WO2012175013A1

WO2012175013A1 - System and method for diagnosing human body with abnormal state

Info

Publication number: WO2012175013A1
Application number: PCT/CN2012/077175
Authority: WO
Inventors: 李伟阳; 曾玺; 徐佳佳; 蒋慧; 杨玲; 易赏; 陈盛培; 管彦芳; 易鑫; 胡学达; 李英睿; 张秀清; 杨焕明
Original assignee: 深圳华大基因科技有限公司; 深圳华大基因研究院
Priority date: 2011-06-24
Filing date: 2012-06-19
Publication date: 2012-12-27
Also published as: CN103797130A; CN103797130B

Abstract

Provided are a system and a method for diagnosing a human body with an abnormal state. The method for determining the human body with an abnormal state comprises: providing nucleotide sequence information about a human sample, wherein the nucleotide sequence information about the human sample is obtained based on human sample testing; and determining whether the human body has an abnormal state based on the nucleotide sequence information about the human sample.

Description

用于确定人体具有异常状态的***和方法优先权信息 System and method for determining abnormal state of the human body

本申请请求 2011 年 6 月 24 日向中国国家知识产权局提交的、专利申请号为 201110174686.2的专利申请，以及 2011年 11月 24 日向中国国家知识产权局提交的、专利申请号为 PCT/CN201 1/082855 的专利申请的优先权和权益，并且通过参照将其全文并入此处。技术领域 This application requests a patent application filed on June 24, 2011 from the State Intellectual Property Office of China, with a patent application number of 201110174686.2, and a patent application number PCT/CN201 1/ submitted to the State Intellectual Property Office of China on November 24, 2011. Priority and interest in the patent application of 082855, the entire disclosure of which is incorporated herein by reference. Technical field

本发明涉及生物医学领域。更具体地，本发明涉及用于确定人体具有异常状态的系统和方法。背景技术 The invention relates to the field of biomedicine. More specifically, the present invention relates to systems and methods for determining an abnormal state of a human body. Background technique

HBV是一种全球性的慢性病毒感染性疾病。我国的乙肝病毒感染率约 60%-70%, 而乙肝表面抗原携带率约占总人口的 7.18% , 以此计算，全国约有 9300万人携带乙肝病毒，其中乙肝患者大约有 3000 万。在全世界，大约有 45%的人群生活在慢性 HBV 感染的高发区， 43%的人群生活在慢性 HBV感染的中发区，并且 HBV为已知的引起肝硬化以及肝癌的主要原因，现已知 HBV基因组与人类基因组整合为肝癌的主要诱因之一。 EBV 现已知为鼻咽癌的主要诱因，初发症状到死亡的自然病程从 3 ~ 13个月不等，放射治疗后 5年生存率为 8% ~ 62%。幽门螺旋杆菌为胃癌的主要诱因，并且慢性胃炎患者的胃粘膜活检标本中幽门螺杆菌检出率可达 80% ~ 90% , 而消化性溃疡患者更高，可达 95%以上，甚至接近 100%。胃癌由于局部上皮细胞已发生异化，因此检出率高低 •ί艮道不一。以上病原体，包括 HBV, HCV, HIV, EBV, 幽门螺旋杆菌等，均能够与宿主基因组进行整合，并且与引起的相关病症直接相关，能够对感染者造成持久和高危性的危害，是高致病性，高致癌性的高危型病原微生物。因此，建立一种无创的诊断及病程追踪监测手段，既能够免除对病原体感染患者进行病理组织取样的伤害，同时可以定期跟踪检测感染者感染程度以及治疗效果，提高感染患者的治愈率以及有利于及时地指导用药，对于由病原体引起的肿瘤晚期病人也可进行及时检测判断治愈效果以及复发的可能性，能够及时有效地对患者进行病情随访并给出最佳建议。 HBV is a global chronic viral infectious disease. The hepatitis B virus infection rate in China is about 60%-70%, and the hepatitis B surface antigen carrying rate is about 7.18% of the total population. According to this calculation, about 93 million people in the country carry hepatitis B virus, of which about 30 million are hepatitis B patients. Around the world, about 45% of people live in high-risk areas of chronic HBV infection, 43% live in the middle-incidence area of chronic HBV infection, and HBV is the leading cause of cirrhosis and liver cancer. It is known that the integration of HBV genome and human genome is one of the main causes of liver cancer. EBV is now known as the main cause of nasopharyngeal carcinoma. The natural course of initial symptoms to death ranges from 3 to 13 months, and the 5-year survival rate after radiation therapy is 8% to 62%. Helicobacter pylori is the main cause of gastric cancer, and the detection rate of Helicobacter pylori in gastric mucosal biopsy specimens of patients with chronic gastritis can reach 80% ~ 90%, while patients with peptic ulcer are higher, more than 95%, even close to 100 %. Gastric cancer has been localized due to localized epithelial cells, so the detection rate is high. The above pathogens, including HBV, HCV, HIV, EBV, Helicobacter pylori, etc., can be integrated with the host genome and directly related to the related diseases, which can cause long-term and high-risk harm to the infected person, which is highly pathogenic. Sexual, highly carcinogenic, high-risk pathogenic microorganism. Therefore, a non-invasive diagnostic and disease course monitoring method can be established, which can eliminate the damage of pathological tissue sampling of patients infected with pathogens, and can regularly track the infection degree and treatment effect of infected persons, improve the cure rate of infected patients and benefit Timely guidance of medication, for patients with advanced tumors caused by pathogens can also timely detect and judge the healing effect and the possibility of recurrence, can timely and effectively follow up the patient's condition and give the best advice.

然而，现有技术并不能满足上述要求。发明内容 However, the prior art does not satisfy the above requirements. Summary of the invention

本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的一个目的在于提出一种具有能够有效确定人体异常状态的方法。本发明的另一目的在于提出一种能够有效确定人体异常状态的***。 The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, an object of the present invention is to provide a method having an ability to effectively determine an abnormal state of a human body. Another object of the present invention is to provide a system capable of effectively determining an abnormal state of a human body.

本发明是基于发明人的下列发现而完成的：在人体样本的检测过程中，对于蛋白盾的检测，通常由于实验条件的限制，无法早期获得人体的状态信息。而利用核酸序列的特性，则可以通过对于离体的人体样本进行核酸分析，尽可能早期地对人体状态进行分析。 The present invention has been completed based on the following findings of the inventors: During the detection of human samples, for protein shields Detection, usually due to limitations of experimental conditions, can not get early state information of the human body. By utilizing the characteristics of the nucleic acid sequence, the human body state can be analyzed as early as possible by performing nucleic acid analysis on the ex vivo human sample.

在本发明的一个方面，本发明提出了一种确定人体具有异常状态的方法。根据本发明的实施例，该方法包括：提供人体样本的核酸序列信息，所述人体样本的核酸序列信息是基于对所述人体样本进行检测而获得的；以及基于所述人体样本的核酸序列信息，确定所述人体是否具有异常状态。根据该实施例的方法，通过对人体样本的核酸序列信息进行分析，可以根据核酸序列中所包含的信息确定人体是否具有异常状态。由于核酸序列信息与原位状态的核酸信息保持一致，因而可以有效地确定人体是否具有异常状态。 In one aspect of the invention, the invention proposes a method of determining an abnormal state of a human body. According to an embodiment of the present invention, the method comprises: providing nucleic acid sequence information of a human sample, the nucleic acid sequence information of the human sample is obtained based on detecting the human sample; and nucleic acid sequence information based on the human sample , determining whether the human body has an abnormal state. According to the method of this embodiment, by analyzing the nucleic acid sequence information of the human sample, it is possible to determine whether or not the human body has an abnormal state based on the information contained in the nucleic acid sequence. Since the nucleic acid sequence information is consistent with the nucleic acid information of the in-situ state, it is possible to effectively determine whether the human body has an abnormal state.

另外，根据本发明上述实施例，上述确定人体具有异常状态的方法，还可以具有如下附加的技术特征： Further, according to the above embodiment of the present invention, the above method of determining the abnormal state of the human body may further have the following additional technical features:

根据本发明的一个实施例，所述人体样本的核酸序列信息是基于对所述人体样本进行核酸序列检测而获得的。由此，可以通过核酸序列检测方法，容易地获得人体样本的核酸序列信息，从而提高了确定人体具有异常状态的效率。根据进一步的实施例，所述核酸序列检测是借助第二代测序技术或第三代测序技术进行的。由此，可以高效地对人体样本进行核酸序列进行检测，并且能够实现高通量深度测序。本发明的发明人发现基于第二代测序技术和第三代测序技术的高效、高精度的性盾 , 可以实现对人体样本核酸序列信息的高效、高精度检测，能够非常灵敏地对人体样本中痕量的核酸进行检测。 According to an embodiment of the present invention, the nucleic acid sequence information of the human sample is obtained based on nucleic acid sequence detection of the human sample. Thereby, the nucleic acid sequence information of the human sample can be easily obtained by the nucleic acid sequence detecting method, thereby improving the efficiency of determining the abnormal state of the human body. According to a further embodiment, the nucleic acid sequence detection is carried out by means of a second generation sequencing technique or a third generation sequencing technique. Thereby, the nucleic acid sequence of the human sample can be efficiently detected, and high-throughput deep sequencing can be realized. The inventors of the present invention have found that an efficient and high-precision shield based on the second-generation sequencing technology and the third-generation sequencing technology can realize efficient and high-precision detection of nucleic acid sequence information of human samples, and can be very sensitive to human samples. Trace amounts of nucleic acid are detected.

根据本发明的一个实施例，所述样本为所述人体的细胞、组织、血液、体液、尿液、 ***物或其组合。由此，根据本发明的实施例的确定人体具有异常状态的方法能够应用于各种人体样本，并且能够根据不同的人体样本的特点，确定多种异常状态。 According to an embodiment of the invention, the sample is cells, tissue, blood, body fluids, urine, feces, or a combination thereof of the human body. Thus, the method of determining an abnormal state of a human body according to an embodiment of the present invention can be applied to various human body samples, and various abnormal states can be determined according to characteristics of different human body samples.

根据本发明的一个实施例，所述人体样本为血浆或者血清。根据该实施例，可以利用常规的方法获得人体的血浆和血清样本，并对其进行核酸序列分析，可以直接对人体的异常状态进行确定。 According to an embodiment of the invention, the human sample is plasma or serum. According to this embodiment, plasma and serum samples of the human body can be obtained by a conventional method, and nucleic acid sequence analysis can be performed thereon, and the abnormal state of the human body can be directly determined.

根据本发明的一个实施例，所述核酸序列信息包括所述人体样本中游离核酸的序列信息。由此，可以根据游离核酸的序列信息，对人体的异常状态进行确定。并且基于游离核酸的序列信息与人体正常的核酸序列或者病原体的核酸序列进行比对分析后，可以获得多种人体的异常状态。 According to an embodiment of the invention, the nucleic acid sequence information comprises sequence information of free nucleic acids in the human sample. Thereby, the abnormal state of the human body can be determined based on the sequence information of the free nucleic acid. Further, after the sequence information of the free nucleic acid is compared with the normal nucleic acid sequence of the human body or the nucleic acid sequence of the pathogen, a plurality of abnormal states of the human body can be obtained.

根据本发明的一个实施例，所述人体样本中游离核酸的序列信息是通过除去所述人体样本中的细胞后，进行测序检测而获得的。由此，可以提高游离核酸的测序检测的精度和准确度，从而能够进一步有效地确定人体具有异常状态。 According to an embodiment of the present invention, the sequence information of the free nucleic acid in the human sample is obtained by performing sequencing detection after removing the cells in the human sample. Thereby, the accuracy and accuracy of the sequencing detection of the free nucleic acid can be improved, so that it is possible to further effectively determine that the human body has an abnormal state.

根据本发明的一个实施例，所述异常状态选自疾病的发生、疾病的发展阶段、疾病的疗效和预后的至少一种。由此，可以确定与人体密切相关的疾病的发生、发展、疗效以及预后，从而有利于制定有效的治疗方案。根据本发明进一步的实施例，所述疾病是肿瘤性疾病、免疫性疾病、遗传性疾病的至少一种。根据本发明的实施例的确定人体异常状态的方法，能够有效地确定人体是否患有肿瘤性疾病、免疫性疾病、遗传性疾病。根据具体的示例，所述肿瘤性疾病是选自肺癌、肝癌、胃癌、食管癌、结直肠癌、胰腺癌、乳腺癌、膀胱癌、肾癌、卵巢癌、 ***、甲状腺癌、鼻咽癌、脑胶盾瘤的至少一种。根据更进一步的示例，如果所述核酸序列信息包含选自下列至少一种的核酸片段序列： HBV、 HPV、 EBV、幽门螺旋杆菌，则确定所述人体患有***、肝癌、鼻咽癌、胃癌的至少一种。由此，根据本发明的实施例的确定人体具有异常状态的方法，可以基于所检测的核酸序列信息，有效地确定人体是否患有肝癌、 ***、鼻咽癌或胃癌。 According to an embodiment of the present invention, the abnormal state is selected from at least one of the occurrence of a disease, a stage of development of the disease, a therapeutic effect of the disease, and a prognosis. Thereby, the occurrence, development, efficacy, and prognosis of diseases closely related to the human body can be determined, thereby facilitating the formulation of an effective treatment plan. According to a further embodiment of the present invention, the disease is at least one of a neoplastic disease, an immune disease, and a hereditary disease. According to the embodiment of the present invention, the method of determining the abnormal state of the human body can effectively determine whether the human body has a neoplastic disease, an immune disease, or a hereditary disease. According to a specific example, the neoplastic disease is selected from the group consisting of lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, breast cancer, At least one of bladder cancer, kidney cancer, ovarian cancer, cervical cancer, thyroid cancer, nasopharyngeal cancer, and brain gelatin. According to still further examples, if the nucleic acid sequence information comprises a nucleic acid fragment sequence selected from at least one of the following: HBV, HPV, EBV, H. pylori, the human body is determined to have cervical cancer, liver cancer, nasopharyngeal cancer, At least one type of gastric cancer. Thus, according to the embodiment of the present invention, the method of determining the abnormal state of the human body can effectively determine whether the human body has liver cancer, cervical cancer, nasopharyngeal cancer or gastric cancer based on the detected nucleic acid sequence information.

根据本发明的一个实施例，对核酸进行测序检测之前，可以利用探针去除含有特定序列的核酸，然后对所述去除后剩余的核酸进行测序检测。由此，可以通过探针除去具有特定序列的核酸，从而能够提高对剩余材料进行序列检测的精度和准确性。根据本发明的具体示例，用于去除特定序列的核酸的探针可以结合人体基因组中的共有序列，或者为可以结合人体基因组中甲基化位点的抗体或蛋白。由此，可以进一步提高检测的精度和准确性。 According to one embodiment of the present invention, prior to sequencing the nucleic acid, the probe may be used to remove the nucleic acid containing the specific sequence, and then the nucleic acid remaining after the removal is subjected to sequencing detection. Thereby, the nucleic acid having a specific sequence can be removed by the probe, so that the accuracy and accuracy of sequence detection of the remaining material can be improved. According to a specific example of the present invention, a probe for removing a nucleic acid of a specific sequence may bind to a consensus sequence in the human genome, or an antibody or protein which can bind to a methylation site in the human genome. Thereby, the accuracy and accuracy of the detection can be further improved.

根据本发明的实施例，对所述核酸进行测序检测之前，还可以利用探针捕获含有特定序列的核酸，然后对所述含有特定序列的核酸进行测序检测。由此，可以通过探针，预先对进行核酸序列分析的核酸进行筛选，从而能够进一步提高确定人体具有异常状态的方法的效率。根据进一步的实施例，所述探针对于选自下列的至少一种是特异性的： HBV、 HPV、 EBV、幽门螺旋杆菌，由此，可以有效地确定人体是否患有肝癌、 ***、鼻咽癌或胃癌。 According to an embodiment of the present invention, before the sequencing of the nucleic acid, a nucleic acid containing a specific sequence can be captured by a probe, and then the nucleic acid containing the specific sequence is subjected to sequencing detection. Thereby, the nucleic acid subjected to nucleic acid sequence analysis can be screened in advance by the probe, and the efficiency of the method for determining the abnormal state of the human body can be further improved. According to a further embodiment, the probe is specific for at least one selected from the group consisting of HBV, HPV, EBV, Helicobacter pylori, thereby enabling effective determination of whether the human body has liver cancer, cervical cancer, nasal Pharyngeal or gastric cancer.

因此，在本发明的另一方面，本发明还提供了一种核酸探针集。根据本发明的实施例，该核酸探针集包括多个探针，且具有以下特征： Accordingly, in another aspect of the invention, the invention also provides a nucleic acid probe set. According to an embodiment of the invention, the set of nucleic acid probes comprises a plurality of probes and has the following characteristics:

( 1 )每个探针上具有 1个或多个生物素标记的 dNTP; 和 /或 (1) having one or more biotinylated dNTPs on each probe; and/or

( 2 )生物素标记的 dNTP在核酸探针集中的丰度为 1 : 6-1 : 2; 和 /或 (2) The abundance of biotinylated dNTPs in a nucleic acid probe set is 1: 6-1 : 2; and / or

( 3 )核酸探针集的全部核酸序列覆盖对应选自 HBV、 HPV、 EBV和幽门螺旋杆菌的至少一种病毒的基因组序列的 70%-100%。 (3) The entire nucleic acid sequence of the nucleic acid probe set covers 70% to 100% of the genomic sequence corresponding to at least one virus selected from the group consisting of HBV, HPV, EBV and H. pylori.

在另一优选例中，本发明的核酸探针集具有 1-20000个核酸探针；较佳地，核酸探针集具有 1000-5000个核酸探针；更佳地，核酸探针集具有 2500个核酸探针。 In another preferred embodiment, the nucleic acid probe set of the present invention has 1 to 20,000 nucleic acid probes; preferably, the nucleic acid probe set has 1000 to 5000 nucleic acid probes; more preferably, the nucleic acid probe set has 2,500 Nucleic acid probes.

在另一优选例中，生物素标记的 dNTP在核酸探针集中的丰度为 1 : 4。 In another preferred embodiment, the abundance of the biotinylated dNTP in the nucleic acid probe set is 1:4.

在另一优选例中，核酸探针集中，探针之间具有部分重叠。 In another preferred embodiment, the nucleic acid probes are concentrated with partial overlap between the probes.

在另一优选例中，核酸探针集（在本文中有时也称为 "探针集"）中的探针长度为 100-500 bp; 较佳地，探针长度为 200-300 bp; 更佳地，探针长度为 250 bp。 In another preferred embodiment, the length of the probe in the set of nucleic acid probes (sometimes referred to herein as "probe sets") is from 100 to 500 bp; preferably, the length of the probe is from 200 to 300 bp; Preferably, the probe is 250 bp long.

在另一优选例中，探针是以病毒基因组作为模板， PCR法扩增获得的，较佳地，扩增模板为乙型肝炎病毒（HBV )基因组、丙型肝炎病毒 ( HCV )基因组、艾滋病病毒（HIV ) 基因组、 ***瘤病毒（HPV )基因组，或其组合；更佳地，扩增模板为 B型 HBV基因组和 /或 C型 HBV基因组。 In another preferred embodiment, the probe is obtained by PCR amplification using a viral genome as a template. Preferably, the amplification template is a hepatitis B virus (HBV) genome, a hepatitis C virus (HCV) genome, and an AIDS virus. The viral (HIV) genome, the papillomavirus (HPV) genome, or a combination thereof; more preferably, the amplification template is the B-type HBV genome and/or the C-type HBV genome.

进一步，在本发明的又一方面，还提供了一种表面固定有本发明的核酸探针集的核酸芯片。 Further, in still another aspect of the present invention, a nucleic acid chip having a surface-immobilized set of nucleic acid probes of the present invention is also provided.

在本发明的再一方面，还提供了本发明的核酸探针集和核酸芯片的用途，用于检测病毒在待测样本中的整合方式；较佳地，整合方式选自下组：重排、异位、 ***、替换，或其组合。在本发明的另一方面，还提供了一种制备本发明的核酸探针的方法，包括步骤： a. 获得探针来源样本； In a further aspect of the invention, there is also provided the use of the nucleic acid probe set and the nucleic acid chip of the invention for detecting the manner in which the virus is integrated in the sample to be tested; preferably, the integration mode is selected from the group consisting of: rearrangement , ectopic, insert, replace, or a combination thereof. In another aspect of the invention, there is also provided a method of preparing a nucleic acid probe of the invention, comprising the steps of: a. obtaining a probe source sample;

b. 对步骤（ a )获得的样本进行 PCR扩增， PCR扩增体系的 dNTP为生物素标记的 dNTP , 以便获得带有生物素标记的 PCR扩增产物； b. PCR amplification of the sample obtained in step (a), the dNTP of the PCR amplification system is biotin-labeled dNTP, in order to obtain a PCR amplification product with biotin label;

c 对步骤（b )获得的生物素标记的 PCR扩增产物进行打断，得到片段化的生物素标记的 PCR扩增产物，即为探针。 c The biotin-labeled PCR amplification product obtained in the step (b) is interrupted to obtain a PCR amplification product of the fragmented biotin label, which is a probe.

在另一优选例中，步骤（ a )所述样本具有以下特征： In another preferred embodiment, the sample of step (a) has the following characteristics:

样本为含有核酸的病毒样本；和 /或 The sample is a virus sample containing nucleic acid; and/or

样本为病毒粒子、血清、血液、组织样本、脱落细胞，上皮细胞，或其组合；和 /或样本选自下组：乙型肝炎病毒（HBV )、丙型肝炎病毒（HCV )、艾滋病病毒（HIV )、 ***瘤病毒（HPV ), 或其组合；和 /或 The sample is virions, serum, blood, tissue samples, exfoliated cells, epithelial cells, or a combination thereof; and/or the sample is selected from the group consisting of hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV ( HIV), papillomavirus (HPV), or a combination thereof; and/or

样本为 B型 HBV和 /或 C型 HBV。 The samples are type B HBV and / or type C HBV.

在另一优选例中，步骤（ b )具有以下特征： In another preferred embodiment, step (b) has the following characteristics:

步骤（b )所述的扩增为对样本中病毒 DNA全长进行扩增；和 /或 The amplification described in step (b) is to amplify the full length of the viral DNA in the sample; and/or

步骤（ b )所述标记的 dNTP为 biotin-dNTP, 且标记的 dNTP能够与链霉素亲和磁珠结合；和 /或 Step (b) wherein the labeled dNTP is a biotin-dNTP, and the labeled dNTP is capable of binding to a streptomycin affinity magnetic bead; and/or

步骤（b )所述标记的 dNTP与非标记 dNTP的比例为 1 : 2-8; 优选比例为 1 : 3-6; 更优选比例为 1 : 4。 The ratio of the labeled dNTP to the unlabeled dNTP in the step (b) is 1: 2-8; the preferred ratio is 1: 3-6; more preferably, the ratio is 1:4.

在另一优选例中，步骤（ c )所述打断为超声法打断。 In another preferred embodiment, the interruption in step (c) is interrupted by ultrasound.

在另一优选例中，还包括步骤（d ): 对步骤（c )获得的探针进行纯化和 /或定量。在另一优选例中，根据本发明的制备核酸探针的方法制备的探针，其长度为 100-500 bp; 较佳地，探针的长度为 200-300 bp, 更佳地，探针的长度为 250 bp。 In another preferred embodiment, step (d) is further included: purifying and/or quantifying the probe obtained in step (c). In another preferred embodiment, the probe prepared according to the method for producing a nucleic acid probe of the present invention has a length of 100 to 500 bp; preferably, the probe has a length of 200 to 300 bp, more preferably, a probe The length is 250 bp.

在本发明的又一方面，本发明还提供了一种检测病毒在待测样本中基因整合方式的方法，包括步骤： In still another aspect of the present invention, the present invention also provides a method for detecting a method of genetic integration of a virus in a sample to be tested, comprising the steps of:

( i )获得待测样本； (i) obtaining a sample to be tested;

( ϋ )对步骤（ i )获得的样本进行文库构建； ( ϋ ) performing library construction on the sample obtained in step ( i );

( iii )将本发明的探针与步骤（ii )获得的文库进行杂交，捕获与病毒基因整合有关的核酸序列； (iii) hybridizing the probe of the present invention to the library obtained in step (ii) to capture a nucleic acid sequence associated with integration of the viral gene;

( iv )对步骤（iii )捕获的核酸序列进行扩增，获得与病毒整合有关的扩增产物； ( V )对步骤（iv )获得的扩增产物进行测序，获得与病毒整合方式有关核酸信息。在另一优选例中，步骤（ i )具有以下特征： (iv) amplifying the nucleic acid sequence captured in step (iii) to obtain an amplification product related to viral integration; (V) sequencing the amplification product obtained in step (iv) to obtain nucleic acid information related to virus integration . In another preferred embodiment, step (i) has the following characteristics:

所述待测样本为组织、血液、脱落细胞，上皮细胞；和 /或 The sample to be tested is tissue, blood, exfoliated cells, epithelial cells; and/or

所述待测样本来源于人或非人哺乳动物，较佳地来源于人；和 /或 The sample to be tested is derived from a human or non-human mammal, preferably from a human; and/or

所述待测样本来源于 HBV感染者或肝癌患者。 The sample to be tested is derived from an HBV infected person or a liver cancer patient.

在另一优选例中，步骤（ iii )具有以下特征： In another preferred embodiment, step (iii) has the following characteristics:

所述探针为变性的单链 DNA; 和 /或在杂交液中加入接头封闭分子和标签封闭分子；和 /或 The probe is a denatured single-stranded DNA; and/or Adding a linker blocking molecule and a tag blocking molecule to the hybridization solution; and/or

所述接头封闭分子的序列如 SEQ ID NO: 8所示；和 /或 The sequence of the linker blocking molecule is set forth in SEQ ID NO: 8; and/or

所述标签封闭分子的序列如 SEQ ID NO: 9和 SEQ ID NO: 10所示。 The sequence of the tag blocking molecule is shown in SEQ ID NO: 9 and SEQ ID NO: 10.

在另一优选例中，在步骤（V ) 中，将所述的扩增产物与固相载体上固定的测序探针进行杂交，进行固相桥式 PCR扩增，形成测序簇；然后对所述测序簇用 "边合成-边测序"法进行测序，从而得到与病毒整合方式有关核酸信息。 In another preferred embodiment, in step (V), the amplification product is hybridized with a sequencing probe immobilized on a solid phase carrier, and subjected to solid phase bridge PCR amplification to form a sequencing cluster; The sequencing clusters were sequenced by the "Synthesis-Side Sequencing" method to obtain nucleic acid information related to the way of virus integration.

在另一优选例中，在步骤（ii ) 中，所述的文库构建为：对打断的基因组 DNA进行末端修复，加入接头，对具有接头的片段进行扩增，获得的带有接头的扩增混合物即为样本文库。 In another preferred embodiment, in the step (ii), the library is constructed as: end-repairing the interrupted genomic DNA, adding a linker, and amplifying the fragment having the linker, and obtaining the expanded with the linker The addition mixture is the sample library.

在另一优选例中，所述的接头具有如 SEQ ID NO: 1和 SEQ ID NO: 2所示的序列；和 In another preferred embodiment, the linker has the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2;

/或，所构建的文库具有如 SEQ ID NO: 3和 SEQ ID NO: 4所示的标签序列。 / or, the constructed library has the tag sequence as shown in SEQ ID NO: 3 and SEQ ID NO: 4.

在本发明的又一方面，本发明还提供了一种可用于本发明前面所述方法的试剂盒，所述试剂盒包括： In yet another aspect of the invention, the invention also provides a kit useful in the methods of the invention described above, the kit comprising:

(1)第一容器以及位于容器内前面所述的核酸芯片，或本发明前面所述的探针； (2)第二容器以及位于容器内的用于构建样本文库的接头； (1) a first container and a nucleic acid chip as described above in the container, or a probe as described above in the present invention; (2) a second container and a linker for constructing a sample library located in the container;

(3)第三容器以及位于容器内的接头封闭分子； (3) a third container and a joint blocking molecule located in the container;

(4)第四容器以及位于容器内的标签封闭分子； (4) a fourth container and a label blocking molecule located within the container;

(5)检测说明书。 (5) Test instructions.

在另一优选例中，所述试剂盒还包括选自下组的试剂： In another preferred embodiment, the kit further comprises a reagent selected from the group consisting of:

用于进行 PCR扩增所需的试剂、 Reagents required for PCR amplification,

用于进行封闭反应所需的试剂、 The reagents needed to carry out the blocking reaction,

用于进行杂交反应所需的试剂、 The reagents needed to carry out the hybridization reaction,

用于进行测序反应所需的试剂、 The reagents needed to perform the sequencing reaction,

或其组合。 Or a combination thereof.

在本发明的另一方面，本发明还提供了一种可用于本发明前面所述方法的试剂盒，所述试剂盒包括： In another aspect of the invention, the invention also provides a kit useful in the methods of the invention described above, the kit comprising:

前面所述的核酸芯片或前面所述的探针；接头；接头封闭分子；以及标签封闭分子。根据本发明的实施例，在该试剂盒中，所包含的成分被设置在不同的容器中，由此，可以方便使用。根据本发明的实施例，该试剂盒还包括选自下组的试剂：用于进行 PCR扩增所需的试剂、用于进行封闭反应所需的试剂、用于进行杂交反应所需的试剂、用于进行测序反应所需的试剂、或其组合。 a nucleic acid chip as described above or a probe as described above; a linker; a linker blocking molecule; and a tag blocking molecule. According to an embodiment of the present invention, in the kit, the contained components are disposed in different containers, whereby it can be conveniently used. According to an embodiment of the present invention, the kit further comprises a reagent selected from the group consisting of: a reagent required for performing PCR amplification, a reagent required for performing a blocking reaction, a reagent required for performing a hybridization reaction, The reagents, or combinations thereof, required to perform the sequencing reaction.

在本发明的另一方面，本发明还提供了一种用于确定人体具有异常状态的***。根据本发明的实施例，该***包括：核酸序列信息接收器，所述核酸序列信息接收器接收人体样本的核酸序列信息；以及核酸序列信息分析器，所述核酸序列信息分析器与所述核酸序列信息接收器相连，并基于所述人体样本的核酸序列信息，确定所述人体是否具有异常状态。利用该***，可以有效地实施根据本发明实施例的确定人体具有异常状态的方案，其具有本发明实施例的确定人体具有异常状态的方法的全部优点，在此不再赘述。另外，根据本发明上述实施例，上述确定人体具有异常状态的***，还可以具有如下附加的技术特征： In another aspect of the invention, the invention also provides a system for determining an abnormal state of a human body. According to an embodiment of the present invention, the system includes: a nucleic acid sequence information receiver that receives nucleic acid sequence information of a human sample; and a nucleic acid sequence information analyzer, the nucleic acid sequence information analyzer and the nucleic acid The sequence information receiver is connected, and based on the nucleic acid sequence information of the human sample, determines whether the human body has an abnormal state. With the system, it is possible to effectively implement a scheme for determining an abnormal state of a human body according to an embodiment of the present invention, All the advantages of the method for determining the abnormal state of the human body in the embodiment of the present invention are not described herein. In addition, according to the above embodiment of the present invention, the above system for determining an abnormal state of the human body may further have the following additional technical features:

根据本发明的一个实施例，所述核酸序列信息分析器内预存有选自下列的至少一种：人体正常状态的基因组序列、病原体的基因组序列、正常人群的基因组序列。从而可以有效地对核酸序列进行分析，提高了用于确定人体具有异常状态的***的效率。根据进一步的实施例，所述病原体为选自 HBV、 HPV、 EBV、幽门螺旋杆菌的至少一种。由此，可以有效地确定人体是否患有肝癌、 ***、鼻咽癌或胃癌。 According to an embodiment of the present invention, the nucleic acid sequence information analyzer prestores at least one selected from the group consisting of: a genomic sequence of a human normal state, a genomic sequence of a pathogen, and a genomic sequence of a normal population. Thereby, the nucleic acid sequence can be effectively analyzed, and the efficiency of the system for determining the abnormal state of the human body is improved. According to a further embodiment, the pathogen is at least one selected from the group consisting of HBV, HPV, EBV, and H. pylori. Thereby, it is possible to effectively determine whether the human body has liver cancer, cervical cancer, nasopharyngeal cancer or gastric cancer.

根据本发明的一个实施例，进一步包括核酸序列检测装置，所述核酸序列检测装置与所述核酸序列信息接收器相连，用于对所述人体样本进行核酸序列检测获得所述核酸序列信息并输送至所述核酸序列信息接收器。由此，可以直接对核酸进行序列检测，并且输送至核酸序列接收器，进而进行核酸序列分析，从而确定人体是否具有异常状态从而提高了确定人体具有异常状态的效率。根据进一步的实施例，所述核酸序列检测装置借助第二代测序技术或第三代测序技术。由此，可以高效地对人体样本进行核酸序列进行检测，并且能够实现高通量深度测序。本发明的发明人发现基于第二代测序技术和第三代测序技术的高效、高精度的性盾，可以实现对人体样本核酸序列信息的高效、高精度检测，能够非常灵敏地对人体样本中痕量的核酸进行检测。 According to an embodiment of the present invention, further comprising a nucleic acid sequence detecting device, wherein the nucleic acid sequence detecting device is connected to the nucleic acid sequence information receiver, configured to perform nucleic acid sequence detection on the human sample to obtain the nucleic acid sequence information and transmit To the nucleic acid sequence information receiver. Thereby, the nucleic acid can be directly detected by the sequence and delivered to the nucleic acid sequence receiver, thereby performing nucleic acid sequence analysis to determine whether the human body has an abnormal state, thereby improving the efficiency of determining the abnormal state of the human body. According to a further embodiment, the nucleic acid sequence detecting device is by means of a second generation sequencing technique or a third generation sequencing technique. Thereby, the nucleic acid sequence of the human sample can be efficiently detected, and high-throughput deep sequencing can be realized. The inventors of the present invention have found that an efficient and high-precision shield based on the second-generation sequencing technology and the third-generation sequencing technology can realize efficient and high-precision detection of nucleic acid sequence information of human samples, and can be very sensitive to human samples. Trace amounts of nucleic acid are detected.

根据本发明的一个实施例，进一步包括游离核酸捕获装置，所述游离核酸捕获装置与所述核酸序列检测装置相连，其中，所述游离核酸捕获装置设置有探针，所述探针适于捕获含有特定序列的核酸，并且将所述含有特定序列的核酸输送至所述核酸序列检测装置进行核酸序列检测；或者所述探针适于除去含有特定序列的核酸，并且将经过所述除去的核酸输送至所述核酸序列检测装置进行核酸序列检测。由此，对所述核酸进行测序检测之前，利用探针捕获含有特定序列的核酸，然后对所述含有特定序列的核酸进行测序检测。由此，可以通过探针，预先对进行核酸序列分析的核酸进行筛选，从而能够进一步提高确定人体具有异常状态的方法的效率。根据进一步的实施例，所述探针对于选自下列的至少一种是特异性的： HBV、 HPV、 EBV、幽门螺旋杆菌，其来源于前面所述的本发明的核酸探针集，具有其全部优点，在此不再赘述。由此，可以有效地确定人体是否患有肝癌、 ***、鼻咽癌或胃癌。或者，可以通过探针除去具有特定序列的核酸，从而能够提高对剩余材料进行序列检测的精度和准确性。根据本发明的具体示例，用于去除特定序列的核酸的探针可以结合人体基因组中的共有序列，或者为可以结合人体基因组中甲基化位点的抗体或蛋白。由此，可以进一步提高检测的精度和准确性。 According to an embodiment of the present invention, further comprising a free nucleic acid capture device connected to the nucleic acid sequence detecting device, wherein the free nucleic acid capture device is provided with a probe, the probe being adapted to capture a nucleic acid containing a specific sequence, and delivering the nucleic acid containing the specific sequence to the nucleic acid sequence detecting device for nucleic acid sequence detection; or the probe is adapted to remove a nucleic acid containing a specific sequence, and the nucleic acid to be removed Delivery to the nucleic acid sequence detecting device for nucleic acid sequence detection. Thus, prior to sequencing the nucleic acid, a nucleic acid containing a specific sequence is captured using a probe, and then the nucleic acid containing the specific sequence is subjected to sequencing detection. Thereby, the nucleic acid subjected to nucleic acid sequence analysis can be screened in advance by the probe, and the efficiency of the method for determining the abnormal state of the human body can be further improved. According to a further embodiment, the probe is specific for at least one selected from the group consisting of HBV, HPV, EBV, H. pylori, which is derived from the set of nucleic acid probes of the invention described above, having All the advantages are not described here. Thereby, it is possible to effectively determine whether the human body has liver cancer, cervical cancer, nasopharyngeal cancer or gastric cancer. Alternatively, the nucleic acid having a specific sequence can be removed by the probe, thereby improving the accuracy and accuracy of sequence detection of the remaining material. According to a specific example of the present invention, a probe for removing a nucleic acid of a specific sequence may bind to a consensus sequence in the human genome, or an antibody or protein which can bind to a methylation site in the human genome. Thereby, the accuracy and accuracy of the detection can be further improved.

本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。附图说明 The additional aspects and advantages of the invention will be set forth in part in the description which follows. DRAWINGS

本发明的上述和 /或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中： The above and/or additional aspects and advantages of the present invention will become apparent from the following description of the embodiments in conjunction with the accompanying drawings Easy to understand, where:

图 1是根据本发明一个实施例的确定人体具有异常状态的方法的流程示意图；图 2是根据本发明另一个实施例的确定人体具有异常状态的方法的流程示意图；图 3是根据本发明又一个实施例的确定人体具有异常状态的方法的流程示意图；图 4是才居本发明一个实施例的用于确定人体具有异常状态的***的示意图；图 5是根据本发明另一个实施例的用于确定人体具有异常状态的***的示意图；图 6是根据本发明又一个实施例的用于确定人体具有异常状态的***的示意图；图 7是根据本发明又一个实施例的对 HBV全基因组 PCR扩增后的电泳检测结果；图 8是根据本发明又一个实施例的对 HB V全长产物打断后的电泳检测结果；图 9是根据本发明又一个实施例的建库杂交后一种文库的片断大小检测结果；以及图 10是根据本发明又一个实施例的建库杂交另一种文库的片断大小检测结果。发明详细描述 1 is a schematic flow chart of a method for determining an abnormal state of a human body according to an embodiment of the present invention; FIG. 2 is a flow chart showing a method for determining an abnormal state of a human body according to another embodiment of the present invention; FIG. 4 is a schematic diagram of a system for determining an abnormal state of a human body according to an embodiment of the present invention; FIG. 5 is a schematic diagram of a system according to another embodiment of the present invention; FIG. FIG. 6 is a schematic diagram of a system for determining an abnormal state of a human body according to still another embodiment of the present invention; FIG. 7 is a partial genome PCR for HBV according to still another embodiment of the present invention. The result of the electrophoresis detection after amplification; FIG. 8 is an electrophoresis detection result after breaking the full length product of HB V according to still another embodiment of the present invention; FIG. 9 is a diagram of the hybridization of the library according to still another embodiment of the present invention. The fragment size detection result of the library; and FIG. 10 is another hybridization method according to another embodiment of the present invention. Fragment size detection result library. Detailed description of the invention

下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。 The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the drawings are intended to be illustrative of the invention and are not to be construed as limiting.

在本发明的描述中，需要说明的是，除非另有明确的规定和限定，术语 "相连"、 "连接，，应做广义理解，例如，可以是固定连接，一体地连接，也可以是可拆卸连接；可以是机械连接或电连接，也可以是两个元件内部的连通；可以是直接相连，也可以通过中间媒介间接相连，对于本领域的普通技术人员而言，可以根据具体情况理解上述术语的具体含义。 In the description of the present invention, it should be noted that the terms "connected" and "connected" should be understood broadly, and may be, for example, a fixed connection, an integral connection, or a Disassembling the connection; it may be a mechanical connection or an electrical connection, or it may be the internal connection of the two components; it may be directly connected or indirectly connected through an intermediate medium, and those skilled in the art may understand the above according to the specific situation. The specific meaning of the term.

本发明是基于本发明的下列发现而完成的：在人体样本的检测过程中，对于蛋白盾的检测，通常由于实验条件的限制，无法直接对应其原位状态的信息，而核酸序列由于其自身性盾比较稳定，因而在对于离体的人体样本进行核酸分析的结果可以直接对应其原位状态的信息，进而可以有效地对人体的状态进行分析。 The present invention has been completed based on the following findings of the present invention: In the detection process of a human sample, the detection of a protein shield is usually unable to directly correspond to the information of its in-situ state due to limitations of experimental conditions, and the nucleic acid sequence is due to itself. The sexual shield is relatively stable, so the results of nucleic acid analysis on the isolated human sample can directly correspond to the information of the in-situ state, and thus can effectively analyze the state of the human body.

下面参考附图，首先对根据本发明实施例的确定人体具有异常状态的方法进行详细描述。 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring now to the drawings, a method for determining an abnormal state of a human body according to an embodiment of the present invention will be described in detail.

参考图 1 , #>据本发明实施例的确定人体具有异常状态的方法包括以下步骤：步骤 100: 提供人体样本的核酸序列信息；以及 Referring to FIG. 1, a method for determining an abnormal state of a human body according to an embodiment of the present invention includes the following steps: Step 100: providing nucleic acid sequence information of a human sample;

步骤 200: 基于人体样本的核酸序列信息，确定人体是否具有异常状态。 Step 200: Determine whether the human body has an abnormal state based on the nucleic acid sequence information of the human sample.

根据该实施例的方法，通过对人体样本的核酸序列信息进行分析，可以根据核酸序列中所包含的信息确定人体是否具有异常状态。由于核酸序列信息与原位状态的核酸信息保持一致，因而可以有效地确定人体是否具有异常状态。 According to the method of this embodiment, by analyzing the nucleic acid sequence information of the human sample, it is possible to determine whether or not the human body has an abnormal state based on the information contained in the nucleic acid sequence. Since the nucleic acid sequence information is consistent with the nucleic acid information in the in-situ state, it is possible to effectively determine whether the human body has an abnormal state.

在本发明中，术语 "人体，，应作广义理解，其并不限于人，其可以是任何能够通过核酸信息预测异常状态的生命体。在本发明中，术语 "核酸，，可以是任何包含脱氧核糖核苷酸或者核糖核苷酸的聚合物，包括但不限于经过修饰的或者未经修饰的 DNA、 RNA, 其长度不受任何特别限制。在本发明中，术语 "核酸序列信息"，是指核酸序列所包含的所有信息，包括但不限于核酸的碱基序列、是否被修饰等信息。在本发明中，术语 "人体样本" 的含义不受特别限制，可以应用于本发明实施例的人体样本的类型，包括但不限于人体的细胞、组织、血液、体液、尿液、 ***物或其组合，更具体的示例包括血浆或血清。更进一步的示例中，釆用血浆作为人体样本。本申请的发明人发现选择血浆作为研究样本，背景噪音会较小，检测结果精度高。本领域技术人员可以根据需要选择进行检测和分析的样本类型。由此，根据本发明的实施例的确定人体具有异常状态的方法能够应用于各种人体样本，并且能够才居不同的人体样本的特点，确定多种异常状态。 In the present invention, the term "human body" is to be understood broadly and is not limited to humans, and may be any living body capable of predicting an abnormal state by nucleic acid information. In the present invention, the term "nucleic acid," may be any inclusion. a polymer of deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, Degree is not subject to any special restrictions. In the present invention, the term "nucleic acid sequence information" means all information contained in a nucleic acid sequence, including but not limited to, a base sequence of a nucleic acid, whether or not it is modified. In the present invention, the meaning of the term "human body sample" is not particularly limited, and may be applied to the type of human body sample of the embodiment of the present invention, including but not limited to cells, tissues, blood, body fluids, urine, excretions or A more specific example of the combination includes plasma or serum. In a further example, plasma is used as a human sample. The inventors of the present application found that plasma was selected as a research sample, the background noise was small, and the detection result was high in accuracy. Those skilled in the art can select the type of sample to be tested and analyzed as needed. Thus, the method of determining an abnormal state of a human body according to an embodiment of the present invention can be applied to various human body samples, and can determine a plurality of abnormal states by taking advantage of the characteristics of different human body samples.

在本发明中，术语 "异常状态" 是指人体与正常状态不同的状态，包括但不限于生理状态、心理状态，例如病理状态。据本发明的一个实施例，异常状态选自疾病的发生、疾病的发展阶段、疾病的疗效和预后的至少一种。由此，可以确定与人体密切相关的疾病的发生、发展、疗效以及预后，从而有利于制定有效的治疗方案。根据本发明进一步的实施例，疾病是肿瘤性疾病、免疫性疾病、遗传性疾病的至少一种。由此，居本发明的实施例的确定人体异常状态的方法，能够有效地确定人体是否患有肿瘤性疾病、免疫性疾病、遗传性疾病。根据具体的示例，所述肿瘤性疾病是选自肺癌、肝癌、胃癌、食管癌、结直肠癌、胰腺癌、乳腺癌、膀胱癌、肾癌、卵巢癌、 ***、甲状腺癌、鼻咽癌、脑胶盾瘤的至少一种。根据更进一步的示例，如果所述核酸序列信息包含选自下列至少一种的核酸片段序列： HBV、 HPV、 EBV、幽门螺旋杆菌，则确定所述人体患有***、肝癌、鼻咽癌、胃癌的至少一种。由此，根据本发明的实施例的确定人体具有异常状态的方法，可以基于所检测的核酸序列信息，有效地确定人体是否患有肝癌、 ***、鼻咽癌或胃癌。 In the present invention, the term "abnormal state" refers to a state in which the human body is different from the normal state, including but not limited to a physiological state, a mental state, such as a pathological state. According to an embodiment of the present invention, the abnormal state is selected from at least one of the occurrence of a disease, a stage of development of the disease, a therapeutic effect of the disease, and a prognosis. As a result, the occurrence, development, efficacy, and prognosis of diseases closely related to the human body can be determined, thereby facilitating the development of an effective treatment plan. According to a further embodiment of the present invention, the disease is at least one of a neoplastic disease, an immunological disease, and a hereditary disease. Thus, the method for determining the abnormal state of the human body in the embodiment of the present invention can effectively determine whether the human body has a neoplastic disease, an immune disease, or a hereditary disease. According to a specific example, the neoplastic disease is selected from the group consisting of lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, thyroid cancer, nasopharyngeal cancer. At least one of the brain gelatin tumors. According to still further examples, if the nucleic acid sequence information comprises a nucleic acid fragment sequence selected from at least one of the following: HBV, HPV, EBV, H. pylori, the human body is determined to have cervical cancer, liver cancer, nasopharyngeal cancer, At least one type of gastric cancer. Thus, according to the embodiment of the present invention, the method of determining the abnormal state of the human body can effectively determine whether the human body has liver cancer, cervical cancer, nasopharyngeal cancer or gastric cancer based on the detected nucleic acid sequence information.

根据本发明的实施例，人体样本的核酸序列信息的来源并不受特别限制。根据本发明的一个实施例，人体样本的核酸序列信息是基于对人体样本进行检测而获得的。根据本发明的实施例，检测人体样本而获得人体样本的核酸序列信息的方法并不受特别限制，可以直接对人体样本进行核酸序列测序分析而获得，也可以通过其他方法例如盾谱等。根据本发明的一些示例，可以基于对人体样本直接进行核酸序列检测而获得人体样本的核酸序列信息。即，如图 2所示，根据本发明实施例，进一步包括步骤 300：对核酸序列进行检测。由此，可以通过核酸序列检测方法，容易地获得人体样本的核酸序列信息，从而提高了确定人体具有异常状态的效率。根据更具体的示例，可以借助第二代测序技术或第三代测序技术进行核酸序列检测。由此，以边合成边测序方法为代表的第二代测序技术，以及以单分子测序为代表的第三代测序方法可以高效地对人体样本进行核酸序列进行检测，并且能够实现高通量深度测序。本发明的发明人发现基于第二代测序技术和第三代测序技术的高效、高精度的性盾，可以实现对人体样本核酸序列信息的高效、高精度检测，能够非常灵敏地对人体样本中痕量的核酸进行检测，从而进一步提高确定人体存在异常状态的效率。这里所使用的术语 "高通量" 是指可以同时对大量的核酸进行测序检测，术语 "深度" 是指可以对核酸进行重复多次检测，例如在实施例 1 中可以进行 100轮测序检测，当然根据样本的不同，重复的次数也可以根据需要进行选择。当然，本领域技术人员能够预见的是，未来可以釆用其他更先进的测序技术。目前可以利用的第二代测序技术包括但不限于 Illumina/HiSeq 2000 ^ Roche/454 ^ ABI/SOLiD。在本发明的一个实施例中，所釆用的测序方法为 Illumina/HiSeq 2000。 According to an embodiment of the present invention, the source of the nucleic acid sequence information of the human sample is not particularly limited. According to an embodiment of the invention, the nucleic acid sequence information of the human sample is obtained based on the detection of the human sample. According to the embodiment of the present invention, the method for detecting the nucleic acid sequence information of the human body sample by detecting the human body sample is not particularly limited, and may be obtained by directly performing nucleic acid sequence sequencing analysis on the human body sample, or may be obtained by other methods such as shield spectrum or the like. According to some examples of the invention, nucleic acid sequence information of a human sample can be obtained based on direct nucleic acid sequence detection of a human sample. That is, as shown in FIG. 2, according to an embodiment of the present invention, further comprising the step 300: detecting the nucleic acid sequence. Thereby, the nucleic acid sequence information of the human sample can be easily obtained by the nucleic acid sequence detecting method, thereby improving the efficiency of determining the abnormal state of the human body. According to a more specific example, nucleic acid sequence detection can be performed by means of a second generation sequencing technique or a third generation sequencing technique. Thus, the second-generation sequencing technology represented by the side-synthesis sequencing method and the third-generation sequencing method represented by single-molecule sequencing can efficiently detect nucleic acid sequences of human samples and achieve high-throughput depth. Sequencing. The inventors of the present invention have found that an efficient and high-precision shield based on the second-generation sequencing technology and the third-generation sequencing technology can realize efficient and high-precision detection of nucleic acid sequence information of human samples, and can be very sensitive to human samples. Trace amounts of nucleic acid are detected to further improve the efficiency of determining abnormal conditions in the human body. The term "high-throughput" as used herein refers to the simultaneous detection of a large number of nucleic acids, and the term "depth" means that the nucleic acid can be repeatedly detected multiple times, for example, in Example 1, 100 rounds of sequencing can be performed. Of course, depending on the sample, the number of repetitions can also be selected as needed. Of course, those skilled in the art can foresee that Other more advanced sequencing technologies can be used in the future. Second generation sequencing technologies currently available include, but are not limited to, Illumina/HiSeq 2000 ^ Roche/454 ^ ABI/SOLiD. In one embodiment of the invention, the sequencing method employed is Illumina/HiSeq 2000.

根据本发明的实施例，对核酸序列信息进行分析，从而确定异常状态的方法，不受特别限制。可以是在获得核酸序列信息后，与人体的正常基因组信息或者病原体的基因组信息进行比对，得到比对结果后，进行判断人体是否具有异常状态。也可以基于，核酸序列在样本中的含量来判断，人体的异常状态。根据本发明的实施例，可以通过分析人体样本中，游离核酸的含量，来确定人体是否存在异常状态。如实施例 1 所示，本发明的发明人发现，在癌症患者的外周血中，游离核酸的含量远高于正常人外周血中游离核酸的含量（为至少大约 10倍）。根据本发明另外的实施例，可以分析人体样本中，游离核酸序列中是否存在突变位点或者经修饰的位点例如甲基化位点，从而判断个体是否存在某些特定的异常状态。具体地，可以釆用常用的比对软件进行操作，根据本发明的一个示例，釆用 SOAP 软件包进行比对分析，由此，能够高效地对核酸序列信息进行分析，并得到准确和精确的结果。 According to an embodiment of the present invention, a method of analyzing nucleic acid sequence information to determine an abnormal state is not particularly limited. After the nucleic acid sequence information is obtained, the normal genomic information of the human body or the genomic information of the pathogen may be compared, and after the comparison result is obtained, it is determined whether the human body has an abnormal state. It is also possible to determine the abnormal state of the human body based on the content of the nucleic acid sequence in the sample. According to an embodiment of the present invention, it is possible to determine whether or not an abnormal state exists in a human body by analyzing the content of free nucleic acid in a human sample. As shown in Example 1, the inventors of the present invention found that the content of free nucleic acid in the peripheral blood of a cancer patient is much higher than that of normal human peripheral blood (at least about 10 times). According to a further embodiment of the present invention, it is possible to analyze whether there is a mutation site or a modified site such as a methylation site in the free nucleic acid sequence in the human sample, thereby judging whether the individual has some specific abnormal state. Specifically, the operation can be performed by using the usual comparison software. According to an example of the present invention, the SOAP software package is used for the comparison analysis, thereby efficiently analyzing the nucleic acid sequence information and obtaining accurate and accurate information. result.

根据本发明的实施例，可以用于本发明实施例的确定人体具有异常状态的方法的核酸类型不受特别限制， #>据本发明的一个实施例，所釆用的核酸序列信息包括人体样本中游离核酸的序列信息。由此，可以根据游离核酸的序列信息，对人体的异常状态进行确定。并且基于游离核酸的序列信息与人体正常的核酸序列或者病原体的核酸序列进行比对分析后，可以获得多种人体的异常状态。为了方便理解，下面对游离核酸进行详细描述。 According to an embodiment of the present invention, the type of nucleic acid which can be used in the method for determining an abnormal state of a human body in the embodiment of the present invention is not particularly limited, and according to an embodiment of the present invention, the nucleic acid sequence information used includes a human body sample. Sequence information of free nucleic acids. Thereby, the abnormal state of the human body can be determined based on the sequence information of the free nucleic acid. Further, after the sequence information of the free nucleic acid is aligned with the normal nucleic acid sequence of the human body or the nucleic acid sequence of the pathogen, various abnormal states of the human body can be obtained. For ease of understanding, the free nucleic acid is described in detail below.

在本发明中，所使用的术语 "游离核酸"是指在细胞外的游离状态的核酸，可以是 DNA、 In the present invention, the term "free nucleic acid" as used herein refers to a nucleic acid in a free state outside the cell, which may be DNA,

RNA或者其他类型的核酸。本发明的发明人发现，正常状态下，会有少量的核酸由于代谢而进入到外周血中而成为游离核酸，而在异常状态下，例如癌症病人体内，游离核酸的含量大大高于正常状态下的游离核酸含量。发明人发现，在癌症病人体内，游离核酸（如游离 DNA ) 的含量取决于肿瘤的生物学特性，即与肿瘤细胞的恶性程度、侵袭程度、是否发生转移、疾病进程等相关。因而，可以通过分析人体样本中游离核酸的含量，来确定人体是否存在异常状态，例如患有肿瘤性疾病，以及确定肿瘤性疾病的进展阶段，侵袭程度等。从而，为选择有效的治疗方案，提供有利的信息。另外，对于某些由于病原体引起的疾病，在人体样本中，可能会存在这样的游离核酸，即，它属于病原体基因组序列的一部分，这样可以确定人体已经被这些病原体侵染并且处于疾病的发生阶段。甚至有些个体样本中的游离核酸既包含病原体基因组的一部分序列，也包含人体基因组的某些序列，这样可以确定这些病原体基因组已经与人体基因组发生了整合重组。由此，可以判断个体的异常状态的阶段。根据本发明的一个实施例，人体样本中游离核酸的序列信息是通过除去人体样本中的细胞后，进行测序检测而获得的。由此，可以提高游离核酸的测序检测的精度和准确度，从而能够进一步有效地确定人体具有异常状态。 RNA or other type of nucleic acid. The inventors of the present invention found that under normal conditions, a small amount of nucleic acid enters into peripheral blood due to metabolism and becomes a free nucleic acid, and in an abnormal state, such as a cancer patient, the content of free nucleic acid is much higher than that under normal conditions. Free nucleic acid content. The inventors have found that in cancer patients, the amount of free nucleic acid (such as free radical DNA) depends on the biological characteristics of the tumor, that is, the degree of malignancy of the tumor cells, the degree of invasion, whether metastasis, disease progression, and the like. Therefore, it is possible to determine whether or not the human body has an abnormal state by analyzing the content of free nucleic acid in the human sample, for example, having a tumor disease, determining the progress stage of the tumor disease, the degree of invasion, and the like. Thus, to provide an effective treatment plan, provide favorable information. In addition, for certain diseases caused by pathogens, in human samples, there may be such free nucleic acids, ie, they are part of the pathogen genome sequence, which can confirm that the human body has been infected by these pathogens and is at the stage of disease. . Even some of the free nucleic acids in individual samples contain both a portion of the sequence of the pathogen genome and certain sequences of the human genome, which confirms that these pathogen genomes have been integrated and recombined with the human genome. Thereby, the stage of the abnormal state of the individual can be judged. According to an embodiment of the present invention, sequence information of free nucleic acid in a human sample is obtained by performing sequencing detection after removing cells in a human sample. Thereby, the accuracy and accuracy of the sequencing detection of the free nucleic acid can be improved, so that it is possible to further effectively determine that the human body has an abnormal state.

另外，根据本发明的实施例，对所述核酸进行测序检测之前，还可以包括步骤 400, 如图 3 所示，即利用探针捕获含有特定序列的核酸，然后对含有特定序列的核酸进行测序检测。由此，可以通过探针，预先对进行核酸序列分析的核酸进行筛选，从而能够进一步提高确定人体具有异常状态的方法的效率。另外，本发明的发明人惊奇地发现，通过此步骤，可以提高检测到与人体基因组发生整合的游离核酸的效率。 In addition, according to an embodiment of the present invention, before performing sequencing detection on the nucleic acid, step 400 may be further included, as shown in FIG. 3, that is, using a probe to capture a nucleic acid containing a specific sequence, and then sequencing the nucleic acid containing the specific sequence. Check Measurement. Thereby, the nucleic acid subjected to nucleic acid sequence analysis can be screened in advance by the probe, and the efficiency of the method for determining the abnormal state of the human body can be further improved. Further, the inventors of the present invention surprisingly found that by this step, the efficiency of detecting free nucleic acid integrated with the human genome can be improved.

本领域技术人员能够理解，所使用的探针类型可以根据检测的目的进行改变，即可以根据所期望的特定序列来选择所使用的探针类型。根据本发明的实施例，特定序列可以是外源核酸序列，也可以是含有突变位点的人基因组部分序列，也可以是含有修饰位点例如甲基化的人基因组部分序列。根据进一步的实施例，所述探针对于选自下列的至少一种是特异性的： HBV、 HPV、 EBV、幽门螺旋杆菌，其可以以包括多个探针的核酸探针集的形式提供，并且其具有以下特征：（1 )每个探针上具有 1 个或多个生物素标记的 dNTP; 和 / 或（2 )生物素标记的 dNTP在核酸探针集中的丰度为 1 : 6-1 : 2; 和 /或（3 )核酸探针集的全部核酸序列覆盖对应选自 HBV、 HPV、 EBV和幽门螺旋杆菌的至少一种病毒的基因组序列的 70%-100%。在另一优选例中，本发明的核酸探针集具有 1-20000个核酸探针；较佳地，核酸探针集具有 1000-5000个核酸探针；更佳地，核酸探针集具有 2500个核酸探针。在另一优选例中，生物素标记的 dNTP在核酸探针集中的丰度为 1 : 4。在另一优选例中，核酸探针集中，探针之间具有部分重叠。在另一优选例中，核酸探针集（在本文中有时也称为 "探针集"）中的探针长度为 100-500 bp; 较佳地，探针长度为 200-300 bp; 更佳地，探针长度为 250 bp。在另一优选例中，探针是以病毒基因组作为模板， PCR法扩增获得的，较佳地，扩增模板为乙型肝炎病毒（HBV )基因组、丙型肝炎病毒（HCV )基因组、艾滋病病毒（HIV )基因组、 ***瘤病毒（HPV )基因组，或其组合；更佳地，扩增模板为 B 型 HBV基因组和 /或 C型 HBV基因组。由此，可以有效地确定人体是否患有肝癌、 ***、鼻咽癌或胃癌。 Those skilled in the art will appreciate that the type of probe used can be varied depending on the purpose of the assay, i.e., the type of probe used can be selected based on the particular sequence desired. According to an embodiment of the present invention, the specific sequence may be an exogenous nucleic acid sequence, a human genome partial sequence containing a mutation site, or a human genome partial sequence containing a modification site such as methylation. According to a further embodiment, the probe is specific for at least one selected from the group consisting of: HBV, HPV, EBV, H. pylori, which may be provided in the form of a set of nucleic acid probes comprising a plurality of probes, And it has the following characteristics: (1) one or more biotin-labeled dNTPs per probe; and/or (2) biotin-labeled dNTPs have abundance in the nucleic acid probe set of 1: 6- The entire nucleic acid sequence of the 1:2; and/or (3) nucleic acid probe set covers 70%-100% of the genomic sequence corresponding to at least one virus selected from the group consisting of HBV, HPV, EBV and H. pylori. In another preferred embodiment, the nucleic acid probe set of the present invention has 1 to 20,000 nucleic acid probes; preferably, the nucleic acid probe set has 1000 to 5000 nucleic acid probes; more preferably, the nucleic acid probe set has 2,500 Nucleic acid probes. In another preferred embodiment, the abundance of the biotinylated dNTP in the nucleic acid probe set is 1:4. In another preferred embodiment, the nucleic acid probes are concentrated with partial overlap between the probes. In another preferred embodiment, the length of the probe in the set of nucleic acid probes (sometimes referred to herein as "probe sets") is from 100 to 500 bp; preferably, the length of the probe is from 200 to 300 bp; Preferably, the probe is 250 bp long. In another preferred embodiment, the probe is obtained by PCR amplification using a viral genome as a template. Preferably, the amplification template is a hepatitis B virus (HBV) genome, a hepatitis C virus (HCV) genome, and an AIDS virus. The viral (HIV) genome, the papillomavirus (HPV) genome, or a combination thereof; more preferably, the amplification template is the B-type HBV genome and/or the C-type HBV genome. Thereby, it is possible to effectively determine whether the human body has liver cancer, cervical cancer, nasopharyngeal cancer or gastric cancer.

另外，根据本发明的实施例，可以在对核酸进行测序之前，通过使用特定的探针除去某些含有特定序列的核酸，例如人类基因组中的共有序列，从而提高确定人类具有异常状态的方法的准确性。具体地，可以在对所述核酸进行测序检测之前，利用探针去除含有特定序列的核酸，然后对所述去除后剩余的核酸进行测序检测。这些用于除去含有特定序列的核酸的探针如同用于捕获含有特定序列的核酸的探针一样，它们的类型不受特别限制，可以为核酸、蛋白盾以及任何小分子，只要其能够特异性地结合特定的序列即可。另外，为了除去含有特定序列的核酸，所釆用的探针能够结合人体基因组中的共有序列，或者也可以是能够结合人体基因组中甲基化位点的抗体或者蛋白。可以根据具体的情况对所釆用的探针的类型，以及是需要对样本进行核酸捕获，还是需要进行特异性去除。 In addition, according to embodiments of the present invention, certain nucleic acid containing a specific sequence, such as a consensus sequence in the human genome, may be removed by using a specific probe prior to sequencing the nucleic acid, thereby improving a method for determining an abnormal state of a human being. accuracy. Specifically, a nucleic acid containing a specific sequence may be removed by a probe prior to sequencing detection of the nucleic acid, and then the remaining nucleic acid after the removal may be subjected to sequencing detection. These probes for removing a nucleic acid containing a specific sequence are the same as those for capturing a nucleic acid containing a specific sequence, and their types are not particularly limited, and may be a nucleic acid, a protein shield, and any small molecule as long as it is capable of specificity. It can be combined with a specific sequence. Further, in order to remove a nucleic acid containing a specific sequence, the probe to be used can bind to a consensus sequence in the human genome, or can be an antibody or protein capable of binding to a methylation site in the human genome. The type of probe to be used, depending on the specific situation, and whether nucleic acid capture is required for the sample, or whether specific removal is required.

在本发明的另一方面，本发明提供了一种用于确定人体具有异常状态的***，其可以有效地实施上述根据本发明实施例的确定人体具有异常状态的方法。根据本发明的实施例，参考图 4, 该***包括：核酸序列信息接收器 500、以及核酸序列信息分析器 600。其中，核酸序列信息接收器 500接收人体样本的核酸序列信息，核酸序列信息分析器 600与核酸序列信息接收器 500相连，并基于人体样本的核酸序列信息，确定人体是否具有异常状态。利用该***，可以有效地实施根据本发明实施例的确定人体具有异常状态的方案，其具有本发明实施例的确定人体具有异常状态的方法的全部优点，在此不再赘述。 In another aspect of the present invention, the present invention provides a system for determining an abnormal state of a human body, which can effectively implement the above-described method of determining an abnormal state of a human body according to an embodiment of the present invention. According to an embodiment of the present invention, referring to FIG. 4, the system includes: a nucleic acid sequence information receiver 500, and a nucleic acid sequence information analyzer 600. The nucleic acid sequence information receiver 500 receives the nucleic acid sequence information of the human body sample, and the nucleic acid sequence information analyzer 600 is connected to the nucleic acid sequence information receiver 500, and determines whether the human body has an abnormal state based on the nucleic acid sequence information of the human body sample. With the system, it is possible to effectively implement a scheme for determining an abnormal state of a human body according to an embodiment of the present invention, which has All the advantages of the method for determining the abnormal state of the human body in the embodiment of the present invention are not described herein again.

如前所述，根据本发明实施例，对核酸序列信息进行分析的方法不受特别限制，根据具体地示例，可以通过将核酸序列信息与人体正常状态的基因组序列、病原体的基因组序列、正常人群的基因组序列进行比对而确定人体是否具有异常状态。人体正常状态的基因组序列、病原体的基因组序列、正常人群的基因组序列的存放位置不受特别限制，可以存储在远程的数据库中。根据本发明的一个实施例，核酸序列信息分析器 500 内可以预存有选自下列的至少一种：人体正常状态的基因组序列、病原体的基因组序列、正常人群的基因组序列。由此，从而可以有效地对核酸序列进行分析，提高了用于确定人体具有异常状态的***的效率。根据进一步的实施例，所述病原体为选自 HBV、 HPV、 EBV、幽门螺旋杆菌的至少一种。由此，可以有效地确定人体是否患有肝癌、 ***、鼻咽癌或胃癌。通过将核酸序列信息与人体正常状态的基因组序列（即将同一身体在不同状态下的基因组信息）进行比对，可以确定人体在一段时间内的状态变化。另外，通过将人体样本的核酸序列信息与正常人群的基因组序列信息进行比对，可以获知与正常人相比的异常状态。 As described above, the method of analyzing the nucleic acid sequence information according to the embodiment of the present invention is not particularly limited, and according to a specific example, the nucleic acid sequence information can be obtained from the genomic sequence of the normal state of the human body, the genomic sequence of the pathogen, and the normal population. The genomic sequences are aligned to determine if the human body has an abnormal state. The genomic sequence of the human body, the genomic sequence of the pathogen, and the location of the genomic sequence of the normal population are not particularly limited and can be stored in a remote database. According to an embodiment of the present invention, at least one selected from the group consisting of: a genomic sequence of a human normal state, a genomic sequence of a pathogen, and a genomic sequence of a normal population may be pre-stored in the nucleic acid sequence information analyzer 500. Thereby, it is possible to efficiently analyze the nucleic acid sequence and improve the efficiency of the system for determining the abnormal state of the human body. According to a further embodiment, the pathogen is at least one selected from the group consisting of HBV, HPV, EBV, and H. pylori. Thereby, it is possible to effectively determine whether the human body has liver cancer, cervical cancer, nasopharyngeal cancer or gastric cancer. By comparing the nucleic acid sequence information with the genomic sequence of the normal state of the human body (that is, the genomic information of the same body in different states), it is possible to determine the state change of the human body over a period of time. Further, by comparing the nucleic acid sequence information of the human sample with the genomic sequence information of the normal population, an abnormal state compared with a normal person can be known.

根据本发明的实施例，进行分析的核酸序列信息的来源不受特别限制。根据本发明的一个实施例，参考图 5 , 本发明的用于确定人体具有异常状态的***可以进一步包括核酸序列检测装置 700。该核酸序列检测装置 700与核酸序列信息接收器 500相连，用于对述人体样本进行核酸序列检测获得核酸序列信息并输送至核酸序列信息接收器 500 ,进而进行分析和确定人体是否存在异常状态。由此，可以直接对核酸进行序列检测，并且输送至核酸序列接收器 500, 进而进行核酸序列分析，从而确定人体是否具有异常状态从而提高了确定人体具有异常状态的效率。根据进一步的实施例，所述核酸序列检测装置借助第二代测序技术或第三代测序技术。由此，可以高效地对人体样本进行核酸序列进行检测，并且能够实现高通量深度测序。本发明的发明人发现基于第二代测序技术和第三代测序技术的高效、高精度的性盾，可以实现对人体样本核酸序列信息的高效、高精度检测，能够非常灵敏地对人体样本中痕量的核酸进行检测。 According to an embodiment of the present invention, the source of the nucleic acid sequence information to be analyzed is not particularly limited. According to an embodiment of the present invention, referring to FIG. 5, the system for determining an abnormal state of a human body of the present invention may further include a nucleic acid sequence detecting device 700. The nucleic acid sequence detecting device 700 is connected to the nucleic acid sequence information receiver 500 for performing nucleic acid sequence detection on the human body sample to obtain nucleic acid sequence information and transporting it to the nucleic acid sequence information receiver 500, thereby performing analysis and determining whether the human body has an abnormal state. Thereby, the nucleic acid can be directly subjected to sequence detection and transported to the nucleic acid sequence receiver 500, thereby performing nucleic acid sequence analysis to determine whether the human body has an abnormal state, thereby improving the efficiency of determining that the human body has an abnormal state. According to a further embodiment, the nucleic acid sequence detecting device utilizes a second generation sequencing technique or a third generation sequencing technique. Thereby, the nucleic acid sequence of the human sample can be efficiently detected, and high-throughput deep sequencing can be realized. The inventors of the present invention have found that an efficient and high-precision shield based on the second-generation sequencing technology and the third-generation sequencing technology can realize efficient and high-precision detection of nucleic acid sequence information of human samples, and can be very sensitive to human samples. Trace amounts of nucleic acid are detected.

根据本发明的一个实施例，参考图 6, 本发明的用于确定人体具有异常状态的***还可以进一步包括游离核酸捕获装置 800,该游离核酸捕获装置 800与核酸序列检测装置 700相连，并且游离核酸捕获装置 800设置有探针，这些探针适于捕获含有特定序列的核酸，并且将含有特定序列的核酸输送至核酸序列检测装置 700进行核酸序列检测。由此，对所述核酸进行测序检测之前，利用探针捕获含有特定序列的核酸，然后对所述含有特定序列的核酸进行测序检测。由此，可以通过探针，预先对进行核酸序列分析的核酸进行筛选，从而能够进一步提高确定人体具有异常状态的方法的效率。另外，本发明的发明人惊奇地发现，通过此步骤，可以提高检测到与人体基因组发生整合的游离核酸的效率。 According to an embodiment of the present invention, referring to FIG. 6, the system for determining an abnormal state of a human body of the present invention may further include a free nucleic acid capture device 800 connected to the nucleic acid sequence detecting device 700 and free The nucleic acid capture device 800 is provided with probes adapted to capture a nucleic acid containing a specific sequence, and deliver the nucleic acid containing the specific sequence to the nucleic acid sequence detecting device 700 for nucleic acid sequence detection. Thus, prior to sequencing the nucleic acid, a nucleic acid containing a specific sequence is captured using a probe, and then the nucleic acid containing the specific sequence is subjected to sequencing detection. Thereby, the nucleic acid subjected to nucleic acid sequence analysis can be screened in advance by the probe, whereby the efficiency of the method for determining the abnormal state of the human body can be further improved. Further, the inventors of the present invention surprisingly found that by this step, the efficiency of detecting free nucleic acid integrated with the human genome can be improved.

本领域技术人员能够理解，所使用的探针类型可以根据检测的目的进行选择，即可以根据所期望的特定序列来选择所使用的探针类型。根据本发明的实施例，特定序列可以是外源核酸序列，也可以是含有突变位点的人基因组部分序列，也可以是含有修饰位点例如甲基化的人基因组部分序列。根据进一步的实施例，所述探针对于选自下列的至少一种是特异性的： HBV、 HPV、 EBV、幽门螺旋杆菌，其可以以包括多个探针的核酸探针集的形式提供，并且其具有以下特征：（1 )每个探针上具有 1 个或多个生物素标记的 dNTP; 和 / 或（2 )生物素标记的 dNTP在核酸探针集中的丰度为 1 : 6-1 : 2; 和 /或（3 )核酸探针集的全部核酸序列覆盖对应选自 HBV、 HPV、 EBV和幽门螺旋杆菌的至少一种病毒的基因组序列的 70%-100%。在另一优选例中，本发明的核酸探针集具有 1-20000个核酸探针；较佳地，核酸探针集具有 1000-5000个核酸探针；更佳地，核酸探针集具有 2500个核酸探针。在另一优选例中，生物素标记的 dNTP在核酸探针集中的丰度为 1 : 4。在另一优选例中，核酸探针集中，探针之间具有部分重叠。在另一优选例中，核酸探针集（在本文中有时也称为 "探针集"）中的探针长度为 100-500 bp; 较佳地，探针长度为 200-300 bp; 更佳地，探针长度为 250 bp。在另一优选例中，探针是以病毒基因组作为模板， PCR法扩增获得的，较佳地，扩增模板为乙型肝炎病毒（HBV )基因组、丙型肝炎病毒（HCV )基因组、艾滋病病毒（HIV )基因组、 ***瘤病毒（HPV )基因组，或其组合；更佳地，扩增模板为 B 型 HBV基因组和 /或 C型 HBV基因组。由此，可以有效地确定人体是否患有肝癌、 ***、鼻咽癌或胃癌。根据本发明的具体实施例，可以釆用 HPV的 E1基因区域的特异性序列作为探针。由此，能够高效准确地确定患者体内的 HPV是否已经与患者体内的基因组发生整合，从而判断个体是否患有***。根据本发明的一些具体示例，可以釆用 HPV的全长序列作为探针，由此，能够高效地准确地确定患者体内的 HPV是否已经与患者体内的基因组发生整合，从而判断个体宫颈病变程度。另外，根据本发明的具体示例，可以釆用 HBV的 X基因和 /或 C基因中的特异性序列作为探针，由此，能够高效地准确地确定患者体内的 HBV 是否已经与患者体内的基因组发生整合，从而判断个体是否患有肝癌。根据本发明的一些实施例，可以釆用 HBV的全长基因区域的特异性序列作为探针，由此，能够高效地准确地确定患者体内的 HBV是否已经与患者体内的基因组发生整合，从而判断个体肝炎病变程度。 Those skilled in the art will appreciate that the type of probe used can be selected based on the purpose of the assay, i.e., the type of probe used can be selected based on the particular sequence desired. According to an embodiment of the present invention, the specific sequence may be an exogenous nucleic acid sequence, a human genome partial sequence containing a mutation site, or a human genome partial sequence containing a modification site such as methylation. According to a further embodiment, the probe is for at least one selected from the group consisting of Specific: HBV, HPV, EBV, H. pylori, which may be provided in the form of a set of nucleic acid probes comprising a plurality of probes, and which have the following characteristics: (1) 1 or more on each probe Biotin-labeled dNTP; and/or (2) biotin-labeled dNTPs have abundance in the nucleic acid probe set of 1: 6-1: 2; and/or (3) full nucleic acid sequence coverage of the nucleic acid probe set 70%-100% of the genomic sequence corresponding to at least one virus selected from the group consisting of HBV, HPV, EBV, and H. pylori. In another preferred embodiment, the nucleic acid probe set of the present invention has 1 to 20,000 nucleic acid probes; preferably, the nucleic acid probe set has 1000 to 5000 nucleic acid probes; more preferably, the nucleic acid probe set has 2,500 Nucleic acid probes. In another preferred embodiment, the abundance of the biotinylated dNTP in the concentration of the nucleic acid probe is 1:4. In another preferred embodiment, the nucleic acid probes are concentrated with partial overlap between the probes. In another preferred embodiment, the length of the probe in the set of nucleic acid probes (sometimes referred to herein as "probe sets") is from 100 to 500 bp; preferably, the length of the probe is from 200 to 300 bp; Preferably, the probe is 250 bp long. In another preferred embodiment, the probe is obtained by PCR amplification using a viral genome as a template. Preferably, the amplification template is a hepatitis B virus (HBV) genome, a hepatitis C virus (HCV) genome, and an AIDS virus. The viral (HIV) genome, the papillomavirus (HPV) genome, or a combination thereof; more preferably, the amplification template is the B-type HBV genome and/or the C-type HBV genome. Thereby, it is possible to effectively determine whether the human body has liver cancer, cervical cancer, nasopharyngeal cancer or gastric cancer. According to a specific embodiment of the present invention, a specific sequence of the E1 gene region of HPV can be used as a probe. Thereby, it is possible to efficiently and accurately determine whether or not the HPV in the patient has been integrated with the genome in the patient, thereby judging whether the individual has cervical cancer. According to some specific examples of the present invention, the full-length sequence of HPV can be used as a probe, thereby enabling efficient and accurate determination of whether or not HPV in a patient has been integrated with the genome in the patient, thereby judging the degree of cervical lesion in the individual. Further, according to a specific example of the present invention, a specific sequence in the X gene and/or the C gene of HBV can be used as a probe, whereby it is possible to efficiently and accurately determine whether or not the HBV in the patient has been associated with the genome in the patient. Integration occurs to determine whether an individual has liver cancer. According to some embodiments of the present invention, a specific sequence of a full-length gene region of HBV can be used as a probe, thereby enabling efficient and accurate determination of whether or not HBV in a patient has been integrated with a genome in a patient, thereby judging The degree of individual hepatitis lesions.

另外，根据本发明的实施例，还可以釆用这样的探针，其能够除去含有特定序列的核酸，从而可以在对核酸进行测序之前，通过使用特定的探针除去某些含有特定序列的核酸，例如人类基因组中的共有序列，从而提高确定人类具有异常状态的方法的准确性。具体地，可以在对所述核酸进行测序检测之前，利用探针去除含有特定序列的核酸，然后对所述去除后剩余的核酸进行测序检测。这些用于除去含有特定序列的核酸的探针如同用于捕获含有特定序列的核酸的探针一样，它们的类型不受特别限制，可以为核酸、蛋白盾以及任何小分子，只要其能够特异性地结合特定的序列即可。另外，为了除去含有特定序列的核酸，所釆用的探针能够结合人体基因组中的共有序列，或者也可以是能够结合人体基因组中甲基化位点的抗体或者蛋白。可以根据具体的情况对所釆用的探针的类型，以及是需要对样本进行核酸捕获，还是需要进行特异性去除。为了方便理解，下面提供具体的实施例，对本发明的技术方案进行解释，需要说明的是，这些实施例仅仅是为了说明目的，而不以任何方式限制本发明的范围。除非特别说明，实施例中未注明具体条件的，均为按照常规条件或制造商建议的条件进行。下列实施例中，所用试剂或仪器未注明生产厂商的，均为可以通过市购获得的常规产品。所使用的测序用的接头和标签序列（ Index )来源于 Illumina公司的 Multiplexing Sample Preparation Oligonutide Kit。实施例 1: In addition, according to an embodiment of the present invention, a probe capable of removing a nucleic acid containing a specific sequence can be used, so that certain nucleic acids containing a specific sequence can be removed by using a specific probe before sequencing the nucleic acid. For example, consensus sequences in the human genome, thereby improving the accuracy of methods for determining abnormal states in humans. Specifically, a nucleic acid containing a specific sequence may be removed using a probe before sequencing detection of the nucleic acid, and then the remaining nucleic acid after the removal may be subjected to sequencing detection. These probes for removing a nucleic acid containing a specific sequence are the same as those for capturing a nucleic acid containing a specific sequence, and their types are not particularly limited, and may be a nucleic acid, a protein shield, and any small molecule as long as it is capable of specificity. It can be combined with a specific sequence. In addition, in order to remove a nucleic acid containing a specific sequence, the probe to be used can bind to a consensus sequence in the human genome, or can be an antibody or protein capable of binding to a methylation site in the human genome. The type of probe to be used, depending on the specific situation, and whether nucleic acid capture is required for the sample, or whether specific removal is required. For the sake of understanding, the specific embodiments are provided below, and the technical solutions of the present invention are explained. It is to be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise stated, the specific conditions are not specified in the examples, and are carried out according to the general conditions or the conditions recommended by the manufacturer. In the following examples, The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained commercially. The linker and tag sequence (Index) used for sequencing was derived from Illumina's Multiplexing Sample Preparation Oligonutide Kit. Example 1:

1. 样本文库制备 Sample library preparation

1.1 样本来源 1.1 Sample source

样品的来源为同一患者的肝癌组织，且此患者肝癌组织有全基因组测序信息。 The source of the sample is liver cancer tissue of the same patient, and the liver cancer tissue of this patient has whole genome sequencing information.

1.2样本文库制备 1.2 sample library preparation

文库构建按照 Illumina公司的标准文库制备流程说明书（ Paired-End Sample Preparation Library construction according to Illumina's standard library preparation procedure ( Paired-End Sample Preparation

Guide )进行构建，具体方法如下： Guide ) to build, the specific method is as follows:

釆用 Covaris s2打断基因组 DNA, 末端补平修复，末端加 A, 加入接头，建库过程所用的接头序列为： Co Interrupt the genomic DNA with Covaris s2, end-filling and repairing, adding A to the end, adding the linker, and the linker sequence used in the database construction process is:

5 ' -GATCGGAAGAGC AC ACGTCTGAACTCC AGTC AC-3 ' ( SEQ ID NO: 1 ); 5 ' -GATCGGAAGAGC AC ACGTCTGAACTCC AGTC AC-3 ' ( SEQ ID NO: 1 );

5 ' -TAC ACTCTTTCCCTAC ACGACGCTCTTCCGATCT-3 ' ( SEQ ID NO: 2 ) 5 ' -TAC ACTCTTTCCCTAC ACGACGCTCTTCCGATCT-3 ' ( SEQ ID NO: 2 )

对加入接头的片段进行 PCR, 得到样本文库，所构建的文库带有 Index标签序列，其中 Index序列如下： The fragment added to the adaptor is subjected to PCR to obtain a sample library, and the constructed library carries an Index tag sequence, wherein the Index sequence is as follows:

5'-CAAGC, 5'-CAAGC,

TCTTCCGATCT-3' ( SEQ ID NO: 3 ); TCTTCCGATCT-3' (SEQ ID NO: 3);

TCTTCCGATCT-3' ( SEQ ID NO: 4 )。 TCTTCCGATCT-3' (SEQ ID NO: 4).

将样本分别做成两种片段大小文库，对构建文库片段进行片段大小检测，主带为 170 bp 左右和 800 bp左右。 2. 制备 HBV探针 The samples were made into two fragment size libraries, and the fragment size of the constructed library fragments was detected. The main bands were about 170 bp and about 800 bp. 2. Preparation of HBV probe

2.1 引物设计 2.1 Primer design

本实施例中，所设计的引物为： In this embodiment, the designed primers are:

PI : TTTTTCACCTCTGCCTAATCA ( SEQ ID NO: 5 ); PI : TTTTTCACCTCTGCCTAATCA ( SEQ ID NO: 5 );

P2: AAAAAGTTGCATGGTGCTGG ( SEQ ID NO: 6 ) P2: AAAAAGTTGCATGGTGCTGG (SEQ ID NO: 6)

2.2 PCR反应体系 2.2 PCR reaction system

PCR反应体系见表 1。表 1

The PCR reaction system is shown in Table 1. Table 1

(注： dNTP中 biotin-dNTP与普通 dNTP的比例是 1 : 4, 总浓度为 2.5 mM ) (Note: The ratio of biotin-dNTP to normal dNTP in dNTP is 1 : 4, total concentration is 2.5 mM)

2.3 PCR反应条件 2.3 PCR reaction conditions

PCR反应在 AB-9700PCR仪上进行，反应程序见表 2。 The PCR reaction was carried out on an AB-9700 PCR machine. The reaction procedure is shown in Table 2.

表 2 Table 2

2.4 PCR产物纯化及电泳检测 2.4 PCR product purification and electrophoresis detection

反应结束后，釆用 1%琼脂糖凝胶电泳检测 PCR产物，并用 1.2-1.5倍体积 AMPURE BEADS纯化，釆用 80 μ1水溶解。然后釆用 25 OMinElute PCR Purification Kit纯化，釆用 60 μΐ水溶解。其中， PCR产物的 1%琼脂糖凝胶电泳检测结果见图 7, 结果表明，扩增并纯化出了大小约 3.2 Κ的 HBV的片段。 After the reaction, the PCR product was detected by 1% agarose gel electrophoresis, purified with 1.2-1.5 volumes of AMPURE BEADS, and dissolved in 80 μl of water. It was then purified using 25 OMinElute PCR Purification Kit and dissolved in 60 μM water. Among them, the results of 1% agarose gel electrophoresis of the PCR product are shown in Fig. 7. The results showed that a fragment of HBV of about 3.2 Κ was amplified and purified.

, HBV基因组序列 ( Hepatitis B virus serotype adr, complete genome )如下： The HBV genome sequence (Hepatitis B virus serotype adr, complete genome ) is as follows:

 

2.5 PCR产物片段化 2.5 PCR product fragmentation

将经过纯化的 PCR产物全部转移至 Covaris打断小管并补加 TE緩冲液至总体积为 80 ^(Nanodrop检测其总量为 5 g), Covaris S2仪器（基因有限公司）进行打断，由此，获得片段化产物，其中打断条件见表 3。 The purified PCR products were all transferred to Covaris interrupted tubules and supplemented with TE buffer to a total volume of 80 ^ (Nanodrop was detected as a total of 5 g), and Covaris S2 instrument (Gene Co., Ltd.) was interrupted by Thus, a fragmented product was obtained in which the breaking conditions are shown in Table 3.

表 3

table 3

2.6 片段化产物电泳检测 2.6 Fragmentation product electrophoresis detection

2%琼脂糖凝胶电泳检测片段化产物的大小，结果见图 8 , 结果显示片段化产物的主带在 250-300 bp, 表明所获得的片段化产物即可用作杂交的探针。 The size of the fragmented product was detected by 2% agarose gel electrophoresis. The results are shown in Fig. 8. The results showed that the main band of the fragmented product was 250-300 bp, indicating that the obtained fragmented product could be used as a probe for hybridization.

2.7探针保存 2.7 probe save

使用 MinElute PCR Purification Kit纯化片段产物，溶于 40 μΐ緩冲液中，用 Nanodrop 仪检测探针 DNA的浓度 , 使得探针的浓度为 120 ng/μΐ左右。得到的探针可以保存在 -20 C 或 -80°C。 The fragment product was purified using a MinElute PCR Purification Kit, dissolved in 40 μM buffer, and the probe DNA concentration was measured with a Nanodrop instrument so that the probe concentration was about 120 ng/μΐ. The resulting probe can be stored at -20 C or -80 °C.

3. HBV探针与样本文库进行杂交 3. Hybridization of the HBV probe to the sample library

3.1 探针变性 3.1 Probe denaturation

探针使用前必须 95 °C变性 10分钟，然后迅速放于水上冷却形成单链。 The probe must be denatured at 95 °C for 10 minutes before use and then rapidly placed on water to form a single strand.

3.2 选用已确定的整合文库，文库用量为 1 g, 探针用量为 600 ng ( Nanodrop定量），加入接头封闭分子，接头封闭分子的量与文库量的比值为 l nmol: l g, 标签封闭分子与文库量的比值为 1 nmol: 1 μg. 3.2 Using the identified integrated library, the amount of the library is 1 g, the amount of the probe is 600 ng (Quantification of Nanodrop), and the blocking molecule is added. The ratio of the amount of the blocking molecule to the amount of the library is l nmol: lg, the tag blocking molecule and The ratio of library amounts is 1 nmol: 1 μg.

接头封闭分子序列为： The closed molecular sequence of the linker is:

5'-AATGATACGGCG. 5'-AATGATACGGCG.

ATCT-3' ( SEQ ID NO: 8 ); ATCT-3' (SEQ ID NO: 8);

标签封闭分子序列为： The tag blocking molecular sequence is:

5'-AAGCAGAAGAC( 5'-AAGCAGAAGAC(

CTTCCGATCT-3' ( SEQ ID NO: 9 ); CTTCCGATCT-3' ( SEQ ID NO: 10 )。 CTTCCGATCT-3' (SEQ ID NO: 9); CTTCCGATCT-3' (SEQ ID NO: 10).

在一个 1.5 mL的 EP管中加入 1 的待杂交文库， 1 nmol接头封闭分子， 1 nmol标签封闭分子， 5 g Cot DNA。盖好管盖，用千净的 50 ml注射器针在分装的 EP管盖上戳一个孔，然后置于 60°C旋蒸仪中蒸千。使用新的离心管管盖替换戳孔的管盖，并做好标记。 EP 管中分别加入 EZ杂交***中的两种试剂： 2 X SC Hybridiation Buffer杂交緩冲液 7.5 μΐ和 1 X SC Hybridiation Component A 3 μΐ, 然后 95 °C变性 10分钟，在上述杂交混合物加入自制探针 600 ng, 探针体积共 5 μ1。震荡混勾后置于离心机上全速离心 10秒，并将样品全部转移到 200 μΙ ΡΟ 小管中。 Add 1 library to be hybridized, 1 nmol linker blocking molecule, 1 nmol tag blocking molecule, 5 g Cot DNA in a 1.5 mL EP tube. Cover the cap and poke a hole in the dispensed EP tube cap with a clean 50 ml syringe needle and place it in a 60 °C rotary evaporator. Replace the capped cap with a new centrifuge tube cap and mark it. Two reagents in the EZ hybridization system were separately added to the EP tube: 2 X SC Hybridiation Buffer hybridization buffer 7.5 μΐ and 1 X SC Hybridiation Component A 3 μΐ, then denatured at 95 °C for 10 minutes, and the self-exploration was added to the above hybridization mixture. The needle is 600 ng and the probe volume is 5 μl. After shaking, mix and place on a centrifuge for 10 seconds at full speed, and transfer all the samples to a 200 μΙ ΡΟ tube.

杂交混合物中含有的成分见表 4。 The components contained in the hybridization mixture are shown in Table 4.

表 4 Table 4

将 200 μΐ PCR小管放置于 PCR仪上， 47 °C条件下杂交 24 h。 A 200 μΐ PCR vial was placed on the PCR machine and hybridized for 24 h at 47 °C.

4. 杂交后洗脱 4. Hybrid after hybridization

4.1 准备链霉亲和素磁珠 (Invitrogen M280) 4.1 Preparing Streptavidin Magnetic Beads (Invitrogen M280)

提前从水箱中拿出链霉亲和素磁珠；漩涡震荡磁珠 1 min, 使其充分混匀；在 1.5 mL 的 EP管中加入 ΙΟΟ μΙ磁珠；将 ΕΡ管置于磁力架上至液体澄清，用移液器小心地去除上清；保持 ΕΡ管在磁力架上，加入 200 μΐ ( 2倍体积）的结合緩冲液（购于 Agilent公司）；从磁力架上取下 EP管，漩涡震荡 10 s, 使其混匀；将 EP管重新放回磁力架至液体澄清，用移液器小心地去除上清；重复清洗两次；用 100 μΐ 的 Agilent 结合緩冲液 r悬浮磁珠；将其转入 0.2 ml的小管中；用磁力架结合磁珠（将小管靠到磁力架上），直到液体澄清，用移液器小心去除上清；现在磁珠可以用来结合捕获的 DNA了。 Remove the streptavidin magnetic beads from the water tank in advance; vortex the magnetic beads for 1 min, and mix them thoroughly; add ΙΟΟ μΙ magnetic beads to the 1.5 mL EP tube; place the manifold on the magnetic stand to the liquid Clarify, carefully remove the supernatant with a pipette; keep the manifold on the magnetic stand, add 200 μΐ (2 volumes) of binding buffer (purchased from Agilent); remove the EP tube from the magnetic stand, swirl Shake for 10 s, mix thoroughly; place the EP tube back into the magnetic stand until the liquid is clear, carefully remove the supernatant with a pipette; repeat the wash twice; suspend the magnetic beads with 100 μΐ of Agilent Binding Buffer r; Transfer it into a 0.2 ml tube; use a magnetic stand to bind the beads (put the tube to the magnetic stand) until the liquid is clear, carefully remove the supernatant with a pipette; now the beads can be used to bind the captured DNA. .

4.2 将捕获到的 DNA结合到链霉素磁珠上 4.2 Bind the captured DNA to the streptomycin magnetic beads

将杂交混合物吸出来，加到上步准备好的磁珠中；用移液器吹打 10次混匀；将小管放在 PCR仪上 47°C孵育 45 min (每隔 15 min拿出来漩涡震荡 3 s以防止磁珠沉淀)；孵育 45 min 后，将混合物从 0.2 mL的小管中转入 1.5 mL的 EP管中。 The hybridization mixture was aspirated and added to the magnetic beads prepared in the previous step; the mixture was pipetted 10 times with a pipette; the tube was placed on a PCR machine and incubated at 47 ° C for 45 min (the vortex was shaken every 15 min 3 s to prevent precipitation of magnetic beads); after incubation for 45 min, the mixture was transferred from a 0.2 mL vial into a 1.5 mL EP tube.

4.3 洗涤结合了捕获 DNA的链霉亲和素磁珠 4.3 Washing the streptavidin magnetic beads combined with the captured DNA

1 )将 EP管置于磁力架上至液体澄清，用移液器小心地去除上清； 1) Place the EP tube on the magnetic stand until the liquid is clear, and carefully remove the supernatant with a pipette;

2 )加 100 μL预热到 47°C的 1 χ清洗緩冲液 I； 2) Add 100 μL of 1 χ Wash Buffer I preheated to 47 ° C;

3 )漩涡震荡 10 s, 使其混匀； 4 )将 EP管置于磁力架上至液体澄清，用移液器小心地去除上清； 3) vortex for 10 s, mix it; 4) Place the EP tube on the magnetic stand until the liquid is clear, and carefully remove the supernatant with a pipette;

5)从磁力架上取下 EP管，加入 200 μΐ预热到 47°C的 1 χ严谨清洗緩冲液，用移液器吹打混匀 10次（该步操作应迅速以尽量使管中液体温度不低于 47°C ); 5) Remove the EP tube from the magnetic stand, add 200 μΐ of 1 χ rigorous cleaning buffer preheated to 47 ° C, and mix by pipetting 10 times (this step should be done quickly to try to make the liquid in the tube The temperature is not lower than 47 ° C);

6 ) 47°C孵育 5 min; 6) Incubate at 47 ° C for 5 min;

7 )重复步骤 5 ) -7 ), 总共用 1 严谨清洗緩冲液洗两次； 7) Repeat steps 5) -7), total 1 wash twice with rigorous wash buffer;

8 )将 EP管置于磁力架上至液体澄清，用移液器小心地去除上清； 8) Place the EP tube on the magnetic stand until the liquid is clear, and carefully remove the supernatant with a pipette;

9 )加 200 μL室温下放置的 1 χ清洗緩冲液 I (不用 47°C预热的），漩涡震荡 2 min, 使其混匀； 9) Add 200 μL of 1 χ Wash Buffer I (not preheated at 47 °C) at room temperature, vortex for 2 min, and mix it;

10 )将 EP管置于磁力架上至液体澄清，用移液器小心地去除上清； 10) Place the EP tube on the magnetic stand until the liquid is clear, and carefully remove the supernatant with a pipette;

11 )加 200 μL室温下放置的 1 χ清洗緩冲液 II, 漩涡震荡 1 min, 使其混匀； 11) Add 200 μL of 1 χ Wash Buffer II at room temperature, vortex for 1 min, and mix well;

12 )将 EP管置于磁力架上至液体澄清，用移液器小心的去除上清； 12) Place the EP tube on the magnetic stand until the liquid is clear, and carefully remove the supernatant with a pipette;

13 ) 加 200 μ 室温下放置的 1 χ清洗緩冲液 III, 漩涡震荡 30 s, 使其混匀； 13) Add 200 μl of Wash Buffer III at room temperature, vortex for 30 s, and mix well;

14) 将 EP管置于磁力架上至液体澄清，用移液器小心地去除上清； 14) Place the EP tube on the magnetic stand until the liquid is clear, and carefully remove the supernatant with a pipette;

15)从磁力架上取下 EP管，加入 76 μΐ超纯水（不用将 DNA从磁珠上洗脱下来，可以直接进行 PCR, 取样 35 μΐ进行后面的 PCR反应）。 15) Remove the EP tube from the magnetic stand and add 76 μM of ultrapure water (you can directly perform PCR without sampling the DNA from the magnetic beads, and sample 35 μΐ for subsequent PCR reaction).

5. PCR反应 5. PCR reaction

预先从 -20°C保存的试剂盒中取出 PFX聚合酶 (购于 Invirtogen公司）， PFX反应緩冲液 (10 X ) , dNTP(10 mM)。引物序列为： PFX polymerase (purchased from Invirtogen), PFX reaction buffer (10 X ), and dNTP (10 mM) were previously taken from the kit stored at -20 °C. The primer sequence is:

PCR Flowcell-Primer F ( 10 pm/μΐ ): PCR Flowcell-Primer F ( 10 pm/μΐ ):

AATGATACGGCGACCACCGAGATC ( SEQ ID NO: 11 )； AATGATACGGCGACCACCGAGATC (SEQ ID NO: 11);

PCR Flowcell-Primer R ( 10 pm/μΐ ): PCR Flowcell-Primer R ( 10 pm/μΐ ):

CAAGCAGAAGACGGCATACGA ( SEQ ID NO: 12 )。 CAAGCAGAAGACGGCATACGA (SEQ ID NO: 12).

在 PCR小管上，每孔按照表格 5配置 PCR反应体系。 On the PCR vial, configure the PCR reaction system according to Table 5 for each well.

表 5 table 5

反应条件见表 6。表 6 The reaction conditions are shown in Table 6. Table 6

PCR结束后，每个样品都用 1.5倍体积的 Amprue Beads纯化，回收的 PCR产物溶于 30 μΐ超纯水中， Nanodrop 1000测浓度。 6. PCR产物上机测序 After the end of the PCR, each sample was purified using 1.5 volumes of Amprue Beads, and the recovered PCR product was dissolved in 30 μl of ultrapure water and measured by Nanodrop 1000. 6. PCR product sequencing

上述纯化后的 PCR产物经 2100 Bioanalyzer (Agilent)确定大小及***片段大小见图 9和图 10, 纯化产物大小分别为 271 bp和 876 bp, QPCR精确定量后上机测序。在本实施例中，上机测序按照 Illumina/Solexa官方公布的 c-Bot和 HISEQ2000Hiseq 2000说明书进行操作。 7. 信息分析 The purified PCR product was determined by 2100 Bioanalyzer (Agilent) and the size of the insert was shown in Figure 9 and Figure 10. The purified product was 271 bp and 876 bp, respectively. QPCR was accurately quantified and sequenced. In this example, the sequencing of the upper machine was carried out in accordance with the specifications of c-Bot and HISEQ2000Hiseq 2000 published by Illumina/Solexa. 7. Information analysis

将下机数据除去重复和被接头污染的 reads,统计下机数据的基本信息（文库长度； reads 长度； reads条数；碱基数；重复率等）；分别截取 PE的两条 reads 前面的 50 bp碱基，形成一对长为 50 bp新 reads,即 PE50 reads„将新的 PE50 reads运用 soap比对软件（ -r 1 -v 2 ) 分别与人的参考序列（hgl9)和 HBV各种参考序列进行比对，从比对结果中挑选出一条 read 比到人的参考序列并且另一条比对到 HBV参考序列的一对 reads; 这样的 reads很有可能跨过 HBV***的位点；统计这部分 reads比对信息，找到在人类基因组的***热点区域。 Remove the duplicate data and the reader contaminated by the linker, and count the basic information of the machine data (library length; reads length; number of reads; number of bases; repetition rate, etc.); respectively intercept 50 of the two reads of PE The bp base forms a pair of new reads of 50 bp long, ie PE50 reads „ use the new PE50 reads using the soap comparison software (-r 1 -v 2 ) with the human reference sequence (hgl9) and various references to HBV The sequences are aligned, and a pair of reads whose read is compared to the human reference sequence and the other aligned to the HBV reference sequence are selected from the alignment results; such reads are likely to cross the HBV insertion site; Part of the reads alignment information is found in the insertion hotspot region of the human genome.

杂交结果见表 7。 The results of the hybridization are shown in Table 7.

表 7 Table 7

表 7的结果为釆用上机数据得到结果，样本 L-170, L-800, Genome均来自同一肝癌样本， L-170为***片段 170 bp, L-800为 800bp文库， genome为全基因组测序。从表 7中可以得出自制探针对于捕获基因片段的准确性，以及片段长度的影响。通过本发明的方法完全可以得到稳定以及可靠的位点，且所需数据量仅为全基因组测序数据的 1%左右。实施例 2: 对***患者外周血样品进行全基因组分析 The results in Table 7 are the results obtained by using the data on the machine. The samples L-170, L-800, Genome are all from the same liver cancer. In this case, L-170 is an insert of 170 bp, L-800 is an 800 bp library, and genome is whole genome sequencing. The accuracy of the self-made probe for capturing the gene fragment, as well as the effect of fragment length, can be derived from Table 7. Stable and reliable sites are fully available by the method of the present invention, and the amount of data required is only about 1% of the genome-wide sequencing data. Example 2: Whole genome analysis of peripheral blood samples from patients with cervical cancer

1、 DN A提取及测序 1. DN A extraction and sequencing

根据常规方法，对***患者进行静脉取血，得到***患者的外周血样本，通过离心得到血浆样本。按照 Tiangen Micro Kit (DP316) 量基因组操作流程从血浆样本提取 DNA,分别用 Qubit ( Invitrogen , the Quant-iT™ dsDNAHS Assay Kit )定量，所提取的 DNA 总量分别为 5 ~ 50 ng。 According to a conventional method, a patient who has cervical cancer is subjected to venous blood sampling to obtain a peripheral blood sample of a patient with cervical cancer, and a plasma sample is obtained by centrifugation. DNA was extracted from plasma samples according to the Tiangen Micro Kit (DP316) genome-wide protocol and quantified using Qubit (Invitrogen, the Quant-iTTM dsDNAHS Assay Kit). The total amount of DNA extracted was 5 to 50 ng.

将所提取到的 DNA , 分别按照制造商提供的标准建库规程（参见 http://www.illumina.com/提供的 Illumina标准建库说明书）建立 DNA文库。筒言之，在 DNA 分子两端加上测序用的接头，并被加上不同的标签序列，然后与测序芯片表面互补接头杂交，使核酸分子成簇生长，然后在 Illumina HiSeq 2000上通过 100轮深度测序循环，得到长度为 lOObp的 DNA片段序列。本实施例中，对于获自肿瘤病人外周血的 DNA样本分批按照制造商提供的操作说明书（参见 Illumina官方公布说明书）进行上机测序操作。 The extracted DNA was constructed according to the standard library-building procedures provided by the manufacturer (see the Illumina standard library specification provided at http://www.illumina.com/). In other words, a linker for sequencing is added to both ends of the DNA molecule, and a different tag sequence is added, and then hybridized with a complementary linker on the surface of the sequencing chip to cluster the nucleic acid molecules, and then pass through 100 rounds on the Illumina HiSeq 2000. The deep sequencing cycle yielded a DNA fragment sequence of length lOObp. In the present example, the DNA samples obtained from the peripheral blood of the tumor patient were subjected to the sequencing operation in batches according to the manufacturer's instructions (see Illumina official publication specification).

2、数据分析 2, data analysis

根据制造商 Illumina提供的 Pipeline操作说明书（参见 http://www.illumina.com/提供的 Pipeline方法说明书），将步骤 DNA测序部分中测得的序列信息经过图形转化获得测序序列信息，去掉测序盾量低的序列之后最终可以获得针对 NCBI版本 36的人类基因组参考序列的 ELA D比对结果。 According to the Pipeline operating instructions provided by the manufacturer Illumina (see the Pipeline method manual provided at http://www.illumina.com/), the sequence information measured in the DNA sequencing part of the step is graphically transformed to obtain sequencing sequence information, and the sequencing shield is removed. The ELA D alignment results for the human genome reference sequence of NCBI version 36 can finally be obtained after a low amount of sequence.

将获得的数据使用 SOAP软件包进行比对分析，使用两个末端测序信息进行比对时去除两个末端均比对至人基因组的序列，保留其中一条链比对至人基因组的序列，将另一末端序列比对至 HPV基因组序列中，获得 HPV在人基因组重组信息，包括人基因组中重组位置以及 HPV类型。 The obtained data were compared using the SOAP software package. When the two end sequencing information was used for comparison, the sequences of both ends were aligned to the human genome, and one of the strands was aligned to the sequence of the human genome, and the other sequence was The one-end sequence is aligned into the HPV genome sequence to obtain information on HPV recombination in the human genome, including recombination locations in the human genome as well as HPV types.

3、分析结果： 3. Analysis results:

根据数据分析部分中的数据分析流程，通过使用高通量测序平台对***样品进行深度测序以及数据分析 , 共检测到 45个超过 10条测序序列支持的 HP V整合基因，发生整合 HPV区域均为 E1区，发生整合的 HPV型别为 HPV16型。 According to the data analysis process in the data analysis section, through the use of high-throughput sequencing platform for deep sequencing and data analysis of cervical cancer samples, 45 HP V integrated genes supported by more than 10 sequencing sequences were detected, and integrated HPV regions were generated. For the E1 area, the integrated HPV type is HPV16 type.

表 8、检测出来的支持数超过 10的 HPV整合情况 Table 8, HPV integration with more than 10 detected support

ARHGEFIOL chrl hpv-9,hpv- 10,hpv- 11 El HPV16 55160

ARHGEFIOL chrl hpv-9, hpv-10, hpv- 11 El HPV16 55160

TMC07 chrl6 hpv-9,hpv- 10,hpv- 11 El HPV16 79613TMC07 chrl6 hpv-9, hpv-10, hpv- 11 El HPV16 79613

PPEF1 chrX hpv-9,hpv- 10,hpv- 11 El HPV16 5475PPEF1 chrX hpv-9, hpv-10, hpv- 11 El HPV16 5475

PRKD2 chrl 9 hpv-9,hpv- 10,hpv- 11 El HPV16 25865PRKD2 chrl 9 hpv-9, hpv- 10, hpv- 11 El HPV16 25865

UNC13A chrl 9 hpv-9,hpv- 10,hpv- 11 El HPV16 23025UNC13A chrl 9 hpv-9, hpv-10, hpv- 11 El HPV16 23025

DENNDIA chr9 hpv-9,hpv- 10,hpv- 11 El HPV16 57706DENNDIA chr9 hpv-9, hpv-10, hpv- 11 El HPV16 57706

ZCCHC7 chr9 hpv-9,hpv- 10,hpv- 11 El HPV16 84186ZCCHC7 chr9 hpv-9, hpv-10, hpv- 11 El HPV16 84186

GLG1 chrl 6 hpv-9,hpv- 10,hpv- 11 El HPV16 2734GLG1 chrl 6 hpv-9, hpv-10, hpv- 11 El HPV16 2734

PCCA chrl 3 hpv-9,hpv- 10,hpv- 11 El HPV16 5095PCCA chrl 3 hpv-9, hpv-10, hpv- 11 El HPV16 5095

SMYD3 chrl hpv-9,hpv- 10,hpv- 11 El HPV16 64754SMYD3 chrl hpv-9, hpv-10, hpv- 11 El HPV16 64754

DOCK1 chrlO hpv-9,hpv- 10,hpv- 11 El HPV16 1793DOCK1 chrlO hpv-9, hpv-10, hpv- 11 El HPV16 1793

INSR chrl 9 hpv-9,hpv- 10,hpv- 11 El HPV16 3643INSR chrl 9 hpv-9, hpv-10, hpv- 11 El HPV16 3643

C2orG4 chr2 hpv-9,hpv- 10,hpv- 11 El HPV16 79823C2orG4 chr2 hpv-9, hpv-10, hpv- 11 El HPV16 79823

JUP chrl 7 hpv-9,hpv- 10,hpv- 11 El HPV16 3728JUP chrl 7 hpv-9, hpv-10, hpv- 11 El HPV16 3728

NSF chrl 7 hpv-9,hpv- 10,hpv- 11 El HPV16 4905NSF chrl 7 hpv-9, hpv-10, hpv- 11 El HPV16 4905

GPC3 chrX hpv-9,hpv- 10,hpv- 11 El HPV16 2719GPC3 chrX hpv-9, hpv-10, hpv- 11 El HPV16 2719

CAC B2 chrlO hpv-9,hpv- 10,hpv- 11 El HPV16 783CAC B2 chrlO hpv-9, hpv-10, hpv- 11 El HPV16 783

CHD9 chrl 6 hpv-9,hpv- 10,hpv- 11 El HPV16 80205CHD9 chrl 6 hpv-9, hpv-10, hpv- 11 El HPV16 80205

ADK chrlO hpv-9,hpv- 10,hpv- 11 El HPV16 132ADK chrlO hpv-9, hpv-10, hpv- 11 El HPV16 132

INTS4 chrl l hpv-9,hpv- 10,hpv- 11 El HPV16 92105INTS4 chrl l hpv-9, hpv-10, hpv- 11 El HPV16 92105

ACVR1B chrl2 hpv-9,hpv- 10,hpv- 11 El HPV16 91ACVR1B chrl2 hpv-9, hpv-10, hpv- 11 El HPV16 91

DT B chr2 hpv-9,hpv- 10,hpv- 11 El HPV16 1838DT B chr2 hpv-9, hpv-10, hpv- 11 El HPV16 1838

BMPR2 chr2 hpv-9,hpv- 10,hpv- 11 El HPV16 659BMPR2 chr2 hpv-9, hpv-10, hpv- 11 El HPV16 659

UVRAG chrl l hpv-9,hpv- 10,hpv- 11 El HPV16 7405UVRAG chrl l hpv-9, hpv-10, hpv- 11 El HPV16 7405

BPHL chr6 hpv-9,hpv- 10,hpv- 11 El HPV16 670BPHL chr6 hpv-9, hpv-10, hpv- 11 El HPV16 670

ADCY2 chr5 hpv-9,hpv- 10,hpv- 11 El HPV16 108ADCY2 chr5 hpv-9, hpv-10, hpv- 11 El HPV16 108

NVL chrl hpv-9,hpv- 10,hpv- 11 El HPV16 4931NVL chrl hpv-9, hpv-10, hpv- 11 El HPV16 4931

SAMHD1 chr20 hpv-9,hpv- 10,hpv- 11 El HPV16 25939SAMHD1 chr20 hpv-9, hpv-10, hpv- 11 El HPV16 25939

VKORCILI chr7 hpv-9,hpv- 10,hpv- 11 El HPV16 154807VKORCILI chr7 hpv-9, hpv-10, hpv- 11 El HPV16 154807

ATP1B3 chr3 hpv-9,hpv-10 El HPV16 483ATP1B3 chr3 hpv-9, hpv-10 El HPV16 483

DE ND2C chrl hpv-9,hpv-10 El HPV16 163259DE ND2C chrl hpv-9, hpv-10 El HPV16 163259

ACACA chrl 7 hpv-9,hpv- 10,hpv- 11 El HPV16 31ACACA chrl 7 hpv-9, hpv-10, hpv- 11 El HPV16 31

STYXL1 chr7 hpv-9,hpv- 10,hpv- 11 El HPV16 51657STYXL1 chr7 hpv-9, hpv-10, hpv- 11 El HPV16 51657

ADD3 chrlO hpv-9,hpv-l l El HPV16 120ADD3 chrlO hpv-9, hpv-l l El HPV16 120

DPP8 chrl 5 hpv-9,hpv- 10,hpv- 11 El HPV16 54878 11 SLC4A5 chr2 hpv-9,hpv- 10,hpv- 11 El HPV16 57835DPP8 chrl 5 hpv-9, hpv-10, hpv- 11 El HPV16 54878 11 SLC4A5 chr2 hpv-9, hpv-10, hpv- 11 El HPV16 57835

10 ABR chrl7 hpv-9,hpv- 10,hpv- 11 El HPV16 2910 ABR chrl7 hpv-9, hpv-10, hpv- 11 El HPV16 29

10 MYOM1 chrl8 hpv-9,hpv- 10,hpv- 11 El HPV16 873610 MYOM1 chrl8 hpv-9, hpv-10, hpv- 11 El HPV16 8736

10 DSTN chr20 hpv-9,hpv- 10,hpv- 11 El HPV16 1103410 DSTN chr20 hpv-9, hpv-10, hpv- 11 El HPV16 11034

10 ASTN2 chr9 hpv-9,hpv- 10,hpv- 11 El HPV16 2324510 ASTN2 chr9 hpv-9, hpv-10, hpv- 11 El HPV16 23245

10 FBX043 chr8 hpv-9,hpv- 10,hpv- 11 El HPV16 28615110 FBX043 chr8 hpv-9, hpv-10, hpv- 11 El HPV16 286151

10 HCN1 chr5 hpv-9,hpv- 10,hpv- 11 El HPV16 348980 本实施例表明通过由于第二代测序技术能够对肿瘤样品进行深度测序，从而能够快速地对病毒与人体基因的整合进行检测，并且能准确提供被整合的人基因组区域信息以及整合外源序列信息。实施例 3: 对肝癌样品外周血通过目标区域分析 10 HCN1 chr5 hpv-9, hpv-10, hpv- 11 El HPV16 348980 This example demonstrates the ability to rapidly detect the integration of viruses and human genes by deep sequencing of tumor samples due to second-generation sequencing technology. And can accurately provide integrated human genomic region information and integrate exogenous sequence information. Example 3: Analysis of peripheral blood passage of target cells in liver cancer samples

实验方法： experimental method:

1、 DNA提取及文库构建： 1. DNA extraction and library construction:

外周血 DNA提取和文库制备方法与实施例 2相同，只是样品来源为多名肝癌患者。 Peripheral blood DNA extraction and library preparation methods are the same as in Example 2 except that the sample source is a plurality of liver cancer patients.

2、捕获游离核酸： 2. Capture free nucleic acids:

本实施例中将使用核酸探针芯片（Mmblegen )对含有外源序列区域的核酸片段进行捕获。实验流程如下： In this example, a nucleic acid probe chip (Mmblegen) will be used to capture a nucleic acid fragment containing a foreign sequence region. The experimental process is as follows:

a. 样品准备： a. Sample preparation:

其中，核酸探针是以具有 SEQ ID NO: 7所示的核酸序列的 HBV基因组（ Hepatitis B virus serotype adr, complete genome ) 为模板，以 60 bp为一个探针长度，每间隔 5 bp设置一个探针，进行设计得到的。 Wherein, the nucleic acid probe is a HBV genome having a nucleic acid sequence of SEQ ID NO: 7 (Hepatitis B virus serotype adr, complete genome) as a template, with a probe length of 60 bp, and a probe at intervals of 5 bp. Needle, designed to get.

PE Block 1.0: PE Block 1.0:

5'-AATGATA' 5'-AATGATA'

ATCT-3' ( SEQ ID NO: 13 ); ATCT-3' (SEQ ID NO: 13);

PE Block 2.0: CTTCCGATCT-3' ( SEQ ID NO: 14 )。 PE Block 2.0: CTTCCGATCT-3' (SEQ ID NO: 14).

b. 将准备好的样品置于 SpeedVac中 60°C蒸千，然后加入 11.2 超纯水溶解样品。 c 全速离心样品 30秒，分别加入以下两种试剂： 18.5 的 2 X SC Hybridiation Buffer ( Roche NimbleGen公司）和 7.3 μL的 SC Hybridiation Component A ( Roche NimbleGen公司）。震荡混匀后置于离心机上全速离心 30秒，然后于 95 °C使 DNA变性 10分钟。 b. Place the prepared sample in SpeedVac at 60 °C and then add 11.2 ultrapure water to dissolve the sample. c Centrifuge the sample at full speed for 30 seconds and add the following two reagents: 18.5 of 2 X SC Hybridiation Buffer (Roche NimbleGen) and 7.3 μL of SC Hybridiation Component A (Roche NimbleGen). After shaking, mix and centrifuge on a centrifuge for 30 seconds at full speed, then denature the DNA at 95 °C for 10 minutes.

d. 根据制造商提供的说明书，将带有相应探针的芯片固定在杂交仪 ( Roche NimbleGen 公司）上，将变性后的样品加入芯片中并封闭芯片，然后设定杂交程序，于 42 °C杂交 64-72 小时。 d. Fix the chip with the corresponding probe on the hybrid instrument (Roche NimbleGen) according to the manufacturer's instructions, add the denatured sample to the chip and block the chip, then set the hybridization program at 42 °C. Hybridize for 64-72 hours.

e. 芯片洗涤与样品洗脱： e. Chip washing and sample elution:

f. 将 NaOH洗脱液回收，并用 32 20%水醋酸中和，得到中和液。 f. The NaOH eluate was recovered and neutralized with 32 20% aqueous acetic acid to obtain a neutralized solution.

g. 将上述中和液用 Qiagen MinElute PCR Purification Kit纯化，捕获后的样品最后溶解于 138 μL纯水中。 g. The above neutralized solution was purified by Qiagen MinElute PCR Purification Kit, and the captured sample was finally dissolved in 138 μL of pure water.

h. PCR扩增捕获的 DNA文库，分为 6管 50 反应进行 PCR, PCR的反应物组成如下: h. PCR-amplified the captured DNA library, which is divided into 6 tubes and 50 reactions for PCR. The composition of the PCR reaction is as follows:

其巾， Its towel,

PE Post Primer 1.0: AATGATACGGCGACCACCGAGATC ( SEQ ID NO: 15 ); PE Post Primer 1.0: AATGATACGGCGACCACCGAGATC (SEQ ID NO: 15);

PE Post Primer 2.0: CAAGCAGAAGACGGCATACGA ( SEQ ID NO: 16 )。 PE Post Primer 2.0: CAAGCAGAAGACGGCATACGA (SEQ ID NO: 16).

PCR的反应条件如下： ( a ) . 98 °C 30 s The reaction conditions of PCR are as follows: ( a ) . 98 °C 30 s

( b ) . 98 °C 15 s ( b ) . 98 °C 15 s

( c ) . 62 °C 30 s ( c ) . 62 °C 30 s

( d ) . 72 °C 30 s ( d ) . 72 °C 30 s

( e ) . 重复（ b ) - ( d ) 步骤 11-19次（共扩增 12-20次） (e) . Repeat (b) - (d) Steps 11-19 (12-20 total amplification)

( f) . 72 °C 5 min (f) . 72 °C 5 min

( g ) . 4°C 静置 ( g ) . 4 ° C standing

i.用 Qiagen QIAquick PCR Purification Kit纯化 PCR产物，最后溶于 30 纯水中。 i. Purify the PCR product using Qiagen QIAquick PCR Purification Kit and finally dissolve in 30 pure water.

3、高通量测序 3. High-throughput sequencing

本实施例中，对于获自肿瘤病人外周血 DNA样本分批按照制造商提供的操作说明书（参见 Illumina/Solexa官方公布的 cBot )进行上机测序操作。通过 100轮测序循环，得到长度为 100 bp的 DNA片段序列。 In this example, the peripheral blood DNA samples obtained from the tumor patient were subjected to a sequencing operation in batches according to the manufacturer's instructions (see the Illumina/Solexa officially published cBot). A DNA fragment sequence of 100 bp in length was obtained by 100 cycles of sequencing.

4、数据分析 4, data analysis

根据制造商 Illumina提供的 Pipeline操作说明书（参见 http://www.illumina.com/提供的 Pipeline 方法说明书），将步骤高通量测序部分中测得的序列信息经过图形转化获得测序序列信息，去掉测序盾量低的序列之后最终可以获得针对 NCBI版本 36的人类基因组参考序列的 ELA D比对结果。 According to the Pipeline operating instructions provided by the manufacturer Illumina (see the Pipeline method manual provided at http://www.illumina.com/), the sequence information measured in the high-throughput sequencing part of the step is graphically transformed to obtain the sequencing sequence information, and removed. The results of the ELA D alignment of the human genome reference sequence for NCBI version 36 were finally obtained after sequencing the low-profile sequence.

获得的数据使用 SOAP软件包进行比对分析，使用两个末端测序信息进行比对时去除两个末端均比对至人基因组的序列，保留其中一条链比对至人基因组的序列，将另一末端序列比对至 HBV基因组序列中，获得 HBV在人基因组重组信息，包括人基因组中重组位置以及 HBV类型。 The obtained data was analyzed by SOAP software package. When the two end sequencing information was used for comparison, the sequences of both ends were aligned to the human genome, and one of the strands was aligned to the sequence of the human genome, and the other was The end sequences are aligned into the HBV genomic sequence to obtain HBV recombination information in the human genome, including recombination positions in the human genome as well as HBV types.

5、数据结果 5, the data results

测到肝癌样品中 HBV病毒整合信息，即一段 733 bp HBV序列整合至 1号染色体区域。实施例 4: 对肝癌样品外周血及组织通过目标区域捕获分析

The HBV virus integration information in the liver cancer samples was measured, that is, a 733 bp HBV sequence was integrated into the chromosome 1 region. Example 4: Capture and analysis of peripheral blood and tissues of liver cancer samples through target regions

实验方法： experimental method:

1、 DN A提取及测序 1. DN A extraction and sequencing

根据常规方法，对肝癌患者进行静脉取血，得到患者的外周血样本，通过离心得到血浆样本。按照 Tiangen Micro Kit (DP316)微量基因组操作流程从血浆样本提取 DNA, 并分别用 Qubit ( Invitrogen , the Quant-iT™ dsDNAHS Assay Kit )定量，所提取的各样本的 DNA 总量均为 5 ~ 50 ng。 According to a conventional method, a liver cancer patient is subjected to venous blood sampling, a peripheral blood sample of the patient is obtained, and a plasma sample is obtained by centrifugation. DNA was extracted from plasma samples according to the Tiangen Micro Kit (DP316) microgenome protocol Do not quantify with Qubit (Invitrogen, the Quant-iTTM dsDNAHS Assay Kit). The total amount of DNA extracted from each sample is 5 to 50 ng.

根据常规方法，取肝癌患者，癌组织样本对组织样本进行全基因组提取，取 3 微克进行常规建库，文库***片段主带为 170bp. According to the conventional method, liver cancer patients and cancer tissue samples were subjected to whole genome extraction of tissue samples, and 3 micrograms were routinely constructed. The main insert of the library insert was 170 bp.

将所提取到的 DNA , 分别按照制造商提供的标准建库规程（参见 http://www.illumina.com/提供的 Illumina标准建库说明书）建立 DNA文库。筒言之，在 DNA 分子两端加上测序用的接头，并被加上不同的标签序列，然后与测序芯片表面互补接头杂交，使核酸分子成簇生长，然后在 Illumina HiSeq 2000上测序 PE101 , 得到长度为 lOObp的 DNA片段序列。 The extracted DNA was constructed according to the standard library-building procedures provided by the manufacturer (see the Illumina standard library specification provided at http://www.illumina.com/). In conclusion, a linker for sequencing is added to both ends of the DNA molecule, and a different tag sequence is added, and then hybridized with a complementary linker on the surface of the sequencing chip to cluster the nucleic acid molecules, and then PE101 is sequenced on an Illumina HiSeq 2000. A DNA fragment sequence of length lOObp was obtained.

2、捕获游离核酸： 2. Capture free nucleic acids:

本实施例中将使用 HBV ( Nimblegen )核酸探针芯片对含有外源序列区域的核酸片段进行捕获。实验流程如下： In this example, a nucleic acid fragment containing a foreign sequence region will be captured using a HBV (Nimblegen) nucleic acid probe chip. The experimental process is as follows:

a. 样品准备： a. Sample preparation:

其中， HBV核酸探针是由 HBV的 A, B , C, D, E, F, G, H八个型别的基因组进行设计得到的，各基因组序列具体可见公知数据库中的 HB V基因组序列。具体地，按照 HB V 基因组长度，每次在基因组上 10 bp滑动移动一次，合成 60-90 bp长度且带 Bio-tin标记的探针。具体探针是委托相应的公司合成的。 Among them, the HBV nucleic acid probe is designed from the eight types of genomes of HBV A, B, C, D, E, F, G, H, and the genome sequence of each is specifically visible in the HB V genome sequence in the well-known database. Specifically, a 60-90 bp-length probe with Bio-tin labeling was synthesized by sliding 10 bp on the genome each time according to the length of the HB V genome. The specific probe is commissioned by the corresponding company.

b. 将准备好的样品置于 SpeedVac中 60°C蒸千。 b. Place the prepared sample in a SpeedVac at 60 °C.

c 全速离心样品 30秒，分别加入以下两种试剂： 7.5 的 2 X SC Hybridiation Buffer ( Roche NimbleGen公司 )和 3.0 μL的 SC Hybridiation Component A ( Roche NimbleGen公司）。震荡混匀后置于离心机上全速离心 30秒，然后于 95 °C使 DNA变性 10分钟，而后加入探针 4.5 μΐ于 47°C杂交 24小时。 c Centrifuge the sample at full speed for 30 seconds and add the following two reagents: 7.5 2 X SC Hybridiation Buffer (Roche NimbleGen) and 3.0 μL SC Hybridiation Component A (Roche NimbleGen). After shaking, the mixture was centrifuged at full speed for 30 seconds, then the DNA was denatured at 95 °C for 10 minutes, and then probed at 4.5 μC for 24 hours at 47 °C.

e. 芯片洗涤与样品洗脱：按照标准的 EZ洗脱流程进行洗脱，回收。 e. Chip washing and sample elution: Elution and recovery according to the standard EZ elution procedure.

f. 预先从 -20°C保存的试剂盒中取出 Pfx酶 (Invirtogen), Pfx bufFer(10*), dNTP(lO mM), PCR Primer F(10 pm/μΐ), PCR Primer R( 10 ριη/μ1)。 f. Remove Pfx enzyme (Invirtogen), Pfx bufFer (10*), dNTP (10 mM), PCR Primer F (10 pm/μΐ), PCR Primer R (10 ριη/) from the kit stored at -20 °C in advance. 11).

在 PCR小管上，每孔按照下面的表格配置 PCR反应体系：

Pfx緩冲液 (10*) 5 μΐ On the PCR vial, configure the PCR reaction system for each well according to the following table:

Pfx Buffer (10*) 5 μΐ

MgS0₄ 2 μΐ MgS0 ₄ 2 μΐ

dNTP(10 mM) 2 μΐ dNTP (10 mM) 2 μΐ

Flowcell-primer-F 1 (10 pm/μΐ) 2.5 μΐ Flowcell-primer-F 1 (10 pm/μΐ) 2.5 μΐ

Flowcell-primer-Rl (10 pm/μΐ) 2.5 μΐ Flowcell-primer-Rl (10 pm/μΐ) 2.5 μΐ

总体积 50 μΐ Total volume 50 μΐ

其巾， Its towel,

Flowcell-primer-F 1 : AATGATACGGCGACCACCGAGATC ( SEQ ID NO: 17 ); Flowcell-primer-F 1 : AATGATACGGCGACCACCGAGATC ( SEQ ID NO: 17 );

Flowcell-primer-Rl : CAAGCAGAAGACGGCATACGA ( SEQ ID NO: 18 )。 Flowcell-primer-Rl: CAAGCAGAAGACGGCATACGA (SEQ ID NO: 18).

程序如下： The procedure is as follows:

94 °C 2min; 94 °C 2min;

94 °C 15s, 58 °C 30s, 72 °C 30s, 14个循环； 94 °C 15s, 58 °C 30s, 72 °C 30s, 14 cycles;

72 °C 5min, 72 °C 5min,

4°C ∞。 4 ° C ∞.

PCR结束后，每个样品都用 1.5倍体积的 Amprue Beads纯化，回收的 PCR产物溶于 30 μΐ超纯水中， NanodroplOOO测其浓度为 10 After the end of PCR, each sample was purified using 1.5 volumes of Amprue Beads. The recovered PCR product was dissolved in 30 μM ultrapure water and the concentration of Nanodrop 100 was measured at 10

3、高通量测序 3. High-throughput sequencing

本实施例中，对于获自肿瘤病人外周血 DNA样本分批按照制造商提供的操作说明书（参见 Illumina/Solexa官方公布的 cBot )进行上机测序操作。 In this example, the peripheral blood DNA samples obtained from the tumor patient were subjected to a sequencing operation in batches according to the manufacturer's instructions (see the Illumina/Solexa officially published cBot).

4、数据分析 4, data analysis

将下机数据除去重复和被接头污染的 reads,统计下机数据的基本信息（文库长度； reads 长度； reads条数；碱基数；重复率等）；分别截取 PE的两条 reads 前面的 50bp碱基，形成一对长为 50bp新 reads, 即 PE50 reads„ 将新的 PE50 reads运用 soap比对软件（ -r 1 -v 2 ) 分别与人的参考序列（hgl9)和 HBV各种参考序列进行比对，从比对结果中挑选出一条 read 比到人的参考序列并且另一条比对到 HBV参考序列的一对 reads; 这样的 reads很有可能跨过 HBV***的位点；组装这部分 reads,釆用 BWA比对找到在人类基因组的***热点区域。 The lower data is removed from the duplicate and the joint contaminated by the joint, and the basic information of the data (the length of the library; the length of the reads; the number of reads; the number of bases; the repetition rate) are counted; and the 50 bp of the two reads of the PE are respectively intercepted. Bases, forming a pair of 50bp new reads, ie PE50 reads„ The new PE50 reads using the soap comparison software (-r 1 -v 2 ) with the human reference sequence (hgl9) and HBV various reference sequences, respectively The alignment selects a pair of reads that are read to the human reference sequence and another to the HBV reference sequence; such reads are likely to cross the HBV insertion site; assemble this part of the reads , using BWA to find the insertion hotspots in the human genome.

5、数据结果 5, the data results

A患者组织 DNA, 经过杂交后 1G (碱基 )数据量结果如下： A patient tissue DNA, after 1G (base) data volume after hybridization, the results are as follows:

chrl7 18778266 8

Chrl7 18778266 8

A患者血浆 DNA, 经过杂交后 5G (碱基 )数据量结果如下: The plasma DNA of patient A, the amount of 5G (base) data after hybridization is as follows:

釆用上述方法对同一患者的血浆与肝癌组织中的整合状态进行查找，发现组织中存在的支持数最高的位置，在血浆中同样存在，此方法不仅证明组织与血浆游离 DNA之间的关系，并且能够准确有效地找出血浆中的整合位置，表明本发明的用于确定人体具有异常状态方法为能够有效地应用于病原体引起染色体整合相关疾病的无创检测。实施例 5: 对 HPV患者子宫颈脱落细胞目标区域捕获分析釆 Using the above method to find the integration status of plasma and liver cancer tissues of the same patient, and found that the position with the highest number of support in the tissue exists in plasma, this method not only proves the relationship between tissue and plasma free DNA, Moreover, it is possible to accurately and efficiently find the integrated position in the plasma, indicating that the method for determining abnormal state of the human body of the present invention is a non-invasive detection capable of being effectively applied to a pathogen-causing chromosomal integration-related disease. Example 5: Capture analysis of target areas of cervical exfoliated cells in HPV patients

实验方法： experimental method:

1、 DN A提取及测序 1. DN A extraction and sequencing

根据常规方法，对 HPV 患者进行子宫颈脱落细胞取样，样本按照 Tiangen Micro Kit According to the conventional method, cervical exfoliated cells were sampled from HPV patients, and the samples were according to Tiangen Micro Kit.

(DP316) 量基因组操作流程从血浆样本提取 DNA, 分别用 Qubit ( Invitrogen , the Quant-iT™ dsDNAHS Assay Kit )定量，所提取的 DNA总量分别为 100 ~ 500 ng。 (DP316) Genome manipulation procedure DNA was extracted from plasma samples and quantified using Qubit (Invitrogen, the Quant-iTTM dsDNAHS Assay Kit), respectively. The total amount of DNA extracted was 100-500 ng.

将所提取到的 DNA , 分别按照制造商提供的标准建库规程（参见 http：〃 www.illumina.com/提供的 Illumina标准建库说明书）建立 170 bp DNA文库。筒言之，在 DNA分子两端加上测序用的接头，并被加上不同的标签序列，然后与测序芯片表面互补接头杂交，使核酸分子成簇生长，然后在 Illumina HiSeq 2000上测序 PE101 , 得到长度为 100 bp的 DNA片段序列。 The extracted DNA was constructed into a 170 bp DNA library according to the manufacturer's standard library protocol (see http: Il www.illumina.com/ for the Illumina standard library specification). In conclusion, a linker for sequencing is added to both ends of the DNA molecule, and a different tag sequence is added, and then hybridized with a complementary linker on the surface of the sequencing chip to cluster the nucleic acid molecules, and then PE101 is sequenced on an Illumina HiSeq 2000. A DNA fragment sequence of 100 bp in length was obtained.

2、捕获游离核酸： 2. Capture free nucleic acids:

本实施例中将使用（Mygenostics公司） HPV核酸探针芯片对含有外源序列区域的核酸片段进行捕获。实验流程如下： In this example, a nucleic acid fragment containing a foreign sequence region will be captured using a (Mygenostics) HPV nucleic acid probe chip. The experimental process is as follows:

a. 样品准备: a. Sample preparation:

总体积

total capacity

65度 22小时 65 degrees 22 hours

其中， HPV核酸探针是由 HPV的 6, 11 , 16, 18, 31 , 33 , 35 , 39, 45 , 52, 56, 58, 59, 66, 68, 69, 82型别的基因组进行设计得到的，各基因组序列具体可见公知数据库中的 HPV基因组。具体地，按照 HPV基因组长度，每次在基因组上 10 bp滑动移动一次，合成 60-90 bp长度且带 Bio-tin标记的探针。具体探针是委托相应的公司合成的。 Among them, HPV nucleic acid probes are designed from the genomes of HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 66, 68, 69, 82 The genomic sequences are specifically visible in the HPV genome in a well-known database. Specifically, according to the length of the HPV genome, each time a 10 bp sliding movement on the genome was performed, a probe of 60-90 bp in length and carrying a Bio-tin label was synthesized. The specific probe is commissioned by the corresponding company.

b. 芯片洗涤与样品洗脱：按照标准（MyGenostics )洗脱流程进行洗脱，回收，具体步骤如下： b. Chip washing and sample elution: elute according to the standard (MyGenostics) elution process, and the specific steps are as follows:

1 )提前将温浴器调至 65 °C ; 1 ) Adjust the bath to 65 °C in advance;

2 )用漩涡混合仪剧烈振荡重悬 Myone beads CI ( Invitrogen ) 至混匀； 2) Resuspend Myone beads CI (Invitrogen) with a vortex mixer and mix thoroughly;

3 )每一个杂交反应取 50 μΐ Myone beads CI磁珠于一新的 1.5 mL离心管中，放于磁力架上，然后去除上清； 3) Take 50 μΐ of Myone beads CI beads in each hybridization reaction in a new 1.5 mL centrifuge tube, place on a magnetic stand, and then remove the supernatant;

4 ) 洗涤磁珠： 4) Wash the magnetic beads:

a) 加入 50 μL 1*结合緩冲液； a) Add 50 μL of 1* binding buffer;

b) 用漩涡混合仪剧烈振荡 5秒钟重悬磁珠; b) Re-suspend the magnetic beads by vigorously shaking with a vortex mixer for 5 seconds;

c) 将离心管放于磁力架上，待液体变澄清; c) Place the centrifuge tube on the magnetic stand until the liquid becomes clear;

d) 去除上清液； d) removing the supernatant;

e) 重复 2次 "步骤 a到步骤 d" ; e) repeat "step a to step d" twice;

5 )加入 80 μL (根据不同大小捕获区域区域加入量不同，具体按照 KIT要求加入） 1* 结合緩冲液重悬磁珠； 5) Add 80 μL (according to different sizes of capture area, add according to KIT requirements) 1* Resuspend magnetic beads in combination with buffer;

6 )加入 64 μL 2*结合緩冲液重悬磁珠（与杂交液体积同等体积），将液体转移到 EP1 后，总体积约为 200 升； 6) Resuspend the magnetic beads (same volume as the hybridization volume) by adding 64 μL of 2* binding buffer, and transfer the liquid to EP1 after a total volume of about 200 liters;

7 )振荡混匀后在室温下放于 ROATER上进行 1小时； 7) After shaking and mixing, put it on ROATER at room temperature for 1 hour;

8 )震荡混匀后将样品放于磁力架上，去除上清； 8) After shaking and mixing, place the sample on the magnetic stand and remove the supernatant;

9 )加入 500 μ1 \¥Β1 , 振荡混匀后旋转混匀 15分钟，然后放于磁力架上去上清； 9) Add 500 μ1 \¥Β1, mix by shaking, rotate and mix for 15 minutes, then place on the magnetic stand to remove the supernatant;

10 )加入 500 μΐ WB3 , 振荡混匀后放于 65度温浴， 850 rpm, 10分钟，放于磁力架上去上清； 10) Add 500 μΐ WB3, mix by shaking, place in a 65-degree warm bath, 850 rpm, 10 minutes, place on the magnetic stand to remove the supernatant;

11 )重复（ 10 ) 5次，最后一次完全去除上清； 11) Repeat (10) 5 times, the last time the supernatant is completely removed;

12 )加入 50 μΐ洗脱緩冲液，振荡混勾，室温放置 10分钟，放于磁力架上取上清转入另一 ΕΡ管（管内包含 70 μΐ Ε緩冲液）； 12) Add 50 μM elution buffer, shake and mix, leave it at room temperature for 10 minutes, place on the magnetic stand, take the supernatant and transfer it to another tube (the tube contains 70 μΐ buffer);

13 )釆用 QIAquick Minelute进行纯化，最终溶解 42 μΐ ΕΒ。 13) Purify with QIAquick Minelute and finally dissolve 42 μΐ.

c. 捕获样品的扩增与纯化 c. Amplification and purification of capture samples

1)从 -20°C水箱中取出 2*PHUSION MASTER, Flowcell primer s( 10 μΜ), 将其置于水上化冻并充分混匀。 1) Remove 2*PHUSION MASTER, Flowcell primer s (10 μΜ) from the -20 °C water tank, place it on the water and freeze it thoroughly.

2)在水上为每个捕获样品配制一份 Mix, 另外加入一个无模板的阴性对照，按以下表格组分配制反应 Mix并用移液器混匀： 2) Prepare a Mix for each captured sample on the water, and add a negative control without template, as shown in the following table. Prepare the reaction Mix and mix with a pipette:

3)在热循环仪中运行下列程序： 3) Run the following program in the thermal cycler:

a. 98 °C 30s a. 98 °C 30s

b. 98 °C 25 s b. 98 °C 25 s

c. 65 °C 30 s c. 65 °C 30 s

d. 72 °C 30 s d. 72 °C 30 s

e. 重复 b-d步骤 (共 15次循环） e. Repeat b-d steps (15 cycles total)

f. 72 °C 5 min f. 72 °C 5 min

g. 4 °C Hold g. 4 °C Hold

PCR结束后，每个样品都用 1.5倍体积的 Amprue Beads纯化，回收的 PCR产物溶于 After the end of PCR, each sample was purified with 1.5 volumes of Amprue Beads and the recovered PCR product was dissolved.

30 μΐ超纯水中， NanodroplOOO测浓度，并记录，其浓度为 10 30 μΐ ultrapure water, NanodroplOOO measured concentration, and recorded, its concentration is 10

3、高通量测序 3. High-throughput sequencing

本实施例中，对于获自 HPV患者的子宫颈脱落细胞 DNA样本分批按照制造商提供的操作说明书（参见 Illumina/Solexa官方公布的 cBot )进行上机测序操作。通过 100轮测序循环，得到长度为 100 bp的 DNA片段序列。 In this example, DNA samples from cervical exfoliated cells obtained from HPV patients were batched in accordance with the manufacturer's instructions (see Illumina/Solexa officially published cBot). The DNA fragment sequence of 100 bp in length was obtained by 100 cycles of sequencing.

4、数据分析 4, data analysis

将下机数据除去重复和被接头污染的 reads,统计下机数据的基本信息（文库长度； reads 长度； reads条数；碱基数；重复率等）；分别截取 PE的两条 reads 前面的 50bp碱基，形成一对长为 50bp新 reads, 即 PE50 reads„ 将新的 PE50 reads运用 Soap比对软件 ( -r 1 -v 2 ) 分别与人的参考序列（hgl9)和 HBV各种参考序列进行比对，从比对结果中挑选出一条 read 比到人的参考序列并且另一条比对到 HBV参考序列的一对 reads; 这样的 reads很有可能跨过 HBV***的位点；组装这部分 reads,釆用 BWA比对找到在人类基因组的***热点区域。 The lower data is removed from the duplicate and the joint contaminated by the joint, and the basic information of the data (the length of the library; the length of the reads; the number of reads; the number of bases; the repetition rate) are counted; and the 50 bp of the two reads of the PE are respectively intercepted. Base, forming a pair of 50bp new reads, ie PE50 reads„ Using the new PE50 reads with Soap alignment software (-r 1 -v 2 ) with the human reference sequence (hgl9) and HBV various reference sequences, respectively The alignment selects a pair of reads that are read to the human reference sequence and another to the HBV reference sequence; such reads are likely to cross the HBV insertion site; assemble this part of the reads , using BWA to find the insertion hotspots in the human genome.

5、数据结果 5, the data results

HPV16型感染且宫颈病变程度为 CIN四期病人，数据量 1G, 结果如下：

chr2 133034596 16 The HPV16 type infection and the degree of cervical lesions were CIN stage 4 patients, the data volume was 1G, and the results were as follows:

Chr2 133034596 16

上述方法釆用宫颈病变程度为 CIN四期病人，感染的宫颈脱落细胞进行检测，结果准确发现高频整合位置，证明了袅明太明 ^用千硇工业实用性 The above method uses the cervical lesions in the fourth stage of CIN patients, and the infected cervical exfoliated cells are tested. The result is that the high-frequency integrated position is accurately found, which proves that the Ming Dynasty is too clear.

本发明的用于确定人体具有异常状态的***和方法，能够有效地应用于人体疾病的无创检测，通过对人体样本的核酸序列信息进行分析，可以根据核酸序列中所包含的信息准确地确定人体是否具有异常状态。尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。 The system and method for determining abnormal state of a human body can be effectively applied to non-invasive detection of human diseases, and by analyzing nucleic acid sequence information of a human sample, the human body can be accurately determined according to information contained in the nucleic acid sequence. Whether there is an abnormal state. Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of those details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

在本说明书的描述中，参考术语 "一个实施例"、 "一些实施例"、 "示意性实施例"、 "示例"、 "具体示例"、或 "一些示例" 等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。 In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Claims

权利要求书 Claim

1、一种确定人体具有异常状态的方法，其特征在于，包括： A method for determining an abnormal state of a human body, comprising:

提供人体样本的核酸序列信息，所述人体样本的核酸序列信息是基于对所述人体样本进行检测而获得的；以及 Providing nucleic acid sequence information of a human sample obtained by detecting the human sample; and

基于所述人体样本的核酸序列信息，确定所述人体是否具有异常状态。 Based on the nucleic acid sequence information of the human sample, it is determined whether the human body has an abnormal state.

2、根据权利要求 1所述的确定人体具有异常状态的方法，其特征在于，所述人体样本的核酸序列信息是基于对所述人体样本进行核酸序列检测而获得的。 2. The method of determining an abnormal state of a human body according to claim 1, wherein the nucleic acid sequence information of the human body sample is obtained based on nucleic acid sequence detection of the human body sample.

3、根据权利要求 2所述的确定人体具有异常状态的方法，其特征在于，所述核酸序列检测是借助第二代测序技术或第三代测序技术进行的。 3. The method of determining an abnormal state of a human body according to claim 2, wherein the nucleic acid sequence detection is performed by means of a second generation sequencing technique or a third generation sequencing technique.

4、根据权利要求 1所述的确定人体具有异常状态的方法，其特征在于，所述样本为所述人体的细胞、组织、血液、体液、尿液、 ***物或其组合。 4. The method of determining an abnormal state of a human body according to claim 1, wherein the sample is cells, tissues, blood, body fluids, urine, excretions, or a combination thereof of the human body.

5、根据权利要求 1所述的确定人体具有异常状态的方法，其特征在于，所述人体样本为血浆或者血清。 5. The method of determining an abnormal state of a human body according to claim 1, wherein the human body sample is plasma or serum.

6、根据权利要求 1所述的确定人体具有异常状态的方法，其特征在于，所述核酸序列信息包括所述人体样本中游离核酸的序列信息。 6. The method of determining an abnormal state of a human body according to claim 1, wherein the nucleic acid sequence information comprises sequence information of free nucleic acids in the human sample.

7、根据权利要求 6所述的确定人体具有异常状态的方法，其特征在于，所述人体样本中游离核酸的序列信息是通过除去所述人体样本中的细胞后，进行测序检测而获得的。 7. The method of determining an abnormal state of a human body according to claim 6, wherein the sequence information of the free nucleic acid in the human sample is obtained by performing sequencing detection after removing cells in the human sample.

8、根据权利要求 1所述的确定人体具有异常状态的方法，其特征在于，所述异常状态选自疾病的发生、疾病的发展阶段、疾病的疗效和预后的至少一种。 The method of determining an abnormal state of a human body according to claim 1, wherein the abnormal state is selected from at least one of a disease occurrence, a disease development stage, a disease efficacy, and a prognosis.

9、根据权利要求 8所述的确定人体具有异常状态的方法，其特征在于，所述疾病是肿瘤性疾病、免疫性疾病、遗传性疾病的至少一种。 The method of determining an abnormal state of a human body according to claim 8, wherein the disease is at least one of a tumor disease, an immune disease, and a hereditary disease.

10、根据权利要求 9 所述的确定人体具有异常状态的方法，其特征在于，所述肿瘤性疾病是选自肺癌、肝癌、胃癌、食管癌、结直肠癌、胰腺癌、乳腺癌、膀胱癌、肾癌、卵巢癌、 ***、甲状腺癌、鼻咽癌、脑胶盾瘤的至少一种。 10. The method for determining an abnormal state of a human body according to claim 9, wherein the neoplastic disease is selected from the group consisting of lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, breast cancer, and bladder cancer. At least one of kidney cancer, ovarian cancer, cervical cancer, thyroid cancer, nasopharyngeal cancer, and brain gelatin.

11、根据权利要求 8 所述的确定人体具有异常状态的方法，其特征在于，如果所述核酸序列信息包含选自下列至少一种的核酸片段序列： HBV、 HPV、 EBV、幽门螺旋杆菌，则确定所述人体患有***、肝癌、鼻咽癌、胃癌的至少一种。 11. The method of determining an abnormal state of a human body according to claim 8, wherein if the nucleic acid sequence information comprises a sequence of a nucleic acid fragment selected from at least one of the following: HBV, HPV, EBV, Helicobacter pylori, It is determined that the human body has at least one of cervical cancer, liver cancer, nasopharyngeal cancer, and gastric cancer.

12、根据权利要求 1所述的确定人体具有异常状态的方法，其特征在于， 12. The method of determining an abnormal state of a human body according to claim 1, wherein:

对所述核酸进行测序检测之前，利用探针捕获含有特定序列的核酸，然后对所述含有特定序列的核酸进行测序检测。 Prior to sequencing the nucleic acid, a nucleic acid containing a specific sequence is captured using a probe, and then the nucleic acid containing the specific sequence is subjected to sequencing detection.

13、根据权利要求 12所述的确定人体具有异常状态的方法，其特征在于， 13. The method of determining an abnormal state of a human body according to claim 12, wherein:

所述探针对于选自下列的至少一种是特异性的： HBV、 HPV、 EBV、幽门螺旋杆菌，任选地，所述探针为核酸探针集，包括多个探针，所述核酸探针集具有以下特征： ( 1 )每个探针上具有 1个或多个生物素标记的 dNTP; 和 /或 The probe is specific for at least one selected from the group consisting of: HBV, HPV, EBV, H. pylori, optionally, the probe is a set of nucleic acid probes comprising a plurality of probes, the nucleic acid The probe set has the following characteristics: (1) one or more biotinylated dNTPs on each probe; and/or

( 2 )所述生物素标记的 dNTP在所述探针集中的丰度为 1 : 6-1 : 2; 和 /或 ( 3 )所述探针集的全部核酸序列覆盖对应选自 HBV、 HPV、 EBV和幽门螺旋杆菌的至少一种病毒的基因组序列的 70%-100%, (2) the biotin-labeled dNTP has an abundance of 1: 6-1 : 2 in the probe set; and/or (3) the entire nucleic acid sequence of the probe set covers 70%-100% of the genomic sequence corresponding to at least one virus selected from the group consisting of HBV, HPV, EBV and H. pylori,

优选地，所述探针集具有 1-20000个核酸探针；较佳地，所述探针集具有 1000-5000个核酸探针；更佳地，所述探针集具有 2500个核酸探针， Preferably, the probe set has 1-20000 nucleic acid probes; preferably, the probe set has 1000-5000 nucleic acid probes; more preferably, the probe set has 2500 nucleic acid probes ,

优选地，所述生物素标记的 dNTP在所述探针集中的丰度为 1 : 4, Preferably, the abundance of the biotin-labeled dNTP in the probe set is 1:4,

优选地，在所述探针集中，所述探针之间具有部分重叠， Preferably, in the concentration of the probes, there is a partial overlap between the probes,

优选地，所述探针长度为 100-500 bp; 较佳地，所述探针长度为 200-300 bp; 更佳地，所述探针长度为 250 bp, Preferably, the probe has a length of 100-500 bp; preferably, the probe has a length of 200-300 bp; more preferably, the probe has a length of 250 bp.

优选地，所述探针是以病毒基因组作为模板， PCR法扩增获得的，较佳地，所述模板为选自 HBV基因组、 HCV基因组、 HIV基因组和 HPV基因组的至少一种；更佳地，所述模板为 B型 HBV基因组和 /或 C型 HBV基因组。 Preferably, the probe is obtained by PCR amplification using a viral genome as a template, preferably, the template is at least one selected from the group consisting of a HBV genome, an HCV genome, an HIV genome, and an HPV genome; more preferably The template is a B-type HBV genome and/or a C-type HBV genome.

14、根据权利要求 1所述的确定人体具有异常状态的方法，其特征在于， 14. The method of determining an abnormal state of a human body according to claim 1, wherein:

在对所述核酸进行测序检测之前，利用探针去除含有特定序列的核酸，然后对所述去除后剩余的核酸进行测序检测。 Prior to sequencing the nucleic acid, the nucleic acid containing the specific sequence is removed using a probe, and then the remaining nucleic acid after the removal is subjected to sequencing detection.

15、根据权利要求 14所述的确定人体具有异常状态的方法，其特征在于， 15. The method of determining an abnormal state of a human body according to claim 14, wherein:

所述探针可以结合人体基因组中的共有序列，或者为可以结合人体基因组中甲基化位点的抗体或者蛋白。 The probe may bind to a consensus sequence in the human genome or be an antibody or protein that binds to a methylation site in the human genome.

16、一种用于确定人体具有异常状态的***，其特征在于，包括： 16. A system for determining an abnormal state of a human body, comprising:

核酸序列信息接收器，所述核酸序列信息接收器接收人体样本的核酸序列信息；以及核酸序列信息分析器，所述核酸序列信息分析器与所述核酸序列信息接收器相连，并基于所述人体样本的核酸序列信息，确定所述人体是否具有异常状态。 a nucleic acid sequence information receiver, the nucleic acid sequence information receiver receiving nucleic acid sequence information of a human sample; and a nucleic acid sequence information analyzer, the nucleic acid sequence information analyzer being connected to the nucleic acid sequence information receiver, and based on the human body The nucleic acid sequence information of the sample determines whether the human body has an abnormal state.

17、根据权利要求 16所述的用于确定人体具有异常状态的***，其特征在于，所述核酸序列信息分析器内预存有选自下列的至少一种：人体正常状态的基因组序列、病原体的基因组序列、正常人群的基因组序列。 17. The system for determining an abnormal state of a human body according to claim 16, wherein the nucleic acid sequence information analyzer prestores at least one selected from the group consisting of: a genomic sequence of a human normal state, and a pathogen. Genomic sequence, genomic sequence of the normal population.

18、根据权利要求 17所述的用于确定人体具有异常状态的***，其特征在于，所述病原体为选自 HBV、 HPV、 EBV、幽门螺旋杆菌的至少一种。 The system for determining an abnormal state of a human body according to claim 17, wherein the pathogen is at least one selected from the group consisting of HBV, HPV, EBV, and Helicobacter pylori.

19、根据权利要求 18所述的用于确定人体具有异常状态的***，其特征在于，进一步包括核酸序列检测装置，所述核酸序列检测装置与所述核酸序列信息接收器相连，用于对所述人体样本进行核酸序列检测获得所述核酸序列信息并输送至所述核酸序列信息接收器。 19. The system for determining an abnormal state of a human body according to claim 18, further comprising a nucleic acid sequence detecting device, wherein said nucleic acid sequence detecting device is connected to said nucleic acid sequence information receiver for use in The human sample is subjected to nucleic acid sequence detection to obtain the nucleic acid sequence information and delivered to the nucleic acid sequence information receiver.

20、根据权利要求 19所述的用于确定人体具有异常状态的***，其特征在于，所述核酸序列检测装置借助第二代测序技术或第三代测序技术。 20. A system for determining an abnormal state of a human body according to claim 19, wherein said nucleic acid sequence detecting means is by means of a second generation sequencing technique or a third generation sequencing technique.

21、根据权利要求 19所述的用于确定人体具有异常状态的***，其特征在于，进一步包括 21. The system for determining an abnormal state of a human body according to claim 19, further comprising

游离核酸捕获装置，所述游离核酸捕获装置与所述核酸序列检测装置相连， a free nucleic acid capture device, the free nucleic acid capture device being coupled to the nucleic acid sequence detection device,

其巾，所述游离核酸捕获装置设置有探针 , Its towel, The free nucleic acid capture device is provided with a probe,

所述探针适于捕获含有特定序列的核酸，并且将所述含有特定序列的核酸输送至所述核酸序列检测装置进行核酸序列检测；或者 The probe is adapted to capture a nucleic acid containing a specific sequence, and deliver the nucleic acid containing the specific sequence to the nucleic acid sequence detecting device for nucleic acid sequence detection; or

所述探针适于去除含有特定序列的核酸，并且将所述去除后的核酸输送至所述核酸序列检测装置进行核酸序列检测。 The probe is adapted to remove a nucleic acid containing a specific sequence, and deliver the removed nucleic acid to the nucleic acid sequence detecting device for nucleic acid sequence detection.

22、根据权利要求 21所述的确定人体具有异常状态的方法，其特征在于， 22. The method of determining an abnormal state of a human body according to claim 21, wherein:

所述适于捕获含有特定序列的核酸的探针对于选自下列的至少一种是特异性的： HBV、 HPV、 EBV、幽门螺旋杆菌， The probe suitable for capturing a nucleic acid containing a specific sequence is specific for at least one selected from the group consisting of: HBV, HPV, EBV, Helicobacter pylori,

任选地，所述探针为核酸探针集，包括多个探针，所述核酸探针集具有以下特征： ( 1 )每个探针上具有 1个或多个生物素标记的 dNTP; 和 /或 Optionally, the probe is a nucleic acid probe set comprising a plurality of probes, the nucleic acid probe set having the following characteristics: (1) having one or more biotin-labeled dNTPs on each probe; and / or

( 2 )所述生物素标记的 dNTP在所述探针集中的丰度为 1 : 6-1 : 2; 和 /或 (2) the abundance of the biotin-labeled dNTP in the probe set is 1: 6-1: 2; and/or

( 3 )所述探针集的全部核酸序列覆盖对应选自 HBV、 HPV, EBV和幽门螺旋杆菌的至少一种病毒的基因组序列的 70%-100%, (3) the entire nucleic acid sequence of the probe set covers 70%-100% of the genomic sequence corresponding to at least one virus selected from the group consisting of HBV, HPV, EBV and H. pylori,

23、根据权利要求 21所述的确定人体具有异常状态的方法，其特征在于， 23. The method of determining an abnormal state of a human body according to claim 21, wherein:

所述适于除去含有特定序列的核酸的探针为可以结合人体基因组中的共有序列，或者为可以结合人体基因组中甲基化位点的抗体或者蛋白。 The probe suitable for removing a nucleic acid containing a specific sequence is an antibody or protein which can bind to a consensus sequence in the human genome or can bind to a methylation site in the human genome.