CN108165633A

CN108165633A - The method that one species specificity suppresses primer system and tumour ct-DNA abrupt climatic changes

Info

Publication number: CN108165633A
Application number: CN201711499676.XA
Authority: CN
Inventors: 高川; 夏昊强; 陶勇; 陈飞钦; 罗玥; 陆才瑞; 阮碧波
Original assignee: Guangzhou Gene Denovo Biotechnology Co ltd
Current assignee: Guangzhou Gene Denovo Biotechnology Co ltd
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2018-06-15
Anticipated expiration: 2037-12-29
Also published as: CN108165633B

Abstract

The invention belongs to biomedical inspection fields, and in particular to a species specificity suppresses the method that primer system and tumour ct DNA mutations detect.The specificity suppresses primer system, it includes OMS specificity and suppresses primer, block probes and detection probe, using the method that the specificity suppresses primer system to carry out the detection of tumour ct DNA mutations is determined including gene order, OMS specificity suppresses primer and probe design, research and development sample prepares, PCR system determines and optimizes and mutation rate calculating, its specificity is high, it is not interfered by wild-type genotype sequence, according to this method can the millesimal abrupt climatic changes of Accurate Determining tumour ct DNA, be suitble to clinically promote and apply.

Description

The method that one species specificity suppresses primer system and tumour ct-DNA abrupt climatic changes

Technical field

The invention belongs to biomedical inspection fields, and in particular to a species specificity suppresses primer system and tumour ct-DNA The method of abrupt climatic change.

Background technology

It is the goldstandard of current cancer diagnosis and a kind of method of oncogene parting, but due to tumour to organize biopsy Heterogeneity and disease progress, tissue biopsy have certain office to the monitoring of the progress of tumour, prognosis, treatment and Genotyping It is sex-limited, it is therefore desirable to which that a kind of detection method more optimized substitutes the progress of tissue biopsy detection tumour.Plasma circulation Tumour DNA (circulating tumor DNA, ctDNA) is found in 1948 for the first time, is the single or double chain being present in blood plasma or serum DNA, clip size about 134bp or so.Grind it is high show ctDNA have the relevant molecular characterization of many cancers, the liquid based on ctDNA Body biopsy has the screening of gene mutation hypersensitivity and specificity, the progress of sustainable dynamic observation tumour.Utilize ctDNA Detection can accurately detect the gene mutation of tumour, be conducive to the early detection of tumour, moreover it is possible to which accurate judgement tumour is in progress, in advance Afterwards and assist targeted therapy.

It grinds at present there are three types of high reports in the source of ctDNA：1. Apoptosis or the tumour cell of necrosis；2nd, tumour living is thin Born of the same parents；3rd, ring tumour cell (circulating tumor cell, CTC), since the amount of ctDNA is few and segment is small, the inspection to it Survey causes very big obstruction.CtDNA detection techniques can be divided into two stages：First stage is the detection skill of based on PCR technology Art, such as direct sequencing (Sanger methods), mutation amplification retarding system (amplification refractory mutation System, ARMS) method, droplet digitlization PCR (droplet digital PCR System, ddPCR), BEAMING digital pcrs Method etc., second stage are the detection method to grow up on the basis of NGS.

Sanger methods extend the primer being incorporated on sequence template undetermined using a kind of archaeal dna polymerase.Until incorporation one Until kind chain termination nucleotide.Sequencing is individually reacted by a set of four and formed each time, and each reaction contains all four Kind deoxynucleotide triphosphoric acid (dNTP), and it is mixed into a kind of different dideoxyribonucleoside triphosphate (ddNTP) of limitation.Due to DdNTP, which lacks, extends required 3-OH groups, and extended oligonucleotide is made selectively to be terminated at G, A, T or C.

ARMS methods are the originals that could must be effectively expanded with its template DNA complementation using the last bit base in 3 ' ends of PCR primer Reason designs ApoE gene amplimer, under strict conditions, only when 3 ' base of primer and template are matched PCR amplification band could occur, so as to detect to be mutated.ARMS method detection sensitivities are apparently higher than its other party such as direct sequencing Method can detect that the mutator of content in sample down to 0.1%~1.0%.This method combines real-time PCR platforms and realizes amplification When stopped pipe operate, it is easy to operate, without the post processing of product, can farthest avoid the pollution of amplified production.Become Tumour individuation Molecular Detection is most important in the world, one of state-of-the-art technology, and the advantage in clinical practice is by the industry Expert is widely recognized as.

Digital pcr (Digital PCR) new technology quantitative as DNA is to be assigned to the PCR reactions of a standard greatly Measure in small reactor, included in each reactor or not comprising one or more copy target molecules (DNA profiling), It realizes unimolecule template PCR amplifications, after amplification, the copy number of target sequence " is counted " by the number of positive reaction device, The advantages of this method is can to carry out absolute quantitation to ctDNA, and sensitivity can reach single nucleic acid molecules, detection limit down to 0.001%, but simultaneously there is also can only detect known mutational site, flux is low the shortcomings that.

BEAMING technologies are to combine digital pcr and streaming technology, and targeted mutagenesis area is expanded using Specific PCR primers, with Magnetic bead (special PCR primer is fixed on magnetic bead) mixing carries out Water-In-Oil unimolecule amplified reaction.After demulsification, utilize PCR product on the fluorescence probe combination magnetic bead of different colours sends out red or green fluorescence, uses flow cytometry analysis magnetic Pearl color determines catastrophe.Such an approach achieves single-molecule DNA absolute quantitation, disadvantage be it is of high cost, complicated for operation, And success rate is not very too high, therefore application at present is not extensive.

It can be seen that not having unified standard to the detection method of ctDNA now, detection method disunity lacks standard Change flow, from the technology of based on PCR to NGS technologies, in addition to ARMS methods, other detection methods do not form the flow of standardization.

Grind it is high show with direct Sequencing hair with ARMS methods detect EGFR genetic mutation in 154 non-small cell lung cancers simultaneously Sensibility, 32 mutation (32/154,20.8%) of PCR sequencing PCR, 52 mutation (53/154,33.8%) of ARMS methods.Two methods are equal Detect mutation person 29, without mutation person 99, direct sequencing, which detects, to be mutated and ARMS methods negative patient 3, otherwise 23 Example, inconsistent rate difference are significant (P ＜ 0.0001).In conclusion detection NSCLC during EGFR catastrophes, ARMS methods are more direct PCR sequencing PCR operating procedure is few, and mutation rate is high.It is another to grind EGFR gene in high 364 corrective surgeries or biopsy tumor tissues sample The catastrophe of 18-21 extrons, wherein there is 344 patients to carry out direct sequencing detection, 188 patients have carried out ARMS Method detects, and the patient that two methods detect simultaneously has 168.Conclusion ARMS methods have higher detection sensitivity than PCR sequencing PCR and dash forward Become recall rate, ARMS methods are protrusion compared with PCR sequencing PCR advantage history in the detection of small sample, and AMRS methods are simpler, fast Victory is suitble to be used widely, and can replace direct sequencing in clinical EGFR abrupt climatic changes.PCR sequencing PCR can detect Rare mutation not to be covered in ARMS kits, it is more fully reliable with reference to two methods testing result.It is another to grind high mutation It expands retarding system (ARMS) method and next-generation sequencing (NGS) method detection non-small cell lung cancer (NSCLC) patient sample drives more Difference on gene alteration instructs clinical individualization to treat.51 NSCLC patient's samples of method are first using ARMS methods, conclusion ARMS methods and NGS are suitable for NSCLC patient mutations and drive genetic test.For DNA point mutation detections, NGS is not only shown The omission of ARMS detections also show mutation abundance, with mutation and unusual regulatory mutant etc., and ARMS, which is detected, supplementary function, ARMS is a kind of easy, quick, economic detection mode.

Invention content

ARMS technological cores are the design of primer, and at present, the whole world only has Liang Jia enterprises to possess ARMS technologies and corresponding production Product, Xiamen Amoydx Bio-Pharmaceutical Technology Co., Ltd. possess the ADx-ARMS patents of independent intellectual property right --- and skill is transformed in primer The multiple design of art, unique buffer system and secondary amplification amplification, scorpions+ARMS primers, be used to detecting EGFR, The gene mutations such as KRAS, BRAF have the advantages that it is simple, easily and fast, it is sensitive, it is only necessary to can obtain accurate result within 90 minutes, Therefore it is easy to carry out in clinic.The core of ABI companies is MGB probe+ARMS primers, can reach mutation one thousandth detection.

The present invention is intended to provide a species specificity suppresses primer system, suppress primer including OMS specificity, block is visited Needle and detection probe include gene sequence using the method that the specificity suppresses primer system to carry out tumour ct-DNA abrupt climatic changes Row determine, OMS specificity suppresses primer and probe designs, detection sample prepares, PCR system determines and optimizes and mutation rate meter Calculate and etc., specificity is high, is not interfered by wild-type genotype sequence, can Accurate Determining tumour ct-DNA thousand according to this method / mono- abrupt climatic change is suitble to clinically promote and apply.The present invention also aims to provide a kind of tumour ct-DNA thousand The new method of/mono- abrupt climatic change, is designed using novel primer, and sensitivity is up to 0.1/1000th, and specificity is high, and detection stream Journey is simple.

The present invention is achieved through the following technical solutions above-mentioned technical purpose：

One species specificity suppresses primer system, suppresses primer, block probes and detection probe including OMS specificity, The wherein described OMS specificity suppresses primer to be made of 5 ' arms, 3 ' arms and centre loop parts, wherein 5 ' 20~50nt of arm, bear Blame anchor primer；3 ' 8~15nt of arm, are responsible for specific amplification, and the non genome sequence that intermediate loop parts are 18~25nt rises Main amplification effect；The block probes suppress primer in the same direction with OMS specificity, and probe 3 ' is held more than SNP 3~8bp of point, 3 ' end progress-NH2 modifications；5 ' ends of detection probe carry out FAM modifications, and 3 ' BHQ1 are quenched, and 3 ' ends of distance block probes are extremely Few 1nt.

Specificity described above is suppressed in amplification system, and the OMS specificity suppresses primer to be forward primer；It is described Block probes block genotype be wild-type genotype.

Specificity described above is suppressed in amplification system, and OMS specificity suppresses the Tm values of primer to be 58~61 DEG C, described The Tm values of block probes are 70~80 DEG C, and the Tm values of the detection probe are 66~68 DEG C.

A kind of method for primer system being suppressed to carry out tumour ct-DNA abrupt climatic changes using above-mentioned specificity, including following Step：

(1) gene order determines：Determine target gene sequence and SNP positions and allelotype；Those skilled in the art Target gene sequence and SNP positions and allelotype can be determined according to ncbi database, and with reference to bibliography.

(2) OMS specificity suppresses primer and probe to design：OMS specificity suppresses primer by 5 ' arms, 3 ' arms and centre loop Part forms, wherein 5 ' 20~50nt of arm, are responsible for anchor primer；3 ' 8~1Snt of arm are responsible for specific amplification, intermediate Ioop portions It is divided into the non genome sequence of 18~25nt, plays main amplification effect；Block probes suppress primer in the same direction with OMS specificity, visit The end of needle 3 ' is more than 3~8bp of SNP points, 3 ' end progress-NH2 modifications；5 ' ends of detection probe carry out FAM modifications, and 3 ' BHQ1 are quenched, 3 ' end at least 1nt of its distance block probes；

(3) detection sample prepares：Clinical test plasma is taken, its ctDNA, as base are extracted using ctDNA extracts kits Because of a group sample；Three parts are classified as, first part of concentration that its mutant DNA is detected using Agilent 2100；It is denoted as concentration 1；Separately take A genome sample adds in the mutated-genotype standard plasmid M for determining concentration, as check sample thereto；Another base Because group sample is sample to be tested；

(4) PCR system is determined and is optimized：PCR system composition and PCR amplification condition are adjusted to sample to be tested and control sample This progress fluorescent quantitative PCR, setting fluorescence signal detection terminal, records sample to be tested and check sample arrives separately at fluorescence The ct values of signal detection terminal, the mutated-genotype standard plasmid M densimeters in the ct differences and check sample of the two Calculate the concentration of mutated-genotype standard plasmid M in sample to be tested, as concentration 2；

(5) mutation rate calculates：Determine the mutated-genotype standard plasmid M's in tumour ctDNA according to concentration 1 and concentration 2 Mutation rate.

In tumour ct-DNA mutation detection methods described above, the target gene is cancer target medication gene position Point EGFR T790M.

In the method for above-mentioned tumour ct-DNA abrupt climatic changes, the PCR amplification system is：

PCR amplification program is：

OMS primers have 4 big advantages in step 2 of the present invention：1. specificity is substantially improved -- OMS primer need 3p arms and 5p arms while specific bond template (template DNA).OMS primers of the present invention can dramatically increase primer compared to conventional primer Specificity.It is calculated according to probabilistic model, specificity is lifted beyond 26,000 times.2. the ability with tolerant template variation -- In certain applications, purposefully 5p arms can be designed it is longer, so that it can hybridize corresponding template.Even if there are one in template A mispairing (for example, a SNP).In view of there is 6,000,000 high equipotential base in the human genome for possessing 3,000,000,000 base-pairs Because of the SNPs of frequency (＞ 1%), average just to have 1 SNP per 50bp, this is a highly useful characteristic.Draw 3. being greatly reduced The formation of object dimer --- amplifiable primer dimer only generates in this case：When the 3p arms of two primers are mutually miscellaneous It hands over, and 3 ' termini-complementaries of at least one primer are attached on another primer.OMS primers are usually carried to be drawn than conventional The shorter 3p arms (16nt) of object (35nt), thus the probability for statistically forming amplifiable primer dimer is much smaller. 4. longer real sequence being accommodated in amplicon --- the sequencing read of 1 amplicon is by real sequence and primer sequence two It is grouped as.The 3p arms of primer, which can be included in amplicon, becomes a part for primer section.Draw since OMS primers have than conventional The shorter 3p arms of object, thus, real sequence section can occupy higher ratio in the amplicon generated by OMS primers.

PCR reaction systems adjust system ionic strength in step 4, further improve amplification efficiency and specificity.

Typically, conventional ion concentration：Magnesium ion 1.5Mm, potassium ion 50Mm, ammonium ion 10-20Mm.It is of the present invention PCR reaction systems adjustment system ionic strength so that amplification efficiency and specificity greatly improve.

The present invention relative to the prior art it is a technical advantage that：

1) tumour ctDNA mutation detection methods of the present invention are simpler relative to biopsy operation, and efficiency is more Height is easier to be easily accepted by the patient in clinic.

2) OMS Primer specificity highers of the present invention, and the formation of primer dimer is greatly reduced, it is applicable in Property is more preferable.

3) present invention determines the optimal system ionic intensity of PCR reaction systems by experimental exploring, can further carry High amplification efficiency and specificity.

Description of the drawings

Fig. 1 present invention has the OMS Primer structure diagrams of 3 functional areas.

The fluorescence intensity of 11-1 in Fig. 2 embodiment of the present invention, 11-2 and 11-3 sample-amplification cycles curve graph.

The fluorescence intensity of G3-1 in Fig. 3 embodiment of the present invention, G3-2 and G3-3 sample-amplification cycles curve graph.

11-1,11-2,11-3 in Fig. 4 embodiment of the present invention, fluorescence intensity-amplification cycles curve of G3-1 and G3-2 samples Figure.

G3-1 in Fig. 5 embodiment of the present invention, fluorescence intensity-amplification cycles curve of G3-2, G3-3,10^7 and 10^5 sample Figure.

The gene order segment distribution map that Fig. 6 is measured using Agilent 2100.

The concentration map for the target gene sequence that Fig. 7 Agilent 2100 measures.

Specific embodiment

The present invention is further described, but the embodiment does not limit this hair in any way below by way of specific embodiment The range of bright patent protection.

Embodiment 1：The method of the present invention is to cancer target medication gene loci EGFR T790M one thousandth abrupt climatic changes

Specific implementation process is as follows：

1st, primer and probe design

(1) specificity suppresses design of primers：Selection forward primer is designed as amplification and suppresses primer, is open country by suppressor gene type Raw genotype, 3 parts of OMS primers point, 5 ' arms, 3 ' arms and centre Ioop parts.5 ' 20~50nt of arm are responsible for anchor primer；3’ 8~15nt of arm, be responsible for specific amplification (shorter primer combine it is unstable, can be from there are one 3 ' arm primers when base mispairing Dissociated in template, so as to prevent to expand), intermediate Ioop parts are non genome sequence, and 18~25nt is universal primer, plays master Want amplification effect.Forward and reverse 58~61 DEG C of present invention of design of primers Tm values have the OMS Primer structural representations of 3 functional areas Figure.

(2) block probes design：In the same direction with forward primer, block genotype is wild-type genotype, and the end of probe 3 ' is more than 3~8bp of SNP points；Block probe Tm values are designed as 70~80 degrees Celsius, 3 ' end progress-NH2 modifications.

(3) detection probe designs：At least 1nt design detection probes are being held from block probes 3 ', the end of probe 5 ' carries out FAM Modification, 3 ' BHQ1 are quenched, 66~68 DEG C of probe Tm values

2. it detects sample to prepare：

(1) 170bp mutant DNAs prepare：Primer is set, makes expanding fragment length about 170bp (practical for 173bp), with prominent Become genotype standard plasmid M and carry out PCR amplification into template, Ago-Gel identification purifies simultaneously 2.0 Accurate Determining concentration of Qubit For 7.5ng, the sequence of amplified fragments and the sequence of mutated-genotype standard plasmid M are as follows：

According to copy number calculation formula：Copies=(6.02*10²³*7.5ng/ul*10^-9)/(173bp*660)= 3.95E10

(2) mutant plasmid is added to plasma sample

1. 5ul mutant DNAs is taken to add 495ul H2O, mixing obtains 3.95*10^8 samples DM1；Take 10ul DM1+90ul H2O, mixing obtain the DM2 of 3.95*10^7；It dilutes successively, obtains the DM3 of 3.95*10^6；3.95*10^5 DM4；3.95* The DM5. of 10^4

2. taking same pipe blood plasma G, 14000rpm centrifugations 5min is spare；3 clean 1.5ml centrifuge tubes are taken, are respectively labeled as G11-1, G11-2, G11-3 take 200ul blood plasma that it is sterile ultrapure to add 2ul DM2,2ul DM4,2ul respectively into centrifuge tube Water, mixing are spare.

3. with the ct-DNA extracts kits that our company researches and develops, ct-DNA extractions are carried out to plasma sample above, finally The ct-DNA volumes of dissolving are 40ul, and when the DM100% recycling of addition, the theoretical concentration of DM is respectively in ct-DNA：

G11-1：C=2*3.95*10^7/40=1.98*10^6copies/ul

G11-2：C=2*3.95*10^5/40=1.98*10^4copies/ul

G11-3：C=0

(3) check sample is set：Same pipe plasma sample G is taken, is carried out with the ct-DNA extracts kits of our company's research and development Ct-DNA is extracted, and labeled as G3, the volume finally dissolved is 50ul.4ul G3 ct-DNA samples are taken respectively into new centrifuge tube In, labeled as G3-1, G3-2, G3-3,1ul DM3,1ul DM5, the sterile ultra-pure waters of 1ul are added respectively, and the DM of check sample is dense It spends and is：

G3-1：C=1*3.95*10^6/5=7.9*10^5copies/ul；

G3-2：C=1*3.95*10^4/5=7.9*10^3copies/ul；

G3-3：C=0

3.Q-PCR detection sample settings

Detect sample：G11-1、G11-2、G11-3；

Check sample one：G3-1、G3-2、G3-3；

Check sample two：DM2(3.95*10^7)、DM4(3.95*10^5)

4. experiment detection

System：

Program：

5th, experimental result：

The specificity of 5.1 detection methods

The fluorescence intensity of 11-1 in Fig. 2 embodiment of the present invention, 11-2 and 11-3 sample-amplification cycles curve graph.Conclusion：Add Add the DM of 3.95*10^5copies, after extracted during theoretical kind of a concentration of 1.98*10^4copies/ul, can effectively detect, G11-3 is compareed without amplification curve, illustrates no nonspecific signals interference.The sensitivity of 5.2 detection methods

The fluorescence intensity of 3-1 in Fig. 3 embodiment of the present invention, 3-2 and 3-3 sample-amplification cycles curve graph.Conclusion：Work as ct- When DM concentration in DNA sample reaches 7.9*10^3copies/ul (G3-2), Ct values are 30.8, and detection efficiency, will such as expand successively When increasing recurring number is set as 45Cyclers, it is 7.9*10copies/ul to understand detection sensitivity；Need subsequent experimental setting verification.

The actual concentrations measuring and calculating of 5.3 measuring samples

Conclusion：1. judging from Ct values, G11-1 differs 0.6 with G3-1, and concentration multiple differs about 1.8 times of (concentration differences 10 Times, when amplification efficiency is consistent and 100% expands, 3.3) Ct values differ；2. thus the actual concentrations of rough calculation G11-1 are about：

G11-1 '=G3-1/1.8=7.9*10^5copies/ul/1.8=4.4*10^5copies/ul；

Interference of 5.4 wild-type genotypes to detection method

Conclusion：1. in terms of amplification curve, G3-1 is overlapped with DM4 starting points, and Ct values G3-1 is slightly less than DM4；G3-1's is dense It spends for 7.9*10^5copies/ul, the DM concentration of DM4 3.9*10^5copies/ul, G3-1 are about twice of DM4, and Ct values are small 0.6, it is completely close with calculated value, it can further verify that the wild-type genotype in negative sample does not affect mutator The detection of type, specificity are good.

6th, mutant copies number detection ratio calculates in sample

(1) 2100 detections (genetic fragment size and Concentration Testing) are carried out to ct-DNA samples G3

G3 sample Qubit concentration：1.12ng/ul.The gene order segment distribution map that Fig. 6 is measured using Agilent 2100.

Wherein the segment of 170bp or so is gene order of the present invention, other long fragments are dirty for genome fragment.Fig. 7 pacifies The concentration map for the target gene sequence that prompt human relations 2100 measure.According to 2100 testing results, G3 sample object fragment concentrations are 28.09pg/ul。

(2) mutant DNA ratio calculates

The amount with DM same clip sizes 173bp in G3-2 (taking 4ul)：

G3-2-CtDNA=4ul*0.02809ng/ul=0.11236ng (takes 4uIG3+1ulDM5 to form G3-2)；

The amount of DM (173bp mutant DNAs) is in G3-2：

DM=7.5ng (3.9*10^10copies) * 10^-6=0.0000075ng (3.9*10^4copies)

The total CtDNA=0.0000075ng/0.11236ng=6.67*10^-5*100%=0.0067 of DM/

Conclusion：It can complete to be mutated cancer target medication gene loci EGFR T790M one thousandths according to the method for the present invention Detection.

Claims

1. a species specificity suppresses primer system, suppress primer, block probes and detection probe including OMS specificity, Described in OMS specificity primer is suppressed to be made of 5 ' arms, 3 ' arms and centre loop parts, it is responsible wherein 5 ' 20~50nt of arm Anchor primer；3 ' 8~15nt of arm, are responsible for specific amplification, and the non genome sequence that intermediate loop parts are 18~25nt plays master Want amplification effect；The block probes suppress primer in the same direction with OMS specificity, and the end of probe 3 ' is more than 3~8bp of SNP points, and 3 ' End progress-NH2 modifications；5 ' ends of detection probe carry out FAM modifications, and 3 ' BHQ1 are quenched, and 3 ' ends of distance block probes are at least 1nt。

2. specificity according to claim 1 suppresses primer system, which is characterized in that the OMS specificity, which is suppressed, draws Object is forward primer.

3. specificity according to claim 1 suppresses primer system, which is characterized in that the block of the block probes Genotype is wild-type genotype.

4. specificity according to claim 1 suppresses primer system, which is characterized in that OMS specificity suppresses the Tm of primer It is 58~61 DEG C to be worth, and the Tm values of the block probes are 70~80 DEG C, and the Tm values of the detection probe are 66~68 DEG C.

5. a kind of specificity using described in claim 1-4 suppresses the side of primer system progress tumour ct-DNA abrupt climatic changes Method includes the following steps：

(1) gene order determines：Determine target gene sequence and SNP positions and allelotype；Those skilled in the art can be with Target gene sequence and SNP positions and allelotype are determined according to ncbi database, and with reference to bibliography.

(2) OMS specificity suppresses primer and probe to design：OMS specificity suppresses primer by 5 ' arms, 3 ' arms and centre loop parts Composition, wherein 5 ' 20~50nt of arm, are responsible for anchor primer；3 ' 8~15nt of arm, are responsible for specific amplification, and intermediate loop parts are The non genome sequence of 18~25nt plays main amplification effect；Block probes suppress primer in the same direction with OMS specificity, probe 3 ' End is more than 3~8bp of SNP points, 3 ' end progress-NH2 modifications；5 ' ends of detection probe carry out FAM modifications, and 3 ' BHQ1 are quenched, away from 3 ' from block probes hold at least 1nt；

(3) research and development sample prepares：Clinical test plasma is taken, its ctDNA, as genome are extracted using ctDNA extracts kits Sample；Three parts are classified as, first part of concentration that its mutant DNA is detected using Agilent 2100；It is denoted as concentration 1；Separately take portion Genome sample adds in the mutated-genotype standard plasmid M for determining concentration, as check sample thereto；Another genome Sample is sample to be tested；

(4) PCR system is determined and is optimized：Adjust PCR system composition and PCR amplification condition to sample to be tested and check sample into Row fluorescent quantitative PCR, setting fluorescence signal detection terminal, records sample to be tested and check sample arrives separately at fluorescence signal The ct values of terminal are detected, the mutated-genotype standard plasmid M concentration calculating in the ct differences and check sample of the two is treated The concentration of mutated-genotype standard plasmid M, as concentration 2 in test sample sheet；

(5) mutation rate of the mutated-genotype standard plasmid M in tumour ctDNA is determined according to concentration 1 and concentration 2.

6. the method for tumour ct-DNA abrupt climatic changes according to claim 5, which is characterized in that the target gene is Cancer target medication gene loci EGFR T790M.

7. the method for tumour ct-DNA abrupt climatic changes according to claim 6, which is characterized in that the PCR amplification body It is to be：

PCR amplification program is：

。