TWI683904B - Method for efficiently detecting ctDNA in samples - Google Patents

Method for efficiently detecting ctDNA in samples Download PDF

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TWI683904B
TWI683904B TW107101197A TW107101197A TWI683904B TW I683904 B TWI683904 B TW I683904B TW 107101197 A TW107101197 A TW 107101197A TW 107101197 A TW107101197 A TW 107101197A TW I683904 B TWI683904 B TW I683904B
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任軍
耿荷芳
陸思嘉
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大陸商上海億康醫學檢驗所有限公司
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Abstract

本發明提供了一種高效檢測樣本中的ctDNA的方法，具體地，本發明的方法可在極低的核酸濃度下，將游離DNA碎片自環化，其中ctDNA因片段較短而有更高的自環化率，隨後用特定環化優先擴增或擴增建庫方法可獲得較高效率的擴增，得到大量的對應於ctDNA的擴增產物，從而獲得非常高的檢測靈敏度和特異性。The present invention provides a method for efficiently detecting ctDNA in a sample. Specifically, the method of the present invention can self-circulate free DNA fragments at a very low nucleic acid concentration, where ctDNA has a higher self-efficiency due to shorter fragments. Circularization rate, followed by specific circularization priority amplification or amplification library construction method can obtain higher efficiency amplification, and obtain a large number of amplification products corresponding to ctDNA, thereby obtaining very high detection sensitivity and specificity.

Description

高效檢測樣本中的ｃｔＤＮＡ的方法Method for efficiently detecting ctDNA in samples

本發明涉及生物技術領域，具體地，涉及一種高效檢測樣本中的ctDNA的方法。The invention relates to the field of biotechnology, in particular, to a method for efficiently detecting ctDNA in a sample.

研究表明，腫瘤病人的血液中存在極其微量的來自於腫瘤細胞的ctDNA，迴圈腫瘤DNA片段，其主要是死亡的腫瘤細胞破裂後所釋放出來的。 　　以乳腺癌為例，人類表皮生長因數受體2(human epidermalgrowth factor receptor-2,HER2)基因,即c-erbB-2基因,定位於染色體17q12-21.32上,編碼相對分子品質為185000的跨膜受體樣蛋白,具有酪氨酸激酶活性。HER2基因的過表達或拷貝數擴增與乳腺癌和胃癌的發生密切相關。約有20%～25%的乳腺癌、15%的胃癌患者過表達HER2，該部分患者的預後較差。 　　Her2基因在部分乳腺癌和胃癌中發生拷貝數擴增僅僅是腫瘤細胞基因拷貝數變異的例子之一。實際上，先前研究中已經發現很多基因或染色體區段在腫瘤細胞中存在著拷貝數擴增或缺失的現象，因此，檢測腫瘤細胞中目標基因的拷貝數變異是癌生物學中的重要技術之一。 　　然而，現有Her2的檢測方法主要是用IHC和FISH方法對腫瘤活檢組織樣本進行檢測。另外，現在已有人嘗試對血漿游離DNA(cfDNA)中存在的游離腫瘤DNA(ctDNA)進行檢測，鑒定是否存在Her2基因的擴增，但效果尚不理想。 　　IHC方法與FISH方法都是對腫瘤活檢組織切片樣本進行檢測，其應用有很大局限性，主要表現在： 　　有些病人身體已經極為虛弱，無法承受活檢手術，即無法獲得病理切片樣本。 　　對腫瘤復發病人，診療原則不主張手術活檢取樣，代之以細針穿刺活檢。細針穿刺活檢僅能獲取很少量的腫瘤組織，經常不足以進行IHC和FISH檢測。對存在Her2基因擴增病人，在使用靶向藥物治療後可能會發生耐藥情況，故此需要在用藥期間持續、動態檢測Her2基因擴增情況，但在臨床現實中，對腫瘤組織反復活檢取樣幾乎是一件不可能的事情。因此，IHC與FISH方法對此無能為力。 　　理論上，用常規二代測序的方法可以對血漿游離DNA(cfDNA)中存在的游離腫瘤DNA(ctDNA)進行檢測，鑒定是否存在Her2基因的擴增，但實際應用中效果尚不理想的主要原因是：首先，cfDNA在血漿中含量非常少，從一毫升血漿中通常只能獲取幾納克DNA。並且，cfDNA的主要來源是白細胞死亡後釋放入血液的DNA，其中真正來源於腫瘤細胞的ctDNA少之又少，用常規基因拷貝數分析的方法難以鑒定是否存在Her2基因擴增。 　　因此，本領域迫切需要開發一種能夠高效富集、檢測樣本中的ctDNA，從而判斷樣本中是否存在目標基因(如HER2)擴增的方法。Studies have shown that there are extremely small amounts of ctDNA from tumor cells in the blood of tumor patients, and loop DNA fragments of tumors, which are mainly released after the rupture of dead tumor cells. Taking breast cancer as an example, the human epidermal growth factor receptor 2 (HER2) gene, or c-erbB-2 gene, is located on chromosome 17q12-21.32 and encodes a transmembrane with a relative molecular mass of 185000 Receptor-like protein with tyrosine kinase activity. Overexpression or copy number amplification of HER2 gene is closely related to the occurrence of breast cancer and gastric cancer. About 20% to 25% of breast cancer and 15% of gastric cancer patients overexpress HER2, and the prognosis of these patients is poor.　　 Her2 gene copy number amplification in some breast and gastric cancers is just one example of tumor cell gene copy number variation. In fact, previous studies have found that many genes or chromosome segments have copy number amplification or deletion in tumor cells. Therefore, detecting the copy number variation of target genes in tumor cells is one of the important techniques in cancer biology. One.　　However, the existing Her2 detection method mainly uses IHC and FISH methods to detect tumor biopsy tissue samples. In addition, there have been attempts 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 yet satisfactory.　　IHC method and FISH method are used to detect biopsy tissue biopsy samples, its application has great limitations, mainly manifested in: 　　 Some patients are already very weak, unable to withstand biopsy surgery, that is, can not obtain pathological biopsy samples.　　For patients with tumor recurrence, the principle of diagnosis and treatment does not advocate surgical biopsy sampling, and replace it with fine needle biopsy. Fine needle aspiration biopsy can only obtain a small amount of tumor tissue, often not enough for IHC and FISH detection. For patients with Her2 gene amplification, drug resistance may occur after treatment with targeted drugs, so it is necessary to continuously and dynamically detect Her2 gene amplification during medication, but in clinical reality, repeated biopsy sampling of tumor tissue It is almost impossible. Therefore, IHC and FISH methods can do nothing about it. In theory, the conventional second-generation sequencing method can be used to detect free tumor DNA (ctDNA) in plasma free DNA (cfDNA) and identify whether there is amplification of the Her2 gene, but the main reason for the actual application is not satisfactory. Yes: First, cfDNA is very low in plasma, and usually only a few nanograms of DNA can be obtained from a milliliter of plasma. In addition, the main source of cfDNA is the DNA released into the blood after the death of white blood cells. Among them, ctDNA that is really derived from tumor cells is very small. It is difficult to identify the presence of Her2 gene amplification by conventional gene copy number analysis.　　 Therefore, there is an urgent need in the art to develop a method that can efficiently enrich and detect ctDNA in samples to determine whether there is amplification of target genes (such as HER2) in the samples.

本發明的目的在於提供一種能夠高效富集、檢測樣本中的ctDNA，從而判斷樣本中是否存在目標基因(如HER2)擴增的方法。 　　本發明的第一方面提供了一種樣品處理方法，包括步驟： 　　(i) 提供一待測樣品，所述樣品含有cfDNA和ctDNA； 　　(ii)稀釋所述待測樣品，獲得經稀釋的樣品，其中所述經稀釋後的樣品中，cfDNA的濃度為0.001-5ng/μl，較佳地，0.01-2 ng/μl，更佳地，0.05-1 ng/μl；和 　　(iii)用環化連接酶對步驟(ii)的經稀釋的樣品進行環化處理，獲得經環化的混合物，其中，所述的經環化的混合物含有環化ctDNA分子。 　　在另一優選例中，所述方法還包括步驟： 　　(iv)以所述經環化的混合物中的環化ctDNA分子為範本，進行擴增，從而得到對應於環化ctDNA的擴增產物。 　　在另一優選例中，所述的擴增為環化優先的PCR擴增(即優先以環化核酸分子為範本進行擴增，而不以或基本上不以線性核酸分子為範本進行擴增)。 　　在另一優選例中，所述的擴增使用鏈置換活性的DNA聚合酶進行擴增。 　　在另一優選例中，所述的擴增包括MALBAC-LAB擴增。 　　在另一優選例中，所述方法還包括步驟： 　　(v)對所述擴增產物進行建庫和測序、或直接進行測序，其中，通過所述測序，獲得所述待測樣本中的ctDNA檢測結果。 　　在另一優選例中，所述方法還包括步驟： 　　(vi)基於步驟(v)的檢測結果，從而判斷目標基因拷貝數的變異情況、或基因序列的突變情況、或其組合。 　　在另一優選例中，在所述的經環化的混合物中，ctDNA環化產物佔優勢。 　　在另一優選例中，在經稀釋的樣品和經環化的混合物中，滿足式I

Figure 02_image001
式中， 　　Ct1為在所述經環化的混合物中，環化ctDNA分子的濃度； 　　Cf1為在所述經環化的混合物中，環化cfDNA分子的濃度； 　　Ct0為在所述經稀釋的樣品中，ctDNA分子的濃度； 　　Cf0為在所述經稀釋的樣品中，cfDNA分子的濃度。 　　在另一優選例中，在經稀釋的樣品和待檢測的樣品中，滿足式II
Figure 02_image003
式中， 　　Ct0為在所述經稀釋的樣品中，ctDNA分子的濃度； 　　Cf0為在所述經稀釋的樣品中，cfDNA分子的濃度； 　　Ct為在所述待檢測的樣品中，ctDNA分子的濃度； 　　Cf為在所述待檢測的樣品中，cfDNA分子的濃度。 　　在另一優選例中，所述的Ct1/Cf1與Ct0/Cf0的比值R1≥10，較佳地≥50，更佳地≥100。 　　在另一優選例中，所述的處理方法是非診斷性和非治療性的方法。 　　在另一優選例中，在所述的擴增產物中，對應於ctDNA環化產物的擴增產物佔優勢。 　　在另一優選例中，所述“對應於ctDNA環化產物的擴增產物佔優勢”指擴增產物中，對應於ctDNA環化產物的擴增產物的濃度(C1)顯著高於對應於cfDNA環化產物的擴增產物濃度(C2)。 　　在另一優選例中，所述“顯著高於”指C1/C2≥5，較佳地≥10，更佳地，≥20。 　　在另一優選例中，所述的樣品選自下組：血液、體液、或其組合。 　　在另一優選例中，所述樣品選自下組：血液、血漿、組織間隙液、淋巴液、尿液、腦脊液、唾液、房水、精液、胃腸道分泌液、或其組合。 　　在另一優選例中，所述樣品選自下組：血液、血漿、或血清。 　　在另一優選例中，所述的樣品為無細胞的樣品。 　　在另一優選例中，所述的樣品不含腫瘤細胞。 　　在另一優選例中，所述ctDNA源自腫瘤細胞。 　　在另一優選例中，所述腫瘤細胞選自下組：乳腺癌、卵巢癌、胃癌、肺癌、結直腸癌、膀胱癌、食管癌、胰腺癌、皮膚癌、前列腺癌、食管癌、膽囊癌、甲狀腺癌、肝癌、喉癌、口咽癌、白血病、或其組合。 　　在另一優選例中，所述環化連接酶選自下組：CircLigase、ThermoPhage ™ssDNA連接酶、或其組合。 　　在另一優選例中，在步驟(iv)中，所述擴增方法選自下組：聚合酶鏈式反應(PCR)、多重鏈置換反應(MDA)、滾環DNA擴增(RCA)、環介導基因恒溫擴增技術(LAMP)、或其組合。 　　在另一優選例中，在步驟(v)中，所述的建庫方法選自下組：MALBAC-LAB建庫、打斷建庫、和/或轉座建庫。 　　在另一優選例中，所述測序用選擇下組的方法進行：Illumina測序、Ion Torrent測序、Roche454測序、SoLID測序、Completed Genomics (CG)測序、NanoPore測序、Pacific Bio測序、或其組合。 　　在另一優選例中，所述目標基因選自下組：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、或其組合。 　　本發明第二方面提供了一種檢測樣本中ctDNA的方法，包括步驟： 　　(i) 提供一待測樣品，所述樣品含有cfDNA和ctDNA； 　　(ii)稀釋所述待測樣品，獲得經稀釋的樣品，其中所述經稀釋後的樣品中，cfDNA的濃度為0.001-5ng/ul,較佳地，0.01-2 ng/ul，更佳的，0.05-1 ng/ul； 　　(iii)用環化連接酶對步驟(ii)的經稀釋的樣品進行環化處理，獲得經環化的混合物，其中，所述的經環化的混合物含有環化ctDNA分子； 　　(iv)以所述經環化的混合物中的環化ctDNA分子為範本，進行擴增，從而得到對應於環化ctDNA的擴增產物；和 　　(v)對所述擴增產物進行檢測，獲得ctDNA的檢測結果。 　　在另一優選例中，步驟(v)中的檢測包括：建庫和測序、或直接進行測序，其中，通過所述測序，獲得所述待測樣本中的ctDNA檢測結果，從而檢測樣本中的ctDNA。 　　在另一優選例中，所述方法還包括步驟： 　　(vi)基於步驟(v)的檢測結果，從而判斷目標基因拷貝數的變異情況、或基因序列的突變情況、或其組合。 　　在另一優選例中，所述的擴增為環化優先的PCR擴增(即優先以環化核酸分子為範本進行擴增，而不以或基本上不以線性核酸分子為範本進行擴增)。 　　在另一優選例中，所述的擴增包括MALBAC-LAB擴增。 　　在另一優選例中，所述的檢測方法是非診斷性和非治療性的方法。 　　應理解，在本發明範圍內中，本發明的上述各技術特徵和在下文(如實施例)中具體描述的各技術特徵之間都可以互相組合，從而構成新的或優選的技術方案。限於篇幅，在此不再一一累述。An object of the present invention is to provide a method capable of efficiently enriching and detecting ctDNA in a sample, thereby determining whether there is amplification of a target gene (such as HER2) in the sample. The first aspect of the present invention provides a sample processing method including the steps of: (i) providing a sample to be tested, the sample containing cfDNA and ctDNA; (ii) diluting the sample to be tested to obtain a diluted sample, wherein In the diluted sample, the concentration of cfDNA is 0.001-5 ng/μl, preferably 0.01-2 ng/μl, more preferably 0.05-1 ng/μl; and (iii) using cyclase The diluted sample of step (ii) is subjected to cyclization to obtain a cyclized mixture, wherein the cyclized mixture contains cyclized ctDNA molecules. In another preferred example, the method further includes the steps of: (iv) using the circularized ctDNA molecule in the circularized mixture as a template, and performing amplification to obtain an amplification product corresponding to the circularized ctDNA. In another preferred example, the amplification is circularized PCR amplification (ie, circularized nucleic acid molecules are preferentially used for amplification, and linear nucleic acid molecules are not or substantially not used for amplification) ). In another preferred example, the amplification uses DNA polymerase with strand displacement activity for amplification. In another preferred example, the amplification includes MALBAC-LAB amplification. In another preferred example, the method further includes the steps of: (v) performing library building and sequencing on the amplified product, or directly sequencing, wherein, through the sequencing, the ctDNA in the sample to be tested is obtained Test results. In another preferred example, the method further includes the steps of: (vi) based on the detection result of step (v), to determine the variation of the target gene copy number, or the mutation of the gene sequence, or a combination thereof. In another preferred example, in the cyclized mixture, the ctDNA cyclization product predominates. In another preferred example, in the diluted sample and the cyclized mixture, the formula I is satisfied
Figure 02_image001
Where Ct1 is the concentration of cyclized ctDNA molecules in the cyclized mixture; Cf1 is the concentration of cyclized cfDNA molecules in the cyclized mixture; Ct0 is the diluted sample Where, the concentration of ctDNA molecules; Cf0 is the concentration of cfDNA molecules in the diluted sample. In another preferred example, in the diluted sample and the sample to be tested, Formula II is satisfied
Figure 02_image003
Where 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 tested. In another preferred example, the ratio R1 of Ct1/Cf1 to Ct0/Cf0 is ≥10, preferably ≥50, more preferably ≥100. In another preferred example, the treatment method is a non-diagnostic and non-therapeutic method. In another preferred example, among the amplification products, the amplification product corresponding to the ctDNA circularization product is dominant. In another preferred example, the "amplification product corresponding to the ctDNA circularization product predominates" means that the concentration (C1) of the amplification product corresponding to the ctDNA circularization product in the amplification product is significantly higher than that corresponding to the cfDNA The amplification product concentration of the circularized product (C2). In another preferred example, the "significantly higher" refers to C1/C2 ≥ 5, preferably ≥ 10, more preferably, ≥ 20. In another preferred example, the sample is selected from the group consisting of blood, body fluids, or a combination thereof. In another preferred example, the sample is selected from the group consisting of blood, plasma, interstitial fluid, lymph fluid, urine, cerebrospinal fluid, saliva, aqueous humor, semen, gastrointestinal secretion, or a combination thereof. In another preferred example, the sample is selected from the group consisting of blood, plasma, or serum. In another preferred example, the sample is a cell-free sample. In another preferred example, the sample does not contain tumor cells. In another preferred example, the ctDNA is derived from tumor cells. In another preferred example, 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. In another preferred example, the circularized ligase is selected from the group consisting of CircLigase, ThermoPhage™ssDNA ligase, or a combination thereof. In another preferred example, in step (iv), 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 constant temperature amplification technology (LAMP), or a combination thereof. In another preferred example, in step (v), the library building method is selected from the group consisting of MALBAC-LAB library building, interrupt library building, and/or transposition library building. In another preferred example, the sequencing is performed by selecting a group of methods: Illumina sequencing, Ion Torrent sequencing, Roche 454 sequencing, SoLID sequencing, Completed Genomics (CG) sequencing, NanoPore sequencing, Pacific Bio sequencing, or a combination thereof. In another preferred example, 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, 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 a combination thereof. The second aspect of the present invention provides a method for detecting ctDNA in a sample, including the steps of: (i) providing a sample to be tested, the sample containing cfDNA and ctDNA; (ii) diluting the sample to be tested to obtain a diluted sample , Wherein the concentration of cfDNA in the diluted sample is 0.001-5ng/ul, preferably 0.01-2 ng/ul, more preferably 0.05-1 ng/ul; (iii) cyclization Enzymes cyclize the diluted sample of step (ii) to obtain a cyclized mixture, wherein the cyclized mixture contains cyclized ctDNA molecules; (iv) with the cyclized mixture The circularized ctDNA molecule in is used as a template to perform amplification to obtain an amplified product corresponding to the circularized ctDNA; and (v) detecting the amplified product to obtain a detection result of ctDNA. In another preferred example, the detection in step (v) includes: library building and sequencing, or direct sequencing, wherein, through the sequencing, the ctDNA detection result in the sample to be tested is obtained, thereby detecting the ctDNA. In another preferred example, the method further includes the steps of: (vi) based on the detection result of step (v), to determine the variation of the target gene copy number, or the mutation of the gene sequence, or a combination thereof. In another preferred example, the amplification is circularized PCR amplification (ie, circularized nucleic acid molecules are preferentially used for amplification, and linear nucleic acid molecules are not or substantially not used for amplification) ). In another preferred example, the amplification includes MALBAC-LAB amplification. In another preferred example, the detection method is a non-diagnostic and non-therapeutic method. It should be understood that, within the scope of the present invention, the above technical features of the present invention and the technical features specifically described in the following (eg, embodiments) can be combined with each other, thereby forming a new or preferred technical solution. Due to space limitations, I will not repeat them here.

經過廣泛而深入的研究，本發明意外的發現，在極低的核酸濃度下，將游離DNA碎片進行自環化，其中ctDNA因片段較短而有異乎尋常的高自環化率，隨後用特定環化優先擴增或擴增建庫方法(如MALBAC-LAB技術)可獲得較高效率的擴增(相較正常細胞來源的cfDNA而言)，得到大量的對應於ctDNA的擴增產物，從而獲得非常高的檢測靈敏度和特異性。在此基礎上，本發明人完成了本發明。ctDNA ctDNA即迴圈腫瘤DNA片段，主要是死亡的腫瘤細胞破裂後所釋放出來的、片段化的基因組DNA。ctDNA的含量低，約占全部游離DNA的1%，甚至只有0.01%，通常，ctDNA比cfDNA片段大小要短20-50bp，約為130-145bp。 　　研究表明，腫瘤細胞來源ctDNA的片段長度小於非腫瘤細胞來源的cfDNA。cfDNA cfDNA是血漿中游離DNA的總稱，主要來源有兩種：細胞凋亡過程中產生的片段化核酸(160-180bp)和組織壞死或免疫殺傷過程中細胞釋放的核酸(接近基因組大小)。環化連接酶 在本發明中，所述“環化連接酶”指一種具有熱穩定性的連接酶，可催化線性單鏈DNA連接成環狀單鏈DNA。 　　在本發明中，所述環化連接酶的選擇沒有特別限制，在一優選實施方式中，所述環化連接酶選自下組：CircLigase、ThermoPhage ™ssDNA連接酶、或其組合。 　　其中，CircLigase為單鏈DNA環化連接酶，可在沒有互補序列存在的情況下，催化具有5’-磷酸和3’-羥基基團的單鏈DNA範本的分子內連接(即環化)。ThermoPhage ™ssDNA連接酶(ThermoPhage ™ single-stranded DNA ligase)為一種單鏈DNA連接酶，可在高溫下連接單鏈DNA或RNA。樣品處理方法 本發明提供了一種樣品處理方法。 　　在一優選實施方式中，本發明的樣品處理方法，包括步驟： 　　(i) 提供一待測樣品，所述樣品含有cfDNA和ctDNA； 　　(ii)稀釋所述待測樣品，獲得經稀釋的樣品，其中所述經稀釋後的樣品中，cfDNA的濃度為0.001-5ng/μl,較佳地，0.01-2 ng/μl，更佳地，0.05-1 ng/μl； 　　(iii)用環化連接酶對步驟(ii)的經稀釋的樣品進行環化處理，獲得經環化的混合物，其中，所述的經環化的混合物含有環化ctDNA分子。檢測樣本中 ctDNA 的方法 本發明提供了一種檢測樣本中ctDNA的方法。 　　在一優選實施方式中，本發明的檢測樣本中ctDNA的方法包括步驟： 　　(i) 提供一待測樣品，所述樣品含有cfDNA和ctDNA； 　　(ii)稀釋所述待測樣品，獲得經稀釋的樣品，其中所述經稀釋後的樣品中，cfDNA的濃度為0.001-5ng/μl,較佳地，0.01-2 ng/μl，更佳地，0.05-1 ng/μl； 　　(iii)用環化連接酶對步驟(ii)的經稀釋的樣品進行環化處理，獲得經環化的混合物，其中，所述的經環化的混合物含有環化ctDNA分子； 　　(iv)以所述經環化的混合物中的環化ctDNA分子為範本，進行擴增，從而得到對應於環化ctDNA的擴增產物； 　　(v)對所述擴增產物進行檢測，獲得ctDNA的檢測結果。 　　在一優選實施方式中，步驟(v)中的檢測包括：建庫和測序、或直接進行測序，其中，通過所述測序，獲得所述待測樣本中的ctDNA檢測結果，從而檢測樣本中的ctDNA。 　　在一優選實施方式中，所述方法還包括步驟： 　　(vi)基於步驟(v)的檢測結果，從而判斷目標基因拷貝數的變異情況、或基因序列的突變情況、或其組合。擴增和建庫 在本發明中，所述擴增及建庫方法不受特別限制，能達到本發明擴增效果的擴增和建庫方法均包括在本發明中。 　　在另一優選實施方式中，使用鏈置換活性的DNA聚合酶對環化分子(環化的ctDNA)進行擴增。 　　在一優選實施方式中，本發明所用的擴增及建庫方法為MALBAC-LAB全基因組擴增方法。 　　MALBAC-LAB擴增建庫方法從原理上對比較大的DNA片段有更高的擴增效率。引物延伸後的產物會因片段兩端存在互補序列而形成髮夾結構。而在下一步的指數擴增中，游離引物序列與髮夾序列相同。因此，在退火階段，游離引物只有在髮夾形成前與範本的3’端結合才能形成鏈延伸擴增(競爭贏過該範本片段的5’端)。範本片段越短，則其3’端與5’端距離越近，從而形成髮夾的機會越高，游離引物競爭勝出的機會也就越小，成功擴增的幾率也就越小。反之，範本片段越長，擴增成功的幾率也就越高，整體擴增效率也就越高。 　　為了高效擴增小片段的cfDNA/ctDNA，先利用DNA連接酶將小片段DNA的兩端自連，使線形DNA轉化成環形DNA。這樣，當擴增引物與環形DNA範本結合後，在具有鏈置換活性的DNA聚合酶的催化作用下，鏈延伸即可沿著環形範本迴圈進行，所獲得的產物較長，在下一步的指數擴增階段即可獲得較高的擴增效率。 　　為了便於理解，本發明人提供以下原理。應理解，本發明的保護範圍並不受所述原理的任何限制。 　　參見圖1。在本發明中，當小片段ctDNA自身環化時可形成高效的擴增範本。而在一個連接體系中，除了片段的自環化以外還可能發生片段之間的連接。片段之間的連接也可以起到增加範本長度，改善擴增效率的效果。但是，為了提高對混雜於cfDNA中的少量ctDNA的檢測效果，採用了特殊的措施(包括稀釋和環化)，從而使建庫擴增更傾向於ctDNA片段。在本發明中，一個突出特點是，在連接反應中使用很低濃度的cfDNA(例如，低於1ng/μl，較佳地，0.1ng/μl)。在低濃度DNA片段情況下，一方面各片段之間距離較遠，在不規則熱運動條件下相遇的幾率顯著降低，從而顯著降低了兩個核酸片段之間發生互相連接的幾率。而另一方面，即使對於同一片段，在稀釋過程中，3’與5’端之間的距離(即該片段的長度)，不因片段的濃度而改變。因此，在低片段濃度的情況下，DNA片段自連環化的幾率就會相對提高，以至於高於片段之間互相連接的幾率。另外，對於樣品中存在的待檢測的ctDNA，這種短片段核酸表現出出乎意料的顯著高於cfDNA的自環化效率(兩者相差至少1-3個數量級或更大，即相差10-1000倍或更大)。一種對於ctDNA自環化率高的原因可能是因為其片段越短，其3’與5’端之間的距離越近，自連的幾率也就越高。因此，在本發明中，經過特定的稀釋和DNA片段自環化連接反應，使得ctDNA的自環化率顯著高於非腫瘤細胞來源的cfDNA(提高了至少1-3個數量級或更大)，進而形成更多的環化分子，從而在後續的擴增中得到更高效的擴增。另一可能的解釋是儘管這一自環化效率的差異在初期較小，但在後續PCR擴增中，這一差異會以指數增長形式在每一迴圈中積累，最終形成顯著的差異。 　　對於擴增產物，可以直接進行檢測(例如電泳、酶切或測序)，也可以先建庫然後進行測序，例如用二代測序方法或其他測序方法進行檢測。目的基因拷貝數變化的檢測 在本發明中，所述待檢測的目的基因的拷貝數不限於HER2，包括(但並不限於)表1中的基因及其染色體。

Figure 02_image005
本發明的主要優點包括： (1)本發明可高效的從腫瘤患者血漿游離DNA中檢測目的基因(如Her2基因)拷貝數的擴增。 　　(2)本發明的檢測方法還可應用於檢測其它體液樣本，如尿液，腦脊液，唾液中腫瘤來源游離DNA的任意基因和DNA片段的拷貝數變異。 　　(3)本發明以血漿和其它體液為生物樣本，高效檢測其中腫瘤來源DNA(ctDNA)以觀察目標基因，特別是Her2基因拷貝數變異的方法。 　　(4)本發明利用MALBAC-LAB擴增建庫技術對，首次在低核酸濃度的條件下將游離DNA碎片自環化，其中ctDNA因片段較短而有更高的自環化率，隨後獲得較高效率的擴增(相較正常細胞來源的cfDNA而言)，從而獲得較高的檢測靈敏度。 　　(5)本發明的檢測方法檢測多個目的基因的拷貝數，且均具有較高的檢測靈敏度。 　　下面結合具體實施例，進一步闡述本發明。應理解，這些實施例僅用於說明本發明而不用於限制本發明的範圍。下列實施例中未注明具體條件的實驗方法，通常按照常規條件，或按照製造廠商所建議的條件。除非另外說明，否則百分比和份數是重量百分比和重量份數。 　　本發明所用的材料和試劑如無特別說明，均為市售產品。實施例 1. 環化對於 ctDNA 檢測的影響 在本實施例以及實施例2中，結合Her2基因檢測，對本發明的技術方案進行具體說明。 　　對一例已知發生Her2基因拷貝數擴增的腫瘤患者的血漿游離DNA按表2的設置進行連接反應。
Figure 02_image007
從表2可見，1為不對血漿游離DNA（包括大量的cfDNA和微量的ctDNA）做連接的反應，2為較低濃度血漿游離DNA的連接反應，3為較高濃度血漿游離DNA的連接反應。各反應在37℃孵育一小時後加熱至75℃，孵育15分鐘使連接酶失活。隨後將反應後的DNA各取0.5ng作為範本，進行MALBAC-LAB擴增及建庫。擴增操作的簡要步驟為：將DNA範本(5μl)加入30μl線性擴增試劑(由序康醫療科技(蘇州)有限公司提供)，試劑主要成份包括，引物混合物，特殊設計的具有熱耐受和鏈置換性質的DNA聚合酶，dNTP，以及Mg2+ 、(NH4 )2+ 、K+, SO4 2- 、Cl- 等。然後將反應置入熱迴圈儀。熱迴圈程式為：
Figure 02_image009
熱迴圈結束後，向反應體系中加入30μl指數擴增試劑(獲自序康醫療科技(蘇州)有限公司)，其中主要包括：引物混合物，特殊設計的具有熱耐受和鏈置換性質的DNA聚合酶以及指數擴增反應緩衝液等。然後將反應再次置入熱迴圈儀。熱迴圈程式為：
Figure 02_image011
擴增完成後，以常規凝膠電泳檢測擴增產物可見，反應2、與3均可得到有效的擴增，而反應1沒有發生顯著的擴增(圖2)。 　　上述結果表明，不經過環化處理，那麼使用環化優先的PCR擴增(即優先以環化核酸分子為範本進行擴增，而不以或基本上不以線性核酸分子為範本進行擴增)時，例如使用鏈置換活性的DNA聚合酶進行擴增時，將無法有效得到擴增產物。實施例 2 稀釋預處理對於 ctDNA 檢測影響 實施例1中“反應2”和“反應3”的MALBAC-LAB擴增產物即是Illumina高通量測序平臺的測序庫，將其進行淺測序(約5%的基因組覆蓋度)後以常規拷貝數變異(copy number variation)分析軟體(獲自序康醫療科技(蘇州)有限公司)對17號染色體上Her2基因所在的約500K區段進行分析。同時，對同一位患者的血漿游離DNA按常規方法直接進行建庫，並進行同樣的測序、分析作為對照。 　　從資料結果可見，在“反應2”中可檢測出顯著的Her2基因拷貝數擴增(圖3A)，而在“反應3”(圖3B)和對照檢測(圖3C)中都無法檢測出拷貝數擴增。 　　上述表明，當不對待測樣品進行稀釋，使得經稀釋後的樣品中cfDNA的濃度沒有大幅下降（如0.001-5ng/μl），則雖然可以擴增，但是擴增產物主要是對應於cfDNA的擴增產物,而對應於ctDNA的擴增產物很少或幾乎無法檢出。實施例 3 稀釋處理和環化處理提高了 ctDNA 的相對水準 在本實施例中，對另一例已知發生Her2基因拷貝數擴增的腫瘤（如乳腺癌）患者的血漿游離DNA按表3的設置進行連接反應。
Figure 02_image013
從表3可以看出，反應1是用CircLigase做連接反應，而反應2是用T4連接酶做連接反應，各反應在37℃孵育一小時後加熱至75℃，孵育15分鐘使連接酶失活。隨後將反應後的DNA各取0.5ng作為範本，進行MALBAC-LAB擴增及建庫。具體操作方法如同實施例1中所述。 　　擴增完成後，以常規凝膠電泳檢測擴增產物可見，反應1可以得到有效的擴增，而反應2不能得到顯著的擴增(圖4)。 　　結果表明，本發明可在低核酸濃度(如0.1ng/μl)的條件下將ctDNA自環化，並可獲得非常高效率的擴增，從而獲得較高的檢測靈敏度。 　　上述結果表明，在本發明方法中，在所述的經環化的混合物中，ctDNA環化產物佔優勢。 　　此外，在經稀釋的樣品和經環化的混合物中，滿足式I
Figure 02_image015
式中， 　　Ct1為在所述經環化的混合物中，環化ctDNA分子的濃度； 　　Cf1為在所述經環化的混合物中，環化cfDNA分子的濃度； 　　Ct0為在所述經稀釋的樣品中，ctDNA分子的濃度； 　　Cf0為在所述經稀釋的樣品中，cfDNA分子的濃度。 　　資料表明，所述的Ct1/Cf1與Ct0/Cf0的比值R1至少≥10（如50-100或更大）。 　　在本發明提及的所有文獻都在本申請中引用作為參考，就如同每一篇文獻被單獨引用作為參考那樣。此外應理解，在閱讀了本發明的上述講授內容之後，本領域技術人員可以對本發明作各種改動或修改，這些等價形式同樣落於本申請所附申請專利範圍所限定的範圍。After extensive and in-depth research, the present invention unexpectedly discovered that at very low nucleic acid concentrations, free DNA fragments are self-cyclized, in which ctDNA has an unusually high self-cyclization rate due to shorter fragments, and then a specific circularization is used Priority amplification or amplification library construction methods (such as MALBAC-LAB technology) can obtain higher efficiency amplification (compared to cfDNA derived from normal cells), and obtain a large number of amplification products corresponding to ctDNA, thereby obtaining very High detection sensitivity and specificity. On this basis, the inventor has completed the present invention. ctDNA ctDNA is the looped tumor DNA fragment, which is 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%. Generally, ctDNA is 20-50bp shorter than cfDNA fragment size, about 130-145bp. Studies have shown that tumor cell-derived ctDNA fragments are shorter than non-tumor cell-derived cfDNA. cfDNA cfDNA is the general term for free DNA in plasma. There are two main sources: fragmented nucleic acid (160-180bp) produced during apoptosis and nucleic acid released by cells during tissue necrosis or immune killing (close to the genome size). Circular ligase In the present invention, the "circular ligase" refers to a thermally stable ligase that catalyzes the ligation of linear single-stranded DNA into circular single-stranded DNA. In the present invention, the selection of the circular ligase is not particularly limited. In a preferred embodiment, the circular ligase is selected from the group consisting of CircLigase, ThermoPhage™ssDNA ligase, or a combination thereof. Among them, CircLigase is a single-stranded DNA circularization ligase, which can catalyze the intramolecular ligation (ie cyclization) of a single-stranded DNA template with 5'-phosphate and 3'-hydroxyl groups in the absence of complementary sequences. ThermoPhage ™ssDNA ligase ( ThermoPhage ™ single-stranded DNA ligase) is a single-stranded DNA ligase that can connect single-stranded DNA or RNA at high temperature. Sample processing method The present invention provides a sample processing method. In a preferred embodiment, the sample processing method of the present invention includes the steps of: (i) providing a sample to be tested, the sample containing cfDNA and ctDNA; (ii) diluting the sample to be tested to obtain a diluted sample, Wherein in the diluted sample, the concentration of cfDNA is 0.001-5ng/μl, preferably, 0.01-2 ng/μl, more preferably, 0.05-1 ng/μl; (iii) using cyclized ligase The diluted sample of step (ii) is subjected to cyclization to obtain a cyclized mixture, wherein the cyclized mixture contains cyclized ctDNA molecules. The method of detecting a sample ctDNA present invention provides a method of detecting in a sample of ctDNA. In a preferred embodiment, the method for detecting ctDNA in a sample of the present invention includes the steps of: (i) providing a sample to be tested, the sample containing cfDNA and ctDNA; (ii) diluting the sample to be tested to obtain a diluted Sample, wherein the concentration of cfDNA in the diluted sample is 0.001-5ng/μl, preferably 0.01-2 ng/μl, more preferably 0.05-1 ng/μl; (iii) cyclization Ligase cyclizes the diluted sample of step (ii) to obtain a cyclized mixture, wherein the cyclized mixture contains cyclized ctDNA molecules; (iv) with the cyclized The circularized ctDNA molecule in the mixture is used as a template to perform amplification to obtain an amplified product corresponding to the circularized ctDNA; (v) detecting the amplified product to obtain a detection result of ctDNA. In a preferred embodiment, the detection in step (v) includes: library building and sequencing, or direct sequencing, wherein, through the sequencing, the ctDNA detection result in the sample to be tested is obtained, thereby detecting the ctDNA. In a preferred embodiment, the method further includes the steps of: (vi) based on the detection result of step (v), to determine the variation of the target gene copy number, or the mutation of the gene sequence, or a combination thereof. Amplification and library construction In the present invention, the amplification and library construction methods are not particularly limited, and the amplification and library construction methods that can achieve the amplification effect of the invention are included in the invention. In another preferred embodiment, the circularized molecule (circularized ctDNA) is amplified using DNA polymerase with strand displacement activity. In a preferred embodiment, the amplification and library 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 than larger DNA fragments. The product after the extension of the primer will form a hairpin structure due to the existence of complementary sequences at both ends of the fragment. In the next exponential amplification, the free primer sequence is the same as the hairpin sequence. Therefore, in the annealing stage, the free primer can only form the chain extension amplification (the competition wins the 5'end of the template fragment) only when it is combined with the 3'end of the template before the hairpin is formed. The shorter the template fragment, the closer the 3'end to the 5'end, and thus the higher the chance of forming a hairpin, the smaller the chance of free primer competition and the smaller the chance of successful amplification. Conversely, the longer the template fragment, the higher the chance of successful amplification and the higher the overall amplification efficiency. In order to efficiently amplify small fragments of cfDNA/ctDNA, first use DNA ligase to connect the two ends of small fragments of DNA to convert linear DNA into circular DNA. In this way, after the amplification primer is combined with the circular DNA template, under the catalysis of DNA polymerase with strand displacement activity, the chain extension can be performed along the circular template loop, the product obtained is longer, the index in the next step Higher amplification efficiency can be obtained during the amplification stage. For ease of understanding, the present inventor provides the following principles. It should be understood that the scope of protection of the present invention is not limited by the principles described. See Figure 1. In the present invention, when a small fragment of ctDNA is circularized by itself, an efficient amplification template can be formed. In a connection system, in addition to the self-cyclization of fragments, the connection between fragments may also occur. The connection between fragments can also increase the length of the template and improve the amplification efficiency. However, in order to improve the detection effect of a small amount of ctDNA mixed in cfDNA, special measures (including dilution and circularization) are adopted, so that the library construction amplification is more inclined to ctDNA fragments. In the present invention, a prominent feature is that a very low concentration of cfDNA is used in the ligation reaction (for example, less than 1 ng/μl, preferably, 0.1 ng/μl). In the case of low-concentration DNA fragments, on the one hand, the distance between the fragments is far, and the chance of encountering under irregular thermal motion conditions is significantly reduced, thereby significantly reducing the chance of interconnection between two nucleic acid fragments. On the other hand, even for the same fragment, during the dilution process, the distance between the 3'and 5'ends (that is, the length of the fragment) does not change due to the concentration of the fragment. Therefore, in the case of low fragment concentration, the chances of DNA fragments self-linking will be relatively increased, so that it is higher than the chances of fragments connecting to each other. In addition, for the ctDNA present in the sample to be detected, this short fragment of nucleic acid exhibits an unexpectedly significantly higher self-cyclization efficiency than cfDNA (the difference between the two is at least 1-3 orders of magnitude or greater, that is, the difference is 10- 1000 times or more). One reason for the high self-cyclization rate of ctDNA may be because the shorter the fragment, the closer the distance between its 3'and 5'ends, the higher the probability of self-connection. Therefore, in the present invention, after specific dilution and DNA fragment self-cyclization ligation reaction, the self-cyclization rate of ctDNA is significantly higher than that of cfDNA derived from non-tumor cells (increased by at least 1-3 orders of magnitude or more), In turn, more circularized molecules are formed, and more efficient amplification is obtained in the subsequent amplification. Another possible explanation is that although this difference in self-cyclization efficiency is small in the early stages, in subsequent PCR amplification, this difference will accumulate in each cycle in the form of exponential growth, and eventually form a significant difference. The amplification products can be directly detected (for example, electrophoresis, digestion or sequencing), or the library can be constructed first and then sequenced, for example, by using next-generation sequencing methods or other sequencing methods. Detection of copy number change of target gene In the present invention, the copy number of the target gene to be detected is not limited to HER2, including (but not limited to) the genes in Table 1 and their chromosomes.
Figure 02_image005
The main advantages of the present invention include: (1) The present invention can efficiently detect the copy number amplification of target genes (such as Her2 gene) from plasma free DNA of tumor patients. (2) The detection method of the present invention can also be applied to detect the copy number variation of any gene and DNA fragments of tumor-derived free DNA in other body fluid samples, such as urine, cerebrospinal fluid, and saliva. (3) The present invention uses plasma and other body fluids as biological samples to efficiently detect tumor-derived DNA (ctDNA) to observe the target gene, especially Her2 gene copy number variation. (4) The present invention utilizes the MALBAC-LAB amplification library construction technology pair to self-circulate free DNA fragments under the condition of low nucleic acid concentration for the first time, in which ctDNA has a higher self-cyclization rate due to the shorter fragments, which is subsequently obtained Higher efficiency amplification (compared to cfDNA derived from normal cells), thus obtaining higher detection sensitivity. (5) The detection method of the present invention detects the copy number of multiple target genes, and all have high detection sensitivity. The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples generally follow the conventional conditions or the conditions recommended by the manufacturer. Unless stated otherwise, percentages and parts are percentages by weight and parts by weight. Unless otherwise specified, the materials and reagents used in the present invention are commercially available products. Example 1. Effect of cyclization on ctDNA detection In this example and Example 2, the technical solution of the present invention is specifically described in conjunction with Her2 gene detection. The plasma free DNA of a tumor patient with known copy number amplification of Her2 gene was ligated according to the settings in Table 2.
Figure 02_image007
It can be seen from Table 2 that 1 is a ligation reaction that does not ligate plasma free DNA (including a large amount of cfDNA and a small amount of ctDNA), 2 is a ligation reaction of lower concentration plasma free DNA, and 3 is a ligation reaction of higher concentration plasma free DNA. 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, 0.5ng of the DNA after the reaction was used as a template for MALBAC-LAB amplification and library construction. The brief steps of the amplification operation are: adding a DNA template (5μl) to 30μl linear amplification reagent (provided by Sukang Medical Technology (Suzhou) Co., Ltd.), the main components of the reagent include, primer mixture, specially designed with heat resistance and Strand-displacing DNA polymerase, dNTP, and Mg 2+ , (NH 4 ) 2+ , K+, SO 4 2- , Cl - etc. Then put the reaction into the thermocycler. The hot loop program is:
Figure 02_image009
After the end of the thermal cycle, add 30 μl of exponential amplification reagents (obtained from Sukang Medical Technology (Suzhou) Co., Ltd.) to the reaction system, which mainly include: primer mixtures, specially designed DNA polymerization with heat resistance and strand displacement properties Enzyme and exponential amplification reaction buffer, etc. Then put the reaction into the thermocycler again. The hot loop program is:
Figure 02_image011
After the completion of the amplification, the amplification products were detected by conventional gel electrophoresis. It can be seen that both reactions 2 and 3 can be effectively amplified, while reaction 1 has no significant amplification (Figure 2). The above results indicate that without circularization, circularization-preferred PCR amplification is used (ie, circularized nucleic acid molecules are preferentially used for amplification, and linear nucleic acid molecules are not or substantially not used for amplification) In this case, for example, when the DNA polymerase with strand displacement activity is used for amplification, the amplification product cannot be efficiently obtained. Example 2 The effect of dilution pretreatment on ctDNA detection The MALBAC-LAB amplification products of “Reaction 2” and “Reaction 3” in Example 1 are the sequencing libraries of the Illumina high-throughput sequencing platform, which are subjected to shallow sequencing (about 5 % Genome coverage) followed by conventional copy number variation analysis software (obtained from Xukang Medical Technology (Suzhou) Co., Ltd.) to analyze about 500K segments of the Her2 gene on chromosome 17. At the same time, the plasma free DNA of the same patient was directly used for library construction, and the same sequencing and analysis were performed as controls. It can be seen from the data results that significant copy number amplification of the Her2 gene can be detected in "Reaction 2" (Figure 3A), while copies cannot be detected in both "Reaction 3" (Figure 3B) and control detection (Figure 3C). Number amplification. The above indicates that when the sample to be tested is not diluted, so that the concentration of cfDNA in the diluted sample does not drop significantly (eg, 0.001-5ng/μl), although amplification is possible, the amplification product mainly corresponds to the expansion of cfDNA. Amplification products, and the amplification products corresponding to ctDNA have little or almost no detection. Example 3 The dilution and circularization treatments increase the relative level of ctDNA . In this example, the plasma free DNA of another patient with tumor (eg breast cancer) known to have Her2 gene copy number amplification is set according to Table 3. Perform the connection reaction.
Figure 02_image013
It can be seen from Table 3 that Reaction 1 is a ligation reaction using CircLigase, and Reaction 2 is a ligation reaction using T4 ligase, each reaction is incubated at 37°C for one hour and then heated to 75°C, and the ligase is inactivated by incubation for 15 minutes . Subsequently, 0.5ng of the DNA after the reaction was used as a template for MALBAC-LAB amplification and library construction. The specific operation method is as described in Example 1. After the amplification is completed, it can be seen by conventional gel electrophoresis that the amplification product is detected. Reaction 1 can be effectively amplified, while reaction 2 cannot be significantly amplified (Figure 4). The results show that the present invention can self-circulate ctDNA under the condition of low nucleic acid concentration (such as 0.1 ng/μl), and can obtain very high-efficiency amplification, thereby obtaining higher detection sensitivity. The above results indicate that in the method of the present invention, ctDNA cyclization products dominate in the cyclized mixture. In addition, in the diluted sample and the cyclized mixture, the formula I is satisfied
Figure 02_image015
Where Ct1 is the concentration of cyclized ctDNA molecules in the cyclized mixture; Cf1 is the concentration of cyclized cfDNA molecules in the cyclized mixture; Ct0 is the diluted sample Where, the concentration of ctDNA molecules; Cf0 is the concentration of cfDNA molecules in the diluted sample. The data indicates that the ratio R1 of Ct1/Cf1 to Ct0/Cf0 is at least ≥10 (such as 50-100 or greater). All documents mentioned in the present invention are cited as references in this application, just as each document is individually cited as a reference. In addition, it should be understood that, after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the scope of the patent application attached to this application.

圖1顯示了本發明的原理與技術流程。 　　圖2顯示了不同連接樣本的擴增建庫結果。 　　圖3顯示了不同技術方案的實驗結果。 　　圖4顯示了用不同的連接酶進行連接後的擴增建庫結果。Figure 1 shows the principle and technical process of the present invention.　　 Figure 2 shows the results of amplification of different connected samples.　　 Figure 3 shows the experimental results of different technical solutions.　　 Figure 4 shows the results of the amplified library after ligation with different ligases.

Claims (14)

一種樣品處理方法，其特徵在於，包括步驟：(i)提供一待測樣品，所述樣品含有cfDNA和ctDNA；(ii)稀釋所述待測樣品，獲得經稀釋的樣品，其中所述經稀釋後的樣品中，cfDNA的濃度為0.001-5ng/μl；和(iii)用環化連接酶對步驟(ii)的經稀釋的樣品進行環化處理，獲得經環化的混合物，其中，所述的經環化的混合物含有環化ctDNA分子，所述環化連接酶為單鏈DNA環化連接酶。 A sample processing method, comprising the steps of: (i) providing a sample to be tested, the sample containing cfDNA and ctDNA; (ii) diluting the sample to be tested to obtain a diluted sample, wherein the diluted In the sample afterwards, the concentration of cfDNA is 0.001-5ng/μl; and (iii) the cyclized ligase is used to cyclize the diluted sample of step (ii) to obtain a cyclized mixture, wherein, the The circularized mixture of contains circularized ctDNA molecules, which are single-stranded DNA circularized ligases. 如請求項1所述的方法，其中，所述經稀釋後的樣品中，cfDNA的濃度為0.01-2ng/μl。 The method according to claim 1, wherein the concentration of cfDNA in the diluted sample is 0.01-2ng/μl. 如請求項2所述的方法，其中，所述經稀釋後的樣品中，cfDNA的濃度為0.05-1ng/μl。 The method according to claim 2, wherein the concentration of cfDNA in the diluted sample is 0.05-1 ng/μl. 如請求項1所述的方法，其中，所述方法還包括步驟：(iv)以所述經環化的混合物中的環化ctDNA分子為範本，進行擴增，從而得到對應於環化ctDNA的擴增產物。 The method according to claim 1, wherein the method further comprises the step of: (iv) using the circularized ctDNA molecule in the circularized mixture as a template, and performing amplification to obtain a product corresponding to the circularized ctDNA Amplification products. 如請求項4所述的方法，其中，所述方法還包括步驟： (v)對所述擴增產物進行建庫和測序、或直接進行測序，其中，通過所述測序，獲得所述待測樣本中的ctDNA檢測結果。 The method according to claim 4, wherein the method further comprises the steps of: (v) performing library building and sequencing on the amplified product, or directly performing sequencing, wherein the ctDNA detection result in the sample to be tested is obtained through the sequencing. 如請求項5所述的方法，其中，所述方法還包括步驟：(vi)基於步驟(v)的檢測結果，從而判斷目標基因拷貝數的變異情況、或基因序列的突變情況、或其組合。 The method according to claim 5, wherein the method further comprises the step of: (vi) based on the detection result of step (v), thereby judging the variation of the target gene copy number, or the mutation of the gene sequence, or a combination thereof . 如請求項1所述的方法，其中，在經稀釋的樣品和經環化的混合物中，滿足式I Ct1/Cf1>Ct0/Cf0 (I)式中，Ct1為在所述經環化的混合物中，環化ctDNA分子的濃度；Cf1為在所述經環化的混合物中，環化cfDNA分子的濃度；Ct0為在所述經稀釋的樣品中，ctDNA分子的濃度；Cf0為在所述經稀釋的樣品中，cfDNA分子的濃度。 The method according to claim 1, wherein, in the diluted sample and the cyclized mixture, formula I is satisfied Ct1/Cf1>Ct0/Cf0 (I) where Ct1 is the cyclized mixture Where, the concentration of circularized ctDNA molecules; Cf1 is the concentration of circularized cfDNA molecules in the circularized mixture; Ct0 is the concentration of ctDNA molecules in the diluted sample; Cf0 is the concentration of The concentration of cfDNA molecules in the diluted sample. 如請求項1所述的方法，其中，在經稀釋的樣品和待檢測樣品中，滿足式II Ct0/Cf0=Ct/Cf (II) 式中，Ct0為在所述經稀釋的樣品中，ctDNA分子的濃度；Cf0為在所述經稀釋的樣品中，cfDNA分子的濃度；Ct為在所述待檢測的樣品中，ctDNA分子的濃度；Cf為在所述待檢測的樣品中，cfDNA分子的濃度。 The method according to claim 1, wherein, in the diluted sample and the sample to be tested, the formula II Ct0/Cf0=Ct/Cf (II) is satisfied Where 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 tested ; Cf is the concentration of cfDNA molecules in the sample to be detected. 如請求項1所述的方法，其中，所述的樣品選自下組：血液、體液、或其組合。 The method of claim 1, wherein the sample is selected from the group consisting of blood, body fluid, or a combination thereof. 一種非診斷性地檢測樣本中ctDNA的方法，其特徵在於，包括步驟：(i)提供一待測樣品，所述樣品含有cfDNA和ctDNA；(ii)稀釋所述待測樣品，獲得經稀釋的樣品，其中所述經稀釋後的樣品中，cfDNA的濃度為0.001-5ng/μl；(iii)用環化連接酶對步驟(ii)的經稀釋的樣品進行環化處理，獲得經環化的混合物，其中，所述的經環化的混合物含有環化ctDNA分子；(iv)以所述經環化的混合物中的環化ctDNA分子為範本，進行擴增，從而得到對應於環化ctDNA的擴增產物；和(v)對所述擴增產物進行檢測，獲得ctDNA的檢測結果，並且所述環化連接酶為單鏈DNA環化連接酶。 A method for non-diagnostic detection of ctDNA in a sample, comprising the steps of: (i) providing a sample to be tested, the sample containing cfDNA and ctDNA; (ii) diluting the sample to be tested to obtain a diluted A sample, wherein the concentration of cfDNA in the diluted sample is 0.001-5ng/μl; (iii) the cyclized ligase is used to cyclize the diluted sample of step (ii) to obtain a cyclized A mixture, wherein the circularized mixture contains circularized ctDNA molecules; (iv) the circularized ctDNA molecules in the circularized mixture are used as templates to perform amplification to obtain the corresponding circularized ctDNA Amplification product; and (v) detecting the amplification product to obtain a detection result of ctDNA, and the circularized ligase is a single-stranded DNA circularized ligase. 如請求項10所述的方法，其中，所述經稀釋後的樣品中，cfDNA的濃度為0.01-2ng/μl。 The method according to claim 10, wherein the concentration of cfDNA in the diluted sample is 0.01-2ng/μl. 如請求項11所述的方法，其中，所述經稀釋後的樣品中，cfDNA的濃度為0.05-1ng/μl。 The method according to claim 11, wherein the concentration of cfDNA in the diluted sample is 0.05-1 ng/μl. 如請求項10所述的方法，其中，步驟(v)中的檢測包括：建庫和測序、或直接進行測序，其中，通過所述測序，獲得所述待測樣本中的ctDNA檢測結果，從而檢測樣本中的ctDNA。 The method according to claim 10, wherein the detection in step (v) includes: library construction and sequencing, or direct sequencing, wherein, through the sequencing, the ctDNA detection result in the sample to be tested is obtained, thereby Detect ctDNA in the sample. 如請求項10所述的方法，其中，所述方法還包括步驟：(vi)基於步驟(v)的檢測結果，從而判斷目標基因拷貝數的變異情況、或基因序列的突變情況、或其組合。 The method according to claim 10, wherein the method further comprises the step of: (vi) based on the detection result of step (v), thereby judging the variation of the target gene copy number, or the mutation of the gene sequence, or a combination thereof .
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