JP2022051123A - Sample analysis method - Google Patents

Abstract

To provide a method for rapidly analyzing a biological sample analysis, the method using a material that can fix a certain amount of the sample as a biological sample fixation material to eliminate a purification step.SOLUTION: Provided is a method for analyzing circulating nucleic acid in plasma and/or serum of a subject. The method comprises the steps of preparing plasma and/or serum from the blood of the subject; placing the plasma and/or serum on a body fluid-fixing device and drying it; and subjecting the body fluid-fixing device containing plasma and/or serum to a genetic analysis.SELECTED DRAWING: None

Description

The present disclosure relates to the collection, storage, storage and measurement of a sample for biological or chemical measurement. More specifically, it relates to the collection, storage, storage, and measurement of a sample for gene analysis in a biological sample.

In the medical field, it is necessary to analyze the sample obtained from the living body when making a diagnosis by investigating the condition in the living body and the presence or absence of illness. For example, by investigating the in vivo condition and the presence of nucleic acid related to the disease using a biological sample of a subject suspected of having an abnormality in the living body or cancer or its recurrence, the condition in the living body or the disease can be obtained. It will be possible to examine and diagnose the presence or absence of illness.

On the other hand, substances obtained from living organisms (for example, blood and saliva) contain a large amount of impurities, and in precise measurement where a trace amount of nucleic acid must be detected, the enzymatic reaction required for the measurement. In addition, it is necessary to go through a complicated purification process in order to analyze the information from the biological sample.

The present disclosure provides a rapid method for analyzing a biological sample without a purification step by using a material capable of fixing a certain amount of the sample as a fixing material for the biological sample.

Accordingly, the present disclosure provides:
(Item 1) A method for analyzing circulating nucleic acids in the plasma and / or serum of a subject.
The step of preparing plasma and / or serum from the blood of the subject, and
The step of placing the plasma and / or serum in a body fluid fixing device and drying it,
A method comprising the step of subjecting a body fluid fixing device containing plasma and / or serum to genetic analysis.
(Item 2) The method according to item 1, wherein the circulating nucleic acid is a tumor circulating nucleic acid.
(Item 3) The method according to item 2, wherein the tumor circulating nucleic acid is cfDNA.
(Item 4) The method according to any one of the above items, wherein the body fluid fixing device contains a porous material.
(Item 5) The porous material comprises a polyvinyl alcohol (PVA) sponge, a urethane sponge, a cellulose sponge, a natural sponge (sponge), a sintered structure obtained by hot-pressing a resin powder, and a fiber aggregate. The method according to any one of the above items, comprising a fiber structure and a porous membrane.
(Item 6) The method according to any one of the above items, wherein the drying is heat drying at about 60 ° C. or higher.
(Item 7) The method according to any one of the above items, wherein the drying is heat drying at about 60 ° C. or higher for at least 30 minutes or longer.
(Item 8) The method according to any one of the above items, wherein the drying is natural drying.
(Item 9) The method according to any one of the above items, wherein the analysis of circulating nucleic acid in plasma and / or serum is a quantitative analysis.
(Item 10) The method according to any one of the above items, wherein the analysis of circulating nucleic acid in plasma and / or serum is an absolute quantitative analysis.
(Item 11) The method according to any one of the above items, wherein the step to be subjected to the gene analysis is performed using the nucleic acid extracted from the body fluid fixing device.
(Item 12) The method according to any one of the above items, wherein the nucleic acid is extracted by dropping distilled water onto the body fluid fixing device.
(Item 13) The step to be subjected to the gene analysis is performed by directly adding the constant volume dry body fluid portion obtained by excising the area corresponding to the constant volume of the body fluid fixing device to the liquid containing the nucleic acid amplification reaction reagent. The method according to any one of the above items.
(Item 14) The method according to claim 10, wherein the excision diameter is about 1 to about 4 mm.
(Item 15) The method according to any one of the above items, wherein the subject is an esophageal cancer patient.
(Item A1) A kit for analyzing circulating nucleic acids in the plasma and / or serum of a subject, including a body fluid fixing device.
The step of preparing plasma and / or serum from the blood of the subject, and
The step of placing the plasma and / or serum in a body fluid fixing device and drying it,
A kit used in a method comprising the step of subjecting a body fluid fixing device containing plasma and / or serum to genetic analysis.
(Item A2) The kit according to any one of the above items, wherein the circulating nucleic acid is a tumor circulating nucleic acid.
(Item A3) The kit according to any one of the above items, wherein the tumor circulating nucleic acid is cfDNA.
(Item A4) The kit according to any one of the above items, wherein the body fluid fixing device contains a porous material.
(Item A5) The porous material comprises a polyvinyl alcohol (PVA) sponge, a urethane sponge, a cellulose sponge, a natural sponge (sponge), a sintered structure obtained by hot-pressing a resin powder, and a fiber aggregate. The kit according to any one of the above items, which comprises a fibrous structure and a porous membrane.
(Item A6) The kit according to any one of the above items, wherein the drying is heat drying at about 60 ° C. or higher.
(Item A7) The kit according to any one of the above items, wherein the drying is heat drying at about 60 ° C. or higher for at least 30 minutes or longer.
(Item A8) The kit according to any one of the above items, wherein the drying is natural drying.
(Item A9) The kit according to any one of the above items, wherein the analysis of circulating nucleic acid in plasma and / or serum is a quantitative analysis.
(Item A10) The kit according to any one of the above items, wherein the analysis of circulating nucleic acid in plasma and / or serum is an absolute quantitative analysis.
(Item A11) The kit according to any one of the above items, wherein the step to be subjected to the gene analysis is performed using the nucleic acid extracted from the body fluid fixing device.
(Item A12) The kit according to any one of the above items, wherein the nucleic acid is extracted by dropping distilled water onto the body fluid fixing device.
(Item A13) The step to be subjected to the gene analysis is performed by directly adding the constant volume dry body fluid portion obtained by excising the area corresponding to the constant volume of the body fluid fixing device to the liquid containing the nucleic acid amplification reaction reagent. The kit according to any one of the above items.
(Item A14) The kit according to any one of the above items, wherein the excision diameter is about 1 to about 4 mm.
(Item A15) The kit according to any one of the above items, wherein the subject is an esophageal cancer patient.
(Item B1) A system for analyzing circulating nucleic acids in the plasma and / or serum of a subject.
Means for preparing plasma and / or serum from the subject's blood,
A body fluid fixing device that retains and dries the plasma and / or serum, and
A system comprising a device for performing genetic analysis using a body fluid fixing device containing plasma and / or serum.
(Item B2) The system according to any one of the above items, wherein the circulating nucleic acid is a tumor circulating nucleic acid.
(Item B3) The system according to any one of the above items, wherein the tumor circulating nucleic acid is cfDNA.
(Item B4) The system according to any one of the above items, wherein the body fluid fixing device includes a porous material.
(Item B5) The porous material comprises a polyvinyl alcohol (PVA) sponge, a urethane sponge, a cellulose sponge, a natural sponge (sponge), a sintered structure obtained by hot-pressing a resin powder, and a fiber aggregate. The system according to any one of the above items, comprising a fibrous structure and a porous membrane.
(Item B6) The system according to any one of the above items, wherein the drying is heat drying at about 60 ° C. or higher.
(Item B7) The system according to any one of the above items, wherein the drying is heat drying at about 60 ° C. or higher for at least 30 minutes or longer.
(Item B8) The system according to any one of the above items, wherein the drying is natural drying.
(Item B9) The system according to any one of the above items, wherein the analysis of circulating nucleic acids in plasma and / or serum is a quantitative analysis.
(Item B10) The system according to any one of the above items, wherein the analysis of circulating nucleic acid in plasma and / or serum is an absolute quantitative analysis.
(Item B11) The system according to any one of the above items, wherein the gene analysis is performed using nucleic acid extracted from the body fluid fixing device.
(Item B12) The system according to any one of the above items, wherein the extraction of the nucleic acid is performed by dropping distilled water onto the body fluid fixing device.
(Item B13) The gene analysis is performed by directly adding a constant-volume dry body fluid portion obtained by excising an area corresponding to a certain volume of the body fluid fixing device to a liquid containing a nucleic acid amplification reaction reagent. The system described in any one of the items.
(Item B14) The system according to any one of the above items, wherein the excision diameter is about 1 to about 4 mm in diameter.
(Item B15) The system according to any one of the above items, wherein the subject is an esophageal cancer patient.
(Item C1) A method for analyzing circulating nucleic acids in the body fluids of cancer patients.
The step of placing the body fluid of the cancer patient on the body fluid fixing device and drying it,
A method comprising the steps of subjecting a body fluid fixing device containing the body fluid to genetic analysis.
(Item C2) The method according to any one of the above items, wherein the circulating nucleic acid is a tumor circulating nucleic acid.
(Item C3) The method according to any one of the above items, wherein the tumor circulating nucleic acid is cfDNA.
(Item C4) The method according to any one of the above items, wherein the body fluid fixing device includes a porous material.
(Item C5) The porous material comprises a polyvinyl alcohol (PVA) sponge, a urethane sponge, a cellulose sponge, a natural sponge (sponge), a sintered structure obtained by hot-pressing a resin powder, and a fiber aggregate. The method according to any one of the above items, comprising a fiber structure and a porous membrane.
(Item C6) The method according to any one of the above items, wherein the drying is heat drying at about 60 ° C. or higher.
(Item C7) The method according to any one of the above items, wherein the drying is heat drying at about 60 ° C. or higher for at least 30 minutes or longer.
(Item C8) The method according to any one of the above items, wherein the drying is natural drying.
(Item C9) The method according to any one of the above items, wherein the analysis of circulating nucleic acid in the body fluid is a quantitative analysis.
(Item C10) The method according to any one of the above items, wherein the analysis of circulating nucleic acid in the body fluid is an absolute quantitative analysis.
(Item C11) The method according to any one of the above items, wherein the step to be subjected to the gene analysis is performed using the nucleic acid extracted from the body fluid fixing device.
(Item C12) The method according to any one of the above items, wherein the nucleic acid is extracted by dropping distilled water onto the body fluid fixing device.
(Item C13) The step to be subjected to the gene analysis is performed by directly adding the constant volume dry body fluid portion obtained by excising the area corresponding to the constant volume of the body fluid fixing device to the liquid containing the nucleic acid amplification reaction reagent. The method according to any one of the above items.
(Item C14) The method according to any one of the above items, wherein the excision diameter is about 1 to about 4 mm.
(Item C15) The method according to any one of the above items, wherein the cancer patient is an esophageal cancer patient.
(Item C16) The method according to any one of the above items, wherein the body fluid is plasma and / or serum.
(Item D1) For in vitro diagnosis for diagnosing whether or not the subject has cancer or whether or not the cancer has recurred by analyzing circulating nucleic acids in the body fluid of the subject, including a body fluid fixing device. It ’s a drug,
The step of placing the body fluid of the subject on the body fluid fixing device and drying it,
An in vitro diagnostic drug used in a method comprising the step of subjecting a body fluid fixing device containing the body fluid to genetic analysis.
(Item D2) The drug according to any one of the above items, wherein the circulating nucleic acid is a tumor circulating nucleic acid.
(Item D3) The drug according to any one of the above items, wherein the tumor circulating nucleic acid is cfDNA.
(Item D4) The pharmaceutical product according to any one of the above items, wherein the body fluid fixing device contains a porous material.
(Item D5) The porous material comprises a polyvinyl alcohol (PVA) sponge, a urethane sponge, a cellulose sponge, a natural sponge (sponge), a sintered structure obtained by hot-pressing a resin powder, and a fiber aggregate. The pharmaceutical agent according to any one of the above items, which comprises a fibrous structure and a porous membrane.
(Item D6) The pharmaceutical product according to any one of the above items, wherein the drying is heat drying at about 60 ° C. or higher.
(Item D7) The pharmaceutical product according to any one of the above items, wherein the drying is heat drying at about 60 ° C. or higher for at least 30 minutes or longer.
(Item D8) The drug according to any one of the above items, wherein the drying is natural drying.
(Item D9) The drug according to any one of the above items, wherein the analysis of the circulating nucleic acid in the body fluid is a quantitative analysis.
(Item D10) The drug according to any one of the above items, wherein the analysis of the circulating nucleic acid in the body fluid is an absolute quantitative analysis.
(Item D11) The drug according to any one of the above items, wherein the step to be subjected to the gene analysis is performed using the nucleic acid extracted from the body fluid fixing device.
(Item D12) The drug according to any one of the above items, wherein the nucleic acid is extracted by dropping distilled water onto the body fluid fixing device.
(Item D13) The step to be subjected to the gene analysis is performed by directly adding the constant volume dry body fluid portion obtained by excising the area corresponding to the constant volume of the body fluid fixing device to the liquid containing the nucleic acid amplification reaction reagent. The drug according to any one of the above items.
(Item D14) The drug according to any one of the above items, wherein the excision diameter is about 1 to about 4 mm.
(Item D15) The drug according to any one of the above items for diagnosing whether or not the subject has esophageal cancer or whether or not the esophageal cancer has recurred.
(Item D16) The drug according to any one of the above items, wherein the body fluid is plasma and / or serum.

In the present disclosure, it is intended that the one or more features described above may be provided in combination in addition to the specified combinations. Further embodiments and advantages of the present disclosure will be appreciated by those of skill in the art upon reading and understanding the following detailed description as appropriate.

The features and remarkable actions / effects of the present disclosure other than those described above will be clarified to those skilled in the art by referring to the sections and drawings of the embodiments of the present invention below.

According to the present disclosure, a simple and highly sensitive nucleic acid test (for example, diagnosis of morbidity to a disease) can be performed. In particular, it is advantageous that nucleic acid derived from cancer, which is present only in a trace amount, can be detected early and easily, and early cancer diagnosis and recurrence monitoring can be performed.

FIG. 1A is a schematic diagram showing a nucleic acid detection protocol by a column method for extracting a nucleic acid sample immobilized on a PVA sponge in one embodiment of the present disclosure. FIG. 1B is a schematic diagram showing a nucleic acid detection protocol by a direct method in which a PVA sponge is directly put into a reaction reagent in one embodiment of the present disclosure. FIG. 2 shows, in one embodiment of the present disclosure, when plasma samples are obtained from healthy subjects, esophageal cancer patients, and cancer patients other than esophageal cancer by using the column method, and plasma cfDNA is detected. It is a schematic diagram which shows the detection protocol. FIG. 3 is an amplification curve of standard genomic DNA when the column method is used in one embodiment of the present disclosure. FIG. 4 is a standard curve of standard genomic DNA in one embodiment of the present disclosure. FIG. 5 is a plasma cfDNA amplification curve (with a standard curve) of a healthy subject when the column method is used in one embodiment of the present disclosure. FIG. 6 is a plasma cfDNA amplification curve (without standard curve) of a healthy subject when the column method is used in one embodiment of the present disclosure. FIG. 7 is a plasma cfDNA amplification curve (with a standard curve) of a patient with esophageal cancer when the column method is used in one embodiment of the present disclosure. FIG. 8 is a plasma cfDNA amplification curve (without standard curve) of a patient with esophageal cancer when the column method is used in one embodiment of the present disclosure. FIG. 9 is a plasma cfDNA amplification curve (with a standard curve) of a cancer patient other than esophageal cancer when the column method is used in one embodiment of the present disclosure. FIG. 10 is a plasma cfDNA amplification curve (without standard curve) of a cancer patient other than esophageal cancer when the column method is used in one embodiment of the present disclosure. FIG. 11 is a result of comparing the plasma cfDNA amount of a healthy subject and an esophageal cancer patient when the column method is used in one embodiment of the present disclosure. FIG. 12 is a result of comparing the amount of cfDNA in plasma of a healthy subject and a cancer patient other than esophageal cancer when the column method is used in one embodiment of the present disclosure. FIG. 13 is a schematic diagram showing a detection protocol of serum-diluted human Genome DNA using the direct method in one embodiment of the present disclosure. FIG. 14 is an amplification curve of standard genomic DNA when the direct method is used in one embodiment of the present disclosure. FIG. 15 is a schematic diagram showing the detection protocol of human Genome DNA diluted with serum or plasma using the direct method in one embodiment of the present disclosure. FIG. 16 is an amplification curve at each concentration of Genome DNA / serum solution when the direct method is used in one embodiment of the present disclosure. FIG. 17 is an amplification curve at each concentration of Genome DNA / Healthy person plasma solution when the direct method is used in one embodiment of the present disclosure. FIG. 18 is a schematic diagram showing a detection protocol for detecting plasma cfDNA by obtaining plasma samples from a healthy person and a patient with esophageal cancer using the direct method in one embodiment of the present disclosure. FIG. 19 is a plasma cfDNA amplification curve of a healthy subject when the direct method is used in one embodiment of the present disclosure. FIG. 20 is a plasma cfDNA amplification curve of an esophageal cancer patient when the direct method is used in one embodiment of the present disclosure. FIG. 21 is a result of comparing the plasma cfDNA amount of a healthy subject and an esophageal cancer patient when the direct method is used in one embodiment of the present disclosure. FIG. 22 is a schematic diagram showing a detection protocol for confirming amplification efficiency by directly feeding a serum solution into an amplification reagent using TTX (Tth DNA polymerase) in one embodiment of the present disclosure. FIG. 23 is an amplification curve in one embodiment of the present disclosure when a serum solution is directly added to an amplification reagent using TTX (Tth DNA polymerase) to amplify Genome DNA.

Hereinafter, the present disclosure will be described with reference to the best form. Throughout the specification, it should be understood that the singular representation also includes its plural concept, unless otherwise noted. Therefore, it should be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the plural concept, unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, this specification (including definitions) takes precedence.

The definitions and / or basic technical contents of terms specifically used in the present specification will be described below as appropriate.

As used herein, "about" means ± 10% of the value that follows.

As used herein, "sampling," "sampling," or "sampling" refers to extracting a sample in a test method such as liquid biopsy, and the agents and materials for that purpose are referred to as sampling agents and sampling materials, respectively. ..

As used herein, the term "porous material" refers to a material containing many pores, and the size of the pores is not particularly limited. Moreover, the size of the pores contained in one material is not always constant. For example, as the porous material, a polyvinyl alcohol (PVA) sponge, a urethane sponge, a cellulose sponge, a natural sponge (spawn), a composite product of a fiber material (for example, woven cloth, non-woven fabric, aggregate) and a sponge (so-called cotton bar) Is included, but is not limited to this. The porous material is, for example, compression-molded with a sintered structure obtained by hot-pressing a resin powder, a woven fabric and / or a non-woven fabric made of a fiber aggregate, and organic / inorganic fine particles together with an excipient. It may also include a porous membrane consisting of the structure, acetyl cellulose or nitrocellulose. Excipients include, but are not limited to, crystalline cellulose, starch, calcium silicate, and the like. Excipients include, for example, those having a single composition as long as they have shape retention by compression molding.

As used herein, the term "PVA sponge" is a porous material cross-linked with PVA, and its structure and manufacturing method are known in the art. Cross-linking can be performed with formaldehyde or the like (see Japanese Patent No. 2949982 for using aldehyde as a cross-linking agent, and Japanese Patent Application Laid-Open No. 2001-302840 for other examples).

As used herein, the term "polyvinyl alcohol (PVA)" refers to any polymer obtained by polymerizing vinyl alcohol <specific formula (-CH ₂ CH (OH)-) _n >, and polyvinyl acetate is acid or alkali. It is a water-soluble polymer having a hydroxyl group, which is obtained by hydrolyzing with. Also called Poval or PVOH. Any PVA can be used to make PVA sponges.

The PVA sponge can be produced, for example, as follows. That is, PVF (polyvinyl formal), which is an insoluble substance, is produced by a formalization reaction in which formaldehyde is bound to water-soluble PVA (polyvinyl alcohol, poval) using an acid as a catalyst, and a pore-forming agent is added in the process to form pores. After the insoluble PVF is completed, this pore-forming agent is extracted, and the completed PVF becomes a porous body and forms three-dimensional dendritic network-like continuous pores. This porous body is a PVA sponge.

Nearly 90% of the volume of PVA sponge can be composed of voids, yet it is possible to maintain the structural strength that can be used as a substrate. Further, the substrate portion forming the voids (pores) forms a three-dimensional network structure, and all the individual pores are continuous. Such an integral structure is the greatest feature of PVA sponge, which brings a number of functions. In addition, unlike general urethane sponges, PVA sponges are extremely hydrophilic, and capillarity occurs due to fine pores in the vertical and horizontal directions, so that PVA sponges are extremely excellent in water absorption and water retention. In addition, it has flexibility and elasticity in a wet state, and can prevent damage to the surface of an object, especially when used as a cleaning material or a wiping material.

As the PVA sponge, a commercially available one (for example, manufactured by Aion Co., Ltd.) can be used. As the parameter of the PVA sponge, the pore diameter (average pore diameter) or porosity of the sponge can be selected. Porosity is the ratio of the volume of voids to the volume of sponge.

In the present specification, the "average pore diameter" is an (arithmetic) average value of the pore diameter and can be measured based on JIS R 1655: 2003.

As used herein, "porosity" is the ratio of the volumes of adsorbable pores and voids to the total volume occupied by a predetermined amount of solid and can be measured based on JIS R 1655: 2003. Porosity can also be measured based on JIS Z 8837: 2018. The porosity (ε) is calculated using, for example, the formula: ε = (1-V / Va) × 100 (%) after measuring the apparent volume (Va) and the true volume (V). The apparent volume (Va) is, for example, the volume of a rectangular parallelepiped obtained by the product of three sides (length, width, height) in the case of a rectangular parallelepiped. Further, the true volume (V) is a volume obtained by subtracting the volumes of pores and voids from the apparent volume (Va), and can be measured by using, for example, a dry automatic densitometer Accubic 1330 manufactured by Shimadzu Corporation. be.

Table 1 below is a table showing the physical properties of the PVA sponge provided by Aion Co., Ltd. In other parts of the specification, when the PVA sponge is referred to by the following product number, it refers to the PVA sponge of the product number manufactured by Aion Co., Ltd.

Since FB and GB have large pore diameters, PVA sponges of other standards are considered to be more suitable for sampling biological samples. In one embodiment of the present disclosure, the pore size of the PVA sponge is about 5 to 700 μm. In one preferred embodiment of the present disclosure, the pore size of the PVA sponge is from about 80 μm to about 200 μm. For example, in the case of a biological sample (for example, saliva, blood) that is relatively easy to penetrate into the sponge, the pore size of the PVA sponge is about 40 to 300 μm, more preferably 80 to 200 μm from the viewpoint of operability. It is effective to have.

The porosity of the PVA sponge can be any value as long as it does not interfere with the absorption and retention of the biological sample. In one embodiment of the present disclosure, a PVA sponge that is about 50-98% and has a large number of openings communicating with the interior on the surface is desirable. In one preferred embodiment of the present disclosure, the porosity of the PVA sponge is about 80-95%, more preferably about 88-92, in consideration of both securing the holding capacity per unit volume and practical strength. %, Most preferably about 89% to 91%. The porosity of the cellulose sponge can be, for example, about 90 to 96%, preferably about 93 to 95%, and the porosity of the urethane sponge can be, for example, about 90 to 98%, which is preferable. Can be about 95-97%.

It is considered that there is not much difference in performance in sampling biological samples between D (D) to EB (D) PVA sponges. From the mechanical characteristics, E (D) and F (D) are considered to be the easiest to handle.

By dropping a biological sample (eg, saliva or blood) onto a PVA sponge and drying it, a sample for biological or chemical analysis of the biological sample can be prepared. In one embodiment, the biological analysis is nucleic acid analysis or genetic analysis.

(sample)
As used herein, "body fluid" refers to any biological specimen that is assumed to contain a specific target nucleic acid, such as nasal discharge, nasal swab, ocular conjunctival swab, pharyngeal swab, saliva. , Feces, blood, serum, plasma, medullary fluid, saliva, urine, sweat, milk, semen, oral swab, interdental swab, wet earlid, vaginal swab, and biological or clinical samples such as cell tissue. Other examples include foods, microsomes, foods, plants and animals. A "body fluid" can include a sample containing DNA and / or RNA contained in an entity such as a cell, fungus, bacterium or virus, or a portion thereof.

The gene or nucleic acid to be amplified or the specific gene or nucleic acid to be detected may be DNA or RNA. In this case, in one embodiment of the present disclosure, a "body fluid fixing device" for holding a body fluid is directly added to a reaction solution containing a reverse transcriptase and a polymerase to carry out a nucleic acid amplification reaction, so that the body fluid is present in the body fluid. Nucleic acid can be amplified directly. The body fluid fixing device may be a material that reduces or eliminates the adverse effects of substances that can inhibit reverse transcriptase and polymerase, such as PVA sponge, which may be contained in a sample containing DNA and / or RNA. Here, "direct addition" means that the process of extracting nucleic acid from the body fluid containing this nucleic acid is unnecessary prior to nucleic acid amplification. Further, in one embodiment of the present disclosure, the body fluid fixing device to be directly added to the reaction solution containing the reverse transcription enzyme and the polymerase can be dried, and the area corresponding to a certain volume of the body fluid fixing device is cut off to obtain a volume. It is also possible to prepare a constant dry body fluid portion and then directly add the constant volume constant dry body fluid portion.

The "constant volume dry body fluid portion" can be obtained by punching and cutting a certain amount of tissue or material packed in a pipe, for example, with a biopsy trepan. The punch diameter of this punch is not particularly limited, and may be, for example, about 1 to about 4 mm in diameter depending on the desired amount of liquid contained in the material portion to be punched. Further, the thickness of the body fluid fixing device to be punched can be appropriately set according to the desired amount of body fluid contained therein.

(Liquid biopsy)
As used herein, the term "liquid biopsy" refers to a liquid biological sample, and includes a sample of body fluid such as blood, urine, saliva, and semen. Liquid biopsy analysis can be performed with less invasiveness than conventional biopsy collection, but must be detected with higher sensitivity (eg, 100-1,000 times) than before. In many cases, it does not. This is because the analysis of liquid biopsy involves the analysis of biomolecules leaked into the liquid. For example, analysis of liquid biopsy includes virus detection, blood circulation cancer cells (CTC), blood circulation abnormal cells (CAG), stem cells, blood circulation cancer gene (CTG), cell-free gene (cfDNA), etc. Includes analysis of microRNA (miRNA), blood trace molecules, peptides, microparticles and the like.

(Gene analysis)
In the present specification, "nucleic acid analysis" refers to examining the state of nucleic acid (DNA, RNA, etc.) in a biological sample, and is used synonymously with gene analysis unless otherwise specified. In one embodiment, nucleic acid analysis may include those utilizing a nucleic acid amplification reaction. Examples of nucleic acid analysis, including these, include sequencing, genotyping / polymorphism analysis (SNP analysis, copy number polymorphism, restricted enzyme fragment length polymorphism, repeat number polymorphism), expression analysis, and fluorescent extinction probe ( Quenching Probe: Q-Probe), SYBR green method, melting curve analysis, real-time PCR, quantitative RT-PCR, digital PCR and the like can be mentioned. One example of nucleic acid analysis is genotyping by the TaqMan probe method. For the fluorescence quenching probe method, see https: // www. aist. go. jp / Portals / 0 / resources_images / aist_j / aistinfo / aist_today / vol08_02 / vol08_02_p18_p19. It is described in pdf and the like.

In such nucleic acid analysis, it is common to involve an amplification reaction of nucleic acid. When a biological sample (eg, blood or saliva) is added directly to the reaction solution of a nucleic acid amplification reaction (eg, polymerase chain reaction (PCR)), the action of the substance in the biological sample causes an amplification reaction (eg, Taq DNA polymerase, etc.). DNA polymerase) may be inhibited. In a sample prepared by dropping a biological sample (for example, blood or saliva) onto a PVA sponge, inhibition of such an amplification reaction is suppressed. Without being bound by theory, it is believed that PVA sponge has the ability to remove substances that inhibit PCR amplification (heme, polysaccharides, polyphenols, fulvic acid, dyes, ions, etc.). Be done.

(Polymerase)
Nucleic acid polymerases are used in the nucleic acid amplification reactions that can be used in one embodiment of the present disclosure. In one embodiment, the DNA polymerase used for nucleic acid analysis can be used without particular limitation as long as it is a polymerase having excellent heat resistance for synthesizing nucleic acid by adding a primer, typified by Taq DNA polymerase. The nucleic acid amplification reaction particularly utilized in the present disclosure uses a polymerase having 5'→ 3'exonuclease activity. I do not want to be bound by the theory, but when KOD polymerase is used, it cannot be detected by labeling like Taqman DNA polymerase because the amplification method is different, and it is forced to analyze with a cleavage pattern with a restriction enzyme. Although it is a complicated method, a simple analysis can be realized by using a polymerase having 5'→ 3'exonuclease activity such as Taq DNA polymerase. Polymerases having 5'→ 3'exonuclease activity include Family A (Pol I type) DNA polymerases.

Examples of such a DNA polymerase include Taq DNA polymerase derived from Thermus aquaticus, Tth DNA polymerase, and at least a mixture of at least one of the above-mentioned DNA polymerases. In addition, Tth DNA polymerase and C.I. Since therm DNA polymerase also has reverse transcription (RT) activity, it has a feature that it can be covered by one kind of enzyme when performing RT-PCR with One tube-One step. That is, in the present disclosure, the polymerase and reverse transcriptase may be realized by separate enzymes or by a single enzyme. In one embodiment, methods using other polymerases may be used, but direct use of the sample with a polymerase having 5'→ 3'exonuclease activity, such as Taq DNA polymerase, is recommended as a convenient method. In one embodiment of the present disclosure, a biological sample such as saliva can be directly applied to an analysis method using a polymerase having 5'→ 3'exonuclease activity (for example, the so-called Taqman method or an equivalent method). can. Such a thing could not be achieved by the prior art.

Examples of the reaction using a polymerase include PCR (Polymerase Chain Reaction) method, LAMP (Loop-Mediated Isothermal Amplification) method, SDA (Strand Displacement Amplification) method, and SDA (Strand Displacement Amplification). -SDA (Reverse Transplication Standard Expression) method, RT-PCR (Reverse Transition Polymerase Chain Reaction: Reverse transcription polymerase chain reaction) method, RT-LAMP Mediated isothermal amplification) method, NASBA (Nucleic Acid Sequence-Based Amplification: amplification based on nucleic acid sequence) method, TMA (Transplication Mediated Amplification) method, RCA (Rolling Cycle) method, RCA (Rolling Cycle) Isothermal and Chemical primer-initiated Amplification of Nucleic acids: Isothermal gene amplification) method, UCAN method, LCR (Ligase Chain Reaction: Ligase Chain Reaction) method, LDR (LigaseReaction) method, LDR (LigaseReaction) method Method, SMAP2 (Smart Expression Process Version 2) method, PCR-Invader method, Multiplex PCR-Based Real-Time Invader Assay (mPCR-RETINA), Fluorescent extinguishing probe (QuenchingPro) Can be mentioned. Preferably, the so-called Taqman method is used. The buffer is not particularly limited, but is EzWay ™ (KOMA Biotechnology), Ampdirect (registered trademark) (manufactured by Shimadzu Corporation), Phaseion (registered trademark) Blood Direct PCR kit buffer (New ENGLAND Bio-Labs). A registered trademark) PCR kit (manufactured by Epicentre) or the like that does not diminish the effect of removing amplification inhibition can be used. The LAMP method can also be preferably used. The LAMP method has high specificity because (1) the gene amplification reaction proceeds at an isothermal temperature, (2) four types of primers that recognize 6 regions are used, and (3) amplification. It is preferable in that it has high efficiency, can be amplified in a short time, (4) has a large amount of amplification product, and is suitable for simple detection. In addition, the RT-LAMP method, in which cDNA is synthesized from the target RNA by reverse transcriptase and the obtained cDNA is amplified by the LAMP method, can also be preferably used. Furthermore, (1) the target sequence can be specifically detected, (2) it can be detected with high sensitivity in a short time by isothermal nucleic acid amplification, (3) there are few reagents to be added to the sample RNA, and (4). The SMAP method, which can be performed in one step from reverse transcription to detection, can also be preferably used.

In one preferred embodiment of the present disclosure, it is advantageous to use a porous material such as PVA sponge. Although it is not desired to be bound by theory, it is advantageous in that detection methods applying various amplification techniques such as PCR, LAMP method, RT-LAMP method, and SMAP method can be directly performed, and it is reproduced especially when a PVA sponge is used. It is excellent in terms of properties and convenience.

The primer used in the reaction in the present disclosure can be appropriately designed by a known method at the time when the gene to be amplified or the specific gene to be detected is determined. The primer used in the reaction in the present disclosure is not particularly limited as long as it can specifically amplify the gene to be amplified or the specific gene to be detected.

The amplification of nucleic acids contained in body fluids or the detection of genes contained in body fluids in the present disclosure is preferably performed on a plate-shaped or tube-shaped insoluble carrier. Examples of such an insoluble carrier include a tube made of plastic, glass, etc., which is insoluble in the reaction solution, and a 96-well well. The tubular shape refers to a hollow state, and may have a shape such as a PCR tube having a bottom or an Eppendorf tube.

Specifically, first, the reaction solution is put into a plate-shaped or tube-shaped insoluble carrier. In the case of a tube-shaped insoluble carrier, a reaction solution containing reagents such as a buffer, a polymerase and a primer is put into the inside thereof, and in the case of a plate-shaped insoluble carrier, the reaction solution is placed on the surface thereof. .. Then, the porous material containing the body fluid to be inspected is directly put into the reaction solution, and the amplification methods such as the PCR method, the LAMP method, the RT-LAMP method, and the SMAP method described above are performed.

As the quantitative PCR used in the present specification, any known method such as known real-time PCR can be used. These methods are methods for detecting the amount of DNA amplification in real time using a label such as a fluorescent reagent, and typically require a device that integrates a thermal cycler and a spectrofluorescence meter. Such devices are commercially available. There are several methods depending on the fluorescent reagent used, and examples thereof include an intercalator method, a TaqMan ^TM probe method, and a Molecular Beacon method. In each case, a fluorescent reagent or fluorescent probe called an intercalator, a TaqMan ^TM probe or a Molecular Beacon probe is added to a PCR reaction system containing a template genomic DNA and a primer pair for amplifying a genomic region containing a target SNP site. It is a thing. In the TaqMan ^TM probe method (also referred to as TaqMan ^TM method) preferably used in the present disclosure, the TaqMan ^TM probe is an oligonucleotide capable of hybridizing to the amplified region of the target nucleic acid in which both ends are modified with a fluorescent substance and a dimming substance, respectively. Although it hybridizes to the target nucleic acid during annealing, it does not fluoresce due to the presence of an extinct substance, and it fluoresces when it is decomposed by the exonuclease activity of the DNA polymerase during the extension reaction and the fluorescent substance is released. Therefore, the amount of amplification product produced can be monitored by measuring the fluorescence intensity, thereby estimating the amount of the original template DNA.

Copy number analysis by the Taqman assay is, for example, a technique known in the art. The Taqman assay is based on the 5'-nuclease assay, also referred to as the fluorescence generation 5'-nuclease assay; Holland et al. , Proc. Natl. Acad. Sci. USA 88: 7276-7280 (1991); and Heid et al. , Genome Research 6: 986-994 (1996).

The TaqMan PCR procedure uses two oligonucleotide primers to generate an amplicon specific for the PCR reaction. A third oligonucleotide (TaqMan probe) is designed to hybridize to the nucleotide sequence in the amplicon located between the two PCR primers. The probe may have a structure that cannot be extended by the DNA polymerase used in the PCR reaction and is usually (but not required) co-labeled with a fluorescent reporter dye and quencher moiety in close proximity to each other. The emission from the reporter dye is extinguished by the quenching moiety when the fluorophore and quencher are in close proximity, as is the case on the probe. In some cases, the probe may be labeled only with a fluorescent reporter dye or another detectable moiety.

In the TaqMan PCR reaction, a thermostable DNA-dependent DNA polymerase having 5'-3'nuclease activity is used. During the PCR amplification reaction, the 5'-3'nuclease activity of the DNA polymerase cleaves the labeled probe that hybridizes to the amplicon in a template-specific manner. The resulting probe fragment dissociates from the primer / template complex and the reporter dye is released from the quenching effect of the quencher moiety. Approximately one reporter dye is released for each newly synthesized amplicon molecule, and by detecting the unquenched reporter dye, the amount of fluorescent reporter dye released is directly proportional to the amount of the amplicon template. It provides a basis for the quantitative interpretation of the data in form.

One measure of TaqMan assay data is usually expressed as a threshold cycle (CT). Fluorescence levels are recorded during each PCR cycle and are proportional to the amount of product amplified up to that point in the amplification reaction. The PCR cycle is the threshold when the fluorescence signal is first recorded as statistically significant, or when the fluorescence signal exceeds some other arbitrary level (eg, arbitrary fluorosense level; AFL). Cycle (CT). Protocols and reagents for the 5'-nuclease assay are known to those of skill in the art and are described in various references. For example, the 5'-nuclease reaction and probe are described in US Pat. No. 6,214,979 (Gelfand et al.); US Pat. No. 5,804,375; US Pat. No. 5,487, No. 972 (Gelfand et al.); Gelfand et al. Nos. 5,210,015 (Gelfand et al.) And the like can be referred to.

TaqMan ^TM PCR can be performed using commercially available kits and devices, such as the ABI PRISM® 7700 Science Detection System (Applied Biosystems, Foster City, CA, USA), LightCyl (registered) Sciences, Mannheim, Germany) and the like. In a preferred embodiment, the 5'-nuclease assay procedure is performed on, but is not limited to, a real-time quantitative PCR device such as the ABI PRISM® 7700 Sequence Detection System. The system typically consists of a thermocycler, a laser, a charge-shift bond (CCD), a camera, and a computer. This system amplifies the sample in a microtiter plate format, such as 96 wells on a thermal cycler. During amplification, for all wells, laser-guided fluorescence signals are collected in real time through fiber optic cables and detected by a CCD camera. The system includes software for operating equipment and analyzing data.

As another example of the specific procedure of the Taqman method, for example, the Taqman assay reported for CYP2D6 can be used (Bodin et al., J Biomed Biotechnol. 3: 248-53 2005). In this case, in order to obtain accurate data as needed, a new reverse primer can be designed by Primer Express or the like, and the copy number analysis can be performed again. A Taqman probe can be used in which the 5'end is labeled with FAM and the 3'end is linked with No Fluorescence Quencher and MGB. As a reference gene, an RNase Passay (Thermo Fisher) labeled with an appropriate label (eg, VIC) can be used. All Taqman assays can be performed according to the reported protocol available from the manufacturer and copy count calculations can be performed by the ΔΔCt method (Bodin et al., 2005). As one example, a sample having a median ΔCt value can be assumed to be 2 copies and used as a calibrator, but is not limited thereto. All samples can be tested in duplicate and average copy numbers can be used in the scatter plot analysis, but the number of samples can be increased or decreased as needed.

As the real-time PCR reagent in the present disclosure, TaqPath ^TM ProAmp ^TM Master Mix can be used. TaqPath ^TM ProAmp ^TM Master Mix features exceptional data quality, which can provide high specificity, dynamic range, and reproducibility in genotyping and copy number variation (CNV) analysis, even in the presence of PCR inhibitors. ) And resistance to PCR inhibitors (capable of responding to prepared samples of human or animal origin (cheek swabs, blood, and card punch)). In one embodiment of the present disclosure, nucleic acids can be subjected to the reaction as they are in the porous material without extracting or purifying the nucleic acid from the body fluid held in the porous material. For example, a porous material contacted with body fluid (blood, serum, plasma, saliva, etc.) can be added directly to the reaction solution. In one embodiment of the present disclosure, the porous material can be brought into contact with the body fluid and then dried, if necessary.

In digital PCR, a mixture of nucleic acids is dispensed into so many reaction wells that one well contains one target molecule while the other well does not contain a target. Normal PCR is performed on each reaction to identify wells that do not contain the target molecule. A correction calculation can be performed with a standard statistical model to obtain the final concentration value. Since digital PCR does not use the Ct value to quantify the number of copies, it is not necessary to compare it with a known standard in absolute quantification.

The techniques of the present disclosure may be for the analysis of circulating nucleic acids (eg, circulating acellular nucleic acids). As used herein, the term "circulating nucleic acid" refers to nucleic acid that circulates in a living body (particularly in blood). Of the circulating nucleic acids, those containing no cells are referred to as "circulating cell-free nucleic acids" in the present specification. As used herein, the circulating nucleic acid can be, for example, tumor circulating DNA or tumor circulating miRNA. Since the circulating nucleic acid may exude to the body fluid (saliva or the like) discharged from the body in some cases, the circulating nucleic acid also includes the nucleic acid contained in the body fluid such as saliva. As used herein, the term "tumor circulating nucleic acid" refers to circulating nucleic acid that is or is suspected to be associated with a tumor, and when the nucleic acid is DNA, it is also referred to as "tumor circulating nucleic acid". When is miRNA, it is also referred to as "tumor circulation miRNA".

As used herein, "blood-circulating tumor cell (CTC)" refers to a cell that is released from the primary tumor tissue or metastatic tumor tissue into the blood via epithelial-mesenchymal transition (EMT) and circulates in the bloodstream. .. After being released from the primary tumor site, the CTC circulates in the blood and invades other organs to form metastatic tumors (metastasis). Since CTC is present in the peripheral blood of cancer patients, it is possible to determine the process of metastasis by detecting it and to use it for predicting the prognosis of treatment.

As used herein, the term "cfDNA (cell-free DNA)" refers to free DNA released from a cell by cell death in blood. Among them, DNA derived from cancer cells is referred to as "blood tumor DNA (ctDNA)".

Currently, ctDNA is used as a clinically useful tumor marker for monitoring the amount of tumor in the body, determining a drug resistance gene, detecting ultra-early cancer, and the like. Serum tumor markers are now often used as the most convenient tool for monitoring in-body tumor mass. In fact, tumor markers are a useful auxiliary diagnostic tool when recurrence has progressed to some extent or when time-series fluctuations reflect treatment. For the detection of ultra-early cancer, which may be effective in cancer screening, it may be possible to provide highly accurate cancer screening only by collecting blood. There are also issues such as restrictions on blood collection and the maturation of technology for accurately measuring ultra-low frequency mutations, which is said to be 0.1% or less. However, in the future, ctDNA may change the daily cancer diagnosis from "what you see" to "what you measure".

However, in order to make unstable ctDNA research a standard technique, plasma centrifugation should be performed within 2 hours after blood collection, bias between patients and experimenters should be minimized, and quality should be corrected for bias correction. There are issues such as management / quantification, simplification of the genetic testing process, and enabling absolute quantification rather than relative quantification for control. Sampling using PVA sponge is quick, simple and inexpensive, high reliability and labor cost reduction by shortening the process, minimizing the DNA quality gap between samples, absolute quantification (essential for monitoring), room temperature after drying. It is useful in solving the above-mentioned problems by enabling the storage and transportation of samples in.

As used herein, "digital PCR" refers to a method for the detection and quantification of nucleic acids, which is realized by directly counting the number of target molecules without relying on internal standards or endogenous controls.

The method of absolute quantification is known in the art, and can be typically realized by a method of creating a calibration curve using a standard sample and measuring the absolute amount of an unknown sample. Usually, a standard sample with a known absolute quantity (number of copies) and the same sequence as the unknown sample is required. For example, Bacteria (tufgene) Quantitative PCR Kit available from Takara Bio can be used, but the present invention is not limited thereto.

(Reverse transcriptase)
As used herein, the term "reverse transcriptase (RT)" is used in its broadest sense and is a reverse transcriptase disclosed herein or measured by methods known in the art. It refers to any enzyme that exhibits activity, and refers to an enzyme that synthesizes a DNA strand (that is, cDNA) using an RNA strand as a template (for example, it may further have a reverse transcriptase or a DNA polymerase). Includes reverse transcriptases from retroviruses, other viruses, and bacteria, as well as DNA polymerases exhibiting reverse transcriptase activity such as Tth DNA polymerase, Taq DNA polymerase, Tne DNA polymerase, and Tma DNA polymerase. Retrovirus-derived RTs include, but are not limited to, Molonee murine leukemia virus (M-MLV) RT, human immunodeficiency virus (HIV) RT, trisarcoma leukemia virus (ASLV) RT, Raus sarcoma virus (RSV) RT, Tori myeloid blastosis virus (AMV) RT, Tori erythroblastosis virus (AEV) helper virus MCAV RT, Tori myelodystrophy virus MC29 helper virus MCAV RT, Tori reticular endothelial disease virus (REV-T) helper virus REV-A RT, Tri-sarcoma virus UR2 helper virus UR2AV RT, Tri-sarcoma virus Y73 helper virus YAV RT, Raus concomitant virus (RAV) RT, and myeloid blast disease concomitant virus (MAV) RT, and (see all in all). Includes those described in US Patent Application No. 2003/0198944 (incorporated herein). For a review, see, for example, Levin, 1997, Cell, 88: 5-8; Brosius et al., 1995, Virus Genes 11: 163-79. Known reverse transcriptases derived from viruses require primers to synthesize DNA transcripts from RNA templates. Reverse transcriptase is primarily used to transcribe RNA into cDNA, which is then cloned into a vector for further manipulation, or polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription. It can be used in a variety of amplification methods such as mediated amplification (TMA) and self-retaining sequence replication (3SR). Includes reverse transcriptases from retroviruses, other viruses, and bacteria, as well as DNA polymerases exhibiting reverse transcriptase activity such as Tth DNA polymerase, Taq DNA polymerase, Tne DNA polymerase, and Tma DNA polymerase. Retrovirus-derived RTs include, but are not limited to, Molonee murine leukemia virus (M-MLV) RT, human immunodeficiency virus (HIV) RT, trisarcoma leukemia virus (ASLV) RT, Raus sarcoma virus (RSV) RT, Tori myeloid blastosis virus (AMV) RT, Tori erythroblastosis virus (AEV) helper virus MCAV RT, Tori myelodystrophy virus MC29 helper virus MCAV RT, Tori reticular endothelial disease virus (REV-T) helper virus REV-A RT, Tri-sarcoma virus UR2 helper virus UR2AV RT, Tri-sarcoma virus Y73 helper virus YAV RT, Raus concomitant virus (RAV) RT, and myeloid blast disease concomitant virus (MAV) RT, and (see all in all). Includes those described in US Patent Application No. 2003/0198944 (incorporated herein). For a review, see, for example, Levin, 1997, Cell, 88: 5-8; Brosius et al., 1995, Virus Genes 11: 163-79. Known reverse transcriptases derived from viruses require primers to synthesize DNA transcripts from RNA templates. Reverse transcriptase is primarily used to transcribe RNA into cDNA, which is then cloned into a vector for further manipulation, or polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription. It can be used in a variety of amplification methods such as mediated amplification (TMA) and self-retaining sequence replication (3SR).

(Preferable embodiment)
Hereinafter, preferred embodiments of the present disclosure will be described. The embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in consideration of the description in the present specification. In addition, the following embodiments of the present disclosure may be used alone or in combination thereof.

(Nucleic acid identification method)
In one aspect of the present disclosure, a method for analyzing circulating nucleic acid in a subject's plasma and / or serum, the step of preparing plasma and / or serum from the subject's blood, and the plasma and / or serum. A method is provided comprising a step of placing and drying on a body fluid fixing device and a step of subjecting the plasma and / or serum-containing body fluid fixing device to genetic analysis.

In the present disclosure, when a body fluid such as plasma and / or serum is placed on a body fluid fixing device and dried, as long as the body fluid can be placed and has a shape or structure capable of drying the body fluid. Any fluid fixing device may be used. Any method may be used for drying, such as natural drying including air drying, heat drying at a high temperature, and the like. The body fluid fixing device may be any device for fixing the body fluid, and as long as the target body fluid can be fixed and dried in that state, any form, material, and combination thereof can be described as described in the present specification. It is understood that the above can be adopted, and it may be provided as an integrated device, or it may be provided as a separate part and then assembled (assembled). Alternatively, when provided as an integrated device, it may be further provided in a package (such as a bag). The material of the body fluid fixing device may be any material that does not interact with the target object such as body fluid, preferably has heat resistance, and is preferably made of paper, but may be made of, for example, plastic. .. The shape of the body fluid fixing device is arbitrary as long as it can form a space for drying the body fluid, and preferably has a shape capable of protecting the place where the fixed body fluid is dried. The body fluid fixing device may have, for example, a shape capable of forming a lid by folding a part thereof, or may have a breathable box shape.

In the present disclosure, when an area corresponding to a certain volume of a body fluid fixing device is excised to prepare a dry body fluid portion having a constant volume, any method can be used as long as a constant volume of the portion containing the body fluid can be obtained. A method may be used. When using a sheet such as PVA sponge, since the sheet thickness is constant and the amount of liquid contained in the sheet is constant, a certain volume of sample should be obtained by excising a certain area. Can be done. For example, as a means capable of acquiring a constant volume, a constant volume excision device such as a punch can be used, and as a constant volume excision device, a biopsy trepan can be mentioned. When a biopsy trepan is used, a pipe with a cutting edge provided at the tip of the device can be pressed against a body fluid fixing device, and the tissue clogged in the pipe can be taken out to prepare a constant volume dry body fluid portion. When using a biopsy trepan, the thickness of the body fluid fixing device and the punch diameter of the cutting edge should be 1.0 mm, 1.5 mm, 2.0 mm, 3.0 mm, 4.0 mm, etc., depending on the required amount of body fluid. can do. An automatic punching device can also be used as a constant volume punching device.

In the present disclosure, when a body fluid fixing device containing plasma and / or serum is used for gene analysis, the enzyme (RNA, DNA, etc.) expected to be contained in the body fluid is required for gene analysis or a nucleic acid amplification reaction reagent (for example, Any method may be used as long as it is in contact with a reverse transcriptase, a polymerase (which may be a strand-substituted DNA synthase), etc., depending on the condition. When using more than one type of enzyme, two enzymes or a single enzyme having two enzyme activities may be contacted at the same time, or two separate enzymes may be contacted. Separately, it is preferred to first contact the reverse transcriptase when amplifying RNA. For contact of the constant volume dry body liquid part with reverse transcriptase and polymerase, add the constant volume dry body liquid part to the solution containing the reverse transcriptase and polymerase, or put the constant volume dry body liquid part in a buffer solution etc. Polymerase may be added.

In one embodiment of the present disclosure, any analysis method may be used for gene analysis, and the amplified nucleic acid is analyzed by determining individual sequences with a sequencing device such as a next-generation sequencer (NGS). It may be an analysis of fragments by enzyme treatment with a restriction enzyme or the like. Alternatively, the presence or absence of a specific sequence may be inspected using a probe or the like, and in the case of real-time PCR, nucleic acid analysis can be performed by analyzing the amplification curve. Examples of the nucleic acid analysis include a nucleic acid amplification reaction, a nucleic acid (for example, DNA) synthesis reaction, and a gene analysis including a reverse transcription reaction. Examples of nucleic acid analysis, including these, include sequencing, genotyping / polymorphism analysis (SNP analysis, copy number polymorphism, restricted enzyme fragment length polymorphism, repeat number polymorphism), expression analysis, and fluorescent extinction probe ( Quenching Probe: Q-Probe), SYBR green method, melting curve analysis, real-time PCR, quantitative RT-PCR, digital PCR and the like can be mentioned. One example of nucleic acid analysis is genotyping by the TaqMan probe method.

In one embodiment, the body fluid fixing device comprises a porous material, which is a polyvinyl alcohol (PVA) sponge, urethane sponge, cellulose sponge, natural sponge (spong), fiber material (eg, woven fabric, non-woven fabric, etc.). A composite product (so-called cotton stick) of an aggregate) and a sponge can be used, but the material is not particularly limited as long as it is a material containing many pores. The porous material is, for example, compression-molded with a sintered structure obtained by hot-pressing a resin powder, a woven fabric and / or a non-woven fabric made of a fiber aggregate, and organic / inorganic fine particles together with an excipient. It may also include a porous membrane consisting of the structure, acetyl cellulose or nitrocellulose. Excipients include, but are not limited to, crystalline cellulose, starch, calcium silicate, and the like. Excipients include, for example, those having a single composition as long as they have shape retention by compression molding.

For example, when a PVA sponge is used as a porous material, it is useful for sampling a biological sample containing a liquid biopsy. The PVA sponge can suppress the inhibition of the polymerase amplification activity inhibitory component in the biological sample. PVA sponges can capture microorganisms (eg, cells) and can also contribute to nucleic acid stabilization.

Sampling using a PVA sponge can be performed quickly, easily, and inexpensively. By using the PVA sponge, the process can be shortened (including reduction of the number of processes and time reduction), whereby reliability can be increased and labor cost can be reduced. Also, the DNA quality gap between the samples is minimized. PVA sponges can also be used for absolute quantification, which is important when making multiple measurements (eg, subject monitoring). In addition, the PVA sponge can be stored and mailed at room temperature after drying, and is excellent in ease of handling.

In one embodiment of the present disclosure, the body fluid can include saliva, blood, urine, semen, or tears, but is not particularly limited as long as it is a sample that can be used for liquid biopsy. For example, the advantage of using PVA sponge for saliva sampling is that saliva has higher detection sensitivity and specificity than nasal swab even when nucleic acids such as pathogens and viruses are present in only trace amounts. Can be mentioned. The method of collecting the nasal swab is not quantitative because the amount of sample to be collected is uncertain. On the other hand, when saliva is sampled using a PVA sponge, it can be quantitatively collected, and for example, the amount of pathogens and viruses in 1 mL of saliva can be quantified.

Further, in one embodiment, when the body fluid is sampled using a PVA sponge, there is an advantage that the body fluid can be naturally dried during transportation after sampling. It is also possible to spray alcohol, sodium hypochlorite, etc., and it is usually possible to heat and dry at about 60 ° C. or higher for about 10 minutes to about 1 hour, and the heating temperature is, for example, about 60 ° C. to about 90. ° C., about 60 ° C. to about 85 ° C., about 60 ° C. to about 80 ° C., or about 60 ° C. to about 75 ° C., about 60 ° C. to about 70 ° C. or about 60 ° C. to about 65 ° C. May be good. The heating time may be about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, and the like.

Further, by using an automatic punching machine for newborn screening, work efficiency is improved, and direct PCR in which a PVA sponge piece is directly put into a test reaction solution to perform a reaction such as RT-PCR becomes possible. Further, since saliva is simply dropped on the PVA sponge and the sponge piece is used, the cost of DNA extraction / purification can be reduced. Moreover, since a certain volume can be punched, RNA / DNA (copy number) in saliva and blood can be absolutely quantified.

In one embodiment of the present disclosure, the body fluid fixing device can be dried after the body fluid is held in the body fluid fixing device. In this case, this drying may be natural drying at room temperature, or may be heat drying at about 60 ° C. or higher for about 10 minutes to about 1 hour. The heating temperature may be, for example, about 60 ° C. to about 90 ° C., about 60 ° C. to about 85 ° C., about 60 ° C. to about 80 ° C., or about 60 ° C. to about 75 ° C., and about 60 ° C. to about 70 ° C. Or about 60 ° C to about 65 ° C. The heating time may be about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, and the like. The means for drying is not particularly limited, but for example, the shape of the body fluid fixing device itself may be configured to dry the body fluid, and in this case, the body fluid fixing device is used to hold the body fluid. The body fluid fixing device includes a fixing portion and a lid portion, and the body fluid fixing device is configured to be foldable so that the lid portion forms a space for drying the body fluid in the body fluid fixing portion. Equipment is provided. According to such a configuration, since a space for drying the body fluid in the body fluid fixing portion is provided, the body fluid held in the body fluid process portion is naturally dried while the body fluid fixing device is being transported. Can be done.

In one embodiment of the present disclosure, as a means of drying, the column or body fluid fixing device may be further provided with a desiccant such as molecular sieve (synthetic zeolite) or anhydrous magnesium sulfate for promoting drying of body fluid. be. Alternatively, a heat source can be installed, and the heat source is preferably one that can be adhered to a body fluid fixing device or a body fluid fixing portion, and may be one that spontaneously generates heat by a chemical reaction or the like, or releases stored heat. It may be something to do. For example, the heat source includes a body warmer that utilizes heat generated by the oxidation reaction of iron. Further, in another embodiment of the present disclosure, as a means of drying, the body fluid fixing device can be usually heated and dried at about 60 ° C. or higher for about 10 minutes to about 1 hour using a dryer or the like. The heating temperature may be, for example, about 60 ° C. to about 90 ° C., about 60 ° C. to about 85 ° C., about 60 ° C. to about 80 ° C., or about 60 ° C. to about 75 ° C., and about 60 ° C. to about 70 ° C. Or about 60 ° C to about 65 ° C. The heating time may be about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, and the like.
In one embodiment, the subject's plasma and / or circulating nucleic acid in serum can be tumor circulating nucleic acid or cfDNA. Tumor circulating nucleic acids and cfDNA are nucleic acids released into the blood from cancer cells in the blood, and cfDNA derived from cancer cells released into the blood due to cell disruption due to apoptosis etc. is used for cancer diagnosis and treatment. It can be applied. These tumor-circulating nucleic acids and cfDNA are expected to be superior biomarkers to conventional tumor markers, and research is underway. Due to the large number of purification steps, the recovery rate of cfDNA is low, and the development of new technology is required to advance research in order to accurately quantify and aim for clinical application. The present disclosure is useful in that such cfDNA can be analyzed and quantified.
In one embodiment, the cancer type to be analyzed may be any cancer, and the above-mentioned tumor circulating nucleic acid and cfDNA are not particularly limited as long as they are released into blood. For example, the cancers analyzed in this disclosure include lung cancer, breast cancer, colon cancer, esophageal cancer, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), and RCC. (Renal cell cancer), GIST (gastrointestinal stromal tumor), etc. can be mentioned.
In another aspect of the present disclosure, a kit for analyzing circulating nucleic acid in a subject's plasma and / or serum, including a body fluid fixing device, the step of preparing plasma and / or serum from the subject's blood. And a kit used in a method comprising the step of placing and drying the plasma and / or serum in a body fluid fixing device and the step of subjecting the body fluid fixing device containing the plasma and / or serum to genetic analysis are provided. ..
In still another aspect, a system for analyzing circulating nucleic acid in a subject's plasma and / or serum, the means for preparing plasma and / or serum from the subject's blood, and the plasma and / or serum. A system is provided that includes a body fluid fixing device to hold and dry, and a device for performing genetic analysis using the body fluid fixing device containing plasma and / or serum.

As the means for preparing plasma and / or serum from the blood of the subject, any means or device capable of preparing plasma and / or serum from blood may be used.

The body fluid fixing device used in the present disclosure for holding and drying the body fluid may be any device as long as it can hold and dry the body fluid. For example, a body fluid fixing device refers to a device for fixing body fluid, and as long as the target body fluid can be fixed and dried in that state, any form, material, or combination thereof is described in the present specification. It is understood that the above can be adopted, and it may be provided as an integrated device, or it may be provided as a separate part and then assembled (assembled). Alternatively, when provided as an integrated device, it may be further provided in a package (such as a bag). The material of the body fluid fixing device may be any material that does not interact with the target object such as body fluid, preferably has heat resistance, and is preferably made of paper, but may be made of, for example, plastic. .. The shape of the body fluid fixing device is arbitrary as long as it can form a space for drying the body fluid, and preferably has a shape capable of protecting the place where the fixed body fluid is dried. The body fluid fixing device may have, for example, a shape capable of forming a lid by folding a part thereof, or may have a breathable box shape.

In one embodiment, an apparatus for performing gene analysis using a body fluid fixing device containing plasma and / or serum is such that a nucleic acid (RNA, DNA, etc.) expected to be contained in the body fluid is used for gene analysis or a nucleic acid amplification reaction reagent. (For example, any device may be used as long as the reaction is carried out by contacting with reverse transcriptase, polymerase (which may be a strand-substituted DNA synthase), etc., if necessary). When two or more enzymes such as a transcriptase and a polymerase are used, they may be contacted with two enzymes or a single enzyme having two enzyme activities at the same time, or they may be contacted with two separate enzymes. Separately, it is preferred to first contact the reverse transcriptase when amplifying RNA. For contact of the constant volume dry body liquid part with reverse transcriptase and polymerase, add the constant volume dry body liquid part to the solution containing the reverse transcriptase and polymerase, or put the constant volume dry body liquid part in a buffer solution etc. Polymerase may be added.

In the present disclosure, nucleic acid amplification reaction reagents (eg, reverse transcriptase, polymerase (which may include strand-substituted DNA synthase), etc., as required), and conditions under which nucleic acid amplification occurs (eg, reverse transcriptase and / or polymerization or In the apparatus for generating DNA synthesis), the nucleic acid amplification reaction reagent such as reverse transcriptase and polymerase produces the reaction necessary for the amplification reaction of DNA, RNA, etc., which is considered to be contained in the sample of the present disclosure. Any enzyme or the like (eg, reverse transcriptase and polymerase suitable for reverse transcription and polymerization of nucleic acids) may be used. In one embodiment, the nucleic acid amplification reaction reagent is a polymerase (DNA polymerase, RNA polymerase, strand-substituted DNA synthase, etc.), a reverse transcription enzyme (eg, a combination of a reverse transcription enzyme and a polymerase, reverse transcription, if necessary). It may be an enzyme and a strand-substituted DNA synthase, or an enzyme having the functions of both a polymerase or a strand-substituted DNA synthase and a reverse transcription enzyme). In one embodiment, the conditions under which nucleic acid amplification occurs may be reverse transcription, polymerization (DNA polymerization, RNA polymerization, etc.), and conditions under which DNA synthesis occurs.

The conditions under which nucleic acid amplification occurs (eg, conditions under which reverse transcription and / or polymerization or DNA synthesis occur) can vary depending on the reagent used (eg, reverse transcriptase, polymerase, strand-substituted DNA synthase, etc.), but those skilled in the art. If so, it can be appropriately determined depending on the reagent used (for example, reverse transcriptase, polymerase, strand-substituted DNA synthase, etc.). For example, the polymerization reaction with the reagents used (eg, reverse transcriptase, polymerase, strand-substituted DNA synthase, etc.) can start with a reverse transcriptase reaction at about 42 ° C to about 60 ° C for about 5 minutes, or various. It is also possible to start with a pre-modification reaction at about 90 ° C. for about 30 seconds before performing the reverse transcription reaction of the condition. Alternatively, when the reverse transcriptase and the polymerase are realized by the same enzyme, if the optimum temperature is the same, it may be started at an appropriate temperature (depending on the enzyme, about 42 ° C to about 60 ° C, about 90 ° C, etc.). can.

The device for analyzing nucleic acid in the present disclosure may be any device as long as the nucleic acid can be analyzed. For example, next-generation sequencer, PCR method, LAMP method, SDA method, RT-SDA method, RT-PCR method, RT-LAMP method, NASBA method, TMA method, RCA method, ICAN method, UCAN method, LCR method, LDR. Devices for performing various reactions such as the method, SMAP method, SMAP2 method, PCR invader method, mPCR-RETINA method, and fluorescence quenching probe method can be used.

To diagnose, in other aspects of the disclosure, whether the subject has cancer or has relapsed by performing an analysis of circulating nucleic acids in the subject's body fluids, including body fluid fixing devices. In-vitro diagnostic drug, which is used in a method including a step of arranging and drying the body fluid of the subject on a body fluid fixing device and a step of subjecting the body fluid fixing device containing the body fluid to genetic analysis. The drug is provided.

In one embodiment, an in vitro diagnostic agent for diagnosing whether a subject has cancer or has relapsed by analyzing circulating nucleic acids in the body fluid of the subject is described herein. It may be provided in the form of a kit containing a fluid fixing device as described elsewhere in the book.

In the present disclosure, the sample used is preferably, but is not limited to, a biological sample. In one embodiment, the biological sample may be anything that is supposed to contain a particular nucleic acid of interest, eg, nasal discharge, nasal swab, ocular conjunctival swab, pharyngeal swab, sputum, feces, blood. , Serum, plasma, spinal fluid, saliva, urine, sweat, milk, semen, oral swab, interdental swab, moist earlid, vaginal swab, and biological or clinical samples such as cell tissue, as well as foods, etc. Can be mentioned. The biological sample can include a sample containing DNA and / or RNA selected from the group consisting of cells, fungi, bacteria and viruses.

In the present disclosure, the treatment may be any operation on the sample, but by changing the treatment using a specific component contained in the sample, another product can be produced or information can be extracted. It means to do. Treatments include, but are not limited to, for example, nucleic acid amplification reactions, enzymatic reactions, gene analysis, and chemical substance measurements in liquid biopsy (eg, drug concentration measurements by HPLC-MS / MS and the like).

The device or material used for drying does not substantially adversely affect substances (eg, enzymes such as polymerases, reverse transcriptases, or antibodies, etc.) required for the treatment intended in the present disclosure (in this case, the device or material). It is understood that if the processing does not ultimately affect the intended situation, it may have some effect).

In one embodiment, the gene analysis is, for example, PCR (Polymerase Chain Reaction) method, LAMP (Loop-Mediated Isothermal Amplification) method, SDA (Strand Displacement Amplification) method. , RT-SDA (Reverse Transition Standard Amplification) method, RT-PCR (Reverse Transition Polymerase Chain Reaction: Reverse transcription polymerase chain reaction) method, RT-Lation Photo-loop-mediated isothermal amplification) method, NASBA (Nucleic Acid Sequence-Based Amplification) method, TMA (Transplication Modified Amplification) method, RCA (Rolling Cycle) method, RCA (Rolling Cycle) method ICAN (Isothermal and Polymerase-initiated Amplification of Nucleic acids: isothermal gene amplification) method, UCAN method, LCR (Ligase Chain Reaction: Ligase Chain Reaction) method, LDR (Ligase Chain Reaction) method, LDR (Ligase Chain Reaction) method, LDR (Ligase Chain Reaction) Process) method, SMAP2 (Smart Expression Process Version 2) method, PCR-Invader method, Multiplex PCR-Based Real-Time Invader Assay (mPCR-RETINA), Fluorescent extinguishing probe (MPCR-RETINA) And so on. Preferably, the so-called Taqman method, LAMP method, SMAP method is used.

In one embodiment, the methods, kits or in vitro diagnostic agents of the present disclosure can be used for monitoring in-body tumor mass, determining drug resistance genes, early detection of cancer, or monitoring recurrence. In addition, or in lieu of it, the circulating nucleic acid can be cfDNA. In such cases, the kits, compositions and methods of the present disclosure may be used for prenatal diagnosis, detection of chromosomal abnormalities, or newborn screening. The methods, kits or in vitro diagnostic agents of the present disclosure may also be used for the detection of viral infections. The methods, kits or in-vitro diagnostic agents of the present disclosure may also be used to perform absolute quantification of nucleic acids. Absolute quantification can be done by digital PCR.

The methods, kits or in vitro diagnostic agents of the present disclosure may be for capturing circulating tumor cells in the blood. Such methods, kits or in vitro diagnostic agents may be used to predict the prognosis of cancer or to reexamine drug therapies. The methods, kits or in vitro diagnostic agents of the present disclosure can also be used to quantify tumor cell numbers using digital PCR. Nucleic acid analysis may include digital PCR.

In a further aspect, the present disclosure may provide a sampling agent or sampling material comprising a PVA sponge for analysis of circulating nucleic acids in plasma or serum. Also provided in the present disclosure are kits comprising such sampling agents or materials for the analysis of circulating nucleic acids in plasma or serum. Analysis of circulating nucleic acids in plasma or serum can be a quantitative analysis. The circulating nucleic acid can be a tumor circulating nucleic acid. After the addition of plasma or serum, the PVA sponge can directly add the PVA sponge to a reaction solution such as PCR without liberating the nucleic acid, and analyze the nucleic acid in plasma or serum (for example, quantitative reaction). .. Not using the step of releasing nucleic acid can more accurately reflect the total amount of nucleic acid to be analyzed and can be very advantageous in quantification.

(General technology)
The molecular biology, biochemical, and microbiological methods used herein are well known and commonly used in the art, eg, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F. et al. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F.M. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Currant Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M. et al. A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. et al. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Currant Biology, Greene Pub. Associates; Ausubel, F.M. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Currant Biology, Greene Pub. Associates; Innis, M. et al. A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F.M. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Biology, Wiley, and annual upgrades. J. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, separate volume Experimental Medicine "Gene transfer & expression analysis experimental method" Yodosha, 1997, etc., which are relevant parts (possibly all) herein. Is used as a reference.

For DNA synthesis techniques and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. (1985). Oligonicleotide Synthesis: A Practical Approach, IRL Press; Gait, M. Gait. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. et al. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R.M. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G.M. M. et al. (1996). Nuclear Acids in Chemistry and Biology, Oxford University Press; Hermanson, G.M. T. (I996). It is described in Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference.

As used herein, "or" is used when "at least one" of the matters listed in the text can be adopted. The same applies to "or". When "within a range" of "two values" is specified in the present specification, the range also includes the two values themselves.
References such as scientific literature, patents, and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as they are specifically described.

The present disclosure has been described above with reference to preferred embodiments for ease of understanding. Hereinafter, the present disclosure will be described based on examples, but the above description and the following examples are provided for purposes of illustration only and not for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments and examples specifically described in the present specification, and is limited only by the scope of claims.

(Example 1: Nucleic acid detection protocol)
In this embodiment, a nucleic acid detection protocol will be described. FIG. 1A shows a schematic diagram showing a detection protocol (column method) in which a plasma sample is fixed on a PVA sponge in a column as a biological sample, then heated and dried, and then nucleic acid is extracted and nucleic acid analysis is performed. FIG. 1B shows a biological sample. A schematic diagram showing a detection protocol (direct method) in the case where a plasma sample is fixed on a PVA sponge, dried by heating, and then the PVA sponge is directly put into a reaction reagent for nucleic acid analysis is shown.

First, in FIG. 1A, a plasma sample obtained from a subject is dropped onto a column containing a PVA sponge. Then, after drying overnight at 60 ° C., distilled water is added dropwise and the mixture is allowed to stand at 95 ° C. for 5 minutes, and then centrifuged at 10000 rpm for 3 minutes to purify the cfDNA in the sample. Then, the column is removed, and the solution containing the extracted nucleic acid is mixed with the reaction reagent to perform nucleic acid analysis.

Next, in FIG. 1B, a plasma sample obtained from a subject is dropped onto a capture equipped with a PVA sponge and held. Then, it is dried at 60 ° C. overnight and then punched with a diameter of 2 mm so that a certain volume of the PVA sponge portion of the capture is cut off. Then, a punched constant volume of PVA sponge is placed directly into a container containing a reagent for real-time PCR.

(Example 2: Quantification of cfDNA by PVA sponge using column method)
Plasma samples were taken from 7 healthy subjects, 7 esophageal cancer patients, and 4 cancer patients (other than esophageal cancer), and standard Genome DNA solution preparation (104, 10 ³ , ^{10 2} , ¹⁰¹ , DW) was taken. 5 mL (n = 3)) was adjusted. In each case, the sample was prepared by plasma 80 mL PVA dropping ⇒ drying ⇒ PVA / DW 160 mL dropping ⇒ 95 ° C for 5 minutes ⇒ centrifugation 3000 rpm ⇒ PCR (n = 3). The composition of the standard Genome DNA solution, as well as the PCR cycle and reagent composition, were as shown in FIG.

The standard genomic DNA amplification curve is shown in FIG. 3, and the standard curve is shown in FIG. The plasma cfDNA amplification curves of healthy subjects are shown in FIGS. 5 (with standard curve) and FIG. 6 (without standard curve). The plasma cfDNA amplification curves of 7 esophageal cancer patients are shown in FIGS. 7 (with standard curve) and FIG. 8 (without standard curve). The plasma cfDNA amplification curves of 4 cancer patients (other than esophageal cancer) are shown in FIGS. 9 (with standard curve) and FIG. 10 (without standard curve).

Based on these amplification curves, the results of quantifying plasma cfDNA of healthy subjects are shown in Table 2, the results of quantifying plasma cfDNA of 7 patients with esophageal cancer are shown in Table 3, and the results of quantifying cfDNA in plasma are shown in Table 3 and 4 cancer patients (esophageal cancer). The results of quantifying plasma cfDNA are shown in Table 4, respectively.

Comparing the Ct values that reflect the amount of DNA in the sample, in any of the results, the 2 or 3 results in each patient gave very close Ct values, and the PVA sponge was fairly accurate in terms of purification efficiency. It was confirmed that it was expensive.

Regarding the amount of cfDNA obtained as described above, the results of comparing the plasma cfDNA levels of 7 healthy subjects and 7 esophageal cancer patients are shown in FIG. 11, showing 7 healthy subjects and 4 cancer patients (esophageal cancer). The results of comparing the amount of cfDNA in plasma are shown in FIG. 12, respectively. The vertical axis shows the number of samples, and the horizontal axis shows the number of copies of cfDNA contained in 1 μL of plasma sample. Looking at FIGS. 11 and 12, the number of cfDNA copies per μL of plasma was clearly higher than that of healthy subjects in the comparison results of 7 patients with esophageal cancer and 4 patients with cancer other than esophageal cancer. It was found that the number of copies per 1 μL is less than 20 in healthy subjects, whereas the number of copies per 1 μL is 21 or more only in cancer patients.

(Example 3: Quantification of cfDNA by PVA sponge using direct method)
Next, cfDNA was quantified using the PVA direct method. In this method, a sample such as plasma was added to the PVA sponge and dried, and then the completely dried PVA sponge was punched to a diameter of 2 mm and added to the PCR mix as it was. In this example, in order to confirm whether the amount of cfDNA can be measured by real-time PCR without inhibiting the serum sample by this direct method, a solution obtained by diluting human Genome DNA with sterile water to a desired concentration and a solution diluted with serum are used. Experiments were conducted on two types. The PCR cycle and reagent composition were as shown in FIG.

The amplification curve at each concentration of Genome DNA / serum solution by the direct method using a PVA sponge (thickness 2 mm × diameter 2 mm) is shown in FIG. Based on this amplification curve, Table 5 shows the quantitative results at each concentration of Genome DNA / serum solution.

In all the samples, Ct values very close to each other were obtained in the results of 3 times, and it was confirmed that the PVA sponge was highly accurate in terms of purification efficiency even when the direct method was used. Further, considering that the serum was directly injected into real-time PCR via the PVA sponge, it is considered that the effect of inhibition is small and the detection can be performed with high sensitivity.

(Example 4: Comparison of plasma sample and serum sample by direct method)
Next, plasma samples and serum samples were compared using the PVA direct method. In this example, a sample such as plasma was added to the PVA sponge and dried, and then the completely dried PVA sponge was punched to a diameter of 2 mm and added to the PCR mix as it was. In order to confirm whether the amount of cfDNA can be measured by real-time PCR without inhibiting serum samples and plasma samples, we targeted three types of solutions: a solution of human Genome DNA diluted with sterile water to the desired concentration, and a solution diluted with serum or plasma. An experiment was conducted. The PCR cycle and reagent composition were as shown in FIG.

The amplification curve at each concentration of Genome DNA / serum solution by the direct method using a PVA sponge (thickness 2 mm × diameter 2 mm) is shown in FIG. 16, and the amplification curve by the direct method using a PVA sponge (thickness 2 mm × diameter 2 mm) is shown in FIG. The amplification curves at each concentration of Genome DNA / healthy person's serum solution are shown in FIG. 17, respectively. Based on this amplification curve, Table 6 shows the quantitative results at each concentration of GenomeDNA / serum solution and GenomeDNA / healthy person plasma solution.

In all the samples, Ct values very close to each other were obtained in the results of 3 times, and it was confirmed that the PVA sponge was highly accurate in terms of purification efficiency even when the direct method was used. In addition, considering that plasma or serum was directly injected into real-time PCR via a PVA sponge, it is considered that the effect of inhibition is small and detection can be performed with high sensitivity.

(Example 5: Quantification of cfDNA by PVA sponge using direct method)
CfDNA was quantified in plasma samples of 10 healthy subjects and 9 patients with esophageal cancer using the direct method. The PCR cycle and reagent composition were as shown in FIG.

The amplification curve of cfDNA in healthy human plasma by the direct method using PVA sponge (thickness 2 mm × diameter 2 mm) is shown in FIG. 19, and the esophagus by the direct method using PVA sponge (thickness 2 mm × diameter 2 mm) is shown in FIG. The amplification curves in the plasma of the patient are shown in FIG. 20, respectively.

Based on these amplification curves, Table 7 shows the results of quantifying cfDNA in plasma of healthy subjects, and Table 8 shows the results of quantifying cfDNA in plasma of esophageal cancer patients.

Further, with respect to the amount of cfDNA obtained as described above, the result of comparing the amount of cfDNA in plasma of a healthy subject and a patient with esophageal cancer is shown in FIG. The vertical axis shows the number of samples, and the horizontal axis shows the number of copies of cfDNA contained in 1 μL of plasma sample. From this figure, it was found that the number of cfDNA copies per 1 μL of plasma was clearly higher in esophageal cancer patients than in healthy subjects.

(Example 6: Quantification of Genome DNA by TTX (Tth DNA polymerase))
In this example, the amplification efficiency of TTX (Tth DNA polymerase) used in the above-mentioned example was confirmed. After blood collection, serum was prepared by centrifugation and diluted to various concentrations to prepare a diluted solution. Then, the serum solution was directly added to the amplification reagent. The PCR cycle and reagent composition were as shown in FIG.

5 μL of GenomeDNA serum solution of each concentration was added to 25 μL of PCR mix for the experiment. The amplification curve at each concentration of Genome DNA serum solution is shown in FIG. Based on this amplification curve, the quantitative results at each concentration of GenomeDNA serum solution are shown in the lower right of FIG. 23. From this result, it was found that TTX (Tth DNA polymerase) showed good amplification efficiency even when the serum solution was used as it was, and was resistant to impurities.

(Note)
As described above, the present disclosure has been exemplified by using the preferred embodiments of the present disclosure, but it is understood that the scope of the present disclosure should be interpreted only by the scope of claims. Patents, patent applications and other documents cited herein are to be incorporated by reference in their content as they are specifically described herein. Is understood.

The present disclosure is useful in industries such as cancer testing and diagnosis.

Claims (77)

A method of analyzing circulating nucleic acids in a subject's plasma and / or serum.
The step of preparing plasma and / or serum from the blood of the subject, and
The step of placing the plasma and / or serum in a body fluid fixing device and drying it,
A method comprising the step of subjecting a body fluid fixing device containing plasma and / or serum to genetic analysis. The method according to claim 1, wherein the circulating nucleic acid is a tumor circulating nucleic acid. The method according to claim 2, wherein the tumor circulating nucleic acid is cfDNA. The method according to any one of claims 1 to 3, wherein the body fluid fixing device includes a porous material. The porous material is a polyvinyl alcohol (PVA) sponge, a urethane sponge, a cellulose sponge, a natural sponge (sponge), a sintered structure obtained by hot-pressing a resin powder, a fiber structure composed of a fiber aggregate, and the like. The method of claim 4, comprising the porous membrane and the porous membrane. The method according to any one of claims 1 to 5, wherein the drying is heat drying at about 60 ° C. or higher. The method according to any one of claims 1 to 6, wherein the drying is heat drying at about 60 ° C. or higher for at least 30 minutes or longer. The method according to any one of claims 1 to 5, wherein the drying is natural drying. The method according to any one of claims 1 to 8, wherein the analysis of circulating nucleic acid in plasma and / or serum is a quantitative analysis. The method according to any one of claims 1 to 9, wherein the analysis of circulating nucleic acid in plasma and / or serum is an absolute quantitative analysis. The method according to any one of claims 1 to 10, wherein the step to be subjected to the gene analysis is performed using nucleic acid extracted from the body fluid fixing device. 11. The method of claim 11, wherein the nucleic acid is extracted by dropping distilled water onto the body fluid fixing device. The step to be subjected to the gene analysis is performed by directly adding a constant-volume dry body fluid portion obtained by excising an area corresponding to a certain volume of the body fluid fixing device to a liquid containing a nucleic acid amplification reaction reagent. The method according to any one of 1 to 10. 13. The method of claim 13, wherein the excision has an excision diameter of about 1 to about 4 mm. The method according to any one of claims 1 to 14, wherein the subject is an esophageal cancer patient. A kit for the analysis of circulating nucleic acids in a subject's plasma and / or serum, including a fluid fixation device.
The step of preparing plasma and / or serum from the blood of the subject, and
The step of placing the plasma and / or serum in a body fluid fixing device and drying it,
A kit used in a method comprising the step of subjecting a body fluid fixing device containing plasma and / or serum to genetic analysis. The kit according to claim 16, wherein the circulating nucleic acid is a tumor circulating nucleic acid. The kit according to claim 17, wherein the tumor circulating nucleic acid is cfDNA. The kit according to any one of claims 16 to 18, wherein the body fluid fixing device includes a porous material. The porous material is a polyvinyl alcohol (PVA) sponge, a urethane sponge, a cellulose sponge, a natural sponge (sponge), a sintered structure obtained by hot-pressing a resin powder, a fiber structure composed of a fiber aggregate, and the like. The kit of claim 19, comprising the porous membrane and the porous membrane. The kit according to any one of claims 16 to 20, wherein the drying is heat drying at about 60 ° C. or higher. The kit according to any one of claims 16 to 21, wherein the drying is heat drying at about 60 ° C. or higher for at least 30 minutes or longer. The kit according to any one of claims 16 to 20, wherein the drying is natural drying. The kit according to any one of claims 16 to 23, wherein the analysis of circulating nucleic acid in plasma and / or serum is a quantitative analysis. The kit according to any one of claims 16 to 24, wherein the analysis of circulating nucleic acid in plasma and / or serum is an absolute quantitative analysis. The kit according to any one of claims 16 to 25, wherein the step to be subjected to the gene analysis is performed using nucleic acid extracted from the body fluid fixing device. The kit according to claim 26, wherein the extraction of the nucleic acid is performed by dropping distilled water onto the body fluid fixing device. The step to be subjected to the gene analysis is performed by directly adding a constant-volume dry body fluid portion obtained by excising an area corresponding to a certain volume of the body fluid fixing device to a liquid containing a nucleic acid amplification reaction reagent. The kit according to any one of 16 to 25. 28. The kit of claim 28, wherein the excision has an excision diameter of about 1 to about 4 mm. The kit according to any one of claims 16 to 29, wherein the subject is an esophageal cancer patient. A system that analyzes circulating nucleic acids in the subject's plasma and / or serum.
Means for preparing plasma and / or serum from the subject's blood,
A body fluid fixing device that retains and dries the plasma and / or serum, and
A system comprising a device for performing genetic analysis using a body fluid fixing device containing plasma and / or serum. 31. The system of claim 31, wherein the circulating nucleic acid is a tumor circulating nucleic acid. 32. The system of claim 32, wherein the tumor circulating nucleic acid is cfDNA. The system according to any one of claims 31 to 33, wherein the body fluid fixing device includes a porous material. The porous material is a polyvinyl alcohol (PVA) sponge, a urethane sponge, a cellulose sponge, a natural sponge (sponge), a sintered structure obtained by hot-pressing a resin powder, a fiber structure composed of a fiber aggregate, and the like. 34. The system of claim 34, comprising a porous membrane and a porous membrane. The system according to any one of claims 31 to 35, wherein the drying is heat drying at about 60 ° C. or higher. The system according to any one of claims 31 to 36, wherein the drying is heat drying at about 60 ° C. or higher for at least 30 minutes or longer. The system according to any one of claims 31 to 35, wherein the drying is natural drying. The system according to any one of claims 31 to 38, wherein the analysis of circulating nucleic acids in plasma and / or serum is a quantitative analysis. The system according to any one of claims 31 to 39, wherein the analysis of circulating nucleic acids in plasma and / or serum is an absolute quantitative analysis. The system according to any one of claims 31 to 40, wherein the gene analysis is performed using nucleic acid extracted from the body fluid fixing device. The system according to claim 41, wherein the extraction of the nucleic acid is performed by dropping distilled water onto the body fluid fixing device. The genetic analysis is performed by directly adding a constant-volume dry body fluid portion obtained by excising an area corresponding to a constant volume of the body fluid fixing device to a liquid containing a nucleic acid amplification reaction reagent, claim 31-40. The system described in any one of the above. 43. The system of claim 43, wherein the excision has an excision diameter of about 1 to about 4 mm. The system according to any one of claims 31 to 44, wherein the subject is an esophageal cancer patient. A method for analyzing circulating nucleic acids in the body fluids of cancer patients.
The step of placing the body fluid of the cancer patient on the body fluid fixing device and drying it,
A method comprising the steps of subjecting a body fluid fixing device containing the body fluid to genetic analysis. 46. The method of claim 46, wherein the circulating nucleic acid is a tumor circulating nucleic acid. 47. The method of claim 47, wherein the tumor circulating nucleic acid is cfDNA. The method according to any one of claims 46 to 48, wherein the body fluid fixing device includes a porous material. The porous material is a polyvinyl alcohol (PVA) sponge, a urethane sponge, a cellulose sponge, a natural sponge (sponge), a sintered structure obtained by hot-pressing a resin powder, a fiber structure composed of a fiber aggregate, and the like. 49. The method of claim 49, comprising the porous membrane and the porous membrane. The method according to any one of claims 46 to 50, wherein the drying is heat drying at about 60 ° C. or higher. The method according to any one of claims 46 to 51, wherein the drying is heat drying at about 60 ° C. or higher for at least 30 minutes or longer. The method according to any one of claims 46 to 50, wherein the drying is natural drying. The method according to any one of claims 46 to 53, wherein the analysis of the circulating nucleic acid in the body fluid is a quantitative analysis. The method according to any one of claims 46 to 54, wherein the analysis of the circulating nucleic acid in the body fluid is an absolute quantitative analysis. The method according to any one of claims 46 to 55, wherein the step to be subjected to the gene analysis is performed using nucleic acid extracted from the body fluid fixing device. 56. The method of claim 56, wherein the nucleic acid is extracted by dropping distilled water onto the body fluid fixing device. The step to be subjected to the gene analysis is performed by directly adding a constant-volume dry body fluid portion obtained by excising an area corresponding to a certain volume of the body fluid fixing device to a liquid containing a nucleic acid amplification reaction reagent. The method according to any one of 46 to 55. 58. The method of claim 58, wherein the excision has an excision diameter of about 1 to about 4 mm. The method according to any one of claims 46 to 59, wherein the cancer patient is an esophageal cancer patient. The method according to any one of claims 46 to 60, wherein the body fluid is plasma and / or serum. An in vitro diagnostic agent for diagnosing whether a subject has cancer or has relapsed by analyzing circulating nucleic acids in the subject's body fluid, including a body fluid fixing device. ,
The step of placing the body fluid of the subject on the body fluid fixing device and drying it,
An in vitro diagnostic drug used in a method comprising the step of subjecting a body fluid fixing device containing the body fluid to genetic analysis. The pharmaceutical product according to claim 62, wherein the circulating nucleic acid is a tumor circulating nucleic acid. The pharmaceutical product according to claim 63, wherein the tumor circulating nucleic acid is cfDNA. The pharmaceutical product according to any one of claims 62 to 64, wherein the body fluid fixing device contains a porous material. The porous material is a polyvinyl alcohol (PVA) sponge, a urethane sponge, a cellulose sponge, a natural sponge (sponge), a sintered structure obtained by hot-pressing a resin powder, a fiber structure composed of a fiber aggregate, and the like. 65. The pharmaceutical agent of claim 65, comprising the porous membrane and the porous membrane. The pharmaceutical product according to any one of claims 62 to 66, wherein the drying is heat drying at about 60 ° C. or higher. The pharmaceutical product according to any one of claims 62 to 67, wherein the drying is heat drying at about 60 ° C. or higher for at least 30 minutes or longer. The pharmaceutical product according to any one of claims 62 to 66, wherein the drying is natural drying. The pharmaceutical product according to any one of claims 62 to 69, wherein the analysis of the circulating nucleic acid in the body fluid is a quantitative analysis. The pharmaceutical product according to any one of claims 62 to 70, wherein the analysis of the circulating nucleic acid in the body fluid is an absolute quantitative analysis. The pharmaceutical product according to any one of claims 62 to 71, wherein the step to be subjected to the gene analysis is performed using the nucleic acid extracted from the body fluid fixing device. The pharmaceutical product according to claim 72, wherein the nucleic acid is extracted by dropping distilled water onto the body fluid fixing device. The step to be subjected to the gene analysis is performed by directly adding a constant-volume dry body fluid portion obtained by excising an area corresponding to a certain volume of the body fluid fixing device to a liquid containing a nucleic acid amplification reaction reagent. The drug according to any one of 62 to 71. The pharmaceutical product according to claim 74, wherein the excision diameter is about 1 to about 4 mm. The pharmaceutical product according to any one of claims 62 to 75 for diagnosing whether or not the subject has esophageal cancer or whether or not the esophageal cancer has recurred. The pharmaceutical product according to any one of claims 62 to 76, wherein the body fluid is plasma and / or serum.

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