Reagent for preparing biological sample, sample container, and method for preparing biological sample

Abstract

LB tests have the following four primary problems: a trace amount of cfDNA that is an LB test subject should be highly efficiently recovered; gDNA that is an LB test inhibition factor should be eliminated; the time of complicated centrifugal separation operations should be reduced to one time; and plasma that contains a trace amount of cfDNA should be recovered without loss. The purpose of the present invention is to solve at least one of the above-mentioned primary problems in LB tests. A biological sample preparation reagent according to the present invention comprises: at least one type of salting-out effect molecule of which the final concentration is 4 wt% or more when mixed with a sample; and at least one type of excluded volume effect molecule of which the final concentration is 5-8 wt% when mixed with a sample (see Fig. 1).

Description

Reagents for preparing biological samples, sample containers, and methods for preparing biological samples.

[0001] This invention relates to a sample preparation reagent used when analyzing, testing, and diagnosing biological samples.

[0002] Among biological samples, technologies for liquid biopsy (LB), which uses bodily fluids such as whole blood and plasma as samples, are being developed. This technology is considered desirable because it efficiently recovers trace amounts of free DNA (cfDNA: cell-free DNA) that are the target of the test, while simultaneously eliminating large amounts of genomic DNA (gDNA: genome DNA) that can interfere with the test. Specific details will be discussed later.

[0003] In recent years, it has become clear that the peripheral blood of cancer patients contains DNA (deoxyribonucleic acid) derived from cancer cells, and this DNA is attracting attention as a target for detecting the genetic information of cancer. In August 2020, Guardant360 CDx (Guardant Health) and FoundationOne Liquid CDx (Foundation Medicine) were approved in the United States as Next Generation Sequencing (NGS) cancer genome testing kits for blood samples. In Japan, FoundationOne Liquid CDx was also approved in March 2021, and research on cancer diagnosis using blood samples is accelerating both domestically and internationally. Furthermore, efforts to standardize sample pre-processing steps for this purpose are also becoming more active.

[0004] Thus, a new technique called LB test for diagnosing diseases such as cancer is to extract DNA from body fluids such as blood and use it as a test subject. Compared with biopsy tissue tests that directly collect tissues during surgery, the LB test has the advantage of being able to obtain test specimens relatively easily by a less invasive method such as blood collection. There is an expectation that cancer can be detected at an early stage by detecting DNA fragments (ctDNA: Circulating tumor DNA) derived from cancer cells leaked into the blood, rather than analyzing the cancer cells themselves at the disease site. In addition, in the future, its application to monitoring tests for prognosis recurrence and treatment effects is also expected. In particular, since the monitoring test using the LB test technology requires repeated tests over a long period, it is suitable to use easily collected blood specimens as the test subjects. For clinical laboratory technicians and doctors who perform tests and diagnoses, making use of the LB test technology also has the advantage of being able to monitor regularly and formulate flexible treatment strategies.

[0005] The primary target components used in LB testing are trace amounts of cfDNA released into the bloodstream from disease sites, including ctDNA. cfDNA is released in nucleosome units (complexes consisting of a histone octameric protein wrapped 1.67 times with double-stranded DNA (dsDNA)), which are the basic building blocks of chromatin. It is short-chain DNA released into the bloodstream in the form of polynucleosomes, including mononucleosomes (monomers) centered around a dsDNA strand of approximately 166 base pairs (bp), dinucleosomes (molecular size approximately 332 bp), and trimer (molecular size approximately 498 bp). Fragment analysis, commonly used in this field, analyzes dsDNA detected in the base length range of 50-700 bp as equivalent to cfDNA. It has been revealed that cfDNA released from cells contains mutations in cancer-related genes in the early stages of various cancers, including lung cancer and breast cancer. However, since cfDNA in the blood is present in trace amounts, highly efficient and purified recovery is crucial for advancing LB testing. Furthermore, research is beginning to explore the relationship between quantitative fluctuations and changes in the distribution ratio of mononucleosomes and other polynucleosomes and disease.

[0006] On the other hand, gDNA derived from leukocytes is also present in the blood. gDNA carries the genetic information of normal human cells and is estimated to be about 3 billion bp in size. However, it is often fragmented due to physical shearing during nucleic acid extraction and changes in various physiological activities at the molecular level within the body. Fragment analysis, which is commonly used in this field, analyzes dsDNA detected in the base length range of 800 bp or more as equivalent to gDNA. Since gDNA has a normal base sequence, there is a method of diagnosing cancer by analyzing and comparing the base sequences of gDNA and cancer cell-derived cfDNA (i.e., ctDNA) separately. However, if gDNA is mixed into the plasma sample intended for cfDNA, it leads to a decrease in the sensitivity of detecting mutations in the cfDNA base sequence, which is a major challenge in the field of LB testing.

[0007] Unstable single-stranded DNA (ssDNA), which lacks hydrogen bonds between base pairs like dsDNA, also exists in the blood. Because ssDNA is easily degraded, it tends to be detected as relatively shorter fragments than cfDNA. The distribution and quantitative fluctuations of this ssDNA are expected to be used in LB testing as indicators for disease and diagnosis.

[0008] The general workflow for LB testing involves collecting whole blood from the patient using various cfDNA collection tubes, centrifuging it, and separating the crude plasma fraction. During this process, the plasma is separated only up to a point where it is at a certain distance from the buffy coat layer, which accumulates at the interface between the plasma and the blood cell layer, to avoid accidentally separating that layer. The separated crude plasma fraction is then purified by centrifugation again (a total of two centrifugation operations). This procedure for separating the purified plasma fraction is evident from the package inserts for various cfDNA collection tubes and the trend towards standardization of sample pretreatment processes for LB testing. Next, the quality of the extracted DNA is checked by quantitative analysis and fragment size analysis of the purified plasma fraction. If there are no abnormalities in quality, the extracted DNA is analyzed for gene sequence mutations using NGS or dPCR (Digital Polymerase Chain Reaction) to diagnose cancer and select therapeutic drugs. NGS genetic testing is a test that detects gene sequence mutations and mutation levels by reading DNA base sequences in a massively parallel manner. On the other hand, dPCR-based genetic testing involves dividing the reaction into thousands of parallel PCR reactions and amplifying specific gene sequences to detect mutations or measure mutation levels.

[0009] Meanwhile, in this field, various cfDNA collection tubes for LB testing have been launched by various companies. All of them primarily aim to protect cells and DNA in the sample, and each company has devised ingenious additive compositions for this purpose. For example, collection tubes that utilize cell immobilization technology using formaldehyde sustained-release agents (Patent Document 4), collection tubes that utilize cell maintenance technology using apoptosis inhibitors (Patent Document 3), collection tubes that utilize cell protection technology using excluded volume polymers (Patent Document 1), and collection tubes aimed at stabilizing extracellular nucleic acids (Patent Document 2) have been patented and commercialized. It is presumed that each of these collection tubes has a composition that blends nuclease inhibitors, osmotic pressure regulators, anticoagulants, buffers, etc.

[0010] For example, regarding cfDNA blood collection tubes that use polyethylene glycol (PEG) as the excluded volume polymer and sodium chloride (NaCl) as the main additive, albeit as an osmotic pressure adjuster (Patent Document 1), the intention is solely to protect cells and nucleic acids in the sample and to suppress nucleic acid leakage from cells. Therefore, it can be seen from the technical documentation of this product that even with mixed treatment with PEG, sufficient cfDNA retention and gDNA exclusion effects cannot be obtained, especially when the final NaCl concentration after blood collection is low.

[0011] One nucleic acid processing method that uses compounds with exclusion volume effect and compounds with salting-out effect is the nucleic acid PEG precipitation method using PEG and NaCl (Non-Patent Literature 1). This method aims to purify long-chain DNA by removing short-chain DNA from the prepared nucleic acid solution. Therefore, the nucleic acid PEG precipitation method is based on the premise of purifying long-chain DNA from a nucleic acid mixture solution that has already been purified to some extent, and does not assume preparation from crude samples such as body fluids. Furthermore, in order to precipitate and recover the long-chain DNA, in addition to cooling treatment after mixing with the sample (on ice, 60 min), high-speed centrifugation under cooling (20,000 × g, 4°C, 30 min) is required.

[0012] Furthermore, for example, there is a method that applies PEG and NaCl in the DNA size fractionation step using magnetic beads during library preparation for NGS analysis (Patent Documents 5 and 6). This method is a technique that fractionates DNA of the required base length by switching the PEG concentration conditions during nucleic acid recovery using magnetic beads. However, similar to the nucleic acid PEG precipitation method, it is not possible to achieve both the preservation of short-chain DNA and the exclusion of long-chain DNA from a crude sample. Also, it uses bead separation by magnetism rather than centrifugation, and its properties differ from the other techniques mentioned above.

[0013] EP 3464588B1 Patent No. 6664332 US 2022 / 0349014US 9926590B2US 2022 / 0340954US 10584327B2

[0014] Molecular Cloning: A Laboratory Manual, Second Edition, J. Sambrook, EF. Fritsch & T. Maniatis, Cold Spring Harbor Laboratory Press, 1989

[0015] In light of the conventional technologies described above, the following four points can be identified as the main challenges of LB testing: efficiently recovering the trace amounts of cfDNA that are the target of LB testing; eliminating gDNA that inhibits LB testing; reducing the complicated centrifugation process to a single step; and recovering plasma containing trace amounts of cfDNA without loss.

[0016] Patent documents 1 to 4 do not address all of these issues. Furthermore, non-patent document 1 aims to purify long-chain DNA by removing short-chain DNA from the prepared nucleic acid solution, which is the exact opposite of removing long-chain DNA (e.g., gDNA) and recovering short-chain DNA (e.g., cfDNA).

[0017] This invention has been made in view of the above-mentioned problems, and aims to resolve at least one of the major problems in LB testing.

[0018] The biological sample preparation reagent according to the present invention comprises at least one salting-out effect molecule having a final concentration of 4 wt% or more when mixed with the sample, and at least one exclusion volume effect molecule having a final concentration of 5 to 8 wt% when mixed with the sample.

[0019] The biological sample preparation reagent according to the present invention can resolve at least one of the major problems in LB testing. Other problems, configurations, and advantages of the present invention will become clear from the following description of embodiments.

[0020] This shows a schematic diagram of the overall workflow of the biological sample testing method according to Embodiment 1. This shows the results of calculations for the conditions under which the constituent components reach the optimal final concentration when the processing solution (additive) is freeze-dried and / or partially freeze-dried. This is a plot graph created based on various information such as electropherograms and nucleic acid quantitative values ​​obtained from fragment analysis. This shows the electropherogram patterns used for evaluation and judgment. This shows the electropherogram patterns used for evaluation and judgment. This shows the electropherogram patterns used for evaluation and judgment. This shows the electropherogram patterns used for evaluation and judgment. This shows the electropherogram patterns used for evaluation and judgment. This shows the electropherogram patterns used for evaluation and judgment. This is a perspective view of the blood collection tube 501 according to Embodiment 2. This shows another example of the blood collection tube 501 configuration. This is a configuration diagram of the post-processing kit 708 according to Embodiment 3. This shows another example of the post-processing kit 708 configuration. This shows the results of nucleic acid extraction and fragment analysis performed on a sample treated with the additive described in Embodiment 1. This shows an example of a whole blood sample treated with the additive according to the present invention.

[0021] <Embodiment 1> Figure 1 shows a schematic diagram of the overall workflow of the biological sample testing method according to Embodiment 1 of the present invention. In general, based on the present invention, a processing solution (additive) mainly composed of excluded volume effect molecules and salting-out effect molecules is prepared, and after mixing both molecules with the collected biological sample so that they reach a predetermined final concentration (concentration when mixed with the sample), the supernatant is collected by centrifugation. After extracting nucleic acids from the supernatant, the results of the analysis are applied clinically. The above is a general overview of the workflow. Regarding the processing solution (additive), options such as freeze-drying, semi-freeze-drying, and post-processing kits are available. Furthermore, it is envisioned that characterization of the supernatant and precipitate will be performed in order to generate supplementary information for final clinical application. These are merely typical general outlines, and the details and applications are not necessarily limited to these.

[0022] To solve the above problems, this invention uses a mixture of at least one excluded volume effect molecule and at least one salting-out effect molecule. The aim is to optimize the concentration combination of both molecules in the mixture so that the final concentrations of both molecules in the reaction solution when tested with a specimen (biological sample) are optimal for solving the problems.

[0023] Compounds that exhibit excluded volume effects include polymers, macromolecules (proteins (hemoglobin, collagen, fibrinogen, etc.), DNA, polysaccharides (hyaluronic acid, cellulose, pectin, glycogen, etc.), ribosomes, etc.), small molecules (gas molecules (noble gases such as helium and neon), liquid molecules (water, ethanol, etc.)), colloidal particles (silica particles, polystyrene latex particles, etc.), and nanoparticles (gold nanoparticles, silver nanoparticles, etc.). In particular, polymers that are easy to handle as reagents include polystyrene, polyethylene, polymethyl methacrylate, polyvinyl alcohol, polycaprolactone, polylactic acid, polyvinylpyrrolidone, polyimide, polyurethane, polytetrafluoroethylene, and polysulfone.

[0024] In particular, polyethylene glycol (PEG) is a polymer obtained by the condensation polymerization of ethylene oxide and water. It has a wide range of molecular sizes (e.g., low molecular weight PEG with molecular weights in the hundreds, medium molecular weight PEG with molecular weights of 1,000 to 5,000, high molecular weight PEG with molecular weights of 5,000 to tens of thousands, and high molecular weight PEG (PEO: polyethylene oxide) with molecular weights of hundreds of thousands or more), exhibits high hydrophilicity, nonvolatility, biocompatibility, and low toxicity, making it frequently used in many biological applications, including pharmaceuticals and cosmetics. PEG has the effect of removing hydration water, thus possessing the property of precipitating impurities in samples. In this invention, after verifying the solubility and stringiness of PEG of various molecular sizes, low to high molecular weight PEG, excluding ultra-high molecular weight PEG (PEO: polyethylene oxide), was considered a candidate. The examples described later mainly show cases using PEG8000, which corresponds to high molecular weight PEG. However, the excluded volume effect molecules applicable to this invention are not limited to these.

[0025] On the other hand, examples of compounds that exhibit a salting-out effect include ammonium sulfate, sodium chloride, magnesium sulfate, sodium sulfate, potassium chloride, potassium sulfate, and magnesium chloride. Among these, sodium chloride (NaCl) is a compound that is frequently used in various biological applications because of its high salting-out effect, solubility, and ease of handling. In this invention, various salting-out molecules have been verified, including confirmation of solubility, and the examples described later will mainly show cases using NaCl. However, the salting-out molecules applicable to this invention are not limited to these.

[0026] <Embodiment 1: Preparation of various stock solutions for processing solutions (additives)> The present invention aims for the final combined concentration (concentration when mixed with the sample) of volume-exclusion effect molecules and salting-out effect molecules in the processing solution (additive) to exhibit both cfDNA retention effect and gDNA exclusion effect. For example, in the case of PEG8000, under high final concentration conditions exceeding 10 wt%, it has an extreme co-precipitating effect on contaminants in the blood, including free hemoglobin, but it also excludes short-chain nucleic acids such as cfDNA that we want to recover as the supernatant fraction. On the other hand, under low final concentration conditions below 4 wt%, sufficient co-precipitation effect cannot be obtained, and gDNA remains in the supernatant fraction, and even with the salting-out effect of NaCl, sufficient gDNA exclusion is not achieved. In order to utilize the strong co-precipitation effect of PEG while leaving short-chain nucleic acids such as cfDNA in the supernatant fraction, the problem was solved by fine-tuning the balance between the two effects by utilizing the salting-out effect of NaCl. Specifically, we found that the problem described above can be solved by preparing a mixed solution with the sample under concentration combination conditions where the final concentration of PEG8000 is 5-8 wt%, and the final concentration of NaCl is 4 wt% or more, preferably 4-26 wt%, and more preferably 4-10 wt%.

[0027] Therefore, in practical operation, the concentration and volume of the excluded volume effect molecule stock solution and the salting-out effect molecule stock solution to be mixed will be determined based on the blood collection volume, so that when the processing solution (additive) is finally mixed with the sample, the final concentration in the mixture of excluded volume effect molecules and salting-out effect molecules falls within the optimal concentration range.

[0028] Furthermore, anticoagulants and nuclease inhibitors such as EDTA and citrate, which are commonly added to blood collection tube additives, and preservatives such as sodium azide (NaN3), may be added as needed. However, when adding these, it is desirable to determine the concentration and volume of these additional reagents so that the application conditions for the excluded volume effect molecule stock solution and the salting-out effect molecule stock solution are matched to the aforementioned optimal final concentration range.

[0029] Furthermore, when applied as a blood collection tube additive, it is desirable to reduce the amount of additive as much as possible, considering the balance between the size of the blood collection tube and the blood collection volume. For this reason, it is desirable to freeze-dry and / or partially freeze-dry the additive.

[0030] Figure 2 shows the results of calculations for the conditions under which the components of the treatment solution (additive) reach the optimal final concentration (concentration when mixed with the sample) after freeze-drying and / or partial freeze-drying. Furthermore, the upper and lower limits of the acceptable blood collection volume at the median optimal final concentration (6.5 wt% PEG8000, 7.0 wt% NaCl) and the final concentrations of each additive composition were calculated. The additive composition at the median optimal final concentration is assumed to be as follows: 1.51 mL of 50 wt% PEG8000 stock solution; 3.12 mL of 26 wt% NaCl stock solution; 0.80 mL of 5 wt% EDTA3K stock solution; 0.04 mL of 1 wt% NaN3 stock solution. Freeze-drying this additive yields a total of 1.61 g of dried additive, and the median optimal final concentration is achieved in a 10 mL blood sample. The specific calculation conditions will be described later.

[0031] Regarding the excluded volume effect molecule, one of the main components, we primarily investigated PEG among various candidate molecules, but this was not the only consideration. Furthermore, within PEG, in addition to the high molecular weight PEGs collectively known as PEG6000, PEG8000, and PEG20000, we also evaluated the ultra-high molecular weight PEG500000, PEG2000000, and PEG4000000, expecting them to exhibit excluded volume effects under low concentration conditions. We assessed their solubility in H2O at room temperature, ease of dispensing, and whether or not precipitation occurred when stored for 1 week or more at 4°C. However, this does not apply to PEG molecular sizes.

[0032] As a result, it was confirmed that PEG6000 could meet three criteria: solubility and ease of dispensing in H2O at room temperature, and no precipitation when stored at 4°C, with upper limits of concentrations of 56 wt%, 50 wt%, and 52 wt% for PEG20000. On the other hand, the ultra-high molecular weight molecules PEG500000, PEG2000000, and PEG4000000 showed extremely low solubility in H2O at room temperature, reaching only about 5 wt%, and exhibited strong stringiness, hindering dispensing. The extremely low solubility in H2O means that additives cannot be prepared at higher concentrations, thus imposing concentration limitations for evaluation. This not only limits the rate-limiting process in narrowing down the optimal concentration range, but also exacerbates the difficulty of concentration control. Furthermore, the extreme stringiness hinders high-precision dispensing operations, so these molecules were judged unsuitable as candidate molecules. Based on the above, the following examples will focus on verification cases using 50 wt% PEG8000 as the stock solution. However, this is not necessarily limited to PEG8000 alone.

[0033] Regarding the other major component, the salting-out effect molecule, we primarily investigated NaCl among various candidate molecules, but this was not the only consideration. We evaluated NaCl from the same perspective as PEG, and confirmed that it could meet three criteria: solubility and ease of separation of H2O at room temperature, and no precipitation during storage at 4°C, up to a maximum concentration of 26 wt%. Therefore, the following examples will focus on verification cases using 26 wt% NaCl as the stock solution. However, this is not necessarily limited to NaCl alone.

[0034] Furthermore, when considering additives for blood collection tubes, additional components such as EDTA to supplement the effects as an anticoagulant and nuclease inhibitor, and NaN3 to supplement the effect as a preservative, are conceivable. The following examples will focus on verification cases where 5 wt% EDTA-3K (ethylenediamine-N,N,N',N'-tetrapotassium acetate dihydrate) and 1 wt% NaN3 were used as stock solutions. However, the examples are not necessarily limited to EDTA-3K or NaN3 alone. Also, since these components are not necessarily essential for the effects of the present invention, their addition may be omitted.

[0035] <Embodiment 1: Sample Preparation> In the workflow shown in Figure 1, a sample is prepared using the processing solution (additive) described above (Figure 1: Mixed Solution). As mentioned above, 50 wt% PEG8000 stock solution, 26 wt% NaCl stock solution, 5 wt% EDTA-3K stock solution, and 1 wt% NaN3 stock solution were used to prepare the processing solution (additive). The amount of each stock solution added was set so that a predetermined final concentration was achieved when mixed with a predetermined amount of sample (whole blood or plasma fraction, etc.), and the sample was prepared in a 15 mL conical tube. However, for the 5 wt% EDTA-3K stock solution and 1 wt% NaN3 stock solution, which are not necessarily essential for the effects of the present invention, a uniform amount was used for simplicity. For example, when using a blood sample equivalent to 10 mL as the sample, 800 μL of 5 wt% EDTA-3K stock solution and 40 μL of 1 wt% NaN3 stock solution were added per sample. However, the amount of addition is not limited to this.

[0036] The samples used were primarily whole blood, with plasma fractions such as Cleared Plasma being used depending on the experiment. Whole blood included commercially available whole blood from healthy individuals imported from the United States for experimental purposes, as well as fresh blood from healthy volunteers and fresh blood from cancer patients. In particular, real samples such as fresh blood from healthy volunteers and fresh blood from cancer patients differed from Cleared Plasma and commercially available whole blood from healthy individuals in that they involved blood collection from the subjects themselves. Therefore, they were only applied to post-processing evaluations of samples collected using existing (approved) vacuum blood collection tubes for cfDNA.

[0037] As a specific example of sample preparation, a typical experimental procedure using commercially available whole blood from healthy individuals is as follows: To minimize physical damage during transport, commercially available whole blood from healthy individuals was obtained in blood bags (approx. 450 mL / bag) rather than in small-volume blood collection tube units, and was acquired under refrigerated conditions to ensure temperature stability. EDTA-3K was used as the anticoagulant during blood collection. Blood samples were collected from healthy individuals of any age, race, or sex, and all tested negative for viruses according to FDA standards.

[0038] To prepare the received whole blood for subsequent experiments, it was dispensed from the blood bag into 50 mL conical tubes on the same day and stored upright at 4°C. Care was taken to avoid inadvertently destroying blood cell components by gently inverting and mixing the blood bag to homogenize the blood components, followed by gentle decantation. Furthermore, when using commercially available whole blood from healthy individuals that had already been dispensed into smaller portions for experiments, the required amount (number of tubes) was taken out beforehand, allowed to acclimate to room temperature, then gently inverted and mixed to eliminate any unevenness in naturally settled blood components. Finally, multiple 100 mL conical tubes of dispensed whole blood were decanted and mixed together to homogenize the mixture before application.

[0039] To the processing solution (additive) prepared in accordance with the above, homogenized whole blood was dispensed to the specified volume using a 5 mL disposable pipette with an opening that was not too narrow. The whole blood and additive were then mixed evenly by gently inverting the pipette 180° 10 times. The prepared mixed solution (simulated sample) was immediately subjected to centrifugation, but depending on the experimental purpose, it was stored in the refrigerator or at room temperature for about one week.

[0040] The mixed sample was separated into plasma and blood cell layers by centrifugation once at 1,900 × g for 15 min under room temperature conditions of 22°C. The plasma fraction was transferred to a new 15 mL conical tube and stored frozen at -80°C until nucleic acid extraction. The sample was also documented by taking photographs during sample preparation as needed.

[0041] When gDNA to be excluded or other contaminants from various biological samples (eg cells, proteins, etc.) were to be analyzed separately, the precipitate was used as the sample for analysis. If immediate analysis was not required, the precipitate was frozen or stored at room temperature depending on the experimental purpose.

[0042] <Embodiment 1: Nucleic Acid Extraction> The process for recovering the nucleic acid eluate in the workflow shown in Figure 1 will be described below. Nucleic acid extraction from the plasma fraction was performed in accordance with the following: 30 μL of 20 mg / mL Proteinase K solution (Kanto Chemical) was added to 2 mL of plasma fraction and mixed, then 100 μL of 20% SDS solution (Serva) was added and mixed, incubated at 60°C for 20 minutes, and then cooled on ice for 5 minutes to perform Proteinase K treatment of the sample. Subsequently, nucleic acid extraction was performed using a MagMAX Cell-free DNA Isolation Kit (Thermo Fisher Scientific) and a KingFisher Duo Prime nucleic acid extractor (Thermo Fisher Scientific). Nucleic acid extraction was performed using the equivalent of 4 mL of plasma per sample, and finally 100 μL of nucleic acid eluate was recovered. For samples with less than 4 mL of plasma, the deficiency was supplemented with 1×PBS buffer (pH 7.0) before being used for nucleic acid extraction. Based on the volume of plasma fraction recovered, the volume of plasma fraction used for nucleic acid extraction, and the nucleic acid concentration in the nucleic acid eluate, the amount of nucleic acid in the total recovered plasma fraction was calculated and compared between samples. The nucleic acid eluate was stored frozen at -80°C until it was used for fragment analysis.

[0043] <Embodiment 1: Nucleic Acid Quality Check> The nucleic acid eluate obtained by nucleic acid extraction was subjected to fragment analysis using a cfDNA ScreenTape (Agilent) and a 4200 TapeStation (Agilent). In addition to analyzing the pattern of the obtained electropherogram, the amount of each nucleic acid in the initially recovered total plasma was calculated and compared from the quantitative values ​​(concentration values) of cfDNA equivalent (50-700 bp) and gDNA equivalent (>800 bp), and the retention of cfDNA and exclusion of gDNA in the plasma were evaluated and judged.

[0044] Figure 3 is a plot graph created based on various information such as the electropherogram obtained from fragment analysis and nucleic acid quantification values. Since the whole blood samples from which each plot data is derived span multiple lots (with different blood donors and characteristics), and the time-course state after blood collection is not necessarily uniform, it is difficult to perform quantitative comparison between different experimental days. Therefore, based on the data comparison between each test section (between samples) obtained using whole blood with the same lot and time-course within the same experimental day, the tendency of the sample was qualitatively evaluated and judged. Groups 1 to 5 shown in Figure 3 will be described later.

[0045] Figures 4A to 4E show, as reference examples, the patterns of the electropherograms used for evaluation and judgment. In the figures, ▼ indicates the LMW marker, ▽ indicates the peak of gDNA, and ★ indicates the peak of mono-, di-, and trinucleosomes. These are merely typical 10 patterns, and the signal intensity, etc. changes as the whole blood lot and time-course change. This is why quantitative comparison between different experimental days was considered inappropriate. However, regarding the tendency shown by the electropherogram pattern and nucleic acid quantification value between each test section (between samples) obtained using whole blood with the same lot and time-course within the same experimental day, it was considered possible to compare and evaluate. Therefore, the acquired data was plotted, and the characteristics were categorized mainly by the pattern of the electropherogram. As a result, each data could be classified into the following five groups roughly.

[0046] Group 1: Figure 4A: A dataset in which both cfDNA and gDNA are maintained in plasma. It is occupied by samples under the condition that the final concentration of PEG in the mixed solution is less than 5 wt%, including the untreated section (NC: Negative control). Under the same final concentration condition of PEG, an unexpected tendency was recognized in the coprecipitation effect of nucleic acids regardless of their molecular size.

[0047] Group 2: Figure 4B: A dataset in which, although the effect of maintaining cfDNA in plasma can be expected, a slight residual amount of gDNA in plasma (a tendency not to be completely eliminated) is observed. Samples are occupied by those with a NaCl concentration exceeding 10 wt% within the range of the final PEG concentration in the mixed solution being 5 to 8 wt%. Regarding the obtained data, the amount of gDNA relative to the amount of cfDNA (residual rate) tends to be as low as several percent, and in some cases, there may be a low possibility of affecting or inhibiting tests and diagnoses (such as mutation analysis).

[0048] Group 3: Figure 4C: A dataset in which gDNA can be excluded from plasma while maintaining cfDNA in plasma. It was observed in samples within the range of the final PEG concentration in the mixed solution being 5 to 8 wt% and the final NaCl concentration being 4 to 10 wt%.

[0049] Group 4: Figure 4D: A dataset in which there is a tendency that cfDNA cannot be sufficiently maintained (lost) in plasma, but there is a tendency that gDNA is excluded from plasma. Samples are occupied by those with a NaCl concentration below 4 wt% within the range of the final PEG concentration in the mixed solution being 5 to 8 wt%. Since the cfDNA in plasma is only trace amounts, it is desirable to avoid concentration conditions where there are concerns about recovery loss as much as possible.

[0050] Group 5: Figure 4E: A dataset in which both cfDNA and gDNA are excluded from plasma. This tendency is observed in samples with a final PEG concentration in the mixed solution exceeding 8 wt%. Under the same PEG conditions, it is considered that they are excluded (co-precipitated) regardless of the molecular size of the nucleic acid.

[0051] <Embodiment 1: Separation of Mono and Polynucleosomes Based on Molecular Size> As observed in the above plot graph, the dataset corresponding to Group 3 showed an ideal electropherogram that achieved both cfDNA retention and gDNA exclusion effects in plasma. Group 3 is the region enclosed by the final PEG concentration conditions in the mixed solution being 5-8 wt% and the final NaCl concentration conditions being 4-10 wt%, suggesting that these are the final concentration conditions most suitable for achieving both effects. Further detailed analysis of the dataset corresponding to Group 3 revealed a tendency for stepwise separation of mono, di, and trinucleosomes constituting cfDNA, based on their molecular size, depending on the combination of final PEG and NaCl concentrations.

[0052] Specifically, in the relatively low concentration range within the target concentration range (e.g., within the final concentration range consisting of PEG final concentration of approximately 5–6 wt% and NaCl final concentration of approximately 4–6 wt%), the co-precipitation effect of nucleic acids was relatively low, resulting in a tendency for mono, di, and tri nucleosome peaks to be easily detected in the cfDNA equivalent region of 50–700 bp on the electropherogram. On the other hand, in the relatively high concentration range within the target concentration range (e.g., within the final concentration range consisting of PEG final concentration of approximately 6–8 wt% and NaCl final concentration of approximately 4.5–10 wt%), the co-precipitation effect of nucleic acids was relatively high, resulting in the exclusion of di and tri nucleosomes in the cfDNA equivalent region of 50–700 bp on the electropherogram, and a tendency for only mono nucleosome peaks to be easily detected. In contrast, in the moderate concentration range within the target concentration range (e.g., within the final concentration range consisting of PEG final concentrations of approximately 5.5–7 wt% and NaCl final concentrations of approximately 4–8 wt%), due to the moderate co-precipitation effect of nucleic acids, trinucleosomes were excluded in the cfDNA equivalent region of 50–700 bp on the electropherogram, and mononucleosome peaks tended to be more easily detected.

[0053] <Embodiment 1: Freeze-drying and semi-freeze-drying of additives> The main components of the additive according to the present invention are excluded volume effect molecules represented by PEG and salting-out effect molecules represented by NaCl. The basic structure involves adding various functional molecules such as EDTA as an anticoagulant / nuclease inhibitor and NaN3 as a preservative to this main composition as needed. In particular, the addition specifications for the main components, PEG and NaCl, are set so that their final concentration falls within the optimal final concentration range for the volume of the biological sample such as whole blood, or so that it reaches a predetermined / target final concentration.

[0054] On the other hand, considering general blood collection practices, accurately controlling the amount of blood collected using a designated vacuum blood collection tube is technically difficult, and variations also occur depending on the patient's physical condition. Therefore, in cases like the present invention, where the final concentration of the main component when the additive and a biological sample such as whole blood are mixed is important for effectiveness, a mechanism is needed that can adjust the blood collection volume so that it remains within the optimal final concentration range or reaches a predetermined / target final concentration, even if the blood collection volume varies.

[0055] Furthermore, regarding vacuum blood collection tubes, if the blood collection volume is frequently up to about 10 mL, the additive volume becomes a trade-off with the blood collection volume, so a small volume is desirable. For example, in the case of existing cfDNA blood collection tubes, the additive volume is actually limited to about 0.5 to 1.5 mL for a blood collection volume equivalent to 10 mL.

[0056] As a reference example, we estimated the allowable blood collection volume for a blood collection tube with an additive composition that achieves target final concentrations of 6.5 wt% PEG and 7.0 wt% NaCl in the mixed solution, corresponding to the median of the optimal final concentration range. Considering its operation as a blood collection tube, the calculation was performed using a case where 0.8 mL of 5 wt% EDTA-3K stock solution and 0.04 mL of 1 wt% NaN3 stock solution were added as anticoagulants, nuclease inhibitors, and preservatives, respectively. Assuming a blood collection volume of 10 mL, in order to match the final PEG concentration of 6.5 wt% and final NaCl concentration of 7.0 wt% after blood collection, 2.36 mL of 50 wt% PEG8000 stock solution and 4.88 mL of 26 wt% NaCl stock solution are required as additives. Adding the EDTA-3K and NaN3 together, the total additive volume is 8.08 mL, and the total sample volume including whole blood is 18.08 mL.

[0057] In this case, for example, if we consider the application of a typical 10 mL vacuum blood collection tube, the additive volume is too large, so volume reduction measures such as freeze-drying or partial freeze-drying of the additive are necessary. Since all the additive components are highly soluble in H2O, freeze-drying is theoretically possible. Alternatively, volume reduction by partial freeze-drying is also acceptable. In the former case, where the additive is completely freeze-dried, the additive composition will be calculated as shown in Figure 2. On the other hand, in the latter case, where partial freeze-drying is used, if we want to control the NaCl concentration of the additive to 20 wt% or more, also serving as a preservative, this can be achieved by controlling the freeze-drying process so that the additive volume is approximately 2.45 mL or less.

[0058] <Embodiment 1: Mutation Frequency Analysis> Nucleic acids after quality check were subjected to analysis using NGS and / or dPCR for mutation analysis. The obtained analysis data was used to determine mutation frequencies, etc., by comparison with negative controls, and was utilized for clinical purposes such as testing, diagnosis, and monitoring.

[0059] <Embodiment 2: Graduated blood collection tube (lower limit to upper limit scale, polynucleosome differentiation scale)> Figure 5 is a perspective view of a blood collection tube 501 according to Embodiment 2 of the present invention. The blood collection tube cap 502 seals the opening at the top of the blood collection tube 501. A blood collection tube label 503 is attached to the side of the blood collection tube 501. Freeze-dried or semi-freeze-dried additive 504 (as described in Embodiment 1) is contained inside the blood collection tube 501. The side of the blood collection tube 501 is marked with a lower limit blood collection scale 505, a median blood collection scale 506 (corresponding to the median of the optimal final concentration of the additive), and an upper limit blood collection scale 507. The blood collection surface range 508 in which the final concentration of the additive falls within the optimal range is as shown in Figure 5.

[0060] Based on the freeze-dried and semi-freeze-dried reference examples of Embodiment 1, when blood is collected using a blood collection tube with the same additive specifications, the allowable blood collection volume at which the final concentrations of PEG and NaCl in the sample fall within the optimal range is estimated, as shown in Figure 2. The lower limit of the blood collection volume is 7.8 mL (i.e., final PEG concentration in the mixed solution is 8.0 wt%, final NaCl concentration is 8.6 wt%), and the upper limit of the blood collection volume is 13.5 mL (i.e., final PEG concentration in the mixed solution is 5.0 wt%, final NaCl concentration is 5.4 wt%). Therefore, it is desirable to mark the blood collection tube body with the lower limit of the blood collection volume, the upper limit of the blood collection volume, and the 10 mL mark corresponding to the median. In particular, marking the lower limit of the blood collection volume is important in practice for blood collection. That is, by using the guideline of collecting blood until it exceeds at least this mark, blood collection personnel can easily obtain a sample that falls within the optimal final concentration range. Furthermore, if the collected blood volume falls short of the indicated scale, the sample can be adjusted to fall within the optimal final concentration range by supplementing with 1x PBS buffer (pH 7.0), which is equivalent to physiological saline. Ideally, but not required, scales indicating the equivalent of 10 mL or the upper limit of the collected blood volume should also be printed as needed. In particular, the latter may effectively exceed the capacity of the blood collection tube itself, and there may be cases where printing is not possible.

[0061] Figure 6 shows another example of the blood collection tube 501 configuration. In addition to the configuration shown in Figure 5, the blood collection tube 601 in Figure 6 is equipped with mononucleosome separation range scales 609, mono-dinucleosome separation range scales 610, and mono-di-trinucleosome separation range scales 611.

[0062] As described above, depending on the combination of final concentrations of PEG and NaCl, it is observed that mono, di, and tri nucleosomes constituting cfDNA can be separated stepwise according to their molecular size (see Figures 3 and 4C). Specifically, in the relatively low concentration range within the optimal concentration range (e.g., within the final concentration range consisting of a final PEG concentration of approximately 5–6 wt% and a final NaCl concentration of approximately 4–6 wt%), peaks for mono, di, and tri nucleosomes were more easily detected. In the relatively high concentration range (e.g., within the final concentration range consisting of a final PEG concentration of approximately 6–8 wt% and a final NaCl concentration of approximately 4.5–10 wt%), peaks for mononucleosomes only were more easily detected. In the intermediate concentration range between the two (e.g., within the final concentration range consisting of a final PEG concentration of approximately 5.5–7 wt% and a final NaCl concentration of approximately 4–8 wt%), peaks for mono and dinucleosomes were more easily detected. By marking the blood collection tube 601 with scales corresponding to these three concentration ranges, it becomes possible to easily recover cfDNA containing mono, di, and tri nucleosomes, cfDNA consisting of mono and di nucleosomes, and cfDNA containing only mononucleosomes. It is expected that by evaluating, for example, the composition ratio at the mono- to polynucleosome level, information that can support testing and diagnosis can be obtained.

[0063] <Embodiment 3: Post-treatment kit (whole blood, plasma)> Figure 7 is a diagram of the configuration of a post-treatment kit 708 according to Embodiment 3 of the present invention. The post-treatment kit 708 is constructed by containing a post-treatment additive 703 (described in Embodiment 1, configured as a treatment solution) in a post-treatment container 701 and sealing it with a post-treatment container cap 702. The post-treatment kit 708 further includes a buffer container 704 containing a make-up buffer 706 and sealing it with a buffer container cap 705. Attached document 707 is an attached document such as an instruction manual.

[0064] The additive described in Embodiment 1 can be used not only as an additive for blood collection tubes, but also as a kit for post-processing target samples, such as whole blood samples collected in existing blood collection tubes, or plasma obtained by centrifugation of whole blood samples. In this case as well, by processing the target sample so that it falls within the optimal final concentration range of PEG and NaCl, it becomes possible to easily prepare a sample that achieves both cfDNA preservation and gDNA exclusion. The kit consists of equipment, for example, a container such as a 15 mL conical tube with a predetermined volume of the additive pre-filled, and possibly 1 × PBS buffer (pH 7.0) for making up the solution, and accompanying documents such as an instruction manual, but is not limited to these. Furthermore, it is desirable to mark the equipment with a scale that serves as a guideline for the appropriate sample addition volume in order to keep it within the optimal final concentration range, as with the blood collection tubes described above, but is not limited to this as the volume of the target sample to be added can be controlled.

[0065] Figure 8 shows an alternative configuration of the post-treatment kit 708. Instead of the post-treatment additive 703, a freeze-dried or semi-freeze-dried post-treatment additive 803 is contained in the post-treatment container 801. The rest is the same as in Figure 7.

[0066] <Embodiment 4: Possibility of ssDNA analysis (signal detection by spike experiment)> Figure 9 shows the results of nucleic acid extraction and fragment analysis performed on a sample treated with the additive described in Embodiment 1. ▼ indicates an LMW marker. ▽ indicates a spiked dsDNA (160 bp) peak. ★ indicates a spiked ssDNA (50 mer) peak.

[0067] The sample recovered using this invention, such as a plasma fraction, can recover not only dsDNA but also ssDNA. cfDNA, which is composed of mono- to polynucleosomes, basically exists as dsDNA, but ssDNA is also present among the nucleic acid molecules in biological samples such as whole blood. It is expected that evaluating the proportion of this ssDNA will provide information that can support testing and diagnosis. Currently, there is no simple technique to accurately identify and quantify only ssDNA from a mixture of ssDNA and dsDNA. However, when a simulated sample was prepared by spiked ssDNA and dsDNA in cleared plasma, and nucleic acid extraction and fragment analysis were performed on the sample subjected to the additive treatment of this invention, a signal considered to be ssDNA was detected in a short-chain region near the LMW marker, which was clearly different from the signal peak of dsDNA. This signal is thought to have been detected secondarily because the fluorescent dye, which is originally added during electrophoresis to detect dsDNA, intercalated into the double-helix region formed by the partial annealing of ssDNA. Although this is not suitable for quantitative evaluation, the fact that a signal could be detected indicates that the additive treatment according to this invention preserves ssDNA along with cfDNA (mono-di-trinucleosome) in the supernatant fraction derived from biological samples such as plasma. Therefore, in the future, it is expected that this invention will enable the efficient recovery of ssDNA and cfDNA while excluding gDNA, and that evaluation of both will contribute to improving the accuracy and reliability of tests and diagnoses.

[0068] <Summary of the Invention> The effect of the present invention is to resolve four major issues in LB testing by optimizing the final concentration combination of excluded volume effect molecules and salting-out effect molecules after sample testing. Specifically, it is possible to achieve the following four points at once: (1) efficient recovery of trace amounts of cfDNA that are the target of LB testing, (2) exclusion of gDNA that inhibits LB testing, (3) reduction of the complicated centrifugation operation to one, and (4) recovery of plasma containing trace amounts of cfDNA without loss. Specifically, the above effects can be achieved by using a biological sample preparation reagent having (a) at least one type of salting-out effect molecule with a final concentration of 4 wt% or more in the mixed solution, and (b) at least one type of excluded volume effect molecule with a final concentration of 5 to 8 wt% in the mixed solution. Achieving (1) and (2) at once means that by centrifugating the mixed solution, short-chain nucleic acids are recovered from the supernatant of the mixed solution, and at the same time, biological components and long-chain nucleic acids are excluded from the mixed solution. Therefore, in the centrifugation process, short-chain nucleic acids can be recovered while long-chain nucleic acids can be excluded.

[0069] Figure 10 shows an example of a whole blood sample treated with the additive according to the present invention. The present invention processes biological samples, including whole blood, by utilizing the drastic co-precipitation effect of contaminants, including nucleic acid molecules, by exclusion volume effect molecules, while simultaneously fine-tuning and balancing the co-precipitation of various components contained in the sample through the salting-out effect of salts, which has a different mechanism of action. As a result, it is possible to achieve two different effects simultaneously: maintaining short-chain nucleic acids (e.g., cfDNA, ssDNA, RNA, etc.) with a molecular size of 700 bp or less without precipitation in the supernatant (e.g., plasma) fraction obtained after centrifugation, while removing long-chain nucleic acids (e.g., gDNA, etc.) with a molecular size of 800 bp or more by co-precipitation together with contaminants. Furthermore, as a secondary effect, the molecular crowding effect due to the exclusion volume effect molecule treatment of biological samples promotes the precipitation of contaminants in crude biological samples and compresses the volume of the precipitate after centrifugation compared to samples that have not been treated with the exclusion volume effect molecule. This results in a highly transparent supernatant (e.g., plasma) fraction free of impurities, which not only reduces the cumbersome centrifugation purification process, usually repeated twice, to just once, but also increases the amount of supernatant recovered due to the compression effect of the precipitate. Therefore, the present invention provides a technology that solves the four main problems associated with LB testing listed at the beginning.

[0070] Furthermore, the supernatant solution becomes more transparent due to the co-precipitation-promoting effect of impurities, and can therefore be used for clinical purposes such as examination, diagnosis, and monitoring based on various information such as its optical, color, and component composition properties (Figure 1).

[0071] Furthermore, in this invention, the precipitate that is to be removed after centrifugation of the mixed solution can also be analyzed separately, if necessary. That is, various biological sample components such as gDNA, cells, and proteins contained in the precipitate can be analyzed, and the various information obtained from them can be used for testing and diagnosis, thereby contributing to an improvement in clinical significance.

[0072] The present invention can be applied, for example, as a blood collection tube and additive for novel LB testing. To achieve anticoagulant and nuclease inhibitor effects, various calcium ion chelating agents such as ethylenediaminetetraacetic acid (EDTA), sodium citrate, glycol etherdiaminetetraacetic acid (EGTA), BAPTA (1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid), hydroxyethylethylenediaminetriacetic acid (HEDTA), and nitrilotriacetic acid (NTA) may be added as appropriate. Alternatively, coagulation factor inhibitors such as heparin, warfarin, dabigatran, rivaroxaban, edoxaban, argatroban, enoxaparin, dalteparin, fondaparinux, and nafamostat may be added as appropriate.

[0073] Furthermore, to enhance its preservative effect, sodium azide (NaN3), thimerosal, phenoxyethanol, potassium sorbate, methylparaben, ethylparaben, propylparaben, sorbic acid, 2-phenoxyethanol, chloroform, EDTA, triclosan, benzyl alcohol, nalidixic acid, amikacin, kanamycin, chloramphenicol, etc. may be added as appropriate. However, since the present invention involves handling both the blood collection tube additive and the blood sample after collection under relatively high salt concentration conditions, the addition of preservatives is not necessarily required.

[0074] Furthermore, when applying the present invention as a blood collection tube for novel LB testing, the final concentrations of the excluded volume effect molecule and salting-out effect molecule, which are the main components of the additive, when mixed with the sample are important. Therefore, it is desirable to mark the blood collection tube with a scale indicating the lower limit of blood collection so that the sample falls within each optimal final concentration range. While it is conceivable to also print a scale indicating the upper limit if necessary, for example, in the case of a typical 10 mL blood collection tube, the acceptable upper limit is expected to exceed 10 mL, so it is considered that printing only the lower limit is practically sufficient. Also, if the collected blood volume falls below the lower limit, it is desirable to add a buffer equivalent to physiological saline, such as 1 × PBS buffer (pH 7.0), to bring the final concentration of the main component of the additive in the sample within the optimal concentration range. In this case, although not essential, it is desirable to mark the tube with a scale indicating the median, upper limit, or lower limit within the optimal concentration range as a guide for adding the buffer.

[0075] In addition, when applying the present invention as a blood collection tube additive, although it is possible to directly use the mixture of each stock solution, such as excluded volume effect molecules and salting-out effect molecules, as an additive, the concentration upper limit of the stock solution arises from, for example, the solubility of each component in H2O, so the additive volume tends to be larger than that of existing blood collection tubes. When matching the scale of existing blood collection tubes (e.g., 2 mL or less of additive per 10 mL blood collection tube as a guideline), it is desirable to reduce the volume of the blood collection tube additive according to the present invention by freeze-drying or semi-freeze-drying so that a sufficient amount of blood can be collected. In that case, the salt concentration conditions in the additive become extremely high due to dehydration, so the addition of a preservative is not necessarily required.

[0076] The present invention is not only applicable as a new blood collection tube additive, but can also be applied to the post-processing of whole blood and biological samples collected in existing blood collection tubes and sample collection containers, for example. That is, the additive treatment according to the present invention can be directly applied to samples collected in existing blood collection tubes, or it can be applied to supernatant fractions such as plasma that have been simply purified by centrifugation of the sample. In either case, it is possible to achieve both the effect of promoting the co-precipitation of impurities in the sample, while leaving cfDNA in the supernatant and removing gDNA from the supernatant. It is also envisioned that this be provided as a post-processing kit, which will consist of at least a 15 mL conical tube-like container that can be mounted on a centrifuge and contains the processing solution (additive), and accompanying documents such as an instruction manual. In some cases, it is also envisioned that a physiological saline-like make-up buffer (e.g., 1 × PBS buffer (pH 7.0)) will be included to replenish the sample volume when it falls short of a predetermined volume corresponding to the optimal final concentration range.

[0077] As a further effect of the present invention, the identification and separation of mononucleosomes and polynucleosomes such as mononucleosomes, dinucleosomes, and trinucleosomes that constitute so-called cfDNA can be adjusted to some extent by the combination of concentrations of exclusion volume effect molecules and salting-out effect molecules, if necessary. Specifically, while mononucleosomes are monomeric (peaking around 200 bp on the electroferogram), dinucleosomes (peaking around 400 bp on the electroferogram) and trinucleosomes (peaking around 600 bp on the electroferogram) are dimers and trimers, respectively, so their molecular size increases sequentially to two and three times that of mononucleosomes. Since nucleic acids with larger molecular sizes tend to be more easily co-precipitated by the action of exclusion volume effect molecules and salting-out effect molecules, identification and separation can be performed to some extent by adjusting the final concentration conditions of both molecules within the optimal concentration range. In other words, by shifting the conditions towards higher concentrations within the optimal concentration range (e.g., around 8.0 wt% final PEG concentration and 10.0 wt% final NaCl concentration in the mixed solution), it becomes possible to co-precipitate larger polynucleosomes while maintaining and recovering only smaller mononucleosomes in the supernatant fraction. On the other hand, by shifting the conditions towards lower concentrations within the optimal concentration range (e.g., around 5.0 wt% final PEG concentration and 4.0 wt% final NaCl concentration in the mixed solution), it becomes possible to maintain and recover both larger poly(di and tri)nucleosomes and mononucleosomes in the supernatant fraction. In contrast, under medium concentration conditions within the optimal concentration range (e.g., around 6.5 wt% final PEG concentration and 7.0 wt% final NaCl concentration in the mixed solution), it becomes possible to co-precipitate only larger trinucleosomes while maintaining and recovering smaller mono and dinucleosomes in the supernatant fraction. Such separate collection of mono and polynucleosomes is expected to enable the effective collection of nucleosome species that are the target of testing and diagnosis, and to enable applications in testing and diagnosis such as the abundance ratio of mono, di, and trinucleosomes.Furthermore, regarding the aforementioned graduated containers (blood collection tubes), it is possible to apply not only scales indicating the upper and lower limits of the blood collection capacity (Figure 5), but also scales indicating the blood collection volume range, which allows for the collection of mononucleosomes only, combinations of mono and dinucleosomes, and combinations of mono, di, and trinucleosomes (Figure 6).

[0078] Furthermore, as a further effect of the present invention, not only is it possible to maintain and recover cfDNA in the supernatant while removing gDNA from it, but ssDNA can also be maintained and recovered in the supernatant fraction together with cfDNA. Since quantitative fluctuations in ssDNA can be used as parameters for testing, diagnosis, or supplementing them, cfDNA and / or ssDNA are expected to be targets for future testing, and the efficient recovery of both according to the present invention is a significant advantage.

[0079] 501: Blood collection tube 502: Blood collection tube cap 503: Blood collection tube label 504: Additives 505: Lower limit blood collection scale 506: Median blood collection scale 507: Upper limit blood collection scale 508: Blood collection surface range 609: Mononucleosome preparative range scale 610: Mono-dinucleosome preparative range scale 611: Mono-di-trinucleosome preparative range scale 701: Post-processing container 702: Post-processing container cap 703: Post-processing additives 704: Buffer container 705: Buffer container cap 706: Buffer for mixing up 707: Instructions for use 708: Post-processing kit 801: Post-processing container 803: Post-processing additives

Claims

1. A biological sample preparation reagent used for preparing biological samples, comprising: at least one salting-out effect molecule having a final concentration of 4 wt% or more when mixed with a sample; and at least one excluded volume effect molecule having a final concentration of 5 to 8 wt% when mixed with a sample.

2. The biological sample preparation reagent according to claim 1, wherein the excluded volume effect molecule is at least one of polymers, macromolecules, gaseous molecules, liquid molecules, colloidal particles, and nanoparticles.

3. The biological sample preparation reagent according to claim 1, wherein the excluded volume effect molecule is a polymer, and the polymer is at least one of polystyrene, polyethylene, polymethyl methacrylate, polyvinyl alcohol, polycaprolactone, polylactic acid, polyvinylpyrrolidone, polyimide, polyurethane, polytetrafluoroethylene, and polysulfone.

4. The excluded volume effect molecule is polyethylene glycol (PEG), and the PEG is at least one of the following: low molecular weight PEG with a molecular weight of less than 1,000, medium molecular weight PEG with a molecular weight of 1,000 to 5,000, or high molecular weight PEG with a molecular weight of 5,000 to less than 100,000, excluding high polymer PEG with a molecular weight of several hundred thousand or more. The biological sample preparation reagent according to claim 1.

5. The biological sample preparation reagent according to claim 1, wherein the salting-out effect molecule is a salt, and the salt is at least one of ammonium sulfate, sodium chloride, magnesium sulfate, sodium sulfate, potassium chloride, potassium sulfate, and magnesium chloride.

6. The biological sample preparation reagent according to claim 1, wherein the final concentration of the salting-out effect molecule is 4 to 26 wt%.

7. The biological sample preparation reagent according to claim 1, further comprising at least one of an anticoagulant or a nuclease inhibitor.

8. The biological sample preparation reagent according to claim 1, further comprising a preservative.

9. The biological sample preparation reagent according to claim 1, wherein the biological sample preparation reagent is freeze-dried or partially freeze-dried.

10. A sample container for containing a biological sample preparation reagent according to claim 1, the sample container having a scale indicating an acceptable storage range for the biological sample such that, when the biological sample preparation reagent and the biological sample are contained in the sample container, the final concentration of the salting-out effect molecule in the sample container is 4 wt% or more and the final concentration of the excluded volume effect molecule is 5 to 8 wt%.

11. The sample container according to claim 10, further comprising, when the biological sample preparation reagent and biological sample are contained in the sample container, at least one of the following scales: a scale indicating the range of the portion of the liquid in the sample container that contains only mononucleosomes; a scale indicating the range of a mixture consisting of a combination of mononucleosomes and dinucleosomes; and a scale indicating the range of a mixture consisting of a combination of mononucleosomes, dinucleosomes, and trinucleosomes.

12. A method for preparing a biological sample, comprising the steps of: preparing a biological sample preparation reagent; and preparing a mixed solution of the biological sample preparation reagent and the biological sample, wherein the biological sample preparation reagent comprises: at least one salting-out effect molecule having a final concentration of 4 wt% or more when mixed with the sample; and at least one excluded volume effect molecule having a final concentration of 5 to 8 wt% when mixed with the sample.

13. The method according to claim 12, further comprising the step of recovering short-chain nucleic acids from the supernatant of the mixed solution by centrifuging the mixed solution, while simultaneously removing biological components and long-chain nucleic acids from the mixed solution.

14. The method according to claim 12, wherein the biological sample is a bodily fluid of biological origin, and the bodily fluid is at least one of the following: blood, plasma, serum, urine, saliva, feces, breast milk, tears, sweat, cerebrospinal fluid, synovial fluid, semen, vaginal secretions, ascites, amniotic fluid, or cell culture medium.

15. The method according to claim 13, wherein the short-chain nucleic acid is DNA and / or RNA with a base length of 700 bp or less, and the DNA is dsDNA and / or ssDNA including ct / cfDNA.

16. The method according to claim 13, wherein the long-chain nucleic acid is DNA and / or RNA with a base length of 800 bp or more, and the DNA is dsDNA and / or ssDNA including gDNA.

17. The method according to claim 13, wherein in the step of recovering the short-chain nucleic acids, at least one of the following is recovered separately: mononucleosomes only, a mixture consisting of a combination of mononucleosomes and dinucleosomes, or a mixture consisting of a combination of mononucleosomes, dinucleosomes, and trinucleosomes.

18. The method according to claim 13, wherein the short-chain nucleic acid is cfDNA and ssDNA consisting of mono, di, and tri nucleosomes that constitute DNA, and the method further comprises the step of obtaining supporting information to be used when analyzing the biological sample based on the quantitative values ​​of each nucleosome and / or the quantitative values ​​and / or distribution and / or distribution ratio of ssDNA.

19. The method according to claim 13, wherein the step of performing the centrifugal separation is performed only once.

20. The method according to claim 13, further comprising the step of analyzing gDNA contained in the precipitate after the centrifugation.

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