EB virus DNA (Deoxyribose Nucleic Acid) and tumor high-frequency mutant gene capturing and sequencing method and application

Abstract

The invention provides an EB (Epstein-Barr) virus DNA (Deoxyribose Nucleic Acid) and tumor high-frequency mutant gene capturing and sequencing method and application, the capturing and sequencing method comprises the following steps: (1) acquiring a plasma sample, and extracting plasma cfDNA; (2) constructing a plasma cfDNA DNA library; (3) constructing a plasma cfDNA DNA capture library; and (4) sequencing. According to the invention, a DNA capture probe mixture aiming at seven EB virus subtypes and a proper number of specific human tumor related genes is creatively used for capturing and sequencing the sample to be detected, and the method has higher sensitivity and specificity for detection of EBV DNA in the sample. Moreover, based on the fragment length ratio of EBV DNA to 100 human tumor related genes cfDNA and the content of EBV DNA, the sensitivity, specificity and accuracy of nasopharyngeal carcinoma diagnosis can be further improved. In addition, the invention not only can be used for screening EBV-related tumors, but also can be used for screening generic cancer.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular biology and tumor detection, and particularly relates to a method for capturing and sequencing Epstein-Barr virus DNA and tumor high-frequency mutant genes and its application. Background Art

[0002] With the progress of comprehensive radiotherapy and chemotherapy, the prognosis of early nasopharyngeal carcinoma has been greatly improved, and the 5-year survival rate can reach over 95%. However, due to the hidden location of the disease, the lack of obvious early clinical symptoms, and the failure to participate in early nasopharyngeal carcinoma screening regularly, the early diagnosis rate of nasopharyngeal carcinoma is only less than 30%. Among the newly diagnosed nasopharyngeal carcinoma cases each year, more than 70% of nasopharyngeal carcinomas are in the advanced stage, and the 5-year survival rate is only about 60%. Moreover, the treatment difficulty increases, the treatment side effects are relatively large, and the overall curative effect is poor. Therefore, early detection and early treatment are the keys to improving the prognosis of nasopharyngeal carcinoma patients. By screening high-risk populations of nasopharyngeal carcinoma using sensitive and accurate methods, the screening efficiency of nasopharyngeal carcinoma can be greatly improved, and the early diagnosis rate, cure rate, and quality of life of patients can be enhanced.

[0003] Epstein-Barr virus (EBV) infection is significantly correlated with the occurrence of nasopharyngeal carcinoma. In China, evidence of EBV infection exists in the tumor tissues of approximately 95% of nasopharyngeal carcinoma patients. Therefore, the levels of EBV-related antibodies and EBV DNA increase to varying degrees in nasopharyngeal carcinoma patients. EBV-related markers are currently the most maturely developed and widely clinically applied diagnostic markers for nasopharyngeal carcinoma.

[0004] Currently, the early screening programs for nasopharyngeal carcinoma mainly include the EBV antibody program in blood and the EBV DNA quantitative PCR program. However, the screening sensitivity, specificity, and positive predictive value of these programs all have varying degrees of deficiencies. Moreover, neither the EBV antibody alone nor the EBV DNA quantitative index can accurately evaluate whether a patient has nasopharyngeal carcinoma. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a method for capturing and sequencing Epstein-Barr virus DNA and tumor high-frequency mutant genes and its application. The capture sequencing method captures and sequences the cfDNA of 7 Epstein-Barr virus subtypes and 100 human tumor-related genes to obtain fragment length ratio and Epstein-Barr virus content data, thereby more accurately evaluating whether a patient has nasopharyngeal carcinoma.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions.

[0007] The first aspect of the present invention provides a method for capturing and sequencing Epstein-Barr virus DNA and tumor-related high-frequency mutant genes, comprising the following steps:

[0008] (1) Obtain a plasma sample and extract plasma cfDNA;

[0009] (2) Construction of a plasma cfDNA DNA library: Perform end repair and A-tailing on cfDNA, ligate adapters, perform PCR amplification, and purify;

[0010] (3) Construction of a plasma cfDNA DNA capture library: Hybridize the cfDNA DNA library obtained by purification in step (2) with a DNA capture probe mixture, elute, perform PCR amplification, and purify;

[0011] The DNA capture probe mixture includes DNA capture probes for 7 Epstein-Barr virus subtypes and 100 human tumor-related genes;

[0012] The 7 Epstein-Barr virus subtypes are NC_007605.1, NC_009334.1, AP015015.1, KF717093.1, KF373730.1, KC207814.1, and HQ020558.1;

[0013] The 100 human tumor-related genes are AKT1, ALK, APC, ARID1A, ATM, AXIN1, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDKN2A, CDKN2B, CHEK2, CYLD, EGFR, EPCAM, ERBB2, ERBB3, EZH2, FANCC, FGFR2, GALNT12, GREM1, HNF1A, HOXB13, HRAS, JAK2, KIT, KRAS, MEN1, MET, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, NF2, NOTCH1, NRAS, NTHL1, PALB2, PDGFRA, PIK3CA, PMS2, POLD1, POLE, PRKAR1A, PTCH1, PTEN, PTPN11, RAD51C, RAD51D, RB1, RET, SDHA, SDHB, SDHC, SDHD, SMAD4, SMARC B1, STK11, TP53, TSC2, VHL, TRAF3, NFKBIA, IDH1, BRAF, GNAQ, GNA11, FLT3, CTNNB1, DNMT3A, FBXW7, ERBB4, FGFR3, IDH2, TSC1, MSH3, PRSS1, WT1, PTCH2, DICER1, XPD, CTNNA1, GNAS, TERT, FH, FLCN, SPINK1, FGFR1, NOTCH2, NOTCH3, and TET2 genes;

[0014] (4) Sequencing.

[0015] In some embodiments, the DNA capture probes for each Epstein - Barr virus subtype include multiple capture probes covering the entire viral region, and the length of each capture probe is 100 - 130 bp.

[0016] In some embodiments, the DNA capture probes for each human tumor - related gene include multiple capture probes covering the genomic sequence of the protein - coding region of the gene, and the length of each capture probe is 100 - 130 bp.

[0017] In some embodiments, in the DNA capture probe mixture, the molar ratio of the DNA capture probes for 7 Epstein - Barr virus subtypes to the DNA capture probes for 100 human tumor - related genes is 100:1.

[0018] The second aspect of the present invention provides the application of the capture sequencing method as described above in the preparation of products for nasopharyngeal carcinoma screening or evaluation of the therapeutic effect of nasopharyngeal carcinoma.

[0019] The third aspect of the present invention provides a nasopharyngeal carcinoma screening system, comprising:

[0020] (1) Detection module: The detection module uses the capture sequencing method as described above to perform capture sequencing on the cfDNA of 7 Epstein - Barr virus subtypes and 100 human tumor - related genes in the test sample;

[0021] (2) Data analysis module: The data analysis module analyzes and processes the sequencing results obtained by the detection module to obtain the following data: ① The fragment length ratio of Epstein - Barr virus DNA to the cfDNA of 100 human tumor - related genes; ② The Epstein - Barr virus DNA content;

[0022] (3) Result judgment module: The result judgment module compares the data obtained in step (2) with a preset threshold to determine whether the test sample is a nasopharyngeal carcinoma sample.

[0023] In some embodiments, the method for the data analysis module to analyze and process the sequencing results obtained by the detection module is as follows: Align the sequencing data to the human reference genome and the EBV genome respectively, use BWA for alignment, allowing a maximum of two nucleotide mismatches in each read alignment; then use samtools to obtain the fragments aligned to 100 human tumor - related genes and the EBV genome; calculate the Epstein - Barr virus DNA content and the fragment length ratio of Epstein - Barr virus DNA to the cfDNA of 100 human tumor - related genes;

[0024] The calculation method of the EBV DNA content is as follows: (the number of fragments mapped to the EBV genome) / (the number of fragments mapped to the EBV genome + the number of fragments mapped to 100 human tumor-related genes);

[0025] The calculation method of the fragment length ratio of EBV DNA to cfDNA of 100 human tumor-related genes is as follows: (the content of fragments with lengths of 80 - 110 bp among the fragments mapped to the EBV genome) / (the content of fragments with lengths of 80 - 110 bp among the fragments mapped to 100 human tumor-related genes).

[0026] In some embodiments, the threshold is: the fragment length ratio of EBV DNA to cfDNA of 100 tumor-related human genes is 8.964; the EBV DNA content is 5.25×10 -6 .

[0027] In some embodiments, the data analysis module also obtains the following data: ③ the mutation status of 100 tumor-related human genes.

[0028] In some embodiments, the result judgment module further includes judging whether the subject has other related tumors according to the mutation status of tumor-related human genes.

[0029] Compared with the prior art, the present invention has the following beneficial effects.

[0030] The present invention creatively uses a DNA capture probe mixture targeting 7 EBV subtypes and a suitable number (100) of specific human tumor-related genes to perform capture sequencing on a test sample, which has higher sensitivity and specificity for the detection of EBV DNA in the sample. Moreover, based on the fragment length ratio of EBV DNA to cfDNA of 100 human tumor-related genes and the EBV DNA content, the sensitivity, specificity, and accuracy of nasopharyngeal carcinoma diagnosis can be further improved.

[0031] In addition, the capture sequencing method of the present invention adds capture probes for 100 specific tumor high-frequency mutation genes. For samples with a low EBV DNA content, it can also provide a pan-cancer screening result for the subject based on the mutation status of the corresponding tumor-related genes. Therefore, the present invention can not only be used for screening EBV-related tumors, but also for pan-cancer screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the detection results of the fragment length ratio of EBV DNA and cfDNA of 100 human tumor-related genes and the EBV DNA content in the plasma samples of 80 healthy controls and 80 nasopharyngeal carcinoma patients in Example 3.

[0033] Figure 2 AUC result graph for Example 3.

[0034] Figure 3 Sensitivity comparison graph between EBV DNA capture sequencing method and traditional EBV DNA qPCR quantification in Example 4.

[0035] Figure 4 Graph showing the detection results of plasma EBV DNA concentration during the treatment and follow-up of 7 nasopharyngeal carcinoma patients in Example 5.

[0036] Figure 5 Diagnostic result graph for EBV-positive tumors in Example 6.

[0037] Figure 6 Detection results of tumor-related gene mutations in plasma samples of 8 breast cancer and 8 lung cancer patients in Example 7. Detailed implementation manners

[0038] For the experimental methods without specific conditions indicated in the following examples of the present invention, they are generally in accordance with conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions recommended by the manufacturer. All common chemical reagents used in the examples are commercially available products.

[0039] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0040] The terms "comprising" and "having" and any variations thereof in the present invention are intended to cover non-exclusive inclusion. For example, a process, method, device, product or equipment that includes a series of steps is not limited to the listed steps or modules, but optionally further includes steps not listed, or optionally further includes other steps inherent to these processes, methods, products or equipment.

[0041] In the present invention, "a plurality of" refers to two or more.

[0042] The following is described in conjunction with specific embodiments.

[0043] Example 1

[0044] This example provides an EBV DNA capture sequencing method.

[0045] Capture probe design

[0046] ①For multiple strains of EBV (including: NC_007605.1, NC_009334.1, AP015015.1, KF717093.1, KF373730.1, KC207814.1, HQ020558.1, a total of 7 subtypes), DNA capture probes covering the entire virus were designed.

[0047] ②Genomic sequence capture probes for the protein-coding regions (CDS) were designed for 100 human tumor-related genes of the host. The 100 human tumor-related genes are: AKT1, ALK, APC, ARID1A, ATM, AXIN1, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDKN2A, CDKN2B, CHEK2, CYLD, EGFR, EPCAM, ERBB2, ERBB3, EZH2, FANCC, FGFR2, GALNT12, GREM1, HNF1A, HOXB13, HRAS, JAK2, KIT, KRAS, MEN1, MET, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, NF2, NOTCH1, NRAS, NTHL1, PALB2, PDGFRA, PIK3CA, PMS2, POLD1, POLE, PRKAR1A, PTCH1, PTEN, PTPN11, RAD51C, RAD51D, RB1, RET, SDHA, SDHB, SDHC, SDHD, SMAD4, SMARCB1, STK11, TP53, TSC2, VHL, TRAF3, NFKBIA, IDH1, BRAF, GNAQ, GNA11, FLT3, CTNNB1, DNMT3A, FBXW7, ERBB4, FGFR3, IDH2, TSC1, MSH3, PRSS1, WT1, PTCH2, DICER1, XPD, CTNNA1, GNAS, TERT, FH, FLCN, SPINK1, FGFR1, NOTCH2, NOTCH3, TET2.

[0048] ③Probe synthesis rules

[0049] Probe design method: Use the online probe design platform XCapert of Nuoanda (http: / / nadprobe.njnad.com / ) to re-design the probes using different strategies. The specific description is as follows:

[0050] EBV: Design probes according to the reference sequences of 7 EBV subtypes and remove redundancy;

[0051] 100gene-CDS: Design 1X tiling probes for the CDS regions of 100 genes in the reference genome hg38.

[0052] The summary of the probes is shown in Table 1 below.

[0053] Table 1

[0054]

[0055]

[0056] During the design process of this design scheme, according to the target sequence characteristics of EBV and 100 genes, the optimal probe design scheme is adopted: this scheme effectively avoids the occurrence of probe complementarity, sequence homology, dimers, and repetitive region sequences, and realizes the optimal library capture efficiency.

[0057] Entrust Nuoanda (Nanjing) Biotechnology Co., Ltd. to carry out probe design and synthesis.

[0058] ④ Mix the synthesized EBV capture probes and 100 human gene capture probes according to a molar ratio of 100:1 to finally obtain a capture probe mixture for subsequent capture sequencing; in the probe mixture, the concentration of each capture probe for 100 human genes is 4 amol / probe / μl; the concentration of each capture probe for EBV virus is 400 amol / probe / μl.

[0059] The EBV DNA capture sequencing method includes the following steps:

[0060] 1. Plasma separation

[0061] ① Centrifuge the whole blood sample in a 10 mL EDTA anticoagulant tube directly at 4°C, 1600g (2000 rpm) for 10 min;

[0062] ② Transfer the supernatant to a 1.5 ml enzyme-free EP tube;

[0063] ③ Centrifuge the above plasma at 4°C, 16000g for 10 min to remove residual cells, and aliquot the supernatant into 1.5 mL enzyme-free centrifuge tubes at a specification of 500 μL / tube, and store at -80°C.

[0064] 2. Plasma cfDNA extraction

[0065] Use the DNA extraction kit (D3396) of Omega company to extract DNA, specifically as follows:

[0066] ① Add 500 ul of BL buffer to 500 ul of plasma and mix well;

[0067] ② Add 50 ul of Proteinase K buffer and mix well;

[0068] ③ Incubate at 70 °C for 10 minutes;

[0069] ④ Add 500 ul of ethanol solution and mix well;

[0070] ⑤ Transfer the above mixture to the HiBind TM DNA Column that has been inserted into a 2 ml collection tube and centrifuge at 13,000 rpm for 1 minute;

[0071] ⑥ Insert the HiBind TM DNA separation column into a new 2 ml collection tube;

[0072] ⑦ Add 500 ul of HBC buffer to the HiBind TM DNA separation column, centrifuge at 13,000 rpm for 1 minute, and discard the liquid in the collection tube;

[0073] ⑧ Add 700 ul of DNA wash buffer to the HiBind TM DNA separation column, centrifuge at 13,000 rpm for 1 minute, and discard the liquid in the collection tube; Repeat once more;

[0074] ⑨ Centrifuge the empty HiBind TM DNA separation column at 13,000 rpm for 2 minutes;

[0075] ⑩ Place the HiBind TM DNA separation column into a new 1.5 ml EP tube, and add 50 ul of enzyme-free water (preheated at 70 °C) dropwise to the center of each HiBind TM DNA separation column. After incubating at room temperature for 3 minutes, centrifuge at 13,000 rpm for 2 minutes. The extracted DNA is aliquoted and stored at -80 °C.

[0076] 3. Construction of plasma cfDNA DNA library

[0077] Use the DNA library construction kit (1002422) from Nuoanda (Nanjing) Biotechnology Co., Ltd. as follows:

[0078] Step 1: Measurement of cfDNA concentration

[0079] Take 1 ul of cfDNA and measure the cfDNA concentration using a qubit instrument.

[0080] Step 2: End repair & A-tailing

[0081] (1) Take out the End Repair&A-Tailing Buffer Pro and thaw it at room temperature. Mix it well and place it on ice for later use.

[0082] (2) Take out the End Repair&A-Tailing Enzyme Pro and let it thaw naturally on ice. Mix it well and centrifuge it briefly for later use.

[0083] (3) According to Table 2 below, prepare the reaction system in a 0.2 mL PCR tube placed on ice:

[0084] Table 2

[0085] Fragmented DNA / CfDNA 40 μL End Repair&A-Tailing Buffer Pro 6 μL End Repair&A-Tailing Enzyme Pro 4 uL Total 50 μL

[0086] (4) Mix well and centrifuge briefly to make all the reaction solution settle at the bottom of the PCR tube.

[0087] (5) According to Table 3 below, start the corresponding reaction program on the PCR instrument according to the sample type. When the temperature stabilizes at 25°C / 20°C, put the reaction tube into the PCR instrument:

[0088] Table 3

[0089]

[0090] Step 3: Adapter Ligation

[0091] (1) Take out the Ligation Buffer and thaw it at room temperature. Mix it well and place it on ice for later use.

[0092] (2) Take out the DNA Ligase and let it thaw naturally on ice. Mix it well and centrifuge it briefly for later use.

[0093] (3) Take out the PCR reaction tube from Step 2 on the PCR instrument and place it on ice. Prepare the reaction system according to Table 4 below:

[0094] Table 4

[0095]

[0096]

[0097] The reagent dosages are shown in Table 5:

[0098] Table 5

[0099] cfDNA Input Volume Adapter Concentration Dilution Factor (160 bp*) Adapter-to-Fragment Molar Ratio 5 ng 15 μM 5 120：1

[0100] (4) Mix well and centrifuge briefly to make all the reaction solution settle at the bottom of the PCR tube.

[0101] (5) Start the reaction program as shown in Table 6 below on a PCR instrument. When the temperature stabilizes at 20°C, place the reaction tube into the PCR instrument:

[0102] Table 6

[0103] 20℃ 15 min 4℃ Hold

[0104] Step Four: Purification of Ligation Products

[0105] (1) Take out the SP Beads in advance, vortex and mix well. After equilibrating at room temperature for 30 min, use them.

[0106] (2) Add 40 μL of SP Beads to the ligation reaction product obtained in Step Three, mix well, and incubate at 25°C for 5 - 10 min.

[0107] (3) Centrifuge the PCR tube briefly and then place it on a magnetic stand for 5 min until the liquid becomes completely clear. Use a pipette to aspirate and discard the supernatant.

[0108] (4) Slowly add 150 μL of 80% ethanol along the side wall of the PCR tube, taking care not to disturb the magnetic beads. Let it stand for 30 sec, and then use a pipette to aspirate and discard the supernatant.

[0109] (5) Repeat Step (4) once.

[0110] (6) Centrifuge the PCR tube briefly and then place it on a magnetic stand. Use a 10 - μL tip to remove a small amount of residual ethanol, taking care not to aspirate the magnetic beads.

[0111] (7) Open the lid of the PCR tube and let it stand at room temperature for about 5 min until the ethanol has completely evaporated.

[0112] (8) Remove the PCR tube, add 21 μL of Nuclease Free Water to the PCR tube, suspend the magnetic beads evenly, and incubate at 25°C for 2 min.

[0113] (9) Centrifuge the PCR tube briefly and then place it on a magnetic stand for 2 min until the liquid becomes completely clear. Carefully use a pipette to aspirate 20 μL of the supernatant and transfer it to a new 0.2 - mL PCR tube, taking care not to aspirate the magnetic beads. Place it on ice for standby.

[0114] Step Five: PCR Amplification

[0115] (1) Take out the 2X HiFi PCR Master Pro Mix and Universal UDI - Index Primer Mix, place them on ice to thaw naturally, mix well, and centrifuge briefly for standby.

[0116] (2) Prepare the reaction system in a 0.2 mL PCR tube placed on ice according to Table 7 below (add from top to bottom):

[0117] Table 7

[0118]

[0119] (3) Place the PCR tube in the PCR instrument and start the program in Table 8 below:

[0120] Table 8

[0121]

[0122]

[0123] Step Six: Purification of the Amplified Library

[0124] (1) Add the corresponding volume of SP Beads to the amplified reaction product in Step Five, mix well, and incubate at 25 °C for 5 - 10 min.

[0125] Table 9

[0126]

[0127] (2) Centrifuge the PCR tube transiently and place it on the magnetic stand for 5 min until the liquid is completely clear. Use a pipette to aspirate and discard the supernatant.

[0128] (3) Slowly add 150 μL of 80% ethanol along the side wall of the PCR tube, taking care not to disturb the magnetic beads. Let it stand for 30 sec, and then use a pipette to aspirate and discard the supernatant.

[0129] (4) Repeat step (3) once.

[0130] (5) After transiently centrifuging the PCR tube, place it on the magnetic stand. Use a 10 μL pipette tip to remove a small amount of residual ethanol, taking care not to aspirate the magnetic beads.

[0131] (6) Open the lid of the PCR tube and let it stand at room temperature for about 5 min until the ethanol has completely evaporated.

[0132] (7) Remove the PCR tube, add 20 μL of TE Solution to the PCR tube, and use a pipette to suspend the magnetic beads evenly. Incubate at 25 °C for 2 min.

[0133] (8) After transiently centrifuging the PCR tube, place it on the magnetic stand for 2 min until the liquid is completely clear. Carefully transfer the supernatant to a new 0.2 mL PCR tube for storage using a pipette, taking care not to aspirate the magnetic beads.

[0134] Step Seven: Library Quantification and Quality Inspection

[0135] (1) Use methods such as a fluorescence quantitative instrument (such as QubitTM 3.0 Fluorometer) or quantitative PCR to perform quantitative quality inspection on the library.

[0136] (2) Use (Agilent), Qsep100 (Bioptic) and other fragment analysis instruments to perform quality inspection on the library fragment distribution. The library fragment distribution is an important indicator for library quality assessment; the measurement criteria include relatively concentrated fragments, no free adapters, no adapter dimers, and the fragment size within the range of the library fragments recommended by the sequencing instrument, etc. etc.

[0137] 4. Construction of plasma cfDNA DNA capture library

[0138] Step 1: Library hybridization

[0139] (1) Take out the hybridization reagents ES Hyb#1 and Hyb#2 in ES Hybrid Capture Reagents in advance and thaw them at room temperature.

[0140] (2) If using the vacuum concentration library method, turn on the power of the vacuum concentrator in advance to preheat and adjust the temperature to 60 °C.

[0141] Table 10

[0142]

[0143] (3) Mix each component in a 0.2 / 1.5 mL low-adsorption centrifuge tube according to Table 10 above, vortex and mix well, and centrifuge briefly.

[0144] (4) Place the centrifuge tube in a vacuum concentrator preheated to 60 °C to dry.

[0145] (5) After all the liquid has evaporated and is completely dry, seal the centrifuge tube and set it aside for later use.

[0146] (6) Take out the NEXome Core Panel and let it thaw naturally on ice. After the first use, aliquot in small amounts as needed.

[0147] (7) Prepare the hybridization reaction solution according to Table 11 below. After mixing evenly with a pipette, add it to the bottom of the centrifuge tube that has been vacuum concentrated and dried. Gently pipette and mix 15 - 20 times, centrifuge briefly, and incubate at 25 °C for 5 - 10 min.

[0148] Table 11

[0149] ES Hyb#1 8.5 μL Hyb#2 2.7 μL Nuclease Free Water 1.8 μL Capture Probe Mixture 4 μL <![CDATA[Sealing Oil * > <![CDATA[10μL * > Total <![CDATA[17 / 27 * μL]]>

[0150] (8) Vortex the hybridization reaction mixture, and after instantaneous centrifugation, transfer all the hybridization reaction mixture in the centrifuge tube to a new 0.2 mL PCR tube, perform instantaneous centrifugation, place it in a PCR instrument, and start the hybridization program as shown in Table 12 below:

[0151] Table 12

[0152]

[0153] Step 2: Library elution

[0154] Preparation work

[0155] (1) Take out Other reagents in the ES Hybrid Capture Reagents are allowed to thaw naturally at room temperature and vortexed to mix evenly.

[0156] (2) Take out the Streptavidin Beads from 4°C, vortex to mix evenly, and allow them to equilibrate at room temperature for 30 min before proceeding with the washing and capture steps of the streptavidin magnetic beads.

[0157] Reagent preparation

[0158] (1) Prepare the elution Buffer according to Table 13 below:

[0159] Table 13

[0160]

[0161] (2) Prepare the magnetic bead suspension according to Table 14 below:

[0162] Table 14

[0163] ES Hyb#1 8.5 μL Hyb#2 2.7 μL Nuclease Free Water 5.8 μL Total 17 μL

[0164] Washing of streptavidin magnetic beads

[0165] (1) Vortex the Streptavidin Beads for 15 sec to ensure complete mixing. Pipette 50 μL of the magnetic beads into a 0.2 / 1.5 mL low-binding centrifuge tube.

[0166] (2) Add 100 μL of room temperature ES Wash Buffer to the centrifuge tube, gently pipette up and down 10 times to mix evenly, perform instantaneous centrifugation, place it on a magnetic stand for 1 - 2 min, wait until the liquid is completely clear, and use a pipette to discard the supernatant. Remove the centrifuge tube from the magnetic stand.

[0167] (3) Repeat step (2) once.

[0168] (4) Add 17 μL of magnetic bead suspension to a centrifuge tube, gently pipette up and down to mix well, and transfer all the magnetic bead suspension to a new 0.2 mL low-binding PCR tube.

[0169] (5) Place the 0.2 mL PCR tube containing the suspended capture magnetic beads in a PCR instrument and incubate at 65 °C for 5 min.

[0170] Streptavidin magnetic bead capture

[0171] (1) After a 0.5 - 4 hr hybridization reaction, proceed to the elution procedure.

[0172] (2) Add the resuspended streptavidin magnetic beads to the hybridization system, and gently pipette up and down or vortex to mix well.

[0173] (3) Incubate at 65 °C for 45 min, gently pipette 8 - 10 times every 10 - 12 min to ensure that the magnetic beads are completely resuspended.

[0174] Thermal elution

[0175] (1) After incubation, open the lid of the PCR tube on the PCR instrument, and add 150 μL of 65 °C ES Wash Buffer to it. Gently pipette 15 - 20 times to mix the hybridization system containing the magnetic beads.

[0176] (2) Place the PCR tube on a magnetic rack for 30 sec. After the liquid is completely clear, use a pipette to aspirate and discard the supernatant.

[0177] (3) Remove the PCR tube from the magnetic rack and put it into the PCR instrument. Add 150 μL of 65 °C ES Wash Buffer again, gently pipette 15 - 20 times to mix evenly, and incubate at 65 °C for 5 min.

[0178] Room temperature elution

[0179] (1) Place the PCR tube on a magnetic rack for 30 sec. After the liquid is completely clear, aspirate and discard the supernatant, centrifuge briefly, then change to a 10 μL pipette tip to remove a small amount of residual Buffer. Add 150 μL of room temperature ES Wash Buffer, gently pipette 10 - 15 times to mix well (do not vortex). Transfer all the reaction solution with magnetic beads to a new 0.2 mL low-binding PCR tube, incubate at room temperature for 2 min, vortex for 30 sec and then let it stand for 30 sec alternately during this period to ensure thorough mixing.

[0180] (2) Centrifuge the PCR tube instantaneously and place it on the magnetic stand for 30 seconds. After the liquid is completely clear, aspirate and discard the supernatant. Centrifuge instantaneously, then change to a 10 μL pipette tip to remove a small amount of residual Buffer. Add 150 μL of room temperature ES Wash Buffer again, gently pipette up and down 10 - 15 times to mix evenly, incubate at room temperature for 2 minutes, during which vortex for 30 seconds and then let it stand for 30 seconds, alternating to ensure thorough mixing.

[0181] (3) Centrifuge the PCR tube instantaneously and place it on the magnetic stand for 30 seconds. After the liquid is completely clear, aspirate and discard the supernatant. Centrifuge instantaneously, then change to a 10 μL pipette tip to remove a small amount of residual Buffer.

[0182] (4) Remove the PCR tube from the magnetic stand, add 22.5 μL of Nuclease Free Water, and carry the magnetic beads into Step Three.

[0183] Step Three: PCR Amplification

[0184] (1) Take out 2X HiFi PCR Master Mix and Amplification Primer Mix II and let them thaw naturally on ice. Gently mix them evenly using a pipette or vortex mixer, and centrifuge instantaneously for standby.

[0185] (2) Prepare the reaction system in a PCR tube placed on ice according to the system shown in Table 15 below:

[0186] Table 15

[0187]

[0188] (3) Place the PCR tube into the PCR instrument and start the program shown in Table 16 below, set the hot lid temperature to 105 °C:

[0189] Table 16

[0190]

[0191]

[0192] Table 17

[0193]

[0194] The capture panel designed in the present invention is 50 Kb - 1 Mb.

[0195] Step Four: Library Purification and Quantification

[0196] (1) After the PCR amplification is completed, take out the PCR tube and place it on the magnetic rack for 2 min. After the liquid is completely clarified, transfer the supernatant to a new 0.2 mL PCR tube.

[0197] (2) Add 75 μL of SP Beads into the PCR tube, mix well using a pipette or vortex mixer, and incubate at 25 °C for 5 - 10 min.

[0198] (3) Centrifuge the PCR tube briefly and then place it on the magnetic rack for 5 min. After the liquid is completely clarified, use a pipette to aspirate and discard the supernatant.

[0199] (4) Slowly add 150 μL of 80% ethanol along the side wall of the PCR tube, taking care not to disturb the magnetic beads. Let it stand for 30 sec, then use a pipette to aspirate and discard the supernatant.

[0200] (5) Repeat step (4) once.

[0201] (6) Centrifuge the PCR tube briefly and then place it on the magnetic rack. Use a 10 μL tip to remove a small amount of residual ethanol, taking care not to aspirate the magnetic beads.

[0202] (7) Open the lid of the PCR tube and let it stand at room temperature for about 2 - 5 min until the ethanol has completely evaporated.

[0203] (8) Remove the PCR tube from the magnetic rack, add 20 μL of TE Solution, and suspend the magnetic beads evenly using a pipette or vortex mixer. Incubate at 25 °C for 2 - 5 min.

[0204] (9) Centrifuge the PCR tube briefly and then place it on the magnetic rack for 1 - 2 min. After the liquid is completely clarified, carefully transfer the supernatant to a new 0.2 mL PCR tube or 1.5 mL centrifuge tube for storage using a pipette, taking care not to aspirate the magnetic beads.

[0205] (10) Quantify the library using a fluorescence dye - based method (Qubit); analyze the library fragment distribution (Agilent 2100 Bioanalyzer or similar products).

[0206] 5. Sequencing of the plasma cfDNA DNA capture library

[0207] Sequence the constructed library on the nova X plus sequencer for high - throughput sequencing. The sequencing mode is PE150 and the data volume is 1 G.

[0208] 6. Analysis of the sequencing results of the plasma cfDNA DNA capture library

[0209] Through bioinformatics analysis, the following were obtained: ① the fragment length ratio of EBV DNA to cfDNA of 100 human tumor-related genes, ② the EBV DNA content, and ③ the mutation status of 100 human tumor-related genes. The specific analysis methods are as follows:

[0210] First, the sequencing data was aligned to the human reference genome (UCSC hg38) and the EBV genome (NC_007605.1) respectively using BWA, allowing a maximum of two nucleotide mismatches in each read alignment. Further, samtools was used to obtain the fragments aligned to 100 human tumor-related genes and the EBV genome.

[0211] Then, the EBV DNA content was calculated: EBV DNA content = (number of fragments aligned to the EBV genome) / (number of fragments aligned to the EBV genome + number of fragments aligned to 100 human tumor-related genes).

[0212] The fragment length ratio of EBV DNA to cfDNA of 100 human tumor-related genes was calculated: ratio = (content of fragments with lengths of 80 - 110 bp in the fragments aligned to the EBV genome) / (content of fragments with lengths of 80 - 110 bp in the fragments aligned to 100 human tumor-related genes).

[0213] Finally, VarScan2 was used to analyze the mutation status in the sequencing data, and ANNOVAR was used to annotate the mutation status on 100 human tumor-related genes.

[0214] Example 2

[0215] This example provides a nasopharyngeal carcinoma screening system, including:

[0216] (1) Detection module: The detection module uses the capture sequencing method described in Example 1 to perform capture sequencing on 7 EBV subtypes and cfDNA of 100 human tumor-related genes in the sample to be tested;

[0217] (2) Data analysis module: The data analysis module analyzes and processes the sequencing results obtained by the detection module to obtain the following data: ① the fragment length ratio of EBV DNA to cfDNA of 100 human tumor-related genes; ② the EBV DNA content;

[0218] (3) Result judgment module: The result judgment module compares the data obtained in step (2) with a preset threshold to determine whether the sample to be tested is a nasopharyngeal carcinoma sample; and determines whether other related tumors are present based on the mutation status of tumor-related human genes.

[0219] Example 3 Nasopharyngeal Carcinoma Screening

[0220] In this example, the method described in Example 1 was used to perform EBV DNA capture sequencing and bioinformatics analysis on plasma samples of 80 healthy controls and 80 nasopharyngeal carcinoma patients. The results of the fragment length ratio of EBV DNA to cfDNA of 100 human tumor-related genes and the content of EBV DNA are shown in Figure 1 and Table 18.

[0221] Table 18

[0222]

[0223]

[0224]

[0225] To effectively distinguish nasopharyngeal carcinoma patients from healthy controls, the fragment length ratio of EBV DNA to 100 human gene cfDNA was set to 8.964, and the content of EBV DNA was set to 5.25218×10 -6 . When the fragment length ratio of the test sample is lower than 8.964 and the content of EBV DNA is higher than 5.25218×10 -6 , it is determined as a nasopharyngeal carcinoma sample. On this basis, when the fragment length ratio and the content of EBV DNA obtained by using the method of the present invention are used for nasopharyngeal carcinoma screening at the same time, on this basis, when the fragment length ratio and the content of EBV DNA obtained by using the method of the present invention are used for nasopharyngeal carcinoma screening at the same time, the AUC can reach 0.99, the sensitivity is 100%, and the specificity is 91.2% (85.1%-97.4%); when using only the fragment length ratio for nasopharyngeal carcinoma screening, the AUC is 0.95, the sensitivity is 100%, and the specificity is 77.5% (95% CI: 68.3%-86.7%); when using only the content of EBV DNA for nasopharyngeal carcinoma screening, the AUC is 0.99, the sensitivity is 100%, and the specificity is 82.5% (95% CI: 74.2%-90.8%) ( Figure 2 ). The results show that using the fragment length ratio and the content of EBV DNA obtained by using the method of the present invention for nasopharyngeal carcinoma screening at the same time can effectively improve the sensitivity and specificity of nasopharyngeal carcinoma detection, thereby improving the accuracy of detection.

[0226] Example 4

[0227] This example compares the sensitivity of the EBV DNA capture sequencing method of the present invention and the traditional EBV DNA qPCR quantitative detection for the diagnosis of nasopharyngeal carcinoma.

[0228] Plasma samples of 24 nasopharyngeal carcinoma patients were simultaneously subjected to EBV DNA capture sequencing (the same method as in Example 1) and traditional EBV DNA qPCR quantitative detection. The traditional EBV DNA qPCR quantitative detection was performed using the in vitro diagnostic reagent of Shengxiang Biology - EBV nucleic acid quantitative detection kit (PCR - fluorescence probe method) (product number: SX - EB001), and the operation was carried out strictly according to the instructions.

[0229] The results showed that the sensitivity of traditional EBV DNA qPCR quantitative detection for diagnosing nasopharyngeal carcinoma was 62.5% (15 / 24), while according to the same analysis criteria, the sensitivity of EBV DNA capture sequencing of the present invention for diagnosing nasopharyngeal carcinoma was 100% ( Figure 3 ). Therefore, the sensitivity of EBV DNA capture sequencing for diagnosing nasopharyngeal carcinoma is significantly better than that of traditional EBV DNA qPCR quantitative detection.

[0230] Example 5 Treatment monitoring of nasopharyngeal carcinoma patients

[0231] Dynamic plasma samples were collected from 7 nasopharyngeal carcinoma patients during treatment, and EBV DNA concentration was detected using the method described in Example 1.

[0232] The results showed that only 2 patients who finally achieved complete imaging remission had EBV DNA concentration in plasma below 3% ( Figure 4 ), indicating that the capture sequencing method of the present invention can also be used to monitor the treatment effect of nasopharyngeal carcinoma patients.

[0233] Example 6 Diagnosis of EBV - positive tumors

[0234] The capture sequencing method described in Example 1 of the present invention was used to diagnose tumors in plasma samples of 80 healthy controls and 20 plasma samples each of 6 clinically diagnosed EBV - positive tumors (including gastric cancer, lymphoepithelioma - like carcinoma, Hodgkin lymphoma, Burkitt lymphoma, diffuse large B - cell lymphoma, NK / T lymphoma). cfDNA capture sequencing and analysis were performed using the method of the present invention: when the fragment length ratio of the test sample was lower than 8.964 and the EBV DNA content was higher than 5.25218×10 -6 , it was determined as a tumor patient sample.

[0235] The results showed that the diagnostic sensitivity was 100% ( Figure 5 ).

[0236] Example 7 Diagnosis of multiple tumors

[0237] Tumor diagnosis was performed on plasma samples of 80 healthy controls, 8 breast cancer patients, and 8 lung cancer patients using the capture sequencing method described in Example 1 of the present invention. cfDNA capture sequencing and analysis were performed using the method of the present invention. When tumor-related gene mutations occurred, the sample was determined to be a tumor patient sample.

[0238] Through EBV DNA analysis, it was found that the EBV DNA content in the plasma samples of these 16 tumor patients was comparable to that of the healthy controls; further tumor-related gene mutation analysis was carried out, and the results showed that: no gene mutations were found in the plasma samples of 80 healthy controls; BRCA1 / 2 mutations were present in 5 breast cancer patients, and TP53 mutations were present in 2 breast cancer patients; EGFR mutations were present in the plasma samples of 4 lung cancer patients, and TP53 mutations were present in the plasma samples of 3 lung cancer patients ( Figure 6 ). It shows that the capture sequencing method of the present invention can be used not only for nasopharyngeal carcinoma screening but also for the diagnosis of tumors such as lung cancer and breast cancer.

[0239] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0240] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A capture and sequencing method for Epstein-Barr virus DNA and tumor-related high-frequency mutation genes, characterized in that: The following steps are involved: (1) Obtain plasma samples and extract plasma cfDNA; (2) Construction of plasma cfDNA library: end-repair and A-tailing of cfDNA, adapter ligation, PCR amplification, and purification; (3) Construction of plasma cfDNA capture library: hybridizing the cfDNA library purified in step (2) with the DNA capture probe mixture, eluting, PCR amplification, and purification; The DNA capture probe mixture includes DNA capture probes targeting 7 Epstein-Barr virus subtypes and 100 human tumor-related genes; The seven EBV subtypes are NC_007605.1, NC_009334.1, AP015015.1, KF717093.1, KF373730.1, KC207814.1, and HQ020558.1; The 100 human tumor-related genes are AKT1, ALK, APC, ARID1A, ATM, AXIN1, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDKN2A, CDKN2B, CHEK2, CYLD, EGFR, EPCAM, ERBB2, ERBB3, EZH2, FANCC, FGFR2, GALNT12, GREM1, HNF1A, HOXB13, HRAS, JAK2, KIT, KRAS, MEN1, MET, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, NF2, NOTCH1, NRAS, NTHL', PALB2, PDGFRA, PIK3CA, PM S2, POLD1, POLE, PRKAR1A, PTCHl, PTEN, PTPN11, RAD51C, RAD51D, RB1, RET, SDHA, SDHB, SDHC, SDHD, SM[AD4, SM[ARCB1, STK11, TP53, TSC2, VHL, TRAF3, NFKBIA, IDH1, BRAF, GNAQ , GNA11, FLT3, CTNNB1, DNMT3A, FBXW7, ERBB4, FGFR3, IDH2, TSC1, MSH3, PRSS1, WT1, PTCH2, DICER1, XPD, CTNNA1, GNAS, TERT, FH, FLCN, SPINK1, FGFR1, NOTCH2, NOTCH3 and TET2 genes; (4) Sequencing.

2. The capture sequencing method according to claim 1, characterized in that: The DNA capture probe for each EB virus subtype includes a plurality of capture probes covering the entire virus region, and the length of each capture probe is 110 to 130 bp.

3. The capture sequencing method according to claim 1, characterized in that: The DNA capture probe for each human tumor-related gene includes a plurality of capture probes covering the genomic sequence of the protein coding region of the gene, and the length of each capture probe is 110-130 bp.

4. The capture sequencing method according to claim 1, characterized in that: In the DNA capture probe mixture, the molar ratio of the DNA capture probes targeting 7 EB virus subtypes to the DNA capture probes targeting 100 human tumor-related genes is 100:

1.

5. Use of the capture sequencing method according to any one of claims 1 to 4 in preparing a product for nasopharyngeal carcinoma screening or nasopharyngeal carcinoma treatment effect evaluation.

6. A nasopharyngeal carcinoma screening system, characterized in that: include: (1) Detection module: The detection module uses the capture sequencing method according to any one of claims 1 to 4 to capture and sequence cfDNA of seven EB virus subtypes and 100 human tumor-related genes in the sample to be tested; (2) Data analysis module: The data analysis module analyzes and processes the sequencing results obtained by the detection module to obtain the following data: ① the fragment length ratio of EB virus DNA to cfDNA of 100 human tumor-related genes; ② the EB virus DNA content; (3) Result judgment module: The result judgment module compares the data obtained in step (2) with a preset threshold value to determine whether the sample to be tested is a nasopharyngeal carcinoma sample.

7. The nasopharyngeal carcinoma screening system according to claim 6, characterized in that: The method for the data analysis module to analyze and process the sequencing results obtained by the detection module is as follows: the sequencing data are aligned to the human reference genome and the EBV genome respectively, and the alignment is performed using BWA, allowing a maximum of two nucleotide mismatches in each read segment alignment; then samtools is used to obtain fragments aligned to 100 human tumor-related genes and the EBV genome; the EB virus DNA content and the fragment length ratio of the EB virus DNA to the cfDNA of the 100 human tumor-related genes are calculated; The calculation method of the EBV DNA content is as follows: (the number of fragments aligned to the EBV genome) / (the number of fragments aligned to the EBV genome + the number of fragments aligned to 100 human tumor-related genes); The calculation method for the fragment length ratio of EB virus DNA to cfDNA of 100 human tumor-related genes is as follows: (the content of 80-110 bp in length mapped to EBV genome fragments) / (the content of 80-110 bp in length mapped to 100 human tumor-related gene fragments).

8. The nasopharyngeal carcinoma screening system according to claim 6, characterized in that: The thresholds are: the fragment length ratio of EBV DNA to cfDNA of 100 tumor-related human genes is 8.964; the EBV DNA content is 5.25×10 -6 .

9. The nasopharyngeal carcinoma screening system according to claim 6, characterized in that: The data analysis module also obtains the following data: ③ The mutation status of 100 tumor-related human genes.

10. The nasopharyngeal carcinoma screening system according to claim 9, characterized in that: The result judgment module also includes judging whether the patient suffers from other related tumors according to the mutation of tumor-related human genes.

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