung, then this culture is uniformly coated on the agar plate containing 100 μ g/ml ampicillins, 37 DEG C is placed and incubates Case is incubated overnight.

(5) positive colony is filtered out with special primer, is seeded to 5ml and contains 100 μ g/ml ampicillin LB culture solutions It is incubated overnight in test tube.

(6) plasmid finished is extracted, is quantitative determined with ultraviolet specrophotometer 260nm.

(7) DNA sequencing is made to the plasmid prepared, determines that sequence complies fully with the plasmid of wild type as experiment in next step Template.

According to the catastrophe that gene occurs, the primer of design PCR fixed point rapid mutations is public with reference to U.S. Stratagene The design of primers of department's fixed point rapid mutation kit is completed, and the annealing temperature of primer is higher than the annealing temperature of Standard PCR primer by 10 DEG C or more.The fixed point rapid mutation primer of design is as follows, sense primer：GGTCTAGCTACAGAGAAATCTCGATGGAGTG；Under Swim primer：CACTCCATCGAGATTTCTCTGTAGCTAGACC synthesizes designed primer sequence.

PCR reaction systems are as follows：

Add ddH₂O to 50 μ L

PCR reaction conditions：95 DEG C of pre-degenerations, 3min；95 DEG C of thermal denaturation 30s, 55 DEG C take off fiery 60s, 72 DEG C of extension 3min, and 15 A thermal cycle；72 DEG C of renaturation 10min；4 DEG C of preservations.

(8) 0.8% Ago-Gel is prepared.

(9) PCR amplification band can be seen in the position of plasmid large fragment by taking 15 μ L of PCR product to carry out electrophoretic analysis.

(10) the Dpnl enzymes (NEB companies of the U.S.) of 1 μ L are added in remaining PCR reaction products, 37 DEG C are placed after mixing It is incubated 1h or more.The 2 μ l of PCR product after being incubated are taken, are added in the DH5 α competent cells of 20 μ l, after ice bath half an hour, 42 DEG C Then heat shock 30sec is added 200 μ l SOC culture solutions, 37 DEG C of shake culture 1h, then this culture is uniformly coated on and is contained On the agar plate of 100 μ g/ml ampicillins, places 37 DEG C of incubators and be incubated overnight.

(11) to the clone to grow on culture plate, several are selected at random, are seeded to 5ml respectively and are contained 100 μ g/ml ammonia benzyls It is incubated overnight in the test tube of penicillin LB culture solutions.To the bacterium solution being incubated overnight, using the QIAprep Spin of Qiagen companies The small pumping kit of Miniprep plasmids, carries out plasmid extraction, and operation by specification carries out.

(12) plasmid finished is extracted, is quantitative determined with ultraviolet specrophotometer 260nm.

(13) DNA sequencing is made to the plasmid prepared, determine sequence comply fully with saltant type plasmid can be used as in next step Prepare the plasmid of virus.

(14) Viral packaging cell 293T cultures are positioned in the culture medium of DMEM (Life companies of the U.S.) 10%FBS 37 DEG C of 5%CO₂Incubator in cultivate, in the day before transfection press 3X10⁶Cell/plate prepares six well culture plates.

(15) packaging plasmid and virus particle with mutation are transfected to ready 293T cells, specifically such as together Under：

1.5ml sterilizing EP pipes are taken, the nothing of 1.0 μ g packaging mixing plasmids and 1.0 μ g mutated viruses plasmids and 250 μ l is added Serum Opti MEM (Life companies of the U.S.).Soft mixing is incubated at room temperature 5min, while taking 1.5ml sterilizing EP pipes, takes 6 μ l fat Plastid Lipofectamine 2000 (U.S.'s Life Products) is dissolved in 250 μ l serum-free Opti-MEM I culture mediums, gently Soft mixing is incubated at room temperature 5min；By plasmid solution and the soft mixing of liposome solutions, it is incubated at room temperature 20min；In six orifice plates, Plasmid liposome complex is added；It is then placed into 37 DEG C of 5%CO₂Incubator in cultivate, for 24 hours afterwards with fresh DMEM contain 10% FBS culture mediums change liquid；Continuing harvest after cultivating 48-72h, containing viral supernatant, 3000rpm centrifuges 20min, removal precipitation, virus - 80 DEG C of storages of supernatant or immediately infection cell.

(16) in preprepared cell line, the viral supernatants prepared in right amount are added in the fresh medium, And the polybrene (Sigma products) of final concentration of 6 μ g/ml is added, it is positioned over 37 DEG C of 5%CO₂Incubator in cultivate for 24 hours after, The cell not infected is killed in puromycin (Sigma products) processing that final concentration of 2 μ g/ml are added.

(17) verification obtains positive gene mutation reference material containing mutational site and embedded gene copy number, and specific steps are such as Under：

It is diluted using limiting dilution method, filters out individual cells in 37 DEG C of 5%CO₂Incubator in cultivated, later simultaneously Continue to expand culture, after these cells cover with, a part is positioned over Liquid nitrogen storage, and a part carries out the extracting of DNA, and carries out The verification in mutational site, and it is embedded in the verification of gene copy number, the methods of sequencing and ARMS-PCR can be used by verification method in the former, The latter is compared verification with single copy gene using quantitative PCR technique, verifies correct cell line and suitable chimeric copy Number is exactly correct positive gene mutation reference material.

Embodiment 2

Intend Retroviral using pMXs and prepares BRAF gene mutation positive reference product as carrier：

Identical structure is carried out using pMXs retrovirus as carrier, the upstream and downstream primer restriction enzyme site of BRAF will occur Variation, upstream primer sequence are：GGAATTCGCATCTCACCTCATCCTAAC；,

Downstream primer sequence is：CCCAAGCTTTAGTAACTCAGCAGCATCTCAG,

Wherein GAATTC and AAGCTT is respectively EcoRI and HindIII restriction enzyme sites, respectively to carrier and primer with this two A identical enzyme carries out digestion, is then purified, is connected, is heat-shock transformed, recombinant plasmid extracting identification, pinpointed and quickly dash forward Become and subsequent virus packaging prepares and the foundation of stable cell lines, it is all identical with 1 step of above-described embodiment.

Embodiment 3

The positive gene mutation reference of the two gene associations mutation of Kras and BRAF is prepared using PCDH slow virus as carrier Product：

(1) the genomic DNA sequence in the corresponding mutational site of Kras genes to be detected is recalled from the Genbank of NCBI network address Row, sequence are as follows：

CCGCAGAACAGCAGTCTGGCTATTTAGATAGAACAACTTGATTTTAAGATAAAAGAACTGTCTATGTAGCATTTATG CATTTTTCTTAAGCGTCGATGGAGGAGTTTGTAAATGAAGTACAGTTCATTACGATACACGTCTGCAGTCAACTGGA ATTTTCATGATTGAATTTTGTAAGGTATTTTGAAATAATTTTTCATATAAAGGTGAGTTTGTATTAAAAGGTACTGG TGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAG GCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAG CTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGT GCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTTCATGAGTACTTATTACAAGATAATTATGCTGA AAGTTAAGTTATCTGAAATGTACCTTGGGTTTCAAGTTATATGTAACCATTAATATGGGAACTTTACTTTCCTTGGG AGTATGTCAGGGTCCATGATGTTCACTCTCTGTGCATTTTGATTGGAAGTGTATTTCAGAGTTTCGTGAGAGGGTAG AAATTTGTATCCTATCTGGACCTAAAAGACAATCTTTTTATTGTAACTTTTATTTTTATGGGTTTCTTGGTATTGTG ,

Wherein sense primer is：GCTCTAGAGCGTCGATGGAGGAGTTTG, downstream primer are： GAGTATGTCAGGGTCCATG, GGTGGC are the position in mutation site, and TCTAGA is XbaI enzyme cutting site, to designing Primer sequence synthesis.

(2) it using people's blood genomic DNA as template and the pairs of amplimer of above-mentioned synthesis, is tried with standard PCR amplification containing Taq enzyme Agent carries out PCR amplification, DNA dosages according to operation instruction：0.1 μ g/ pipes；Every primer dosage：10pm/ is managed, reaction volume 30 μ l/ pipes, reaction condition are：94 DEG C of 3min pre-degenerations, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 32 amplification cycles, most 72 DEG C of extension 10min afterwards, respectively take 5 μ l of PCR product to be identified into row agarose gel electrophoresis after completion of the reaction.The single item of electrophoresis showed Band, molecular weight are consistent with expected molecular weight, then by PCR product be positioned over -20 DEG C it is spare.

(3) two gene orders of the Kras of pcr amplified fragment and BRAF are merged, sequence is as follows：

GCGTCGATGGAGGAGTTTGTAAATGAAGTACAGTTCATTACGATACACGTCTGCAGTCAACTGGAATTTTCATGATT GAATTTTGTAAGGTATTTTGAAATAATTTTTCATATAAAGGTGAGTTTGTATTAAAAGGTACTGGTGGAGTATTTGA TAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAA TGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAAT CATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTC TTTGATACAGATAAAGGTTTCTCTGACCATTTTCATGAGTACTTATTACAAGATAATTATGCTGAAAGTTAAGTTAT CTGAAATGTACCTTGGGTTTCAAGTTATATGTAACCATTAATATGGGAACTTTACTTTCCTTGGGAGTATGTCAGGG TCCATG ACATTTCAAGCCCCAAAAATCTTAAAAGCAGGTTATATAG GCTAAATAGAACTAATCATTGTTTTAGACATACTTATTGACTCTAAGAGGAAAGATGAAGTACTATGTTTTAAAGAA TATTATATTACAGAATTATAGAAATTAGATCTCTTACCTAAACTCTTCATAATGCTTGCTCTGATAGGAAAATGAGA TCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTG GTCTAGCTACAGTGAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTA AGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCTGAGATGCTGCTGAGTTACTAG

Wherein overlapping sense primers：GGGTCCATGGCATCTCACCTCATCCTAAC；The downstreams overlapping Primer：GAGGTGAGATGCCATGGACCCTGACATACTC.

The sense primer of Kras and overlapping downstream primers are matched, under overlapping sense primers and BRAF Primer pairing is swum, the template of amplification is PCR product above-mentioned respectively, after amplified production is by overlapping, then uses Kras Sense primer and BRAF downstream primers match PCR amplification carried out to the product after overlapping, clone two required enzymes Point of contact is XbaI and EcoRI respectively, is then cloned into PCDH carriers, and respective various mutational sites are mutated by Fast Fixed-point Method is completed, and method is as described above.

(4) virus and subsequent stable cell lines screening and identification are prepared with above-described embodiment 1.

Embodiment 4

Detection mutation stable cell lines

It is mutated stable cell lines 10 with BRAF⁵A cell/pipe, respectively with normal 293T cells by 5 times of gradient dilutions at containing Mutant cell 2X10⁴、4X10³、8X10²And 1.6X10²A cell/pipe, cell number total amount are still maintained at 10⁵A cell/pipe；

DNA extractings are carried out to this five solencyte using DNA extraction kit；

Its concentration is measured with ultraviolet specrophotometer, concentration is adjusted to 50ng/ μ l；

With the BRAF V600E abrupt climatic change ARMS-PCR fluorescence probe method kits prepared through the invention, It is verified on ABI7500 type fluorescence quantitative PCR instruments；

Wherein, fluoroscopic examination uses Taqman probes, mutant probe to be marked with FAM, and internal reference gene GAPDH uses VIC Label probe；

Reagent dosage：Various primer dosages are respectively 10pm/tube, and various probe dosages are respectively 5pm/tube, Taq enzyme 1.5U/tube, reaction volume are 25 μ l；

Reaction condition：95 DEG C of 2min pre-degenerations, 95 DEG C of 5sec, 65 DEG C of 32sec, 15 cycles do not collect fluorescence, and then 95 DEG C 5sec, 62 DEG C of 32sec, 40 cycles, collect fluorescence.

After reaction, the amplification situation in mutational site is shown in that Fig. 1, corresponding wild type site amplification situation are shown in Fig. 2, Internal reference gene GAPDH amplification situations are shown in Fig. 3, and curve 1,2,3,4,5 points of tables indicate 10 in figure⁵A mutant cell, 2X10⁴It is a prominent Become cell, 4X10³A mutant cell, 8X10²A mutant cell and 1.6X10²The amplification curve of a mutant cell.It can be with from figure Find out, mutator group is still very stable in low concentration, and the amplification situation of internal reference and External reference is also detected and expanded simultaneously Curve is preferable, fully meets the requirement of kit.

Embodiment 5

Stable cell line of the comparison with mutational site and the naturally cell strain containing gene mutation site

It is as follows：

Using the cell line A549 in lung cancer source, the mutational sites the Gly12Ser of gene containing Kras, 10⁵A A549 cells paving In 10cm culture dishes, the DMEM culture mediums containing 10%FBS are added, are positioned over 37 DEG C of 5%CO₂Incubator in cultivate it is 6-7 days thin Born of the same parents cover with, collected by trypsinisation cell, then carry out nucleic acid extraction with nucleic acid extraction kit, operate by specification, then purple Outer spectroscope measures DNA concentration, and a 10cm culture dishes cell can extract general 75-100 μ g DNA, and concentration is adjusted with pure water For 50ng/ μ l be placed in -20 DEG C it is spare.

To the 10 of the artificial constructed stabilization in the mutational sites the Gly12Ser of gene containing Kras⁵A 293T cells are layered on 10cm cultures In ware, the DMEM culture mediums containing 10%FBS are added, is positioned in the incubator of 37 DEG C of 5%CO2 and cultivates, cell is long after culture 5-6 days It is full, cell is directly harvested, nucleic acid extraction is carried out with nucleic acid extraction kit, operates by specification, then ultraviolet spectrometer measures DNA concentration, a 10cm culture dishes cell can extract general 100-150 μ g DNA, and adjusting a concentration of 50ng/ μ l with pure water sets It is spare in -20 DEG C.

The ARMS-PCR fluorescence probe method kits in the mutational sites Kras gene Gly12Ser, A549 are prepared using the present invention It is detected with the nucleic acid of the 293T cells of the mutational site containing Gly12Ser, as a result shows the nucleic acid of two cell strains regardless of concentration is high Low, the two CT values are completely the same, and wherein Fig. 4 is that 50ng/ μ are added in A549 cells and the 293T cells of the mutational site containing Gly12Ser L detection of nucleic acids results；The nucleic acid concentration that Fig. 5 both is dilutes the testing result after 100 times simultaneously, and 1 indicates in Fig. 4 and Fig. 5 The detection of nucleic acids of A549 cells is as a result, 2 indicate the detection of nucleic acids result of the 293T cells of the mutational site containing Gly12Ser.

Embodiment 6

Fusion positive reference product are prepared using PCDH slow virus as carrier, to lead to chronic granulocytic leukemia (CML) for fusion BCR-ABL：

5 milliliters of blood are extracted from the peripheral blood of patient CML, are detached karyocyte with lymphocyte separation medium, are being detached 1 milliliter of Trizol reagents are added in karyocyte out and extract total serum IgE, operation by specification carries out, then ultraviolet spectrometry Instrument measures RNA concentration and is taken 1 μ gRNA to do reverse transcription reaction with random primer using NEB companies of U.S. Reverse Transcriptase kit, obtained The libraries cDNA, operation by specification carry out.

It is as follows that BCR-ABL fusion AF113911.1 sequences are recalled from gene pool：

Gtaccagccctaccagagcatctacgtcgggggcatgatggaaggggagggcaagggcccgctcctgcgcagccaga gcacctctgagcaggagaagcgccttacctggccccgcaggtcctactccccccggagttttgaggattgcggaggc ggctataccccggactgcagctccaatgagaacctcacctccagcgaggaggacttctcctctggccagtccagccg cgtgtccccaagccccaccacctaccgcatgttccgggacaaaagccgctctccctcgcagaactcgcaacagtcct tcgacagcagcagtccccccacgccgcagtgccataagcggcaccggcactgcccggttgtcgtgtccgaggccacc atcgtgggcgtccgcaagaccgggcagatctggcccaacgatggcgagggcgccttccatggagacgcaga ggccagtagcatctgactttgagcctcagggtctgagtgaagccgctcgttggaactccaaggaaa accttctcgctggacccagtgaaaatgaccccaaccttttcgttgcactgtatgattttgtggccagtggagataac actctaagcataactaaaggtgaaaagctccgggtcttaggctataatcacaatggggaatggtgtgaagcccaaac caaaaatggccaaggctgggtcccaagcaactacatcacgccagtcaacagtctggagaaacactcctggtaccatg ggcctgtgtcccgcaatgccgctgagtatctgctgagcagcgggatcaatggcagcttcttggtgcgtgagagtgag agcagtcctggccagaggtccatctcgctgagatacgaagggagggtgtaccattacaggatcaacactgcttctga tggcaagctctacgtctcctccgagagccgcttcaacaccctggccgagttggttcatcatcattcaacggtggccg acgggctcatcaccacgctccattatccagccccaaagcgcaacaagcccactgtctatggtgtgtcccccaactac gacaagt

WhereingagacgcagaWithJunction be exactly sense primer at the fusion of two genes： CGGAATTCgagcaggagaagcgccttac, downstream primer：CGGGATCCtgctctcactctcacgcacca draws in upstream 5 ' ends of object have added the restriction enzyme site of EcoRI, BamHI restriction enzyme sites have been added at 5 ' ends of downstream primer, with this to primer The libraries cDNA of above-mentioned acquisition are expanded, this section of cDNA sequence directly can also synthesize to obtain by gene chemical synthesis company.

Reaction system：10XPCR buffer 5μl,cDNA 1μl；Every primer dosage：15pm/ is managed, reaction volume 50 μ l, Pfu archaeal dna polymerase dosages are 1.5U；

Reaction condition：94 DEG C of 3min pre-degenerations, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 amplification cycles, most 72 DEG C of extension 10min afterwards；

Respectively take after completion of the reaction 5 μ l of PCR product into row agarose gel electrophoresis identify, behind purified pcr product and fusion The construction method and above-described embodiment 1 of gene stable cell lines are identical.

1. SEQUENCE LISTING

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3. <110>Liaoning bio tech ltd Qi Run

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5. <120>A method of preparing gene mutation and fusion positive reference product

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19. atctattagt ccctttcaga cctctgacct tgctcagtgg tagttgagat ataactgaag 60

20.

21. actctaaatt atataacaat gaggtgagaa aaacataata tttctcttcc ctaagtgcag 120

22.

23. actaagatac tatctgcagc atcttcattc caatgaagag cctttactgc tcgcccagga 180

24.

25. gtgccaagag aatatctggg cctacattgc taaaatctaa tgggaaagtt ttaggttctc 240

26.

27. ctataaactt aggaaagcat ctcacctcat cctaacacat ttcaagcccc aaaaatctta 300

28.

29. aaagcaggtt atataggcta aatagaacta atcattgttt tagacatact tattgactct 360

30.

31. aagaggaaag atgaagtact atgttttaaa gaatattata ttacagaatt atagaaatta 420

32.

33. gatctcttac ctaaactctt cataatgctt gctctgatag gaaaatgaga tctactgttt 480

34.

35. tcctttactt actacacctc agatatattt cttcatgaag acctcacagt aaaaataggt 540

36.

37. gattttggtc tagctacagt gaaatctcga tggagtgggt cccatcagtt tgaacagttg 600

38.

39. tctggatcca ttttgtggat ggtaagaatt gaggctattt ttccactgat taaatttttg 660

40.

41. gccctgagat gctgctgagt tactagaaag tcattgaagg tctcaactat agtattttca 720

42.

43. tagttcccag tattcacaaa aatcagtgtt cttatttttt atgtaaatag attttttaac 780

44.

45. ttttttcttt acccttaaaa cgaatatttt gaaaccagtt tcagtgtatt tcaaacaaaa 840

46.

47. atatatgtct tataaacagt gtttcatatt ttattcttaa ataaatatga acccttaaaa 900

48.

49. cgaatatttt gaaaccagtt tcagtgtatt tcaaacaaaa atatatgtct tataaacagt 960

50.

51. gtttcatatt ttattctaaa ttgtttaaag tattttgtgt tcaaaatgtt ctgtgtaccc 1020

52.

53. tgttgaaaaa aaaaacaggt atgcaattta aggcaggtgt gatccacagc cattattatg 1080

54.

55. gttttgctaa gagaactact ccttttaaca gagaagctgt 1120

56. SEQUENCE LISTING

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60. <120>A method of preparing gene mutation and fusion positive reference product

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74. gctctagagc atctcacctc atcctaac 28

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91. ggaattctag taactcagca gcatctcag 29

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108. ggtctagcta cagagaaatc tcgatggagt g 31

109. <110>Liaoning bio tech ltd Qi Run

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125. cactccatcg agatttctct gtagctagac c 31

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137. <211> 27

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142. ggaattcgca tctcacctca tcctaac 27

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155. <212> DNA

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159. cccaagcttt agtaactcag cagcatctca g 31

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176. ccgcagaaca gcagtctggc tatttagata gaacaacttg attttaagat aaaagaactg 60

177.

178. tctatgtagc atttatgcat ttttcttaag cgtcgatgga ggagtttgta aatgaagtac 120

179.

180. agttcattac gatacacgtc tgcagtcaac tggaattttc atgattgaat tttgtaaggt 180

181.

182. attttgaaat aatttttcat ataaaggtga gtttgtatta aaaggtactg gtggagtatt 240

183.

184. tgatagtgta ttaaccttat gtgtgacatg ttctaatata gtcacatttt cattattttt 300

185.

186. attataaggc ctgctgaaaa tgactgaata taaacttgtg gtagttggag ctggtggcgt 360

187.

188. aggcaagagt gccttgacga tacagctaat tcagaatcat tttgtggacg aatatgatcc 420

189.

190. aacaatagag gtaaatcttg ttttaatatg catattactg gtgcaggacc attctttgat 480

191.

192. acagataaag gtttctctga ccattttcat gagtacttat tacaagataa ttatgctgaa 540

193.

194. agttaagtta tctgaaatgt accttgggtt tcaagttata tgtaaccatt aatatgggaa 600

195.

196. ctttactttc cttgggagta tgtcagggtc catgatgttc actctctgtg cattttgatt 660

197.

198. ggaagtgtat ttcagagttt cgtgagaggg tagaaatttg tatcctatct ggacctaaaa 720

199.

200. gacaatcttt ttattgtaac ttttattttt atgggtttct tggtattgtg 770

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217. gcgtcgatgg aggagtttgt aaatgaagta cagttcatta cgatacacgt ctgcagtcaa 60

218.

219. ctggaatttt catgattgaa ttttgtaagg tattttgaaa taatttttca tataaaggtg 120

220.

221. agtttgtatt aaaaggtact ggtggagtat ttgatagtgt attaacctta tgtgtgacat 180

222.

223. gttctaatat agtcacattt tcattatttt tattataagg cctgctgaaa atgactgaat 240

224.

225. ataaacttgt ggtagttgga gctggtggcg taggcaagag tgccttgacg atacagctaa 300

226.

227. ttcagaatca ttttgtggac gaatatgatc caacaataga ggtaaatctt gttttaatat 360

228.

229. gcatattact ggtgcaggac cattctttga tacagataaa ggtttctctg accattttca 420

230.

231. tgagtactta ttacaagata attatgctga aagttaagtt atctgaaatg taccttgggt 480

232.

233. ttcaagttat atgtaaccat taatatggga actttacttt ccttgggagt atgtcagggt 540

234.

235. ccatggcatc tcacctcatc ctaacacatt tcaagcccca aaaatcttaa aagcaggtta 600

236.

237. tataggctaa atagaactaa tcattgtttt agacatactt attgactcta agaggaaaga 660

238.

239. tgaagtacta tgttttaaag aatattatat tacagaatta tagaaattag atctcttacc 720

240.

241. taaactcttc ataatgcttg ctctgatagg aaaatgagat ctactgtttt cctttactta 780

242.

243. ctacacctca gatatatttc ttcatgaaga cctcacagta aaaataggtg attttggtct 840

244.

245. agctacagtg aaatctcgat ggagtgggtc ccatcagttt gaacagttgt ctggatccat 900

246.

247. tttgtggatg gtaagaattg aggctatttt tccactgatt aaatttttgg ccctgagatg 960

248.

249. ctgctgagtt actag 975

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266. gggtccatgg catctcacct catcctaac 29

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283. gaggtgagat gccatggacc ctgacatact c 31

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300. gtaccagccc taccagagca tctacgtcgg gggcatgatg gaaggggagg gcaagggccc 60

301.

302. gctcctgcgc agccagagca cctctgagca ggagaagcgc cttacctggc cccgcaggtc 120

303.

304. ctactccccc cggagttttg aggattgcgg aggcggctat accccggact gcagctccaa 180

305.

306. tgagaacctc acctccagcg aggaggactt ctcctctggc cagtccagcc gcgtgtcccc 240

307.

308. aagccccacc acctaccgca tgttccggga caaaagccgc tctccctcgc agaactcgca 300

309.

310. acagtccttc gacagcagca gtccccccac gccgcagtgc cataagcggc accggcactg 360

311.

312. cccggttgtc gtgtccgagg ccaccatcgt gggcgtccgc aagaccgggc agatctggcc 420

313.

314. caacgatggc gagggcgcct tccatggaga cgcagaagcc cttcagcggc cagtagcatc 480

315.

316. tgactttgag cctcagggtc tgagtgaagc cgctcgttgg aactccaagg aaaaccttct 540

317.

318. cgctggaccc agtgaaaatg accccaacct tttcgttgca ctgtatgatt ttgtggccag 600

319.

320. tggagataac actctaagca taactaaagg tgaaaagctc cgggtcttag gctataatca 660

321.

322. caatggggaa tggtgtgaag cccaaaccaa aaatggccaa ggctgggtcc caagcaacta 720

323.

324. catcacgcca gtcaacagtc tggagaaaca ctcctggtac catgggcctg tgtcccgcaa 780

325.

326. tgccgctgag tatctgctga gcagcgggat caatggcagc ttcttggtgc gtgagagtga 840

327.

328. gagcagtcct ggccagaggt ccatctcgct gagatacgaa gggagggtgt accattacag 900

329.

330. gatcaacact gcttctgatg gcaagctcta cgtctcctcc gagagccgct tcaacaccct 960

331.

332. ggccgagttg gttcatcatc attcaacggt ggccgacggg ctcatcacca cgctccatta 1020

333.

334. tccagcccca aagcgcaaca agcccactgt ctatggtgtg tcccccaact acgacaagt 1079

335. <110>Liaoning bio tech ltd Qi Run

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351. cggaattcga gcaggagaag cgccttac 28

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368. cgggatcctg ctctcactct cacgcacca 29

Claims

1. the preparation method of a kind of gene mutation and fusion positive reference product, it is characterised in that：It is as follows：

(1) structure accordingly meets the virus particle of gene mutation or fusion gene sequence

A. the genomic dna sequence of the corresponding mutational site upstream and downstream of gene to be detected or the mRNA sequence of fusion are recalled；

B. amplimer is selected in the upstream and downstream of gene mutation site or fusion, and is added respectively and disease at the both ends of primer Corresponding digestion point sequence on poisonous carrier；

C. PCR amplification is carried out to gene mutation site or fusion, it is pure to carry out digestion to PCR product and corresponding viral vectors Change；

D. digestion PCR product after purification and virus particle are attached reaction, are converted by heat shock bacterium, positive colony sieve Choosing, plasmid extraction and gene sequencing obtain the recombinant plasmid that sequence complies fully with gene mutation and fusion gene sequence；

(2) stable cell lines of the structure with gene mutation or fusion

A. transfected virus incasing cells is prepared in virus the recombinant virus plasmid that prepared by viral packaging plasmid and above-mentioned (1) together Clearly；

B. drug sieve selects the cell line of embedded recombinant viral genome sequence；

C. mutational site or fusion are verified, gene mutation or fusion positive reference product are obtained.

2. the preparation method of gene mutation and fusion positive reference product according to claim 1, it is characterised in that：(1) It is as follows：

Take 2 μ l of PCR product, 1 μ l of digestion restrovirus carrier, T4DNA after quick 3 μ l of 2XT4DNA connections enzyme reaction buffer solution, digestion 1 μ l of ligase centrifuge mixing, place room temperature 30 minutes, then take 2 μ l of product after connection, and the DH5 α competent cells of 20 μ l are added In, after ice bath half an hour, then 42 DEG C of heat shock 30sec are added 200 μ l SOC culture solutions, 37 DEG C of shake cultures 1 hour, then This culture is uniformly coated on the agar plate containing 100 μ g/ml ampicillins, 37 DEG C of incubators is placed and is incubated overnight.

3. the preparation method of gene mutation and fusion positive reference product according to claim 1, it is characterised in that：(1) It is as follows：

By viral packaging plasmid and slow virus with mutational site or fusion or retroviral plasmid transfect together to Ready 293T cells, it is specific as follows：

1.0 μ g viruses are packed mixing plasmid, 1.0 μ g recombination mutations virus particles, 250 μ l serum-free Opti MEM to be added Softly mixing and room temperature incubate and give 5min in 1.5ml sterilizing EP pipes；

6 μ l liposomes Lipofectamine 2000 are dissolved in 250 μ l serum-free Opti-MEM I culture mediums, soft mixing is simultaneously It is incubated at room temperature 5min；

By plasmid solution and the soft mixing of liposome solutions and it is incubated at room temperature 20min；

In six orifice plates, plasmid liposome complex is added；It is positioned over 37 DEG C of 5%CO₂Incubator in cultivate, for 24 hours afterwards with fresh DMEM culture mediums containing 10%FBS change liquid, and for harvest containing viral supernatant, 3000rpm centrifuges 20min, removal precipitation, disease after 48-72h - 80 DEG C of storages of malicious supernatant or immediately infection cell.

4. the preparation method of gene mutation and fusion positive reference product according to claim 1, it is characterised in that：(2) It is as follows：

The viral supernatants prepared in right amount are added in the fresh medium, and the polybrene of final concentration of 6 μ g/ml is added, It is positioned over 37 DEG C of 5%CO₂Incubator in cultivate and the puromycin processing of final concentration of 2 μ g/ml is added for 24 hours kills and be uninfected by Cell.

5. the preparation method of gene mutation and fusion positive reference product according to claim 1, it is characterised in that：It is described Carrier be the viral vectors that alien gene can be integrated into genome, including slow virus carrier and retroviral vector.

6. the preparation method of gene mutation and fusion positive reference product according to claim 1, it is characterised in that：It is described Cell line be 293T cell lines.

7. the preparation method of gene mutation and fusion positive reference product according to claim 1, it is characterised in that：Pass through The individual cells that limiting dilution method filters out are in 37 DEG C of 5%CO₂Incubator in cultivated, and to the correct cell of sequence verification System is enlarged culture, obtains positive reference product.

8. the preparation method of gene mutation and fusion positive reference product according to claim 1, it is characterised in that：It is described Cell line be the cell line in the single mutational site of term single gene, the cell line in the multiple mutational sites of term single gene and multiple genes The cell line in multiple mutational sites, the cell line of single fusion and a variety of fusions.

9. the preparation method of gene mutation and fusion positive reference product according to claim 1, it is characterised in that：Pass through The present invention prepares the positive of base replacement, frameshit, missing, insertion mutation, fusion, chromosome shift and its rare mutation Reference material.