CN106544341A - The method of the ctDNA in efficient detection sample - Google Patents

The method of the ctDNA in efficient detection sample Download PDF

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CN106544341A
CN106544341A CN201710034984.9A CN201710034984A CN106544341A CN 106544341 A CN106544341 A CN 106544341A CN 201710034984 A CN201710034984 A CN 201710034984A CN 106544341 A CN106544341 A CN 106544341A
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sample
ctdna
cyclisation
jing
molecules
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任军
耿荷芳
陆思嘉
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Shanghai Yikang Medical Laboratory Co Ltd
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Abstract

The invention provides a kind of method of the ctDNA in efficient detection sample, specifically, the method of the present invention can be under extremely low nucleic acid concentration, by dissociative DNA fragment from cyclisation, wherein ctDNA has higher from cyclisation rate because fragment is shorter, the amplification of greater efficiency subsequently can be obtained with specific cyclisation preferential amplification or amplification banking process, the substantial amounts of amplified production corresponding to ctDNA is obtained, so as to obtain very high detection sensitivity and specificity.

Description

The method of the ctDNA in efficient detection sample
Technical field
The present invention relates to biological technical field, in particular it relates to a kind of method of the ctDNA in efficient detection sample.
Background technology
Research shows there is the extremely micro ctDNA for coming from tumor cell, circulating tumor in the blood of tumour patient Discharged after DNA fragmentation, its mainly dead tumor cell rupture.
By taking breast carcinoma as an example, ErbB-2 (human epidermalgrowth factor Receptor-2, HER2) gene, i.e. c-erbB-2 genes, it is positioned on chromosome 17q12-21.32, encodes average molecular matter The transmembrane receptor sample albumen for 185000 is measured, with tyrosine kinase activity.The overexpression of HER2 genes or copy number amplification with Breast carcinoma is closely related with the generation of gastric cancer.20%～25% breast carcinoma, 15% patients with gastric cancer overexpression HER2 is there are about, should The prognosis of some patientss is poor.
It is only that tumor cell gene copy number becomes that Her2 genes occur copy number amplification in partial breast cancer and gastric cancer One of different example.In fact, having been found that in previous research that many genes or chromosome segment are existed in tumor cell The phenomenon that copy number is expanded or lacked, therefore, in detecting that the copy number variation of target gene in tumor cell is cancer biology One of important technology.
However, the detection method of existing Her2 is mainly examined to tumor biopsy tissue sample with IHC and FISH methods Survey.In addition, had tried to now detect the Tumour DNA (ctDNA) that dissociates present in plasma DNA (cfDNA), Amplification of the identification with the presence or absence of Her2 genes, but effect is still undesirable.
IHC methods and FISH methods are all to detect that its application has very big limitation to tumor biopsy tissue section sample Property, it is mainly manifested in：
Some patient bodies are extremely weak, it is impossible to bear biopsy procedure, i.e., cannot obtain pathological section sample.
To tumor recurrence patient, diagnosis and treatment principle does not advocate that open surgical biopsy is sampled, instead fine needle aspiration biopsy.Fine needle aspiration Biopsy is only capable of obtaining minimal amount of tumor tissues, and Jing is often not enough to carry out IHC and FISH detections.To there is Her2 gene amplifications disease People, it may happen that drug resistance situation after being treated using targeted drug, so need to continue during medication, dynamic detection Her2 Gene amplification situation, but in clinical reality, to tumor tissues, biopsy sampling is almost an impossible thing repeatedly.Cause This, IHC and FISH methods are helpless to this.
In theory, can be to tumor of dissociating present in plasma DNA (cfDNA) with the method for conventional secondary sequencing DNA (ctDNA) detected, amplification of the identification with the presence or absence of Her2 genes, but effect is still undesirable main in practical application Reason is：First, cfDNA contents in blood plasma are considerably less, are typically only capable to obtain a few nanogram DNA from one milliliter of blood plasma.Also, The main source of cfDNA is to be released into the DNA of blood after leukocyte death, wherein the ctDNA really from tumor cell is few It is again few, the method analyzed with conventional gene copy number is difficult to identification with the presence or absence of Her2 gene amplifications.
Therefore, this area in the urgent need to develop it is a kind of can efficiently concentrating, detection sample in ctDNA, so as to judge sample The method expanded with the presence or absence of target gene (such as HER2) in this.
The content of the invention
It is an object of the invention to provide it is a kind of can efficiently concentrating, detection sample in ctDNA, so as in judgment sample With the presence or absence of the method that target gene (such as HER2) is expanded.
A first aspect of the present invention provides a kind of sample treatment, including step：
I () provides a testing sample, the sample contains cfDNA and ctDNA；
(ii) testing sample is diluted, obtains diluted sample, wherein in the sample Jing after dilution, cfDNA Concentration be 0.001-5ng/ μ l, it is preferred that 0.01-2ng/ μ l, more preferably, 0.05-1ng/ μ l；With
(iii) cyclisation process is carried out to the diluted sample of step (ii) with cyclisation ligase, obtains the mixed of Jing cyclisation Compound, wherein, the mixture of described Jing cyclisation contains cyclisation ctDNA molecules.
In another preference, methods described also includes step：
(iv) with the cyclisation ctDNA molecules in the mixture of Jing cyclisation as template, expanded, so as to obtain correspondence In the amplified production of cyclisation ctDNA.
In another preference, described amplification is expanded (i.e. preferentially with circularized nucleic acid molecule as mould for the preferential PCR of cyclisation Plate is expanded, and not with or substantially do not expanded by template of linear nucleic acid molecule).
In another preference, described amplification is expanded using the archaeal dna polymerase of strand-displacement activity.
In another preference, described amplification includes that MALBAC-LAB is expanded.
In another preference, methods described also includes step：
V () carries out building storehouse and sequencing, or is directly sequenced to the amplified production, wherein, by the sequencing, obtain CtDNA testing results in the sample to be tested.
In another preference, methods described also includes step：
(vi) testing result based on step (v), so as to judge the variation situation or gene order of copy number of target genes Catastrophe or its combination.
In another preference, in the mixture that described Jing is cyclized, ctDNA cyclisation products are dominant.
In another preference, in the mixture that diluted sample and Jing are cyclized, Formulas I is met
Ct1/Cf1 ＞ Ct0/Cf0 (I)
In formula,
Ct1 is, in the mixture of Jing cyclisation, to be cyclized the concentration of ctDNA molecules；
Cf1 is, in the mixture of Jing cyclisation, to be cyclized the concentration of cfDNA molecules；
Ct0 is the concentration of ctDNA molecules in the diluted sample；
Cf0 is the concentration of cfDNA molecules in the diluted sample.
In another preference, in diluted sample and sample to be detected, Formula II is met
Ct0/Cf0=Ct/Cf (II)
In formula,
Ct0 is the concentration of ctDNA molecules in the diluted sample；
Cf0 is the concentration of cfDNA molecules in the diluted sample；
Ct is the concentration of ctDNA molecules in the sample to be detected；
Cf is the concentration of cfDNA molecules in the sample to be detected.
In another preference, the ratio R 1 >=10, preferably >=50 of described Ct1/Cf1 and Ct0/Cf0, more preferably >= 100。
In another preference, described processing method is the method for nondiagnostic and non-therapeutic.
In another preference, in described amplified production, it is dominant corresponding to the amplified production of ctDNA cyclisation products Gesture.
In another preference, during " dominant corresponding to the amplified production of ctDNA cyclisation products " refers to amplified production, Concentration (C1) corresponding to the amplified production of ctDNA cyclisation products is significantly higher than the amplified production corresponding to cfDNA cyclisation products Concentration (C2).
In another preference, " being significantly higher than " refers to C1/C2 >=5, preferably >=10, more preferably, >=20.
In another preference, described sample is selected from the group：Blood, body fluid or its combination.
In another preference, the sample is selected from the group：Blood, blood plasma, interstitial fluid, lymph fluid, urine, brain ridge Liquid, saliva, aqueous humor, seminal fluid, gastro-intestinal secretion liquid or its combination.
In another preference, the sample is selected from the group：Blood, blood plasma or serum.
In another preference, described sample is acellular sample.
In another preference, described sample does not contain tumor cell.
In another preference, the ctDNA is derived from tumor cell.
In another preference, the tumor cell is selected from the group：Breast carcinoma, ovarian cancer, gastric cancer, pulmonary carcinoma, colorectal cancer, Bladder cancer, the esophageal carcinoma, cancer of pancreas, skin carcinoma, carcinoma of prostate, the esophageal carcinoma, carcinoma of gallbladder, thyroid carcinoma, hepatocarcinoma, laryngeal carcinoma, oropharyngeal cancer, Leukemia or its combination.
In another preference, the cyclisation ligase is selected from the group：CircLigase、ThermoPhageTMSsDNA connects Connect enzyme or its combination.
In another preference, in step (iv), the amplification method is selected from the group：Polymerase chain reaction (PCR), Multiple strand replacement reaction (MDA), rolling circle DNA amplification (RCA), ring mediated gene isothermal amplification technology (LAMP), or its combination.
In another preference, in step (v), described banking process is selected from the group：MALBAC-LAB builds storehouse, interrupts Build storehouse, and/or swivel base builds storehouse.
In another preference, the sequencing is carried out with the method for selecting the following group：Illumina sequencings, Ion Torrent Sequencing, Roche 454 are sequenced, SoLID is sequenced, Completed Genomics (CG) are sequenced, NanoPore is sequenced, Pacific Bio sequencing, or its combination.
In another preference, the target gene is selected from the group：Her2、IGF1R/IGFIR/JTK13、Chr17、 17q22、20p13、chr3、17q25.3、MDM4/MDMX、chr7、MET/AUTS9/HGFR、FGFR1/BFGFR/CEK、8q、 HRAS/HRAS1/K-ras、AKT1/AKT1_NEW/AKT、MAP2K4/JNKK/MEK4、TOP2A/TOP2/TP2A、DCC/ CRC18/CRCR1、GSTM1/GST1/GSTM1-1、MYCN/MODED/N-myc、PDGFRA/PDGFR2、CDK6/MGC59692/ PLSTIRE、CDKN2A/p16/ARF、CCND1/BCL1/PRAD1、CSP12/chr12、CDK4/CMM3、NF1/NFNS/ P21359、GNAS/AHO/C20orf45、AKT3/PKBG/RAC-gamma、FGFR3/ACH/CEK2、KDR/CD309/FLK1、 GNAQ/G-ALPHA-q/GAQ、TSC1/LAM/TSC、ERBB2/HER-2/neu、STK11/LKB1/PJS/GSTT1、AR/AIS/ DHTR、NRAS/N-ras/NRAS1、1q21/PMVK/HUMPMKI、UGT1A1/GNT1/HUG-BR1、VHL/HRCA1/RCA1、 MECOM/A1L4F3/A8KA00、KIT/C-Kit/SCFR、PIK3R1/GRB1/p85-ALPHA、BRAF/BRAF1、8p、DOK2/ p56DOK/p56dok-2、FGFR2/BEK/BFR-1、KRAS/C-K-RAS/K-RAS2A、MDM2/HDM2/HDMX、TSC2/LAM/ TSC4、CDH1/ECAD/CDHE、BRCA1/BRCAI/BRCC1、SIRPB1、TMPRSS2/PRSS10、ARID1A/B120/ BAF250、ALK/TFG、MSH2/COCA1/FCC1、3q29、MYC/c-Myc、CDKN2B/MTS2/P15、NTRK2/GP145- TrkB/TRKB、ASS1/ASS/CTLN1、RET/CDHF12/HSCR1、PTEN/BZS/MHAM、IGF2/C11orf43/INSIGF、 RB1/OSRC/RB、D13S319(FISH_probe)、NFKBIA/IKBA/MAD-3、TP53/p53/LFS1、CCNE1/CCNE、 ZMYND8/PRKCBP1、CYP2D6/CPD6/CYP2D、DDR2/NTRKR3/TYRO10、MSH2/GTBP/HNPCC5、RAC1/ MIG5/TC-25、EGFR/ERBB/ERBB1、NANS/SAS、NOTCH1/TAN1/hN1、D13S25(FISH_probe)、 MAP2K1/MAPKK1/MEK1、NCOA3/ACTR/AIB-1、ZNF217/ZABC1、TERC、ATM/AT1/ATA、NKX2-1/ TITF1/BCH、SMAD4/DPC4/JIP、GNA11/GNA-11、TERT/EST2/TCS1、BRCA2/BRCC2/FACD、NTRK3/ TRKC/gp145(trkC)、PIK3CA/PI3K、POU5F1B、CYP17A1/CPT7/CYP17、AKT2/PRKBB/RAC-BETA、 CYP19A1/ARO/ARO1 or its combination.
The method that second aspect present invention provides ctDNA in a kind of detection sample, including step：
I () provides a testing sample, the sample contains cfDNA and ctDNA；
(ii) testing sample is diluted, obtains diluted sample, wherein in the sample Jing after dilution, cfDNA Concentration be 0.001-5ng/ul, it is preferred that 0.01-2ng/ul, more preferably, 0.05-1ng/ul；
(iii) cyclisation process is carried out to the diluted sample of step (ii) with cyclisation ligase, obtains the mixed of Jing cyclisation Compound, wherein, the mixture of described Jing cyclisation contains cyclisation ctDNA molecules；
(iv) with the cyclisation ctDNA molecules in the mixture of Jing cyclisation as template, expanded, so as to obtain correspondence In the amplified production of cyclisation ctDNA；With
V () is detected to the amplified production, obtain the testing result of ctDNA.
In another preference, the detection in step (v) includes：Build storehouse and sequencing, or be directly sequenced, wherein, lead to The sequencing is crossed, the ctDNA testing results in the sample to be tested is obtained, so as to detect the ctDNA in sample.
In another preference, methods described also includes step：
(vi) testing result based on step (v), so as to judge the variation situation or gene order of copy number of target genes Catastrophe or its combination.
In another preference, described amplification is expanded (i.e. preferentially with circularized nucleic acid molecule as mould for the preferential PCR of cyclisation Plate is expanded, and not with or substantially do not expanded by template of linear nucleic acid molecule).
In another preference, described amplification includes that MALBAC-LAB is expanded.
In another preference, described detection method is the method for nondiagnostic and non-therapeutic.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and have in below (eg embodiment) Can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist This no longer tires out one by one states.
Description of the drawings
Fig. 1 shows the philosophy and technique flow process of the present invention.
Fig. 2 shows that storehouse result is built in the amplification of different connection samples.
Fig. 3 shows the experimental result of different technologies scheme.
Fig. 4 shows that storehouse result is built in the amplification after being attached with different ligases.
Specific embodiment
Through extensive and in-depth study, the present invention is found surprisingly that, under extremely low nucleic acid concentration, dissociative DNA is broken Piece is carried out from cyclisation, and wherein ctDNA has thundering high from cyclisation rate because fragment is shorter, subsequently expanded with specific cyclisation is preferential Increasing or amplification banking process (such as MALBAC-LAB technologies) can obtain the amplification of greater efficiency and (compare normal cell source For cfDNA), the substantial amounts of amplified production corresponding to ctDNA is obtained, so as to obtain very high detection sensitivity and special Property.On this basis, the present inventor completes the present invention.
ctDNA
CtDNA is Circulating tumor DNA fragment, discharged after mainly dead tumor cell rupture, fragmentation Genomic DNA.The content of ctDNA is low, accounts for the 1% of whole dissociative DNAs, or even only 0.01%, generally, ctDNA ratios CfDNA clip sizes want short 20-50bp, about 130-145bp.
Research shows that the fragment length of tumor cell source ctDNA is less than the cfDNA that non-tumor cell is originated.
cfDNA
CfDNA is the general name of dissociative DNA in blood plasma, and main source has two kinds：The fragmentation produced in apoptosis process The nucleic acid (being close to Genome Size) of cell release during nucleic acid (160-180bp) and tissue necrosiss or immunologic cytotoxicity.
Cyclisation ligase
In the present invention, described " cyclisation ligase " refers to a kind of ligase with heat stability, can catalyzed linear it is single-stranded DNA connects into single-stranded cyclic DNA.
In the present invention, the selection of the cyclisation ligase is not particularly limited, in a preferred embodiment, the ring Change ligase to be selected from the group：CircLigase、ThermoPhageTMSsDNA ligases or its combination.
Wherein, CircLigase is that single stranded DNA is cyclized ligase, can be catalyzed in the presence of no complementary seriess The connection of intramolecular with 5 '-phosphoric acid and the single-stranded DNA templates of 3 '-oh group (is cyclized).
ThermoPhageTMSsDNA ligase (ThermoPhageTMSingle-stranded DNA ligase) be A kind of single stranded DNA ligase, can connect single stranded DNA or RNA at high temperature.
Sample treatment
The invention provides a kind of sample treatment.
In a preferred embodiment, sample treatment of the invention, including step：
I () provides a testing sample, the sample contains cfDNA and ctDNA；
(ii) testing sample is diluted, obtains diluted sample, wherein in the sample Jing after dilution, cfDNA Concentration be 0.001-5ng/ μ l, it is preferred that 0.01-2ng/ μ l, more preferably, 0.05-1ng/ μ l；
(iii) cyclisation process is carried out to the diluted sample of step (ii) with cyclisation ligase, obtains the mixed of Jing cyclisation Compound, wherein, the mixture of described Jing cyclisation contains cyclisation ctDNA molecules.
The method of ctDNA in detection sample
The invention provides a kind of method for detecting ctDNA in sample.
In a preferred embodiment, in detection sample of the invention, the method for ctDNA includes step：
I () provides a testing sample, the sample contains cfDNA and ctDNA；
(ii) testing sample is diluted, obtains diluted sample, wherein in the sample Jing after dilution, cfDNA Concentration be 0.001-5ng/ μ l, it is preferred that 0.01-2ng/ μ l, more preferably, 0.05-1ng/ μ l；
(iii) cyclisation process is carried out to the diluted sample of step (ii) with cyclisation ligase, obtains the mixed of Jing cyclisation Compound, wherein, the mixture of described Jing cyclisation contains cyclisation ctDNA molecules；
(iv) with the cyclisation ctDNA molecules in the mixture of Jing cyclisation as template, expanded, so as to obtain correspondence In the amplified production of cyclisation ctDNA；
V () is detected to the amplified production, obtain the testing result of ctDNA.
In a preferred embodiment, the detection in step (v) includes：Build storehouse and sequencing, or be directly sequenced, its In, by the sequencing, the ctDNA testing results in the sample to be tested are obtained, so as to detect the ctDNA in sample.
In a preferred embodiment, methods described also includes step：
(vi) testing result based on step (v), so as to judge the variation situation or gene order of copy number of target genes Catastrophe or its combination.
Expand and build storehouse
In the present invention, the amplification and banking process are not particularly limited, and can reach the amplification of expanding effect of the present invention It is included in the present invention with banking process.
In another preferred embodiment, using the archaeal dna polymerase of strand-displacement activity to cyclisation molecule (ctDNA of cyclisation) Expanded.
In a preferred embodiment, used by the present invention amplification and banking process is MALBAC-LAB whole genome amplifications Method.
MALBAC-LAB amplification banking process contrasts larger DNA fragmentation from principle higher amplification efficiency.Primer Product after extension can form hairpin structure because fragment two ends have complementary seriess.And in the exponential amplification of next step, trip It is identical with hairpin from primer sequence.Therefore, in annealing stage, free primer is held with the 3 ' of template only before hair clip is formed (5 ' ends that the template segments were won in competition) are expanded with reference to chain extension could be formed.Template segments are shorter, then its 3 ' end and 5 ' ends Distance is nearer, and so as to the chance for forming hair clip is higher, the chance that free primer competition is won is also less, the probability of Successful amplification Also it is less.Conversely, template segments are longer, the successful probability of amplification is also higher, and overall amplification efficiency is also higher.
For the cfDNA/ctDNA of efficient amplification small fragment, the two ends of small pieces segment DNA are connected certainly first with DNA ligase, Linear DNA is made to change into annular DNA.So, after amplimer is combined with annular DNA template, with strand-displacement activity Under the catalytic action of archaeal dna polymerase, chain extension can be carried out along the circulation of annular template, and the product for being obtained is longer, next The exponential amplification stage of step can obtain higher amplification efficiency.
In order to make it easy to understand, the present inventor provides following principle.It should be understood that protection scope of the present invention does not receive the original Any restriction of reason.
Referring to Fig. 1.In the present invention, can be formed when small fragment ctDNA recirculations and efficiently expand template.And one In individual linked system, except fragment in addition to be cyclized it may also happen that connection between fragment.Connection between fragment also may be used To play increase template length, improve the effect of amplification efficiency.But, in order to improve to mixing a small amount of ctDNA in cfDNA Detection results, employ special measure (include dilution and be cyclized), so that building storehouse amplification is more likely to ctDNA fragments. In the present invention, an outstanding feature is, the cfDNA of the very low concentrations used in coupled reaction (for example, less than 1ng/ μ l, compared with Goodly, 0.1ng/ μ l).It is in the case of low concentration DNA fragmentation, on the one hand distant between each fragment, move in irregular fever Under the conditions of the probability that meets significantly reduce, there is the probability for interconnecting between two nucleic acid fragments so as to significantly reduce.And On the other hand, even for same fragment, in dilution, the distance between 3 ' and 5 ' ends (i.e. the length of the fragment), no Change because of the concentration of fragment.Therefore, in the case of low fragment concentrations, DNA fragmentation relative will be carried from the probability of concatemerization Height, so that higher than the probability interconnected between fragment.In addition, for ctDNA to be detected present in sample, it is this short Nucleic acids show it is unexpected be significantly higher than cfDNA from cyclisation efficiency (both difference at least 1-3 orders of magnitude or more Greatly, that is, differ 10-1000 times or bigger).It is a kind of for ctDNA from cyclisation rate it is high the reason for be possibly due to its fragment shorter, Its 3 ' with 5 ' end the distance between it is nearer, probability certainly is also higher.Therefore, in the present invention, through specific dilution and DNA fragmentation is cyclized coupled reaction certainly so that the cfDNA for being significantly higher than non-tumor cell source from cyclisation rate of ctDNA (improves At least 1-3 order of magnitude or bigger), and then more cyclisation molecules are formed, so as to obtain more efficient in follow-up amplification Amplification.Another possible explanation is that while that this is less in the early stage from the difference for being cyclized efficiency, but in subsequent PCR amplification, this One difference can be accumulated in each circulation with exponential growth patterns, ultimately form significant difference.
For amplified production, (such as electrophoresis, enzyme action or sequencing) can be directly detected, it is also possible to first build storehouse and then enter Row sequencing, for example, detected with secondary sequence measurement or other sequence measurements.
The detection of genes of interest copy number change
In the present invention, the copy number of the genes of interest to be detected is not limited to HER2, including (but being not limited to) table 1 In gene and its chromosome.
Table 1
Main advantages of the present invention include：
(1) present invention efficiently can be copied from testing goal gene (such as Her2 genes) in tumor patient blood plasma dissociative DNA Several amplifications.
(2) detection method of the invention applies also for detecting other body fluid samples, such as urine that cerebrospinal fluid swells in saliva The copy number variation of any gene and DNA fragmentation of tumor source dissociative DNA.
(3) present invention is with blood plasma and other body fluid as biological specimen, efficient detection wherein tumor carry out source DNA (ctDNA) with Object observing gene, the particularly method of Her2 gene copy number variations.
(4) present invention expands database technology pair using MALBAC-LAB, first will be free under conditions of low nucleic acid concentration From being cyclized, wherein ctDNA has higher from cyclisation rate because fragment is shorter to DNA fragments, subsequently obtains the amplification (phase of greater efficiency For the cfDNA of more normal cell derived), so as to obtain higher detection sensitivity.
(5) detection method of the invention detects the copy number of multiple genes of interest, and is respectively provided with higher detection sensitivity.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limit the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip Part, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage ratio and number are percentage by weight and weight Number.
Material and reagent used by the present invention if no special instructions, is commercially available prod.
Embodiment 1.
It is cyclized for the impact of ctDNA detections
In the present embodiment and embodiment 2, with reference to Her2 gene tests, technical scheme is carried out specifically It is bright.
It is known to occur that to one the plasma DNA of the tumor patient of Her2 gene copy numbers amplification is entered by the setting of table 2 Row coupled reaction.
Table 2
As seen from Table 2,1 not do connect anti-to plasma DNA (include substantial amounts of cfDNA and micro ctDNA) Should, 2 is the coupled reaction of low concentration plasma DNA, and 3 is the coupled reaction of higher concentration plasma DNA.Each reaction exists To 75 DEG C, incubation inactivates ligase in 15 minutes to 37 DEG C of incubations, one hour post-heating.Reacted DNA is respectively taken into 0.5ng subsequently As template, carry out MALBAC-LAB amplifications and build storehouse.Expanding the brief step for operating is：DNA profiling (5 μ l) is added into 30 μ l Linear amplification reagent (is provided by sequence health medical science and technology (Suzhou) company limited), and reagent Main Ingredients and Appearance includes, primer mixture, special The archaeal dna polymerase with heat tolerance and strand displacement property of different design, dNTP, and Mg2+、(NH4)2+、K+,SO4 2-、Cl-Deng. Then thermal cycler is inserted in reaction.Thermocycling program is：
After thermal cycle terminates, 30 μ l exponential amplification reagents are added in reaction system (available from sequence health medical science and technology (Suzhou) Company limited), wherein mainly including：Primer mixture, the archaeal dna polymerase with heat tolerance and strand displacement property of particular design And exponential amplification reaction buffer etc..Then thermal cycler is inserted in reaction again.Thermocycling program is：
It is after the completion of amplification, visible with conventional gel electrophoresis detection amplified production, reaction 2, and 3 effectively expansion is obtained Increase, and 1 is reacted without the significant amplification (Fig. 2) of generation.
The above results show, process without cyclisation, then (be preferentially cyclized core using the PCR amplifications for being cyclized preferential Acid molecule is expanded for template, and not with or substantially do not expanded by template of linear nucleic acid molecule) when, for example use When the archaeal dna polymerase of strand-displacement activity is expanded, will be unable to effectively obtain amplified production.
Embodiment 2
Dilution pretreatment is affected for ctDNA detections
In embodiment 1, the MALBAC-LAB amplified productions of " reaction 2 " and " reaction 3 " are that Illumina high-flux sequences are put down The sequencing storehouse of platform, copies number variation (copy number with conventional after being carried out shallow sequencing (about 5% genome coverage) Variation) analysis software (available from sequence health medical science and technology (Suzhou) company limited) is located to Her2 genes on No. 17 chromosomes About 500K sections be analyzed.Meanwhile, the plasma DNA of same position patient is directly carried out building storehouse according to a conventional method, and Same sequencing, analysis are carried out as control.
From data result, can detect that significant Her2 gene copy numbers expand (Fig. 3 A) in " reaction 2 ", and Cannot all detect that copy number is expanded in " reaction 3 " (Fig. 3 B) and control test (Fig. 3 C).
It is above-mentioned to show, when not being diluted to testing sample so that in the sample Jing after dilution, the concentration of cfDNA is without big Width declines (such as 0.001-5ng/ μ l), although can then expand, amplified production is mainly produced corresponding to the amplification of cfDNA Thing, and the amplified production for corresponding to ctDNA cannot seldom or almost be detected.
Embodiment 3
Dilution is processed and cyclisation processes the relative level that improve ctDNA
In the present embodiment, tumor (such as the breast carcinoma) patient of Her2 gene copy numbers amplification is known to occur to another example Plasma DNA is attached reaction by the setting of table 3.
Table 3
Composition Reaction 1 Reaction 2
DNA 1ng 1ng
10 × ligase buffer solution 1μl 1μl
CircLigase 1μl 0
T4 ligases 0 1μl
Add water to 10μl 10μl
From table 3 it can be seen that reaction 1 is to do coupled reaction with CircLigase, and it is to make to connect of T4 ligases to react 2 Reaction, each reaction are incubated one hour post-heating to 75 DEG C at 37 DEG C, and incubation inactivates ligase in 15 minutes.Subsequently will be reacted DNA respectively takes 0.5ng as template, carries out MALBAC-LAB amplifications and builds storehouse.Concrete operation method is as described in embodiment 1.
After the completion of amplification, visible with conventional gel electrophoresis detection amplified production, reaction 1 effectively can be expanded, and Reaction 2 can not significantly be expanded (Fig. 4).
As a result show, ctDNA can be cyclized under conditions of low nucleic acid concentration (such as 0.1ng/ μ l) by the present invention certainly, and can obtain Very efficient amplification is obtained, so as to obtain higher detection sensitivity.
The above results show that in the methods of the invention, in the mixture that described Jing is cyclized, ctDNA cyclisation products are accounted for Advantage.
Additionally, in the mixture that diluted sample and Jing are cyclized, meeting Formulas I
Ct1/Cf1 ＞ Ct0/Cf0 (I)
In formula,
Ct1 is, in the mixture of Jing cyclisation, to be cyclized the concentration of ctDNA molecules；
Cf1 is, in the mixture of Jing cyclisation, to be cyclized the concentration of cfDNA molecules；
Ct0 is the concentration of ctDNA molecules in the diluted sample；
Cf0 is the concentration of cfDNA molecules in the diluted sample.
As shown by data, the ratio R 1 at least >=10 (such as 50-100 or bigger) of described Ct1/Cf1 and Ct0/Cf0.
The all documents referred in the present invention are all incorporated as reference in this application, independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned teachings for having read the present invention, those skilled in the art can To make various changes or modifications to the present invention, these equivalent form of values equally fall within the model limited by the application appended claims Enclose.

Claims (10)

1. a kind of sample treatment, it is characterised in that including step：
I () provides a testing sample, the sample contains cfDNA and ctDNA；
(ii) testing sample is diluted, obtains diluted sample, wherein in the sample Jing after dilution, cfDNA's is dense Spend for 0.001-5ng/ μ l, it is preferred that 0.01-2ng/ μ l, more preferably, 0.05-1ng/ μ l；With
(iii) cyclisation process is carried out to the diluted sample of step (ii) with cyclisation ligase, obtains the mixture of Jing cyclisation, Wherein, the mixture of described Jing cyclisation contains cyclisation ctDNA molecules.
2. the method for claim 1, it is characterised in that methods described also includes step：
(iv) with the cyclisation ctDNA molecules in the mixture of Jing cyclisation as template, expanded, so as to obtain corresponding to ring Change the amplified production of ctDNA.
3. method as claimed in claim 2, it is characterised in that methods described also includes step：
V () carries out building storehouse and sequencing, or is directly sequenced to the amplified production, wherein, by the sequencing, obtain described CtDNA testing results in sample to be tested.
4. method as claimed in claim 3, it is characterised in that methods described also includes step：
(vi) testing result based on step (v), so as to judge the prominent of the variation situation or gene order of copy number of target genes Change situation or its combination.
5. the method for claim 1, it is characterised in that in the mixture that diluted sample and Jing are cyclized, meet Formulas I
Ct1/Cf1 ＞ Ct0/Cf0 (I)
In formula,
Ct1 is, in the mixture of Jing cyclisation, to be cyclized the concentration of ctDNA molecules；
Cf1 is, in the mixture of Jing cyclisation, to be cyclized the concentration of cfDNA molecules；
Ct0 is the concentration of ctDNA molecules in the diluted sample；
Cf0 is the concentration of cfDNA molecules in the diluted sample.
6. the method for claim 1, it is characterised in that in diluted sample and detected sample, meet Formula II
Ct0/Cf0=Ct/Cf (II)
In formula,
Ct0 is the concentration of ctDNA molecules in the diluted sample；
Cf0 is the concentration of cfDNA molecules in the diluted sample；
Ct is the concentration of ctDNA molecules in the sample to be detected；
Cf is the concentration of cfDNA molecules in the sample to be detected.
7. the method for claim 1, it is characterised in that described sample is selected from the group：Blood, body fluid or its combination.
8. a kind of method that nondiagnostic ground detects ctDNA in sample, it is characterised in that including step：
I () provides a testing sample, the sample contains cfDNA and ctDNA；
(ii) testing sample is diluted, obtains diluted sample, wherein in the sample Jing after dilution, cfDNA's is dense Spend for 0.001-5ng/ μ l, it is preferred that 0.01-2ng/ μ l, more preferably, 0.05-1ng/ μ l；
(iii) cyclisation process is carried out to the diluted sample of step (ii) with cyclisation ligase, obtains the mixture of Jing cyclisation, Wherein, the mixture of described Jing cyclisation contains cyclisation ctDNA molecules；
(iv) with the cyclisation ctDNA molecules in the mixture of Jing cyclisation as template, expanded, so as to obtain corresponding to ring Change the amplified production of ctDNA；With
V () is detected to the amplified production, obtain the testing result of ctDNA.
9. method as claimed in claim 8, it is characterised in that the detection in step (v) includes：Build storehouse and sequencing, or directly It is sequenced, wherein, by the sequencing, the ctDNA testing results in the sample to be tested are obtained, so as to detect in sample ctDNA。
10. method as claimed in claim 8, it is characterised in that methods described also includes step：
(vi) testing result based on step (v), so as to judge the prominent of the variation situation or gene order of copy number of target genes Change situation or its combination.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107523640A (en) * 2017-10-12 2017-12-29 厦门燕旭安生物科技有限公司 The amplicon library constructing method that a kind of ctDNA is precisely sequenced
CN108315321A (en) * 2017-03-31 2018-07-24 索真（北京）医学科技有限公司 The detection of K-ras gene mutation sites in urine ctDNA
WO2018133773A1 (en) * 2017-01-17 2018-07-26 上海亿康医学检验所有限公司 Method for efficiently detecting ctdna in sample
CN110885879A (en) * 2019-12-13 2020-03-17 广州金域医学检验集团股份有限公司 Joint detection method for lymphangioleiomyomatosis and application thereof
CN112877441A (en) * 2021-04-27 2021-06-01 苏州仁端生物医药科技有限公司 Application of bladder urothelial cancer detection combined marker

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014008635A1 (en) * 2012-07-11 2014-01-16 北京贝瑞和康生物技术有限公司 Detection method and detection kit for dna fragments, and use thereof
CN104611410A (en) * 2013-11-04 2015-05-13 北京贝瑞和康生物技术有限公司 Noninvasive cancer detection method and its kit
CN104745679A (en) * 2013-12-31 2015-07-01 北京贝瑞和康生物技术有限公司 Method and kit for non-invasive detection of EGFR (epidermal growth factor receptor) gene mutation
CN105087756A (en) * 2014-04-23 2015-11-25 北京贝瑞和康生物技术有限公司 Method and kit for non-invasive measurement on fetus deaf pathogenic gene mutation
CN105986030A (en) * 2016-02-03 2016-10-05 广州市基准医疗有限责任公司 Methylated DNA detection method
CN106062214A (en) * 2013-12-28 2016-10-26 夸登特健康公司 Methods and systems for detecting genetic variants
CN106148323A (en) * 2015-04-22 2016-11-23 北京贝瑞和康生物技术股份有限公司 A kind of method and test kit merging abrupt climatic change library for building ALK gene
CN108064314A (en) * 2015-01-18 2018-05-22 加利福尼亚大学董事会 Judge the method and system of cancerous state

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2010213510B2 (en) * 2009-02-16 2014-05-01 Epicentre Technologies Corporation Template-independent ligation of single-stranded DNA
CN105518151B (en) * 2013-03-15 2021-05-25 莱兰斯坦福初级大学评议会 Identification and use of circulating nucleic acid tumor markers
JP6721599B2 (en) * 2014-10-29 2020-07-15 ベルジアン ボリション エスピーアールエル Method for concentrating circulating tumor DNA
CN106544341A (en) * 2017-01-17 2017-03-29 上海亿康医学检验所有限公司 The method of the ctDNA in efficient detection sample

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014008635A1 (en) * 2012-07-11 2014-01-16 北京贝瑞和康生物技术有限公司 Detection method and detection kit for dna fragments, and use thereof
CN104611410A (en) * 2013-11-04 2015-05-13 北京贝瑞和康生物技术有限公司 Noninvasive cancer detection method and its kit
CN106062214A (en) * 2013-12-28 2016-10-26 夸登特健康公司 Methods and systems for detecting genetic variants
CN104745679A (en) * 2013-12-31 2015-07-01 北京贝瑞和康生物技术有限公司 Method and kit for non-invasive detection of EGFR (epidermal growth factor receptor) gene mutation
CN105087756A (en) * 2014-04-23 2015-11-25 北京贝瑞和康生物技术有限公司 Method and kit for non-invasive measurement on fetus deaf pathogenic gene mutation
CN108064314A (en) * 2015-01-18 2018-05-22 加利福尼亚大学董事会 Judge the method and system of cancerous state
CN106148323A (en) * 2015-04-22 2016-11-23 北京贝瑞和康生物技术股份有限公司 A kind of method and test kit merging abrupt climatic change library for building ALK gene
CN105986030A (en) * 2016-02-03 2016-10-05 广州市基准医疗有限责任公司 Methylated DNA detection method

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
刘丽荣: "乳腺癌患者血浆循环DNA含量分析及其临床意义", 《细胞与分子免疫学杂志》 *
叶长生 等: "检测循环肿瘤DNA中ESR1突变在晚期乳腺癌治疗中的价值", 《循证医学》 *
屠红等: "肿瘤患者循环DNA的定量研究", 《中华肿瘤杂志》 *
蒋春雨等: "血浆循环ＤＮＡ 定量检测在食管鳞癌诊断中的意义", 《实用医学杂志》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018133773A1 (en) * 2017-01-17 2018-07-26 上海亿康医学检验所有限公司 Method for efficiently detecting ctdna in sample
CN108315321A (en) * 2017-03-31 2018-07-24 索真（北京）医学科技有限公司 The detection of K-ras gene mutation sites in urine ctDNA
CN107523640A (en) * 2017-10-12 2017-12-29 厦门燕旭安生物科技有限公司 The amplicon library constructing method that a kind of ctDNA is precisely sequenced
CN110885879A (en) * 2019-12-13 2020-03-17 广州金域医学检验集团股份有限公司 Joint detection method for lymphangioleiomyomatosis and application thereof
CN110885879B (en) * 2019-12-13 2020-11-13 广州金域医学检验集团股份有限公司 Joint detection method for lymphangioleiomyomatosis and application thereof
CN112877441A (en) * 2021-04-27 2021-06-01 苏州仁端生物医药科技有限公司 Application of bladder urothelial cancer detection combined marker

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