WO2018133773A1 - 高效检测样本中的ctDNA的方法 - Google Patents

高效检测样本中的ctDNA的方法 Download PDF

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WO2018133773A1
WO2018133773A1 PCT/CN2018/072831 CN2018072831W WO2018133773A1 WO 2018133773 A1 WO2018133773 A1 WO 2018133773A1 CN 2018072831 W CN2018072831 W CN 2018072831W WO 2018133773 A1 WO2018133773 A1 WO 2018133773A1
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sample
ctdna
amplification
molecule
concentration
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任军
耿荷芳
陆思嘉
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上海亿康医学检验所有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
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    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

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  • the present invention relates to the field of biotechnology, and in particular to a method for efficiently detecting ctDNA in a sample.
  • the human epidermal growth factor receptor-2 (HER2) gene the c-erbB-2 gene, is located on chromosome 17q12-21.32 and encodes a transmembrane with a relative molecular mass of 185,000.
  • a receptor-like protein with tyrosine kinase activity is closely related to the occurrence of breast cancer and gastric cancer. About 20% to 25% of breast cancers and 15% of patients with gastric cancer overexpress HER2, and the prognosis of this group of patients is poor.
  • Copy number amplification of Her2 gene in some breast cancers and gastric cancer is only one example of tumor cell gene copy number variation.
  • many studies have found that many genes or chromosome segments have copy number amplification or deletion in tumor cells. Therefore, detecting copy number variation of target genes in tumor cells is an important technology in cancer biology. One.
  • the existing Her2 detection method mainly uses the IHC and FISH methods to detect tumor biopsy tissue samples.
  • attempts have been made to detect free tumor DNA (ctDNA) present in plasma free DNA (cfDNA) to identify the presence of Her2 gene amplification, but the effect is not satisfactory.
  • Both the IHC method and the FISH method are used to detect tumor biopsy tissue sections, and their application has great limitations, mainly in:
  • Some patients are extremely weak and cannot undergo biopsy surgery, that is, they cannot obtain a pathological slice.
  • Fine needle aspiration biopsy can only obtain very small amounts of tumor tissue and is often insufficient for IHC and FISH testing.
  • drug resistance may occur after treatment with targeted drugs. Therefore, it is necessary to continuously and dynamically detect Her2 gene amplification during drug administration.
  • repeated biopsy of tumor tissue is almost sampled. It is an impossible thing. Therefore, the IHC and FISH methods are powerless.
  • the free second-generation sequencing method can detect the free tumor DNA (ctDNA) present in plasma free DNA (cfDNA) to identify the presence of Her2 gene amplification, but the main reason for the unsatisfactory effect in practical applications Yes: First, cfDNA is very low in plasma, and only a few nanograms of DNA is usually obtained from one milliliter of plasma. Moreover, the main source of cfDNA is DNA released into the blood after death of leukocytes, and ctDNA derived from tumor cells is rare. It is difficult to identify the presence of Her2 gene amplification by conventional gene copy number analysis.
  • a target gene e.g., HER2
  • a first aspect of the invention provides a sample processing method comprising the steps of:
  • diluted sample has a concentration of cfDNA of 0.001 to 5 ng/ ⁇ l, preferably 0.01 to 2 ng/ ⁇ l, and more preferably, 0.05-1 ng/ ⁇ l;
  • step (iii) cyclizing the diluted sample of step (ii) with a cyclase to obtain a cyclized mixture, wherein the cyclized mixture contains a circularized ctDNA molecule.
  • the method further includes the steps of:
  • Amplification is carried out using the circularized ctDNA molecule in the cyclized mixture as a template to obtain an amplification product corresponding to the circularized ctDNA.
  • the amplification is a circularization-preferred PCR amplification (ie, preferentially using a circularized nucleic acid molecule as a template for amplification, without or substantially not using a linear nucleic acid molecule as a template for amplification. ).
  • the amplification is amplified using a strand displacement active DNA polymerase.
  • the amplification comprises MALBAC-LAB amplification.
  • the method further includes the steps of:
  • the method further includes the steps of:
  • step (vi) Based on the detection result of the step (v), thereby determining the variation of the copy number of the target gene, or the mutation of the gene sequence, or a combination thereof.
  • the ctDNA cyclization product predominates in the cyclized mixture.
  • Formula I is satisfied in the diluted sample and the cyclized mixture
  • Ct1 is the concentration of the cyclized ctDNA molecule in the cyclized mixture
  • Cf1 is the concentration of the cyclized cfDNA molecule in the cyclized mixture
  • Ct0 is the concentration of the ctDNA molecule in the diluted sample
  • Cf0 is the concentration of the cfDNA molecule in the diluted sample.
  • Ct0 is the concentration of the ctDNA molecule in the diluted sample
  • Cf0 is the concentration of the cfDNA molecule in the diluted sample
  • Ct is the concentration of the ctDNA molecule in the sample to be detected
  • Cf is the concentration of the cfDNA molecule in the sample to be detected.
  • the ratio of Ct1/Cf1 to Ct0/Cf0 is R1 ⁇ 10, preferably ⁇ 50, more preferably ⁇ 100.
  • the treatment method is a non-diagnostic and non-therapeutic method.
  • an amplification product corresponding to the ctDNA cyclization product is dominant.
  • the "amplification product corresponding to the ctDNA cyclization product predominates" means that the concentration (C1) of the amplification product corresponding to the ctDNA cyclization product in the amplification product is significantly higher than that corresponding to cfDNA Amplification product concentration (C2) of the cyclized product.
  • the "significantly higher" means C1/C2 ⁇ 5, preferably ⁇ 10, more preferably ⁇ 20.
  • the sample is selected from the group consisting of blood, body fluids, or a combination thereof.
  • the sample is selected from the group consisting of blood, plasma, interstitial fluid, lymph, urine, cerebrospinal fluid, saliva, aqueous humor, semen, gastrointestinal secretions, or a combination thereof.
  • the sample is selected from the group consisting of blood, plasma, or serum.
  • the sample is a cell free sample.
  • the sample is free of tumor cells.
  • the ctDNA is derived from a tumor cell.
  • the tumor cells are selected from the group consisting of breast cancer, ovarian cancer, gastric cancer, lung cancer, colorectal cancer, bladder cancer, esophageal cancer, pancreatic cancer, skin cancer, prostate cancer, esophageal cancer, gallbladder cancer. , thyroid cancer, liver cancer, laryngeal cancer, oropharyngeal cancer, leukemia, or a combination thereof.
  • the cyclization ligase selected from the group: CircLigase, ThermoPhage TM ssDNA ligase, or a combination thereof.
  • the amplification method is selected from the group consisting of polymerase chain reaction (PCR), multiple strand displacement reaction (MDA), rolling circle DNA amplification (RCA), Loop-mediated gene isothermal amplification technique (LAMP), or a combination thereof.
  • PCR polymerase chain reaction
  • MDA multiple strand displacement reaction
  • RCA rolling circle DNA amplification
  • LAMP Loop-mediated gene isothermal amplification technique
  • step (v) the method of building the library is selected from the group consisting of: MALBAC-LAB building, interrupting building, and/or transposition.
  • the sequencing is performed by the method of selecting the next group: Illumina sequencing, Ion Torrent sequencing, Roche 454 sequencing, SoLID sequencing, Completed Genomics (CG) sequencing, NanoPore sequencing, Pacific Bio sequencing, or a combination thereof.
  • the target gene is selected from the group consisting of 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, AKT
  • a second aspect of the invention provides a method of detecting ctDNA in a sample, comprising the steps of:
  • step (iii) cyclizing the diluted sample of step (ii) with a cyclase to obtain a cyclized mixture, wherein the cyclized mixture contains a circularized ctDNA molecule;
  • the detecting in the step (v) comprises: building and sequencing, or directly performing sequencing, wherein the ctDNA detection result in the sample to be tested is obtained by the sequencing, thereby detecting the sample in the sample. ctDNA.
  • the method further includes the steps of:
  • step (vi) Based on the detection result of the step (v), thereby determining the variation of the copy number of the target gene, or the mutation of the gene sequence, or a combination thereof.
  • the amplification is a circularization-preferred PCR amplification (ie, preferentially using a circularized nucleic acid molecule as a template for amplification, without or substantially not using a linear nucleic acid molecule as a template for amplification. ).
  • the amplification comprises MALBAC-LAB amplification.
  • the detection method is a non-diagnostic and non-therapeutic method.
  • FIG 1 shows the principle and technical flow of the present invention.
  • Figure 2 shows the results of the expansion of the different connected samples.
  • FIG. 3 shows the experimental results of different technical solutions.
  • Figure 4 shows the results of amplification and construction after ligation with different ligases.
  • ctDNA is a circulating tumor DNA fragment, mainly fragmented genomic DNA released after the rupture of dead tumor cells.
  • the content of ctDNA is low, accounting for about 1% of all free DNA, or even only 0.01%.
  • ctDNA is 20-50 bp shorter than cfDNA fragment, about 130-145 bp.
  • cfDNA is a general term for free DNA in plasma. There are two main sources: fragmented nucleic acids (160-180 bp) produced during apoptosis and nucleic acids released by cells during tissue necrosis or immune killing (close to genome size).
  • the "cyclized ligase” refers to a thermostable ligase that catalyzes the ligation of linear single-stranded DNA into circular single-stranded DNA.
  • the selection ring ligase is not particularly limited, in a preferred embodiment, the cyclization ligase selected from the group: CircLigase, ThermoPhage TM ssDNA ligase, or a combination thereof.
  • CircLigase is a single-stranded DNA circular ligase which catalyzes intramolecular ligation (i.e., cyclization) of a single-stranded DNA template having a 5'-phosphate and a 3'-hydroxy group in the absence of a complementary sequence.
  • ThermoPhage TM ssDNA Ligase (ThermoPhage TM single-stranded DNA ligase ) into a single stranded DNA ligase, can be connected to a single-stranded DNA or RNA at a high temperature.
  • the present invention provides a sample processing method.
  • the sample processing method of the present invention comprises the steps of:
  • diluted sample has a concentration of cfDNA of 0.001 to 5 ng/ ⁇ l, preferably 0.01 to 2 ng/ ⁇ l, and more preferably, 0.05-1ng/ ⁇ l;
  • step (iii) cyclizing the diluted sample of step (ii) with a cyclase to obtain a cyclized mixture, wherein the cyclized mixture contains a circularized ctDNA molecule.
  • the present invention provides a method of detecting ctDNA in a sample.
  • the method of detecting ctDNA in a sample of the present invention comprises the steps of:
  • diluted sample has a concentration of cfDNA of 0.001 to 5 ng/ ⁇ l, preferably 0.01 to 2 ng/ ⁇ l, and more preferably, 0.05-1ng/ ⁇ l;
  • step (iii) cyclizing the diluted sample of step (ii) with a cyclase to obtain a cyclized mixture, wherein the cyclized mixture contains a circularized ctDNA molecule;
  • the detecting in the step (v) comprises: building and sequencing, or directly performing sequencing, wherein the ctDNA detection result in the sample to be tested is obtained by the sequencing, thereby detecting the sample in the sample. ctDNA.
  • the method further comprises the steps of:
  • step (vi) Based on the detection result of the step (v), thereby determining the variation of the copy number of the target gene, or the mutation of the gene sequence, or a combination thereof.
  • the amplification and database construction method is not particularly limited, and amplification and colonization methods capable of achieving the amplification effect of the present invention are all included in the present invention.
  • the cyclized molecule (cyclized ctDNA) is amplified using a strand displacement active DNA polymerase.
  • the amplification and construction method used in the present invention is the MALBAC-LAB whole genome amplification method.
  • the MALBAC-LAB amplification library construction method has higher amplification efficiency in principle compared with larger DNA fragments.
  • the product after extension of the primer will form a hairpin structure due to the presence of complementary sequences at both ends of the fragment.
  • the free primer sequence is identical to the hairpin sequence.
  • the free primer binds to the 3' end of the template only before the hairpin is formed to form strand extension amplification (competition wins over the 5' end of the template fragment).
  • the shorter the template fragment the closer the 3' end to the 5' end, so the higher the chance of forming a hairpin, the smaller the chance of free primer competition and the smaller the chance of successful amplification.
  • the longer the template fragment the higher the probability of successful amplification and the higher the overall amplification efficiency.
  • DNA ligase is used to self-ligate the ends of the small fragment DNA to convert the linear DNA into circular DNA.
  • the chain extension can be carried out along the circular template, and the obtained product is longer, and the index is expanded in the next step. Higher amplification efficiency can be obtained in the addition phase.
  • a highly efficient amplification template can be formed when the small fragment ctDNA is cyclized by itself.
  • the connections between the segments may occur.
  • the connection between the fragments can also increase the length of the template and improve the amplification efficiency.
  • special measures including dilution and cyclization
  • a salient feature is the use of very low concentrations of cfDNA (e.g., less than 1 ng/ ⁇ l, preferably 0.1 ng/ ⁇ l) in the ligation reaction.
  • concentrations of cfDNA e.g., less than 1 ng/ ⁇ l, preferably 0.1 ng/ ⁇ l
  • the distance between the fragments is far, and the probability of encountering under irregular thermal motion conditions is significantly reduced, thereby significantly reducing the probability of interconnection between the two nucleic acid fragments.
  • the distance between the 3' and 5' ends i.e., the length of the segment) during the dilution process is not changed by the concentration of the segment.
  • this short fragment nucleic acid exhibits an unexpectedly higher self-cyclization efficiency than cfDNA (the difference between the two is at least 1-3 orders of magnitude or greater, ie, a difference of 10) 1000 times or more).
  • One reason for the high rate of self-cyclization of ctDNA may be that the shorter the fragment, the closer the distance between the 3' and 5' ends, and the higher the probability of self-ligation.
  • the specific dilution and DNA fragment self-cyclization ligation reaction makes the self-cyclization rate of ctDNA significantly higher than that of non-tumor cell-derived cfDNA (increased by at least 1-3 orders of magnitude or more). Further, more cyclized molecules are formed, resulting in more efficient amplification in subsequent amplification.
  • Another possible explanation is that although the difference in efficiency of this self-cyclization is small at an early stage, in subsequent PCR amplification, this difference accumulates in each cycle in an exponentially increasing form, eventually forming a significant difference.
  • the amplification product For the amplification product, it can be directly detected (for example, electrophoresis, enzymatic digestion or sequencing), or the library can be first constructed and then sequenced, for example, by a second-generation sequencing method or other sequencing methods.
  • directly detected for example, electrophoresis, enzymatic digestion or sequencing
  • the library can be first constructed and then sequenced, for example, by a second-generation sequencing method or other sequencing methods.
  • the copy number of the gene of interest to be detected is not limited to HER2, and includes, but is not limited to, the gene in Table 1 and its chromosome.
  • IGF1R/IGFIR/JTK13 STK11/LKB1/PJS IGF2/C11orf43/INSIGF Chr17 GSTT1 RB1/OSRC/RB 17q22 AR/AIS/DHTR D13S319 (FISH_probe) 20p13 NRAS/N-ras/NRAS1 NFKBIA/IKBA/MAD-3 Chr3 1q21/PMVK/HUMPMKI TP53/p53/LFS1 17q25.3
  • UGT1A1/GNT1/HUG-BR1 CCNE1/CCNE MDM4/MDMX
  • VHL/HRCA1/RCA1 ZMYND8/PRKCBP1 Chr7
  • MECOM/A1L4F3/A8KA00 CYP2D6/CPD6/CYP2D MET/AUTS9/HGFR KIT/C-Kit/SCFR
  • DDR2/NTRKR3/TYRO10 FGFR1/BFGFR/CEK PIK3R1/GRB1/
  • the present invention can efficiently detect amplification of a copy number of a gene of interest (e.g., Her2 gene) from plasma free DNA of a tumor patient.
  • a gene of interest e.g., Her2 gene
  • the detection method of the present invention can also be applied to the detection of copy number variation of any gene and DNA fragment of tumor-derived free DNA in other body fluid samples such as urine, cerebrospinal fluid, and saliva.
  • the present invention uses plasma and other body fluids as biological samples to efficiently detect tumor-derived DNA (ctDNA) to observe target gene, particularly the Her2 gene copy number variation.
  • ctDNA tumor-derived DNA
  • the present invention utilizes the MALBAC-LAB amplification library construction technique to self-circulate free DNA fragments for the first time under conditions of low nucleic acid concentration, wherein ctDNA has a higher self-cyclization rate due to the shorter fragment, and is subsequently obtained. Higher efficiency amplification (compared to normal cell-derived cfDNA) results in higher detection sensitivity.
  • the detection method of the present invention detects the copy number of a plurality of genes of interest, and both have high detection sensitivity.
  • the materials and reagents used in the present invention are all commercially available products unless otherwise specified.
  • the plasma free DNA of a tumor patient who is known to have a copy of the Her2 gene copy number was subjected to a ligation reaction according to the setting of Table 2.
  • 1 is a reaction that does not link plasma free DNA (including a large amount of cfDNA and a trace amount of ctDNA)
  • 2 is a ligation reaction of a plasma plasma of a lower concentration
  • 3 is a ligation reaction of a plasma ion of a higher concentration.
  • Each reaction was incubated at 37 ° C for one hour, then heated to 75 ° C and incubated for 15 minutes to inactivate the ligase. Subsequently, 0.5 ng of each of the reacted DNA was used as a template, and MALBAC-LAB amplification and database construction were performed.
  • the brief procedure for the amplification procedure is to add the DNA template (5 ⁇ l) to 30 ⁇ l of linear amplification reagent (provided by Sikang Medical Technology (Suzhou) Co., Ltd.), the main components of the reagent include, primer mixture, specially designed for heat tolerance and A strand-displacing DNA polymerase, dNTP, and Mg 2+ , (NH 4 ) 2+ , K+, SO 4 2- , Cl - and the like.
  • the reaction is then placed in a thermal cycler.
  • the thermal cycle program is:
  • the amplified products were visualized by conventional gel electrophoresis, and both of the reactions 2 and 3 were efficiently amplified, and the reaction 1 was not significantly amplified (Fig. 2).
  • cyclization-preferred PCR amplification is used (ie, amplification is preferably performed using a circularized nucleic acid molecule as a template, without or substantially not using a linear nucleic acid molecule as a template).
  • amplification using, for example, a DNA polymerase having a strand displacement activity, an amplification product cannot be efficiently obtained.
  • the MALBAC-LAB amplification product of "Reaction 2" and “Reaction 3" in Example 1 is the sequencing library of the Illumina high-throughput sequencing platform, which is subjected to shallow sequencing (about 5% of genome coverage) followed by a regular copy.
  • the copy number variation analysis software obtained from Prekang Medical Technology (Suzhou) Co., Ltd.) analyzed the approximately 500K segment of the Her2 gene on chromosome 17.
  • the plasma free DNA of the same patient was directly constructed according to the conventional method, and the same sequencing and analysis were performed as a control.
  • the amplification product mainly corresponds to the expansion of cfDNA.
  • the product is increased, and the amplification product corresponding to ctDNA is little or almost impossible to detect.
  • the plasma free DNA of another patient who is known to have a copy of the Her2 gene copy number (such as breast cancer) is subjected to a ligation reaction according to the settings of Table 3.
  • Reaction 1 DNA 1ng 1ng 10 ⁇ ligase buffer 1 ⁇ l 1 ⁇ l CircLigase 1 ⁇ l 0 T4 ligase 0 1 ⁇ l Add water to 10 ⁇ l 10 ⁇ l
  • reaction 1 was ligated with CircLigase
  • reaction 2 was ligated with T4 ligase.
  • Each reaction was incubated at 37 ° C for one hour, then heated to 75 ° C, and incubated for 15 minutes to inactivate the ligase. .
  • 0.5 ng of each of the reacted DNA was used as a template, and MALBAC-LAB amplification and database construction were performed.
  • the specific operation method is as described in Embodiment 1.
  • the amplified product is detected by conventional gel electrophoresis, and the reaction 1 can be efficiently amplified, while the reaction 2 cannot be significantly amplified (Fig. 4).
  • the present invention can self-cyclize ctDNA under conditions of low nucleic acid concentration (e.g., 0.1 ng/ ⁇ l), and can obtain very high-efficiency amplification, thereby obtaining high detection sensitivity.
  • low nucleic acid concentration e.g., 0.1 ng/ ⁇ l
  • Ct1 is the concentration of the cyclized ctDNA molecule in the cyclized mixture
  • Cf1 is the concentration of the cyclized cfDNA molecule in the cyclized mixture
  • Ct0 is the concentration of the ctDNA molecule in the diluted sample
  • Cf0 is the concentration of the cfDNA molecule in the diluted sample.
  • the data indicates that the ratio R1 of Ct1/Cf1 to Ct0/Cf0 is at least ⁇ 10 (e.g., 50-100 or more).

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Abstract

一种高效检测样本中的ctDNA的方法，所述方法可在极低的核酸浓度下，将游离DNA碎片自环化，其中ctDNA因片段较短而有更高的自环化率，随后用特定环化优先扩增或扩增建库方法可获得较高效率的扩增，得到大量的对应于ctDNA的扩增产物，从而获得非常高的检测灵敏度和特异性。

Description

高效检测样本中的ctDNA的方法 技术领域
本发明涉及生物技术领域，具体地，涉及一种高效检测样本中的ctDNA的方法。
背景技术
研究表明，肿瘤病人的血液中存在极其微量的来自于肿瘤细胞的ctDNA，循环肿瘤DNA片段，其主要是死亡的肿瘤细胞破裂后所释放出来的。
以乳腺癌为例，人类表皮生长因子受体2(human epidermalgrowth factor receptor-2,HER2)基因,即c-erbB-2基因,定位于染色体17q12-21.32上,编码相对分子质量为185000的跨膜受体样蛋白,具有酪氨酸激酶活性。HER2基因的过表达或拷贝数扩增与乳腺癌和胃癌的发生密切相关。约有20％～25％的乳腺癌、15％的胃癌患者过表达HER2，该部分患者的预后较差。
Her2基因在部分乳腺癌和胃癌中发生拷贝数扩增仅仅是肿瘤细胞基因拷贝数变异的例子之一。实际上，先前研究中已经发现很多基因或染色体区段在肿瘤细胞中存在着拷贝数扩增或缺失的现象，因此，检测肿瘤细胞中目标基因的拷贝数变异是癌生物学中的重要技术之一。
然而，现有Her2的检测方法主要是用IHC和FISH方法对肿瘤活检组织样本进行检测。另外，现在已有人尝试对血浆游离DNA(cfDNA)中存在的游离肿瘤DNA(ctDNA)进行检测，鉴定是否存在Her2基因的扩增，但效果尚不理想。
IHC方法与FISH方法都是对肿瘤活检组织切片样本进行检测，其应用有很大局限性，主要表现在：
有些病人身体已经极为虚弱，无法承受活检手术，即无法获得病理切片样本。
对肿瘤复发病人，诊疗原则不主张手术活检取样，代之以细针穿刺活检。细针穿刺活检仅能获取很少量的肿瘤组织，经常不足以进行IHC和FISH检测。对存在Her2基因扩增病人，在使用靶向药物治疗后可能会发生耐药情况，故此需要在用药期间持续、动态检测Her2基因扩增情况，但在临床现实中，对肿瘤组织反复活检取样几乎是一件不可能的事情。因此，IHC与FISH方法对此无能为力。
理论上，用常规二代测序的方法可以对血浆游离DNA(cfDNA)中存在的游离肿瘤DNA(ctDNA)进行检测，鉴定是否存在Her2基因的扩增，但实际应用中效果尚不理想的主要原因是：首先，cfDNA在血浆中含量非常少，从一毫升血浆中通常只能获取几纳克DNA。并且，cfDNA的主要来源是白细胞死亡后释放入血液的DNA，其中真正来源于肿瘤细胞的ctDNA少之又少，用常规基因拷贝数分析的方法难以鉴定是否存在Her2基因扩增。
因此，本领域迫切需要开发一种能够高效富集、检测样本中的ctDNA，从而判断样本中是否存在目标基因(如HER2)扩增的方法。
发明内容
本发明的目的在于提供一种能够高效富集、检测样本中的ctDNA，从而判断样本中是否存在目标基因(如HER2)扩增的方法。
本发明的第一方面提供了一种样品处理方法，包括步骤：
(i)提供一待测样品，所述样品含有cfDNA和ctDNA；
(ii)稀释所述待测样品，获得经稀释的样品，其中所述经稀释后的样品中，cfDNA的浓度为0.001-5ng/μl，较佳地，0.01-2ng/μl，更佳地，0.05-1ng/μl；和
(iii)用环化连接酶对步骤(ii)的经稀释的样品进行环化处理，获得经环化的混合物，其中，所述的经环化的混合物含有环化ctDNA分子。
在另一优选例中，所述方法还包括步骤：
(iv)以所述经环化的混合物中的环化ctDNA分子为模板，进行扩增，从而得到对应于环化ctDNA的扩增产物。
在另一优选例中，所述的扩增为环化优先的PCR扩增(即优先以环化核酸分子为模板进行扩增，而不以或基本上不以线性核酸分子为模板进行扩增)。
在另一优选例中，所述的扩增使用链置换活性的DNA聚合酶进行扩增。
在另一优选例中，所述的扩增包括MALBAC-LAB扩增。
在另一优选例中，所述方法还包括步骤：
(v)对所述扩增产物进行建库和测序、或直接进行测序，其中，通过所述测序，获得所述待测样本中的ctDNA检测结果。
在另一优选例中，所述方法还包括步骤：
(vi)基于步骤(v)的检测结果，从而判断目标基因拷贝数的变异情况、或基因序列的突变情况、或其组合。
在另一优选例中，在所述的经环化的混合物中，ctDNA环化产物占优势。
在另一优选例中，在经稀释的样品和经环化的混合物中，满足式I
Ct1/Cf1＞Ct0/Cf0     (I)
式中，
Ct1为在所述经环化的混合物中，环化ctDNA分子的浓度；
Cf1为在所述经环化的混合物中，环化cfDNA分子的浓度；
Ct0为在所述经稀释的样品中，ctDNA分子的浓度；
Cf0为在所述经稀释的样品中，cfDNA分子的浓度。
在另一优选例中，在经稀释的样品和待检测的样品中，满足式II
Ct0/Cf0＝Ct/Cf       (II)
式中，
Ct0为在所述经稀释的样品中，ctDNA分子的浓度；
Cf0为在所述经稀释的样品中，cfDNA分子的浓度；
Ct为在所述待检测的样品中，ctDNA分子的浓度；
Cf为在所述待检测的样品中，cfDNA分子的浓度。
在另一优选例中，所述的Ct1/Cf1与Ct0/Cf0的比值R1≥10，较佳地≥50，更佳地≥100。
在另一优选例中，所述的处理方法是非诊断性和非治疗性的方法。
在另一优选例中，在所述的扩增产物中，对应于ctDNA环化产物的扩增产物占优势。
在另一优选例中，所述“对应于ctDNA环化产物的扩增产物占优势”指扩增产物中，对应于ctDNA环化产物的扩增产物的浓度(C1)显著高于对应于cfDNA环化产物的扩增产物浓度(C2)。
在另一优选例中，所述“显著高于”指C1/C2≥5，较佳地≥10，更佳地，≥20。
在另一优选例中，所述的样品选自下组：血液、体液、或其组合。
在另一优选例中，所述样品选自下组：血液、血浆、组织间隙液、淋巴液、尿液、脑脊液、唾液、房水、精液、胃肠道分泌液、或其组合。
在另一优选例中，所述样品选自下组：血液、血浆、或血清。
在另一优选例中，所述的样品为无细胞的样品。
在另一优选例中，所述的样品不含肿瘤细胞。
在另一优选例中，所述ctDNA源自肿瘤细胞。
在另一优选例中，所述肿瘤细胞选自下组：乳腺癌、卵巢癌、胃癌、肺癌、结直肠癌、膀胱癌、食管癌、胰腺癌、皮肤癌、前列腺癌、食管癌、胆囊癌、甲状腺癌、肝癌、喉癌、口咽癌、白血病、或其组合。
在另一优选例中，所述环化连接酶选自下组：CircLigase、ThermoPhage TM ssDNA连接酶、或其组合。
在另一优选例中，在步骤(iv)中，所述扩增方法选自下组：聚合酶链式反应(PCR)、多重链置换反应(MDA)、滚环DNA扩增(RCA)、环介导基因恒温扩增技术(LAMP)、或其组合。
在另一优选例中，在步骤(v)中，所述的建库方法选自下组：MALBAC-LAB建库、打断建库、和/或转座建库。
在另一优选例中，所述测序用选择下组的方法进行：Illumina测序、Ion Torrent测序、Roche 454测序、SoLID测序、Completed Genomics(CG)测序、NanoPore测序、Pacific Bio测序、或其组合。
在另一优选例中，所述目标基因选自下组：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、或其组合。
本发明第二方面提供了一种检测样本中ctDNA的方法，包括步骤：
(i)提供一待测样品，所述样品含有cfDNA和ctDNA；
(ii)稀释所述待测样品，获得经稀释的样品，其中所述经稀释后的样品中，cfDNA的浓度为0.001-5ng/ul,较佳地，0.01-2ng/ul，更佳的，0.05-1ng/ul；
(iii)用环化连接酶对步骤(ii)的经稀释的样品进行环化处理，获得经环化的混合物，其中，所述的经环化的混合物含有环化ctDNA分子；
(iv)以所述经环化的混合物中的环化ctDNA分子为模板，进行扩增，从而得到对应于环化ctDNA的扩增产物；和
(v)对所述扩增产物进行检测，获得ctDNA的检测结果。
在另一优选例中，步骤(v)中的检测包括：建库和测序、或直接进行测序，其中，通过所述测序，获得所述待测样本中的ctDNA检测结果，从而检测样本中的ctDNA。
在另一优选例中，所述方法还包括步骤：
(vi)基于步骤(v)的检测结果，从而判断目标基因拷贝数的变异情况、或基因序列的突变情况、或其组合。
在另一优选例中，所述的扩增为环化优先的PCR扩增(即优先以环化核酸分子为模板进行扩增，而不以或基本上不以线性核酸分子为模板进行扩增)。
在另一优选例中，所述的扩增包括MALBAC-LAB扩增。
在另一优选例中，所述的检测方法是非诊断性和非治疗性的方法。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明的原理与技术流程。
图2显示了不同连接样本的扩增建库结果。
图3显示了不同技术方案的实验结果。
图4显示了用不同的连接酶进行连接后的扩增建库结果。
具体实施方式
经过广泛而深入的研究，本发明意外的发现，在极低的核酸浓度下，将游离DNA碎片进行自环化，其中ctDNA因片段较短而有异乎寻常的高自环化率，随后用特定环化优先扩增或扩增建库方法(如MALBAC-LAB技术)可获得较高效率的扩增(相较正常细胞来源的cfDNA而言)，得到大量的对应于ctDNA的扩增产物，从而获得非常高的检测灵敏度和特异性。在此基础上，本发明人完成了本发明。
ctDNA
ctDNA即循环肿瘤DNA片段，主要是死亡的肿瘤细胞破裂后所释放出来的、片段化的基因组DNA。ctDNA的含量低，约占全部游离DNA的1％，甚至只有0.01％，通常，ctDNA比cfDNA片段大小要短20-50bp，约为130-145bp。
研究表明，肿瘤细胞来源ctDNA的片段长度小于非肿瘤细胞来源的cfDNA。
cfDNA
cfDNA是血浆中游离DNA的总称，主要来源有两种：细胞凋亡过程中产生的片段化核酸(160-180bp)和组织坏死或免疫杀伤过程中细胞释放的核酸(接近基因组大小)。
环化连接酶
在本发明中，所述“环化连接酶”指一种具有热稳定性的连接酶，可催化线性单链DNA连接成环状单链DNA。
在本发明中，所述环化连接酶的选择没有特别限制，在一优选实施方式中，所述环化连接酶选自下组：CircLigase、ThermoPhage TM ssDNA连接酶、或其组 合。
其中，CircLigase为单链DNA环化连接酶，可在没有互补序列存在的情况下，催化具有5’-磷酸和3’-羟基基团的单链DNA模板的分子内连接(即环化)。
ThermoPhage TM ssDNA连接酶(ThermoPhage TM single-stranded DNA ligase)为一种单链DNA连接酶，可在高温下连接单链DNA或RNA。
样品处理方法
本发明提供了一种样品处理方法。
在一优选实施方式中，本发明的样品处理方法，包括步骤：
(i)提供一待测样品，所述样品含有cfDNA和ctDNA；
(ii)稀释所述待测样品，获得经稀释的样品，其中所述经稀释后的样品中，cfDNA的浓度为0.001-5ng/μl,较佳地，0.01-2ng/μl，更佳地，0.05-1ng/μl；
(iii)用环化连接酶对步骤(ii)的经稀释的样品进行环化处理，获得经环化的混合物，其中，所述的经环化的混合物含有环化ctDNA分子。
检测样本中ctDNA的方法
本发明提供了一种检测样本中ctDNA的方法。
在一优选实施方式中，本发明的检测样本中ctDNA的方法包括步骤：
(i)提供一待测样品，所述样品含有cfDNA和ctDNA；
(ii)稀释所述待测样品，获得经稀释的样品，其中所述经稀释后的样品中，cfDNA的浓度为0.001-5ng/μl,较佳地，0.01-2ng/μl，更佳地，0.05-1ng/μl；
(iii)用环化连接酶对步骤(ii)的经稀释的样品进行环化处理，获得经环化的混合物，其中，所述的经环化的混合物含有环化ctDNA分子；
(iv)以所述经环化的混合物中的环化ctDNA分子为模板，进行扩增，从而得到对应于环化ctDNA的扩增产物；
(v)对所述扩增产物进行检测，获得ctDNA的检测结果。
在一优选实施方式中，步骤(v)中的检测包括：建库和测序、或直接进行测序，其中，通过所述测序，获得所述待测样本中的ctDNA检测结果，从而检测样本中的ctDNA。
在一优选实施方式中，所述方法还包括步骤：
(vi)基于步骤(v)的检测结果，从而判断目标基因拷贝数的变异情况、或基因 序列的突变情况、或其组合。
扩增和建库
在本发明中，所述扩增及建库方法不受特别限制，能达到本发明扩增效果的扩增和建库方法均包括在本发明中。
在另一优选实施方式中，使用链置换活性的DNA聚合酶对环化分子(环化的ctDNA)进行扩增。
在一优选实施方式中，本发明所用的扩增及建库方法为MALBAC-LAB全基因组扩增方法。
MALBAC-LAB扩增建库方法从原理上对比较大的DNA片段有更高的扩增效率。引物延伸后的产物会因片段两端存在互补序列而形成发夹结构。而在下一步的指数扩增中，游离引物序列与发夹序列相同。因此，在退火阶段，游离引物只有在发夹形成前与模板的3’端结合才能形成链延伸扩增(竞争赢过该模板片段的5’端)。模板片段越短，则其3’端与5’端距离越近，从而形成发夹的机会越高，游离引物竞争胜出的机会也就越小，成功扩增的几率也就越小。反之，模板片段越长，扩增成功的几率也就越高，整体扩增效率也就越高。
为了高效扩增小片段的cfDNA/ctDNA，先利用DNA连接酶将小片段DNA的两端自连，使线形DNA转化成环形DNA。这样，当扩增引物与环形DNA模板结合后，在具有链置换活性的DNA聚合酶的催化作用下，链延伸即可沿着环形模板循环进行，所获得的产物较长，在下一步的指数扩增阶段即可获得较高的扩增效率。
为了便于理解，本发明人提供以下原理。应理解，本发明的保护范围并不受所述原理的任何限制。
参见图1。在本发明中，当小片段ctDNA自身环化时可形成高效的扩增模板。而在一个连接体系中，除了片段的自环化以外还可能发生片段之间的连接。片段之间的连接也可以起到增加模板长度，改善扩增效率的效果。但是，为了提高对混杂于cfDNA中的少量ctDNA的检测效果，采用了特殊的措施(包括稀释和环化)，从而使建库扩增更倾向于ctDNA片段。在本发明中，一个突出特点是，在连接反应中使用很低浓度的cfDNA(例如，低于1ng/μl，较佳地，0.1ng/μl)。在低浓度DNA片段情况下，一方面各片段之间距离较远，在不规则热运动条件下相遇的几率显著降低，从而显著降低了两个核酸片段之间发生互相连接的几率。而另一方面，即使对于同一片段，在稀释过程中，3’与5’端之间的距离(即该片段的长度)，不因 片段的浓度而改变。因此，在低片段浓度的情况下，DNA片段自连环化的几率就会相对提高，以至于高于片段之间互相连接的几率。另外，对于样品中存在的待检测的ctDNA，这种短片段核酸表现出出乎意料的显著高于cfDNA的自环化效率(两者相差至少1-3个数量级或更大，即相差10-1000倍或更大)。一种对于ctDNA自环化率高的原因可能是因为其片段越短，其3’与5’端之间的距离越近，自连的几率也就越高。因此，在本发明中，经过特定的稀释和DNA片段自环化连接反应，使得ctDNA的自环化率显著高于非肿瘤细胞来源的cfDNA(提高了至少1-3个数量级或更大)，进而形成更多的环化分子，从而在后续的扩增中得到更高效的扩增。另一可能的解释是尽管这一自环化效率的差异在初期较小，但在后续PCR扩增中，这一差异会以指数增长形式在每一循环中积累，最终形成显著的差异。
对于扩增产物，可以直接进行检测(例如电泳、酶切或测序)，也可以先建库然后进行测序，例如用二代测序方法或其他测序方法进行检测。
目的基因拷贝数变化的检测
在本发明中，所述待检测的目的基因的拷贝数不限于HER2，包括(但并不限于)表1中的基因及其染色体。
表1
IGF1R/IGFIR/JTK13 STK11/LKB1/PJS IGF2/C11orf43/INSIGF
Chr17 GSTT1 RB1/OSRC/RB
17q22 AR/AIS/DHTR D13S319(FISH_probe)
20p13 NRAS/N-ras/NRAS1 NFKBIA/IKBA/MAD-3
chr3 1q21/PMVK/HUMPMKI TP53/p53/LFS1
17q25.3 UGT1A1/GNT1/HUG-BR1 CCNE1/CCNE
MDM4/MDMX VHL/HRCA1/RCA1 ZMYND8/PRKCBP1
chr7 MECOM/A1L4F3/A8KA00 CYP2D6/CPD6/CYP2D
MET/AUTS9/HGFR KIT/C-Kit/SCFR DDR2/NTRKR3/TYRO10
FGFR1/BFGFR/CEK PIK3R1/GRB1/p85-ALPHA MSH2/GTBP/HNPCC5
8q BRAF/BRAF1 RAC1/MIG5/TC-25
HRAS/HRAS1/K-ras 8p EGFR/ERBB/ERBB1
AKT1/AKT1_NEW/AKT DOK2/p56DOK/p56dok-2 NANS/SAS
MAP2K4/JNKK/MEK4 FGFR2/BEK/BFR-1 NOTCH1/TAN1/hN1
TOP2A/TOP2/TP2A KRAS/C-K-RAS/K-RAS2A D13S25(FISH_probe)
DCC/CRC18/CRCR1 MDM2/HDM2/HDMX MAP2K1/MAPKK1/MEK1
GSTM1/GST1/GSTM1-1 TSC2/LAM/TSC4 NCOA3/ACTR/AIB-1
MYCN/MODED/N-myc CDH1/ECAD/CDHE ZNF217/ZABC1
PDGFRA/PDGFR2 BRCA1/BRCAI/BRCC1 TERC
CDK6/MGC59692/PLSTIRE SIRPB1 ATM/AT1/ATA
CDKN2A/p16/ARF TMPRSS2/PRSS10 NKX2-1/TITF1/BCH
CCND1/BCL1/PRAD1 ARID1A/B120/BAF250 SMAD4/DPC4/JIP
CSP12/chr12 ALK/TFG GNA11/GNA-11
CDK4/CMM3 MSH2/COCA1/FCC1 TERT/EST2/TCS1
NF1/NFNS/P21359 3q29 BRCA2/BRCC2/FACD
GNAS/AHO/C20orf45 MYC/c-Myc NTRK3/TRKC/gp145(trkC)
AKT3/PKBG/RAC-gamma CDKN2B/MTS2/P15 PIK3CA/PI3K
FGFR3/ACH/CEK2 NTRK2/GP145-TrkB/TRKB POU5F1B
KDR/CD309/FLK1 ASS1/ASS/CTLN1 CYP17A1/CPT7/CYP17
GNAQ/G-ALPHA-q/GAQ RET/CDHF12/HSCR1 AKT2/PRKBB/RAC-BETA
TSC1/LAM/TSC PTEN/BZS/MHAM CYP19A1/ARO/ARO1
ERBB2/HER-2/neu    
本发明的主要优点包括：
(1)本发明可高效的从肿瘤患者血浆游离DNA中检测目的基因(如Her2基因)拷贝数的扩增。
(2)本发明的检测方法还可应用于检测其它体液样本，如尿液，脑脊液，唾液中肿瘤来源游离DNA的任意基因和DNA片段的拷贝数变异。
(3)本发明以血浆和其它体液为生物样本，高效检测其中肿瘤来源DNA(ctDNA)以观察目标基因，特别是Her2基因拷贝数变异的方法。
(4)本发明利用MALBAC-LAB扩增建库技术对，首次在低核酸浓度的条件下将游离DNA碎片自环化，其中ctDNA因片段较短而有更高的自环化率，随后获得较高效率的扩增(相较正常细胞来源的cfDNA而言)，从而获得较高的检测灵 敏度。
(5)本发明的检测方法检测多个目的基因的拷贝数，且均具有较高的检测灵敏度。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
本发明所用的材料和试剂如无特别说明，均为市售产品。
实施例1.
环化对于ctDNA检测的影响
在本实施例以及实施例2中，结合Her2基因检测，对本发明的技术方案进行具体说明。
对一例已知发生Her2基因拷贝数扩增的肿瘤患者的血浆游离DNA按表2的设置进行连接反应。
表2
Figure PCTCN2018072831-appb-000001
从表2可见，1为不对血浆游离DNA(包括大量的cfDNA和微量的ctDNA)做连接的反应，2为较低浓度血浆游离DNA的连接反应，3为较高浓度血浆游离DNA的连接反应。各反应在37℃孵育一小时后加热至75℃，孵育15分钟使连接酶失活。随后将反应后的DNA各取0.5ng作为模板，进行MALBAC-LAB扩增及建库。扩增操作的简要步骤为：将DNA模板(5μl)加入30μl线性扩增试剂(由序康医疗科技(苏州)有限公司提供)，试剂主要成份包括，引物混合物，特殊设计的具有热耐受和链置换性质的DNA聚合酶，dNTP，以及Mg 2+、(NH 4) 2+、K+,SO 4 2-、Cl -等。然后将反应置入热循环仪。热循环程序为：
Figure PCTCN2018072831-appb-000002
热循环结束后，向反应体系中加入30μl指数扩增试剂(获自序康医疗科技(苏州)有限公司)，其中主要包括：引物混合物，特殊设计的具有热耐受和链置换性质的DNA聚合酶以及指数扩增反应缓冲液等。然后将反应再次置入热循环仪。热循环程序为：
Figure PCTCN2018072831-appb-000003
扩增完成后，以常规凝胶电泳检测扩增产物可见，反应2、与3均可得到有效的扩增，而反应1没有发生显著的扩增(图2)。
上述结果表明，不经过环化处理，那么使用环化优先的PCR扩增(即优先以环化核酸分子为模板进行扩增，而不以或基本上不以线性核酸分子为模板进行扩增)时，例如使用链置换活性的DNA聚合酶进行扩增时，将无法有效得到扩增产物。
实施例2
稀释预处理对于ctDNA检测影响
实施例1中“反应2”和“反应3”的MALBAC-LAB扩增产物即是Illumina高通量测序平台的测序库，将其进行浅测序(约5％的基因组覆盖度)后以常规拷贝数变异(copy number variation)分析软件(获自序康医疗科技(苏州)有限公司)对17号染色体上Her2基因所在的约500K区段进行分析。同时，对同一位患者的血浆游离DNA按常规方法直接进行建库，并进行同样的测序、分析作为对照。
从数据结果可见，在“反应2”中可检测出显著的Her2基因拷贝数扩增(图 3A)，而在“反应3”(图3B)和对照检测(图3C)中都无法检测出拷贝数扩增。
上述表明，当不对待测样品进行稀释，使得经稀释后的样品中cfDNA的浓度没有大幅下降(如0.001-5ng/μl)，则虽然可以扩增，但是扩增产物主要是对应于cfDNA的扩增产物,而对应于ctDNA的扩增产物很少或几乎无法检出。
实施例3
稀释处理和环化处理提高了ctDNA的相对水平
在本实施例中，对另一例已知发生Her2基因拷贝数扩增的肿瘤(如乳腺癌)患者的血浆游离DNA按表3的设置进行连接反应。
表3
成份 反应1 反应2
DNA 1ng 1ng
10×连接酶缓冲液 1μl 1μl
CircLigase 1μl 0
T4连接酶 0 1μl
加水至 10μl 10μl
从表3可以看出，反应1是用CircLigase做连接反应，而反应2是用T4连接酶做连接反应，各反应在37℃孵育一小时后加热至75℃，孵育15分钟使连接酶失活。随后将反应后的DNA各取0.5ng作为模板，进行MALBAC-LAB扩增及建库。具体操作方法如同实施例1中所述。
扩增完成后，以常规凝胶电泳检测扩增产物可见，反应1可以得到有效的扩增，而反应2不能得到显著的扩增(图4)。
结果表明，本发明可在低核酸浓度(如0.1ng/μl)的条件下将ctDNA自环化，并可获得非常高效率的扩增，从而获得较高的检测灵敏度。
上述结果表明，在本发明方法中，在所述的经环化的混合物中，ctDNA环化产物占优势。
此外，在经稀释的样品和经环化的混合物中，满足式I
Ct1/Cf1＞Ct0/Cf0     (I)
式中，
Ct1为在所述经环化的混合物中，环化ctDNA分子的浓度；
Cf1为在所述经环化的混合物中，环化cfDNA分子的浓度；
Ct0为在所述经稀释的样品中，ctDNA分子的浓度；
Cf0为在所述经稀释的样品中，cfDNA分子的浓度。
数据表明，所述的Ct1/Cf1与Ct0/Cf0的比值R1至少≥10(如50-100或更大)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种样品处理方法，其特征在于，包括步骤：
    (i)提供一待测样品，所述样品含有cfDNA和ctDNA；
    (ii)稀释所述待测样品，获得经稀释的样品，其中所述经稀释后的样品中，cfDNA的浓度为0.001-5ng/μl，较佳地，0.01-2ng/μl，更佳的，0.05-1ng/μl；和
    (iii)用环化连接酶对步骤(ii)的经稀释的样品进行环化处理，获得经环化的混合物，其中，所述的经环化的混合物含有环化ctDNA分子。
  2. 如权利要求1所述的方法，其特征在于，所述方法还包括步骤：
    (iv)以所述经环化的混合物中的环化ctDNA分子为模板，进行扩增，从而得到对应于环化ctDNA的扩增产物。
  3. 如权利要求2所述的方法，其特征在于，所述方法还包括步骤：
    (v)对所述扩增产物进行建库和测序、或直接进行测序，其中，通过所述测序，获得所述待测样本中的ctDNA检测结果。
  4. 如权利要求3所述的方法，其特征在于，所述方法还包括步骤：
    (vi)基于步骤(v)的检测结果，从而判断目标基因拷贝数的变异情况、或基因序列的突变情况、或其组合。
  5. 如权利要求1所述的方法，其特征在于，在经稀释的样品和经环化的混合物中，满足式I
    Ct1/Cf1＞Ct0/Cf0    (I)
    式中，
    Ct1为在所述经环化的混合物中，环化ctDNA分子的浓度；
    Cf1为在所述经环化的混合物中，环化cfDNA分子的浓度；
    Ct0为在所述经稀释的样品中，ctDNA分子的浓度；
    Cf0为在所述经稀释的样品中，cfDNA分子的浓度。
  6. 如权利要求1所述的方法，其特征在于，在经稀释的样品和待检测样品中，满足式II
    Ct0/Cf0＝Ct/Cf    (II)
    式中，
    Ct0为在所述经稀释的样品中，ctDNA分子的浓度；
    Cf0为在所述经稀释的样品中，cfDNA分子的浓度；
    Ct为在所述待检测的样品中，ctDNA分子的浓度；
    Cf为在所述待检测的样品中，cfDNA分子的浓度。
  7. 如权利要求1所述的方法，其特征在于，所述的样品选自下组：血液、体液、或其组合。
  8. 一种非诊断性地检测样本中ctDNA的方法，其特征在于，包括步骤：
    (i)提供一待测样品，所述样品含有cfDNA和ctDNA；
    (ii)稀释所述待测样品，获得经稀释的样品，其中所述经稀释后的样品中，cfDNA的浓度为0.001-5ng/μl,较佳地，0.01-2ng/μl，更佳的，0.05-1ng/μl；
    (iii)用环化连接酶对步骤(ii)的经稀释的样品进行环化处理，获得经环化的混合物，其中，所述的经环化的混合物含有环化ctDNA分子；
    (iv)以所述经环化的混合物中的环化ctDNA分子为模板，进行扩增，从而得到对应于环化ctDNA的扩增产物；和
    (v)对所述扩增产物进行检测，获得ctDNA的检测结果。
  9. 如权利要求8所述的方法，其特征在于，步骤(v)中的检测包括：建库和测序、或直接进行测序，其中，通过所述测序，获得所述待测样本中的ctDNA检测结果，从而检测样本中的ctDNA。
  10. 如权利要求8所述的方法，其特征在于，所述方法还包括步骤：
    (vi)基于步骤(v)的检测结果，从而判断目标基因拷贝数的变异情况、或基因序列的突变情况、或其组合。
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