WO2023066271A1 - Gene modification detection method and application thereof - Google Patents

Abstract

A gene modification detection method and an application thereof. The method comprises: obtaining a substance to be detected which has a diameter of 200 nanometers or more in a sample to be detected, and detecting the amount and/or presence of hydroxymethylcytosine in said substance.

Description

A kind of genetic modification detection method and its application technical field

This application relates to the field of biomedicine, in particular to a gene modification detection method and its application.

Background technique

Extracellular vesicles (Extracellular Vesicles) are membrane vesicles of 50-1000nm secreted by most cells, which exist in biological fluids such as cell culture medium, plasma, serum, saliva, urine, amniotic fluid, and malignant ascites. Among them, the size of large extracellular vesicles is >200nm. Mounting evidence suggests that these vesicles act as cellular messengers, delivering messages to cells and tissues throughout the body. Extracellular vesicles contain cell-specific proteins, lipids and RNA, which are transported to other cells and alter the function or physiology of other cells. These extracellular vesicles may play a role in mediating adaptive immune responses to infectious pathogens and tumors, tissue repair, neural communication, and transfer of pathogenic proteins.

Clinically, the difficulty of tissue sampling is a major problem that limits the molecular diagnosis and precise treatment of tumors. Liquid biopsy samples such as blood and urine are a reliable source of tumor DNA because of their easy-to-obtain and non-invasive characteristics, and they are also a hot spot in the molecular diagnosis of tumors. Liquid biopsy samples mainly include plasma cell-free DNA (cfDNA), circulating tumor cells (CTCs) and extracellular vesicles (Extracellular Vesicles). At present, the application of cfDNA gene detection is relatively mature and extensive. However, cfDNA is the DNA fragment after tumor cell apoptosis or death, which may not accurately reflect the real-time genetic information of tumor cells. Conversely, plasma, urine, and other extracellular vesicle-derived nucleic acids (exoRN) are actively released by living tumor cells, which may better reflect the real-time dynamics of tumors.

DNA hydroxymethylation is an important form of epigenetic modification, which has been shown to be a stable epigenetic mark in the human genome, positively correlated with gene transcription levels, and plays a crucial role in cell development, differentiation, maturation and self-renewal important role. As a promising biomarker, 5-hydroxymethylcytosine (5hmC) is closely related to the occurrence and development of various human diseases. Therefore, the development of 5hmC sequencing technology based on extracellular vesicle DNA has important clinical significance.

Contents of the invention

The present application provides a gene modification detection method and its application, which may include a 5hmC sequencing technology method based on extracellular vesicle DNA. Liquid biopsy samples such as blood and urine are easy to obtain and non-invasive, and are a reliable source of tumor DNA, and are also a hot spot in tumor molecular diagnosis. However, cfDNA is the DNA fragment after tumor cell apoptosis or death, which may not be accurate. Reflect real-time genetic information of tumor cells. Extracellular vesicle-derived nucleic acids (exoRN) provided herein are actively released by living tumor cells, wherein preferred large extracellular vesicles are >200nm in size, which can act as cellular messengers to transmit information to cells in the body And tissue, can also better reflect the real-time dynamics of the tumor.

In one aspect, the present application provides an analysis method, comprising (S1) obtaining a test substance with a diameter of about 200 nanometers or more in the test sample, (S2) detecting the amount and / or exists.

In another aspect, the present application provides a method for confirming the existence of a disease, assessing the formation or risk of the disease, assessing the progress and/or prognosis of the disease, and/or screening the population corresponding to the treatment, comprising (S1) obtaining the (S2) Detecting the quantity and/or presence of hydroxymethylcytosine in the analyte region of the analyte in the sample with a diameter of about 200 nanometers or more.

In another aspect, the present application provides an analysis method, comprising detecting the quantity and/or presence of hydroxymethylcytosine in a substance to be tested with a diameter of about 200 nanometers or more in the sample to be tested.

In another aspect, the present application provides a method for confirming the existence of a disease, assessing the formation or risk of the disease, assessing the progress and/or prognosis of the disease and/or screening the population corresponding to the treatment, comprising detecting the diameter The amount and/or presence of hydroxymethylcytosine in the analyte above about 200 nm.

On the other hand, the present application provides a nucleic acid, which comprises a sequence capable of binding to the region to be tested in the substance to be tested with a diameter of about 200 nanometers or more in the sample to be tested, or its complementary region, or the above-mentioned fragments.

On the other hand, the present application provides a method for preparing nucleic acid, which includes the region to be tested in the sample to be tested, or its complementary region, or the sequence of the above-mentioned fragments in the substance to be tested with a diameter of about 200 nanometers or more, and the design can A nucleic acid that binds to the region to be tested, or its complementary region, or the above-mentioned fragments.

In another aspect, the present application provides a kit comprising the nucleic acid of the present application.

On the other hand, the present application provides the application of the nucleic acid in the present application, and/or the kit in the present application, in the preparation of disease detection products.

On the other hand, the present application provides the nucleic acid in the present application, and/or the kit in the present application, in the preparation of confirming the existence of the disease, assessing the formation or risk of forming the disease, assessing the progress and/or prognosis of the disease and/or screening The application of products for the treatment of appropriate populations.

On the other hand, the present application provides a database, which includes the sequence of the region to be tested, or its complementary region, or the above-mentioned fragments in the substance to be tested with a diameter of about 200 nanometers or more in the sample to be tested.

On the other hand, the present application provides a storage medium, which records a program capable of running the method in the present application.

In another aspect, the present application provides a device comprising the storage medium of the present application.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

Figure 1 shows the particle size distribution of extracellular vesicles (>200nm) isolated by the present application.

Figure 2 shows the particle size distribution of exosomes (<200nm) isolated by the present application.

Figure 3 shows the results of quality control of the 5-hydroxymethylcytosine (5hmC) library of DNA in extracellular vesicles (>200nm) isolated in this application.

Figure 4 shows the particle size distribution of extracellular vesicles isolated from plasma measured by dynamic light scattering. Extracellular vesicle size (>200nm) isolated from plasma.

Figure 5 shows the particle size distribution of plasma-isolated exosomes measured by dynamic light scattering. Size of exosomes isolated from plasma (<200nm).

Figure 6 shows the quality control of the 5-hydroxymethylcytosine (5hmC) library of extracellular vesicle (>200nm) DNA: the results of the automatic capillary electrophoresis system show that the library size is about 306bp.

Figures 7A-7D show the detection results of lung cancer patients and healthy people. Figure 7A: PCA graph of lung cancer and healthy people: based on 5hmC sequencing of exosomes (<200nm) from 30 lung cancer patients and 30 healthy people; Figure 7B: ROC curve of lung cancer and healthy people: based on 5hmC markers in exosomal DNA Distinguish between 30 lung cancer patients and 30 healthy people; Figure 7C: PCA graph of lung cancer and healthy people: based on 5hmC sequencing of extracellular vesicles (>200nm) in 30 lung cancer patients and 30 healthy people; Figure 7D: ROC of lung cancer and healthy people Graph: 30 cases of lung cancer patients and 30 cases of healthy people were distinguished based on 5hmC markers in extracellular vesicle DNA.

Figures 8A-8D show the detection results of colorectal cancer patients and healthy people. Figure 8A: PCA graph of colorectal cancer and healthy people: based on 5hmC sequencing of exosomes (<200nm) from 25 patients with colorectal cancer and 30 healthy people; Figure 8B: ROC curve of colorectal cancer and healthy people: based on exocytosis 5hmC markers in body DNA distinguish 25 colorectal cancer patients from 30 healthy people; Figure 8C: PCA plot of colorectal cancer and healthy people: based on exosomes (>200nm) from 25 colorectal cancer patients and 30 healthy people 5hmC sequencing; Figure 8D: ROC curves of colorectal cancer and healthy people: 25 colorectal cancer patients and 30 healthy people were distinguished based on 5hmC markers in extracellular vesicle DNA.

Detailed ways

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the term "sample" generally refers to a material or mixture of materials, usually in liquid or other form, which may contain one or more analytes of interest.

In this application, the term "extracellular vesicle" generally refers to a type of extracellular particle. For example, extracellular particles may refer to membranous particles released by cells into the extracellular matrix. For example, extracellular particles can be from about 30 nanometers to about 1000 nanometers in diameter. For example, an extracellular vesicle can be a type of extracellular particle of a specific size, for example, an extracellular vesicle can have a diameter of about 200 nanometers or more.

In this application, the term "organoid" generally refers to a cell mass. For example, the organoid can have self-renewal ability and self-organization ability, for example, the organoid can have corresponding tissue and organ functions. For example, the organoids can be cultured in Matrigel to have a 3D structure.

In this application, the terms "determine", "measure", "evaluate", "evaluate", "determine" and "analyze" are used interchangeably herein and generally refer to any form of measurement, including determining the presence or absence of an element. These terms can include both quantitative and/or qualitative aspects. Evaluations can be relative or absolute. "Assessing the presence of" can include determining the amount of something present, as well as determining its presence or absence.

For example, the simple steps of gene mutation detection may include taking patient tumor tissue samples, extracting DNA, and testing with kits and other methods to determine whether the patient has a mutation; those with mutations are regarded as positive samples in clinical tests; those without mutations are used as clinical tests. Negative samples.

For example, methods for genetic detection may include: polymerase chain reaction-restriction fragment length polymorphism analysis technology, pyrosequencing method, single-strand conformational polymorphism analysis technology, probe amplification block mutation system, high-performance liquid Phase chromatography, micro-digital polymerase chain reaction, and high-resolution melting curve analysis techniques, etc.

For example, the detection of gene expression can include selecting kits and other methods for detection according to the conditions of genes and tumors to determine whether the patient has gene expression variation; those with gene expression variation are regarded as positive samples in clinical tests; those without gene expression variation as clinically negative samples.

For example, the genes for predicting the occurrence of diseases and related symptoms can include the genes related to diseases and related symptoms obtained through the existing sequencing data, or sequencing of clinical patient samples (including DNA, RNA and other sequencing methods), and data analysis. Genes, and genes that can predict the occurrence of diseases and their related symptoms through their expression levels in the absence of occurrence. Samples that are predicted to be highly correlated with diseases and their related symptoms can be regarded as positive samples of clinical tests, and samples that are predicted to be highly correlated with diseases and their related symptoms can be regarded as negative samples of clinical tests.

For example, the blood test for tumor markers may include blood collection to check the levels of tumor markers in the blood. Alternatively, the pleural effusion or ascites can be drained and sent for examination of tumor markers. The changes in the content of tumor markers in the pleural effusion and ascites can be compared with the changes in the tumor markers in the blood of the patient, thereby strengthening the monitoring and control of the disease. diagnosis. Samples that are predicted to be highly correlated with diseases and their related symptoms can be regarded as positive samples of clinical tests, and samples that are predicted to be highly correlated with diseases and their related symptoms can be regarded as negative samples of clinical tests.

In this application, the term "sequencing" generally refers to a method by which the identity of at least 10 contiguous nucleotides (e.g., at least 20, at least 50, at least 100, or at least 200 nucleotides) of a polynucleotide can be obtained. identities of one or more consecutive nucleotides).

In this application, the term "UDP glucose modified with chemoselective groups" generally refers to UDP glucose that has been functionalized, possibly at the 6-hydroxyl position, to include the ability to participate in 1,3- Groups for cycloaddition (or "click") reactions. Such groups may include azido and alkynyl (eg cyclooctyne). For example, UDP-6-N3-Glu can be UDP glucose modified with chemoselective groups.

In this application, the term "biotin moiety" generally refers to biotin or moieties such as desthiobiotin, oxidized biotin, 2-iminobiotin, diaminobiotin, biotin sulfoxide, biotin Affinity tags for biotin analogs such as Cytin. The biotin moiety can bind streptavidin.

In this application, the terms "cycloaddition reaction" and "click reaction" are generally interchangeably described terms, generally referring to the 1,3-cycloaddition between an azide and an alkynyl to form a five-membered heterocycle. In some embodiments, the alkynyl group can be strained (eg, in a ring such as cyclooctyne), and the cycloaddition reaction can be performed under copper-free conditions. Dibenzocyclooctyne (DBCO) and difluorooctyne (DIFO) may be examples of alkynes capable of participating in copper-free cycloaddition reactions.

In this application, the term "biotin-bound carrier" generally refers to a carrier (eg a bead, which may be magnetic) attached to streptavidin or avidin or a functional equivalent thereof.

In this application, the term "amplification" generally refers to the use of a target nucleic acid as a template to produce one or more copies of the target nucleic acid.

In this application, the term "copy of a fragment" generally refers to an amplified product, wherein the copy of a fragment may be the reverse complement of the strand of the fragment, or may have the same sequence as the strand of the fragment.

In this application, the term "enrichment" generally refers to the separation of analytes with a certain characteristic (such as nucleic acids containing hydroxymethylcytosine) from analytes without that characteristic (such as nucleic acids containing hydroxymethylcytosine). partially purified. For example, enrichment generally increases the concentration of an analyte having the characteristic (e.g., a nucleic acid comprising hydroxymethylcytosine) by at least 2-fold, at least 5-fold, or at least 10-fold relative to an analyte without the characteristic. Following enrichment, at least 10%, at least 20%, at least 50%, at least 80%, or at least 90% of the analytes in the sample may have the characteristic used for the enrichment. For example, at least 10%, at least 20%, at least 50%, at least 80%, or at least 90% of the nucleic acid molecules in the enriched composition may comprise chain of methylcytosine.

Detailed description of the invention

In one aspect, the present application provides an analysis method, which may include obtaining a test substance with a diameter of about 200 nanometers or more in the test sample, and detecting the quantity and/or presence of hydroxymethylcytosine in the test substance.

On the other hand, the present application provides a method for confirming the existence of a disease, assessing the formation of a disease or the risk of forming a disease, assessing the progress and/or prognosis of a disease, and/or screening a population corresponding to treatment, which may include obtaining the diameter The analyte is about 200 nanometers larger, and the amount and/or presence of hydroxymethylcytosine in the analyte is detected.

For example, the sample to be tested in the present application may include plasma, urine, tissue culture fluid, cell culture fluid and/or organoid culture fluid. For example, the substance to be tested in the present application may comprise extracellular vesicles. For example, the extracellular vesicles of the present application may have a diameter of about 200 nanometers or more. For example, the extracellular vesicle diameter of the present application can be about 200 nm or more, 250 nm or more, 300 nm or more, 350 nm or more, 400 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 750 nm or more, 800 nm or more Above nanometers, above 900 nanometers, or above 1000 nanometers. For example, the diameter of extracellular vesicles in the present application can be the average diameter of extracellular vesicles in the sample. For example, the substance to be tested in the present application may contain nucleic acid. For example, the test substance of the present application may contain DNA.

For example, the test sample can be derived from animals, plants and/or fungi. For example, the sample to be tested may include body fluid, tissue, cell and/or organoid culture supernatant. For example, the body fluid in the sample to be tested may include plasma and/or urine. For example, the tissue may include stomach tissue, liver tissue, brain tissue, kidney tissue, breast tissue, lung tissue, lymph node tissue, peritoneal tissue and/or heart tissue.

For example, the methods of the present application may include the step of isolating extracellular vesicles. For example, the separation of extracellular vesicles described therein may include centrifugation, ultrafiltration, size exclusion chromatography, polymer precipitation and/or affinity capture. For example, the centrifugation in the extracellular vesicle isolation described therein may include ultracentrifugation, density gradient centrifugation, sedimentation centrifugation, differential centrifugation, centrifugal elutriation and/or zonal centrifugation.

For example, the centrifugation in the extracellular vesicle isolation may include the first centrifugation, and the first centrifugation speed is about 300g to about 1000g. For example, the centrifugation in the extracellular vesicle isolation may include the first centrifugation, and the temperature of the first centrifugation is about 2°C to about 10°C. For example, the centrifugation in the extracellular vesicle isolation may include the first centrifugation, and the first centrifugation time is about 5 minutes to about 20 minutes. For example, the centrifugation in the extracellular vesicle isolation described therein may include the first centrifugation, and the first centrifugation is performed at a speed of about 300g to about 1000g at a temperature of about 2°C to about 10°C 5 minutes to about 20 minutes.

For example, the centrifugation in the extracellular vesicle isolation described therein may include a second centrifugation, which uses the supernatant obtained from the first centrifugation of the present application for centrifugation. For example, the centrifugation in the extracellular vesicle isolation may include a second centrifugation, and the second centrifugation speed is about 1500g to about 5000g. For example, the centrifugation in the extracellular vesicle isolation may include a second centrifugation, and the temperature of the second centrifugation is about 2°C to about 10°C. For example, the centrifugation in the extracellular vesicle isolation may include a second centrifugation, and the second centrifugation time is about 10 minutes to about 30 minutes.

For example, the centrifugation in the extracellular vesicle isolation described therein may include a third centrifugation, which uses the supernatant obtained from the second centrifugation described in the present application for centrifugation. For example, the centrifugation in the extracellular vesicle isolation may include a third centrifugation, and the third centrifugation speed is about 10000g to about 15000g. For example, the centrifugation in the extracellular vesicle isolation may include a third centrifugation, and the temperature of the third centrifugation is about 2°C to about 10°C. For example, the centrifugation in the extracellular vesicle isolation may include a third centrifugation, and the third centrifugation time is about 10 minutes to about 30 minutes. For example, wherein the third centrifugation can obtain a sediment at the bottom of the tube, and the sediment can be a large vesicle. For example, wherein the diameter of the large vesicles in the third centrifugation can be greater than about 200 nm.

For example, the centrifugation in the separation of extracellular vesicles may include a fourth centrifugation, and the supernatant obtained by the above three centrifugations may be centrifuged. For example, the centrifugation in the extracellular vesicle isolation may include a fourth centrifugation, and the fourth centrifugation speed is about 50000g to about 150000g. For example, the centrifugation in the extracellular vesicle isolation may include a fourth centrifugation, and the temperature of the fourth centrifugation is about 2°C to about 10°C. For example, the centrifugation in the extracellular vesicle isolation may include a fourth centrifugation, and the fourth centrifugation time is about 60 minutes to about 100 minutes. For example, wherein the fourth centrifugation obtains a precipitate at the bottom of the tube, the diameter of the precipitate may be less than about 200 nm.

For example, the method of the present application may include the following steps: (S1-1) centrifuging the sample to be tested at about 500g to obtain a supernatant; (S1-2) centrifuging the sample obtained in the step (S1-1) at about 3000g supernatant, to obtain a supernatant; (S1-3) centrifuging the supernatant obtained in the step (S1-2) at about 12000 g, to obtain a precipitate containing the substance to be tested. For example, the duration of the step (S1-1) may be about 10 minutes. For example, the duration of the step (S1-2) may be about 20 minutes. For example, the duration of the step (S1-3) may be about 20 minutes.

For example, the method of the present application may include the following steps: (S1-1) centrifuging the sample to be tested at about 1350g to obtain a supernatant; (S1-2) centrifuging the sample obtained in the step (S1-1) at about 3000g supernatant, obtaining the supernatant; (S1-3) contacting the supernatant obtained in the step (S1-2) with a size-exclusion filler to separate components containing the substance to be tested. For example, the duration of the step (S1-1) may be about 15 minutes. For example, the duration of the step (S1-2) may be about 10 minutes. For example, the size exclusion filler may comprise a filler having a diameter of about 22 microns to 44 microns. For example, the step (S1-3) can collect two or more tubes of size exclusion eluted fractions, and determine the fractions containing the substance to be tested by dynamic light scattering.

For example, the hydroxymethylation sequencing method of the present application may include the following steps: (S2a) extracting nucleic acid fragments in the sample, (S2b) labeling nucleic acid fragments containing hydroxymethylcytosine in the nucleic acid fragments, (S2c) Enriching the nucleic acid fragments containing hydroxymethylcytosine, (S2d) performing sequencing on the enriched nucleic acid fragments containing hydroxymethylcytosine.

For example, the labeling described herein comprises contacting the nucleic acid fragment with DNA β-glucosyltransferase and UDP glucose modified with a chemoselective group. For example, the label of the present application may be that the chemoselective group is attached to a nucleic acid fragment containing hydroxymethylcytosine in the sample.

For example, a chemoselective group described herein may comprise an azido group. For example, the chemoselective groups of the present application may comprise any chemical click group.

For example, labeling as described herein may also comprise contacting said nucleic acid fragment bearing a chemoselective group with biotin comprising biotin capable of reacting with said chemoselective group. For example, the biotin capable of reacting with the chemoselective group may comprise biotin containing a dibenzocyclooctyne modification. For example, the label may be that a first label is linked to a nucleic acid fragment containing hydroxymethylcytosine in the sample, and may include linking the nucleic acid fragment to which the first label is linked with a second label, the The second label is selectively reactive with said first label.

For example, said enriching can comprise contacting said nucleic acid fragments bearing biotin with magnetic beads comprising streptavidin. For example, the labeled nucleic acid fragments containing hydroxymethylcytosine can be combined with magnetic beads containing streptavidin. For example, said enrichment may comprise separating magnetic beads bearing said hydroxymethylcytosine nucleic acid fragments by magnetic force.

For example, the method of the present application may include detecting the quantity and/or presence of the nucleic acid fragments carrying the hydroxymethylcytosine in the test sample by sequencing. For example, the present application may sequence the sample to be tested by a method selected from the following group: digital PCR and high-throughput sequencing. For example, the sequencing method of the present application can be selected from any sequencing method known in the art.

On the other hand, the present application also provides an analysis method, which may include detecting the quantity and/or presence of hydroxymethylcytosine in the substance to be tested with a diameter of about 200 nanometers or more in the sample to be tested.

On the other hand, the present application also provides a method for confirming the existence of the disease, assessing the formation or risk of the disease, assessing the progress and/or prognosis of the disease and/or screening the corresponding population for treatment, which may include detecting the The amount and/or presence of hydroxymethylcytosine in a substance to be measured with a diameter greater than about 200 nanometers.

On the other hand, the present application provides a nucleic acid, which may comprise a sequence capable of binding to the region to be tested in the substance to be tested with a diameter of about 200 nanometers or more in the sample to be tested, or its complementary region, or the above-mentioned fragments .

On the other hand, the present application provides a method for preparing nucleic acid, which may include the region to be tested in the substance to be tested with a diameter of about 200 nanometers or more in the sample to be tested, or its complementary region, or the sequence of the above-mentioned fragments, designed A nucleic acid capable of binding to the region to be tested, or its complementary region, or the above-mentioned fragments.

In another aspect, the present application provides a kit, which may comprise the nucleic acid described in the present application. For example, the kit may also include blood collection tubes. For example, the kit can be used with a centrifuge. For example, the kit may also include reagents comprising a glycosylation group, for example, the glycosylation group may comprise a UDP glucose group or a derivative thereof, a reagent comprising a labeling group, such as the labeling group Biotin groups or derivatives thereof, linkers, media, amplification primers and/or buffers may be included. For example, the kit can be used with a sequencing instrument.

On the other hand, the present application provides the application of the nucleic acid in the present application, and/or the kit in the present application, in the preparation of disease detection products.

On the other hand, the present application provides the nucleic acid in the present application, and/or the kit in the present application, which can be prepared to confirm the existence of the disease, assess the formation or risk of the disease, assess the progress and/or prognosis of the disease and/or Screening for use in products with appropriate populations for treatment.

On the other hand, the present application provides the nucleic acid of the present application, and/or the kit of the present application, which can be used for disease detection.

In another aspect, the present application provides the nucleic acid in the present application, and/or the kit in the present application, which can be used to confirm the existence of the disease, assess the disease formation or risk of formation, assess the progress and/or prognosis of the disease and/or Or screen the corresponding population for treatment.

On the other hand, a method for detecting a disease of the present application may include providing the nucleic acid of the present application, and/or the kit of the present application.

In another aspect, the method of confirming the existence of a disease, assessing the formation or risk of developing a disease, assessing the progress and/or prognosis of a disease and/or screening a population corresponding to treatment may comprise providing the nucleic acid in the present application, and /or kits in this application.

For example, the diseases of the present application may comprise tumors.

On the other hand, the present application provides a database, which can include the sequence of the region to be tested, or its complementary region, or the above-mentioned fragments in the substance to be tested with a diameter of about 200 nanometers or more in the sample to be tested.

On the other hand, the present application provides a storage medium, which can record a program capable of running the method in the present application.

In another aspect, the present application provides a device, which may include the storage medium in the present application. For example, the non-transitory computer readable storage medium may include a floppy disk, a flexible disk, a hard disk, a solid state storage (SSS) (such as a solid state drive (SSD)), a solid state card (SSC), a solid state module (SSM)), an enterprise high-grade flash drives, tape, or any other non-transitory magnetic media, etc. Non-transitory computer readable storage media may also include punched cards, paper tape, cursor sheets (or any other physical media having a pattern of holes or other optically identifiable markings), compact disc read only memory (CD-ROM) , Rewritable Disc (CD-RW), Digital Versatile Disc (DVD), Blu-ray Disc (BD) and/or any other non-transitory optical media.

For example, the device as described in the present application further includes a processor coupled to the storage medium, and the processor is configured to execute based on the program stored in the storage medium to implement the method described in the present application. For example, the database system may implement various mechanisms to ensure that the methods described herein performed on the database system produce correct results. In this application, the database system may use disks as permanent data storage. In this application, the database system can provide database storage and processing services for multiple database clients. The database client may store database data across multiple shared storage devices, and/or may utilize one or more execution platforms with multiple execution nodes. The database system can be organized such that storage and computing resources can be effectively scaled indefinitely.

Not intending to be limited by any theory, the following examples are only for explaining the methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1 Detection method of extracellular vesicle DNA 5-hydroxymethylcytosine

(1) 5-Hydroxymethylcytosine (5hmC) library establishment

Extracellular Vesicle Isolation

Extracellular vesicles can be isolated from plasma samples:

1. Take whole blood (8mL) in 10mL Kangwei Company (CW2815M) cfDTM free nucleic acid blood collection tube (please operate at room temperature, store at room temperature after blood collection;

2. For the first centrifugation, centrifuge at 500g at 4°C for 10 minutes at low temperature, carefully remove the light yellow supernatant (removing blood cells and platelets), and transfer to a 2mL DNase free sterile centrifuge tube;

3. The second centrifugation, 3000g, 4 ℃ low temperature centrifugation for 20min, carefully remove the supernatant (remove white blood cells and cell debris), transfer to 2-3 tubes of 2mL DNase free sterile centrifuge tube;

4. Centrifuge for the third time at 12,000g, 4°C for 20min at low temperature, carefully remove the supernatant, transfer the supernatant to 2-3 tubes of 2mL DNase free sterile centrifuge tubes, and collect the precipitate at the bottom of the tube (large vesicles with a diameter >200nm ), add 1mL LPBS (1X), mix well and store in -80° refrigerator;

5. For the fourth centrifugation, centrifuge at 100,000g at 4°C for 70 minutes at low temperature, remove the supernatant, collect the sediment at the bottom of the tube (exosomes with a diameter <200nm), add 1mL LPBS (1X), mix well and store in a -80°refrigerator;

Extracellular vesicles can be isolated from urine samples:

1. Take 10mL of urine;

2. For the first centrifugation, centrifuge at 500g at 4°C for 10 minutes at low temperature, carefully remove the light yellow supernatant (to remove cells), and transfer to a 2mL DNase free sterile centrifuge tube;

3. The second centrifugation, 3000g, 4 ℃ low temperature centrifugation for 20min, carefully remove the supernatant (to remove cell debris), transfer to a 15mL sterile centrifuge tube;

4. Centrifuge for the third time at 12000g, 4°C for 20min at low temperature, carefully remove the supernatant, transfer the supernatant to a 15mL sterile centrifuge tube, collect the precipitate at the bottom of the tube (large vesicles with a diameter >200nm), add 1mL PBS (1X ), mix well and store in -80° refrigerator;

5. For the fourth centrifugation, centrifuge at 100,000g at 4°C for 70 minutes at low temperature, remove the supernatant, collect the sediment at the bottom of the tube (exosomes with a diameter <200nm), add 1mL LPBS (1X), mix well and store in a -80°refrigerator;

Extracellular vesicles can be isolated from tissue, cell, primary cell, organoid culture supernatant samples:

1. Take 10ml tissue, cell, primary cell and organoid culture supernatant into a 15mL centrifuge tube;

2. For the first centrifugation, centrifuge at 500g at 4°C for 10 minutes at low temperature, carefully remove the supernatant (remove cells), and transfer to a 15mL sterile centrifuge tube;

3. The second centrifugation, 3000g, 4 ℃ low temperature centrifugation for 20min, carefully remove the supernatant (to remove cell debris), transfer to a 15mL sterile centrifuge tube;

4. Centrifuge for the third time at 12000g, 4°C for 20min at low temperature, carefully remove the supernatant, transfer the supernatant to a 15mL sterile centrifuge tube, collect the precipitate at the bottom of the tube (large vesicles with a diameter >200nm), add 1mL PBS (1X ), mix well and store in -80° refrigerator;

5. For the fourth centrifugation, centrifuge at 100,000g at 4°C for 70 minutes at low temperature, remove the supernatant, collect the sediment at the bottom of the tube (exosomes with a diameter <200nm), add 1mL LPBS (1X), mix well, and store in a -80°refrigerator.

Extracellular vesicles can be isolated by size exclusion chromatography:

1. Place the collected non-anticoagulated whole blood in a 4-degree refrigerator for 4 hours;

2. Centrifuge at 1350g for 15 minutes to separate the plasma sample;

3. Centrifuge the plasma sample at 3000g for 10 minutes to remove cell debris;

4. Use Sigma's superdex filler to fill the size exclusion column;

5. Equilibrate the prepacked column with PBS buffer;

6. Add the plasma sample with decellularized debris into the size exclusion column, and elute with PBS. Collect the eluted fraction in 0.5mL per tube. The large extracellular vesicles are eluted first, and the small extracellular vesicles are eluted after Elution;

7. Perform dynamic light scattering to characterize the particle size of each tube, that is, to distinguish between large extracellular vesicles (>200nm) and exosomes (<200nm).

Extracellular Vesicle (>200nm) gDNA Extraction

1. Use the Quick-DNA miniprep plus kit (ZYMO, D4069) kit to extract about 20ng of genomic DNA from the extracellular vesicle samples prepared by any of the above methods;

2. Use Qubit3.0 to measure the gDNA concentration of extracellular vesicles and granules.

Fragmentation, end-filling, and ligation of gDNA with sequencing adapters

According to the manual of KAPA HyperPlus Library Preparation Kit (KK8514), the brief operation is as follows:

1. Interrupt large fragments of gDNA. The input amount of gDNA is 10ng, the sample volume is 12μL, and the reactants are 2μL 0.6% FCS, 2μL Frag Buffer, 4μL Frag Enzyme; 37℃ water bath for 20 minutes;

2. Prepare 20 μL of a reaction mixture containing 10 ng of genomic DNA, 2.8 μL of End Repair&A-Tailing Buffer and 1.2 μL of End Repair&A-Tailing Enzyme mix (total volume is 24 μL); incubate at 20°C for 30 minutes, then incubate at 65°C for 30 minutes ;

3. Configure the following ligation reaction mixture in a .5mL low-adsorption EP tube: 2μL Nuclease free water (nuclease-free water), 12μL Ligation Buffer and 4μL DNA Ligase; add 2μL sequencing KAPA index (PKR2015, PKR2016 and PKR2017), mixed, added to 24 μL reaction sample, and heated at 20°C for 4 hours;

4. Use the DNA Clean&Concentrator 5 (ZYMO, D4014) purification kit to purify the reaction product, and use 20 μL of elution buffer to elute to obtain the final DNA ligation sample.

5hmC labeling

1. Prepare a labeling reaction mixture with a total volume of 4 μL: 1 μL 50uM UDP-N3-Glu (hmC labeling substrate), 2.5 μL βGT enzyme (NEB) and 2.5 μL HEPES buffer (pH 8.0, final concentration is 50 mM). Add 6 μL labeling mix to 20 μL DNA ligation sample. Place the mixture in a water bath at 37°C for 1 hour;

2. Take out the mixture and purify the reaction product with DNA Clean&Concentrator 5 (ZYMO, D4014) purification kit to obtain 30 μL of purified DNA;

3. Then add 1 μL 45uM DBCO-PEG4-Biotin (Click Chemistry Tools) to the above-mentioned purified 30 μL DNA, and bathe in water at 37°C for 1 hour;

4. Purify the reaction product with DNA Clean&Concentrator 5 (ZYMO, D4014) purification kit to obtain 30 μL of purified labeled product.

5hmC enrichment

1. Equilibrate the bound magnetic beads according to the following steps: take out 2.5 μL Dynabeads (Invitrogen, 65306) and add 100 μL washing buffer (5mM Tris (pH 7.5), 1M NaCl and 0.02% Tween20), blow and mix, place in 1.5mL magnetic On the rack, wash the bound magnetic beads with 100 μL washing buffer repeatedly for 3 times, finally add 100 uL washing buffer, mix the magnetic beads, and vortex for 30 min;

2. After 30min, wash the magnetic beads with 100uL washing buffer 3 times, and finally add 32μL binding buffer (10mM Tris (pH 7.5), 2M NaCl and 0.04% Tween20), and mix well;

3. Add the purified labeled product obtained in the above steps to the magnetic bead mixture, and mix in a rotary mixer for 30 minutes to fully combine;

4. Finally, wash the magnetic beads 5 times with 100 μL washing buffer, and add 23.8 μL RNase-Free water.

PCR amplification

1. Add 25 μL 2×PCR master mix and 1.25 μL PCR primers (total volume is 50 μL) to the final system of the above steps, and perform amplification according to the temperature and conditions of the PCR reaction cycle as described below:

2. The amplified product was purified with AmpureXP beads (KAPA, KK8001), and finally eluted with 20uL eluent to obtain the final 5hmC library. Library concentration determination can be performed with Qubit 3.0.

High-throughput sequencing after quality control of the 5hmC library

1. Perform quality control of the obtained 5hmC library on the Fragment AnalyzerTM automatic capillary electrophoresis system (kit: DNF-900; use software: Fragment Analyzer instrument control software, PROSize data analysis software) to determine the size of the DNA fragments in the library and whether it contains impurities (Library size is about 300bp);

2. Perform qPCR concentration determination (kit: KAPA SYBR FAST Universal qPCR Kit (KK4601)) to determine whether the sample library meets the sequencing standards on the machine;

Specific steps:

a. Preparation of samples: prepare five 1.5mL EP tubes, and mix every 4 samples to be sequenced into one EP tube. There are 20 samples in 5 tubes (index cannot be repeated); each 5hmC library sample absorbs 5ng, and each EP The total volume of the tube is 20uL, and the final concentration is 1ng/uL;

b. The sample reaction system (20uL) is as follows: draw 4uL of the library in each EP tube for qPCR quantification.

Reactant	Volume (uL)
KAPA SYBR FAST qPCR Master Mix(2X)	10

Library Quantification Primer Premix(10X)	1.6
ROX Reference Dye High(50X)	0.4
RNase-Free water	4
5hmC library	4

c.qPCR program settings:

d. Result analysis: analyze according to the qPCR operating software to determine whether the 5hmC library is degraded and whether it meets the sequencing requirements;

3. The library (16uL) that passed the quality inspection was sequenced with Illumina NextSeq500, the sequencing kit used was High Output Kit v2 (75cycles), the sequencing throughput of each sample was 1.5Gb, and the sequencing band size was 75bp.

Raw sequencing data comparison

1. Each original sequencing FASTQ data is first trimmed with low-quality data using Trimmomatic software, and then compared to the human genome hg19 using Bowtie2 software;

2. Use MACS software to identify the peak number of reads containing 5hmC, the parameters are: effective genome size = 2.72e+09; tag size = 38; band width = 100; model fold = 10; P value cutoff = 1.00e-05, And use this to call peaks and generate counts files;

3. Use DEseq2 software to compare the counts files from positive samples and negative samples, find the 5hmC peak area with more than 50 reads, press |log2FoldChange|>=0.5, pvalue<0.05, and get the differential biomarkers of 5hmC up and down respectively thing.

Clinical Sample Testing

Collect clinically positive patient samples and clinically negative patient samples, and detect the hydroxyl groups of differential biomarkers in extracellular vesicles (>200nm) from different sample sources through the 5hmC sequencing technology method based on extracellular vesicle DNA of this application. Copy number of methylated sites. The results show that the 5hmC sequencing technology method based on extracellular vesicle DNA of the present application can more accurately distinguish clinically positive patients from clinically negative patients.

Example 2

1. Sample collection and extracellular vesicle extraction:

Collect a blood sample (8-10mL) from a healthy person, and separate the plasma (4-5mL); extract extracellular vesicles (>200nm) from the plasma by gradient centrifugation; then extract the DNA in the extracellular vesicles for 5hmC library construction; finally, use I1lumina NextSeq500 to sequence the 5hmC library, the sequencing throughput of each sample is 1.5Gb, and the sequencing band size is 75bp;

2. Characterization of extracellular vesicles (>200nm) and exosomes (<200nm):

The extracted extracellular vesicles and exosomes were diluted into PBS at a concentration of 0.2 mg/mL, and then the size of extracellular large vesicles and exosomes was determined by dynamic light scattering using Zeta sizer Nano ZS90 from Malven Company.

Figure 1 shows the particle size distribution of extracellular vesicles (>200nm) isolated in this application, and Figure 2 shows the particle size distribution of exosomes (<200nm) isolated in this application. The results show that the method of the present application can isolate extracellular vesicles (>200nm).

3. 5hmC library quality control:

The obtained 5hmC library was subjected to Fragment AnalyzerTM automatic capillary electrophoresis system quality control (kit: DNF-900; software: Fragment Analyzer instrument control software, PROSize data analysis software), to determine the size of DNA fragments in the library and whether it contained impurities (library The size is about 300bp). Figure 3 shows the quality control results of the 5-hydroxymethylcytosine (5hmC) library of DNA in extracellular vesicles (>200nm) isolated by this application. The results of the automatic capillary electrophoresis system show that the library size is about 306bp . The results show that the detection method based on extracellular vesicles (>200nm) of this application can obtain a qualified 5hmC library.

Example 3

For the detection of samples, the corresponding sites to be tested can be screened through the hydroxymethylation sites.

1. Determine the grouping of clinical samples: According to the results of clinical immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and other methods to detect drug treatment targets (such as: MET), clinical samples are divided into MET positive and negative groups;

2. Through the comparative analysis of the positive group and the negative group, filter the 5hmC differential markers according to the screening conditions of |log2FoldChange|>=0.5, pvalue<0.05, and find the hydroxymethylation site corresponding to MET (MET, etc.);

3. Synthesis of MET hydroxymethylation site primers (Qingke Biotechnology Co., Ltd.)

Digital PCR detection based on cfDNA hydroxymethylation sites

(1) Sample system preparation (20uL)

1. The amount of 5hmC library added to the sample is 10ng, the standard primer concentration is 10uM, and the dye method master mix (Yongnuo, S0200020301);

Reactant	Volume (μL)
sample	x
nuclease free water	9.2 minus X
Primer F1	0.4
Primer F2	0.4
dye master mix	10

(1) Droplet generation

1. Use Yongnuo sample preparation general consumables (S0100010101) for chip preparation, add 50uL droplet generating oil to the first row of 8 holes of the chip, add 20uL sample system to the second row of 8 holes of the chip, and then add 5uL of sealant Added to the second row of sample wells;

Reactant	Volume (μL)	chip location
droplet generating oil	50	first row
sample system	20	second row
Sealants	5	second row

2. Use Yongnuo MicroDrop-100 digital PCR system for microdroplet generation;

(2) Sealing film

1. Transfer the prepared microdroplet (50uL) to a 96-well PCR plate (S0100030101), and attach a membrane;

2. Use Yongnuo MicroDrop-100 digital PCR system to seal the film at 190°C high temperature;

(3) PCR amplification

1. PCR amplification procedure:

2. Perform PCR amplification on the sample;

(4) Digital PCR detection

1. Use Yongnuo MicroDrop-100 digital PCR system to detect and analyze PCR products.

The hydroxymethylation detection of the sample to be tested may not be limited to a specific hydroxymethylation site, and any hydroxymethylation site on the chromosome can be detected by single-gene digital PCR.

Example 4

Detection of samples of extracellular vesicles and exosomes

1. Sample collection and extracellular vesicle extraction:

Collect a blood sample (8-10mL) from a healthy person, and separate the plasma (4-5mL); extract extracellular vesicles (>200nm) from the plasma by gradient centrifugation; then extract the DNA in the extracellular vesicles for 5hmC library construction; finally, use I1lumina NextSeq500 to sequence the 5hmC library, the sequencing throughput of each sample is 1.5Gb, and the sequencing band size is 75bp;

2. Characterization of extracellular vesicles (>200nm) and exosomes (<200nm):

The extracted extracellular vesicles and exosomes were diluted into PBS at a concentration of 0.2 mg/mL, and then the size of extracellular large vesicles and exosomes was determined by dynamic light scattering using Zeta sizer Nano ZS90 from Malven Company.

3. 5hmC library quality control:

The obtained 5hmC library was subjected to Fragment AnalyzerTM automatic capillary electrophoresis system quality control (kit: DNF-900; software: Fragment Analyzer instrument control software, PROSize data analysis software), to determine the size of DNA fragments in the library and whether it contained impurities (library The size is about 300bp);

4. Results

The results shown in Figure 4 and Figure 5 show that the size of extracellular vesicles isolated from plasma (>200nm) and the size of exosomes isolated from plasma (<200nm) were isolated by this application. As shown in Figure 6, the library constructed by extracellular vesicles in this application.

Example 5

For the detection of lung cancer samples

Sample collection and 5hmC-Seal sequencing:

Blood samples (8-10 mL) were collected from 60 lung cancer patients and 60 healthy people, and plasma (4-5 mL) was separated. Extracellular vesicles (>200nm) and exosomes (<200nm) were isolated from plasma by gradient centrifugation, and DNA was extracted for 5hmC-Seal sequencing. Among them, extracellular vesicles (>200nm) were isolated from 30 lung cancer samples and 30 healthy human samples; extracellular vesicles (<200nm) were isolated from 30 lung cancer samples and 30 healthy human samples. The sequencing throughput of each sample is 1.5Gb, and the sequencing band size is 75bp; Figures 7A-7D show the detection results of lung cancer patients and healthy people. The results show that the application has significantly improved accuracy for the detection of 5hmC in extracellular vesicles, and the detection sensitivity and specificity are higher than those for exosomes.

Example 6

For the detection of colorectal cancer samples

Sample collection and 5hmC-Seal sequencing:

Blood samples (8-10 mL) were collected from 50 patients with colorectal cancer and 60 healthy people, and plasma (4-5 mL) was separated. Extracellular vesicles (>200nm) and exosomes (<200nm) were isolated from plasma by gradient centrifugation, and DNA was extracted for 5hmC-Seal sequencing. Among them, extracellular vesicles (>200nm) were isolated from samples from 25 cases of colorectal cancer and 30 cases of healthy individuals; extracellular vesicles (<200nm) were isolated from samples from 25 cases of colorectal cancer and 30 cases of healthy individuals. The sequencing throughput of each sample is 1.5Gb, and the sequencing band size is 75bp; Figures 8A-8D show the detection results of colorectal cancer patients and healthy people. The results show that the application has significantly improved accuracy for the detection of 5hmC in extracellular vesicles, and the detection sensitivity and specificity are higher than those for exosomes.

The foregoing detailed description has been offered by way of explanation and example, not to limit the scope of the appended claims. Variations on the presently recited embodiments of this application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims (35)

1. An analysis method, comprising (S1) obtaining a test substance with a diameter of about 200 nanometers or more in a test sample, (S2) detecting the quantity and/or presence of hydroxymethylcytosine in the test substance.
2. A method for confirming the existence of a disease, assessing the formation or risk of a disease, assessing the progress and/or prognosis of a disease, and/or screening a population corresponding to treatment, comprising (S1) obtaining a sample with a diameter of about 200 nanometers or more The analyte of the analyte, (S2) detecting the quantity and/or presence of hydroxymethylcytosine in the analyte region in the analyte.
3. The method according to any one of claims 1-2, wherein the sample to be tested comprises plasma, urine, tissue culture fluid, cell culture fluid and/or organoid culture fluid.
4. The method according to any one of claims 1-3, wherein the substance to be tested comprises extracellular vesicles.
5. The method according to any one of claims 1-4, wherein the test substance comprises nucleic acid.
6. The method according to any one of claims 1-5, wherein the test substance comprises DNA.
7. The method according to any one of claims 1-6, wherein said step (S1) comprises the following steps: (S1-1) centrifuging said test sample at about 500g to obtain a supernatant; (S1-2) centrifuging the supernatant obtained in the step (S1-1) at about 3000g to obtain the supernatant; (S1-3) centrifuging the supernatant obtained in the step (S1-2) at about 12000g to obtain the supernatant containing the Precipitation of the substance to be tested.
8. The method according to claim 7, the duration of the step (S1-1) is about 10 minutes.
9. The method according to any one of claims 7-8, the duration of the step (S1-2) is about 20 minutes.
10. The method according to any one of claims 7-9, the duration of the step (S1-3) is about 20 minutes.
11. The method according to any one of claims 1-6, wherein said step (S1) comprises the following steps: (S1-1) centrifuging said sample to be tested at about 1350g to obtain a supernatant; (S1-2) centrifuging the supernatant obtained in the step (S1-1) at about 3000g to obtain the supernatant; (S1-3) contacting the supernatant obtained in the step (S1-2) with a size exclusion filler, separating the Components of the substance to be tested.
12. The method according to claim 11, the duration of the step (S1-1) is about 15 minutes.
13. The method according to any one of claims 11-12, the duration of the step (S1-2) is about 10 minutes.
14. The method of any one of claims 11-13, the size exclusion filler comprising a filler having a diameter of about 22 microns to 44 microns.
15. The method according to any one of claims 11-14, said step (S1-3) collects the components of size exclusion elution of two or more tubes, and determines the component containing said substance to be tested by dynamic light scattering the components.
16. The method according to any one of claims 1-15, wherein said step (S2) comprises determining the number and/or number of nucleic acid fragments comprising hydroxymethylcytosine in said test substance by hydroxymethylation sequencing method exist.
17. The method according to claim 16, wherein the hydroxymethylation sequencing method comprises the following steps: (S2a) extracting nucleic acid fragments in the substance to be tested, (S2b) marking the nucleic acid fragments containing hydroxymethylcytosine (S2c) enriching the nucleic acid fragments containing hydroxymethylcytosine, (S2d) sequencing the enriched nucleic acid fragments containing hydroxymethylcytosine.
18. The method of claim 17, said labeling comprising contacting said nucleic acid fragment with DNA β-glucosyltransferase and UDP glucose modified with a chemoselective group.
19. The method of claim 18, said chemoselective group comprising an azido group.
20. The method of any one of claims 18-19, said labeling further comprising contacting said nucleic acid fragment bearing a chemoselective group with biotin comprising biotin capable of reacting with said chemoselective group.
21. The method of claim 20, said biotin capable of reacting with said chemoselective group comprising dibenzocyclooctyne modified biotin.
22. The method of any one of claims 17-21, said enriching comprising contacting said nucleic acid fragments bearing biotin with magnetic beads comprising streptavidin.
23. The method according to any one of claims 17-22, wherein said enriching comprises magnetically separating magnetic beads bearing said hydroxymethylcytosine nucleic acid fragments.
24. The method according to any one of claims 1-23, wherein the substance to be tested is sequenced by a method selected from the following group: digital PCR and high-throughput sequencing.
25. An analytical method comprising detecting the quantity and/or presence of hydroxymethylcytosine in a test substance having a diameter of about 200 nanometers or more in a test sample.
26. A method for confirming the existence of a disease, assessing the formation or risk of a disease, assessing the progress and/or prognosis of a disease, and/or screening people who are responding to treatment, comprising detecting a test sample with a diameter of about 200 nanometers or more The amount and/or presence of hydroxymethylcytosine in a substance.
27. A nucleic acid, which comprises a sequence capable of binding to the region to be tested in the substance to be tested with a diameter of about 200 nanometers or more in the sample to be tested, or its complementary region, or the above-mentioned fragments.
28. A method for preparing nucleic acid, comprising a region to be tested in a substance to be tested with a diameter of about 200 nanometers or more in a sample to be tested, or a complementary region thereof, or a sequence of the above-mentioned fragment, designed to be able to bind to the region to be tested, or The nucleic acid of its complementary region, or the above-mentioned fragment.
29. A kit comprising the nucleic acid according to claim 27.
30. The use of the nucleic acid according to claim 27, and/or the kit according to claim 29, in the preparation of disease detection products.
31. Nucleic acid as claimed in claim 27, and/or test kit as claimed in claim 29, in the preparation confirming the existence of disease, assessing disease formation or forming risk, assessing the progression and/or prognosis of disease and/or screening for treatment There are applications in products for corresponding groups of people.
32. A database, including the sequence of the region to be tested in the substance to be tested with a diameter of about 200 nanometers or more in the sample to be tested, or its complementary region, or the above-mentioned fragments.
33. A storage medium recording a program capable of executing the method according to any one of claims 1-26.
34. A device comprising the storage medium of claim 33.
35. The device according to claim 34, further comprising a processor coupled to the storage medium, the processor configured to execute based on a program stored in the storage medium to implement any one of claims 1-26 method described in the item.

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