Method for capturing nucleotide chain, capturing membrane filter, capturing membrane filter unit, and capturing kit

Abstract

One of the problems addressed by the present invention is to provide a novel method for capturing a nucleotide chain. The problem is solved by a method for capturing a nucleotide chain from a liquid specimen collected from a subject, the method comprising bringing a liquid specimen into contact with capture probe-bound cationic polymer-modified PLGA nanoparticles and recovering a nucleotide chain from the capture probe-bound cationic polymer-modified PLGA nanoparticles that have been brought into contact with the liquid specimen. The capture probe-bound cationic polymer-modified PLGA nanoparticles each include a nanoparticle core containing PLGA, a coating layer containing a cationic polymer and covering the surface of the core, and a capture probe bound to the coating layer, wherein the capture probe is an oligonucleotide.

Description

Method for capturing nucleotide chains, capture membrane filter, capture membrane filter unit, and capture kit

[0001] Disclosed herein are a method for capturing nucleotide chains, a capturing membrane filter, a capturing membrane filter unit, and a capturing kit.

[0002] Patent Document 1 discloses a method for producing polylactic acid / co-glycolic acid (PLGA) nanoparticles encapsulating fat-soluble ascorbyl tetrahexyldecanoate. Patent Document 2 discloses a method for producing PLGA nanoparticles having a particle size that allows passage through a membrane filter for sterilization. Patent Document 3 discloses PLGA nanoparticles encapsulating asORN corresponding to a specific region of antisense RNA for interferon-α. Non-Patent Document 1 describes a method for extracting DNA and RNA using Urine Conditioning Buffer (trademark).

[0003] JP 2005-213170 A JP 2011-111429 A JP 2016-130227 A

[0004] A set of reagents and containers that allows you to store DNA and RNA in urine samples at room temperature | Urine Collection Kit with Urine Conditioning Buffer (UCB) | Funakoshi (funakoshi.co.jp) Posted: May 29, 2017

[0005] Nucleotide chains contained in liquid samples such as urine collected from subjects are being collected and analyzed as non-invasive urinary biomarkers for the early diagnosis of cancer and tuberculosis.

[0006] An object of the present invention is to provide a new method for capturing nucleotide chains.

[0007] The present invention includes the following embodiments: Item 1. A method for capturing nucleotide chains from a liquid sample collected from a subject, comprising: contacting the liquid sample with capture probe-bound cationic polymer-modified PLGA nanoparticles; and recovering nucleotide chains from the capture probe-bound cationic polymer-modified PLGA nanoparticles that have come into contact with the liquid sample, wherein the capture probe-bound cationic polymer-modified PLGA nanoparticles have a nanoparticle core comprising PLGA, a coating layer comprising a cationic polymer that covers the surface of the core, and a capture probe bound to the coating layer, the capture probe being an oligonucleotide. Item 2. The method of Item 1, wherein the cationic polymer is chitosan. Item 3. The method of Item 1, wherein the oligonucleotide comprises a random sequence or a sequence specific to a target nucleotide chain. Item 4. Item 5. A collecting membrane filter for collecting nucleotide chains from a liquid sample, the collecting membrane filter carrying capture probe-bound cationic polymer-modified PLGA nanoparticles, the capture probe-bound cationic polymer-modified PLGA nanoparticles comprising a nanoparticle core containing PLGA, a coating layer containing a cationic polymer covering the surface of the core, and a capture probe bound to the coating layer, the capture probe being an oligonucleotide. Item 6. A collecting membrane filter unit for collecting nucleotide chains from a liquid sample, the collecting membrane filter unit comprising: a filter holder incorporating the collecting membrane filter according to Item 4; an injection tube; and a discharge tube. Item 7. A kit for collecting nucleotide chains from a liquid sample, the kit comprising: the collecting membrane filter unit according to Item 5; and an injector for injecting the liquid sample into the collecting membrane filter unit.

[0008] A new method for capturing nucleotide chains can be provided.

[0009] 1 shows an overview of a method for producing capture probe-bound cationic polymer-modified PLGA nanoparticles. 2 shows the structure of a membrane filter unit 1 for capturing nucleotide chains. (A) shows the appearance of a membrane filter unit 1 for capturing nucleotide chains. (B) shows a midline cross-section of a membrane filter unit 1 for capturing nucleotide chains. 3 shows an overview of a method for capturing nucleotide chains in a liquid sample using capture probe-bound cationic polymer-modified PLGA nanoparticles. (A) shows an overview of a first embodiment. (B) shows an overview of a second embodiment. 4 shows the recovery amounts of small RNA and miRNA in urine. (A) shows the recovery amounts of small RNA and miRNA for a control sample to which no PLGA nanoparticles have been added. (B) shows the recovery amounts of small RNA and miRNA recovered directly from urine without using chitosan-modified PLGA nanoparticles to which no capture probes have been bound. (C) shows the recovery amounts of small RNA and miRNA when Urine Conditioning Buffer (trademark) is used. (D) shows the recovery amounts of small RNA and miRNA recovered directly from urine using capture probe-bound chitosan-modified PLGA nanoparticles. Electron microscopy images of chitosan-modified PLGA nanoparticles are shown. (A) is a scanning electron microscopy image of chitosan-modified PLGA nanoparticles loaded on a nitrocellulose filter. (B) is a scanning electron microscopy image of chitosan-modified PLGA nanoparticles loaded on a PVDF filter. Results of RNA detection are shown. (A) shows the results of RT-PCR detection of 18s rRNA in nucleotide chains repaired with a PLGA-loaded filter or Trizol. (B) shows the results of RT-PCR detection of beta actin mRNA in nucleotide chains repaired with a PLGA-loaded filter or Trizol. (C) shows the results of RT-PCR detection of mitochondrial CO1 mRNA in nucleotide chains repaired with a PLGA-loaded filter or Trizol. In (A) to (C), lane 1 shows the amplification product obtained by amplifying saliva as a sample using total RNA recovered through a PLGA-loaded filter as a template.Lane 2 shows the amplification product of serum samples amplified using total RNA recovered using a PLGA-loaded filter as a template. Lane 3 shows the amplification product of urine samples amplified using total RNA recovered using a PLGA-loaded filter as a template. Lane 4 shows the amplification product of saliva samples amplified using total RNA recovered using Trizol as a template. Lane 4 shows the amplification product of serum samples amplified using total RNA recovered using Trizol as a template. The following shows the results of detection of various RNAs. (A) shows the results of PCR detection of the 18s rRNA gene in nucleotide chains repaired using a PLGA-loaded filter. (B) shows the results of PCR detection of the beta actin gene in nucleotide chains repaired using a PLGA-loaded filter. (C) shows the results of PCR detection of the mitochondrial CO1 gene in nucleotide chains repaired using a PLGA-loaded filter. In (A) to (C), lane 1 shows the amplified product obtained using total DNA collected from serum samples as a template. Lane 2 shows the amplified product obtained using total DNA collected from saliva samples as a template. Lane 3 shows the amplified product obtained using total DNA collected from urine samples as a template. This figure shows the results of detecting dengue virus-derived RNA in the serum of three dengue fever patients. This figure shows the results of detecting Epstein-Barr virus-derived DNA in the saliva of two healthy individuals. This figure shows the results of miRNA collection from human urine samples using a PLGA-loaded filter coupled with random primers. (A) shows the amplification curve. (B) shows the melting curve. This figure shows the results of miRNA collection using a PLAG RP filter, PLGA_30C-5p, and UCB. (A) shows the amplification curve, and (B) shows the Ct value. This figure shows the results of miRNA collection using a PLAG RP filter, PLGA_30C-5p, and UCB. (A) shows the amplification curve, and (B) shows the Ct value.

[0010] 1. Capture Probe-Bound Cationic Polymer-Modified PLGA Nanoparticles One embodiment relates to capture probe-bound cationic polymer-modified PLGA (polylactic acid-co-glycolic acid) nanoparticles.

[0011] Figure 1 shows a schematic diagram of the preparation method for capture probe-bound cationic polymer-modified PLGA nanoparticles, as well as the prepared capture probe-bound cationic polymer-modified PLGA nanoparticles. The core of the cationic polymer-modified PLGA nanoparticles is a nanoparticle containing PLGA. The surface of the core is coated with a cationic polymer.

[0012] The molecular weight of the PLGA used as the core is preferably within the range of 5,000 to 200,000, and more preferably within the range of 15,000 to 25,000.The composition ratio of lactic acid to glycolic acid may be 1:99 to 99:1, and preferably 1 part lactic acid to 0.3 to 0.4 parts glycolic acid.

[0013] Preferred examples of cationic polymers include chitosan and chitosan derivatives, cationized cellulose in which multiple cationic groups are bound to cellulose, polyamino compounds such as polyethyleneimine, polyvinylamine, and polyallylamine, polyamino acids such as polyornithine and polylysine, polyvinylimidazole, polyvinylpyridinium chloride, alkylamino methacrylate quaternary salt polymers (DAM), alkylamino methacrylate quaternary salt-acrylamide copolymers (DAA), and cationic polymers in which a cationic group such as a quaternary ammonium salt is bound to a polymer containing 2-methacryloyloxyethylphosphorcholine (MPC) as a structural unit, which combines a phospholipid polar group (phosphorylcholine group) that is a component of cell membranes (biomembranes) with a highly polymerizable methacryloyl group (e.g., a copolymer of MPC and 2-hydroxy-3-methacryloyloxypropyltrimethylammonium chloride). Chitosan or its derivatives are particularly preferred. An example of a chitosan derivative is hydroxypropyl chitosan (cationized chitosan). The amount of cationic polymer coated is not limited, but is usually about 1 to 10 parts by weight, preferably about 3 to 8 parts by weight, per 100 parts by weight of PLGA.

[0014] (Nanoparticle Formation Process) PLGA nanoparticles are produced by electrospray deposition (ESD). This method involves forming an emulsion and then crystallizing the polymer into spherical particles by utilizing the interdiffusion between a good solvent (organic solvent) and a poor solvent (hydrophilic solvent containing a cationic polymer and polyvinyl alcohol). The procedure involves first dissolving PLGA in a good solvent and then dropping it into a poor solvent under stirring. The good solvent in the mixture rapidly diffuses into the poor solvent. This results in self-emulsification of the good solvent in the poor solvent, forming submicron-sized emulsion droplets of the good solvent. Furthermore, as the interdiffusion between the good and poor solvents progresses, the solubility of PLGA in the emulsion droplets decreases, ultimately resulting in the formation of spherical crystalline PLGA nanoparticles. Furthermore, by adding polyvinyl alcohol and a cationic polymer to the poor solvent, the surface of the PLGA nanoparticles is coated with polyvinyl alcohol and a cationic polymer, resulting in the formation of cationic polymer-modified PLGA nanoparticles. That is, PLGA nanoparticles have a structure in which PLGA is used as a core, which is coated with polyvinyl alcohol, and the outer layer of which is coated with a cationic polymer.

[0015] The concentration of PLGA in the good solvent is approximately 8 mg / mL to 15 mg / mL, preferably approximately 10 mg / mL to 13 mg / mL. The good solvent preferably contains acetone and ethanol. There are no particular restrictions on the mixing ratio of acetone and ethanol in the good solvent, but it is preferable to set the ethanol concentration to 10% by volume or more. If necessary, a solvent other than acetone and ethanol, such as water, may be mixed into the good solvent. Water can be used as the poor solvent. The concentration of polyvinyl alcohol in the poor solvent can be in the range of 0.05% by weight to 10% by weight.

[0016] (Distillation Step) After the cationic polymer-modified PLGA nanoparticles are produced, the organic solvent is distilled off under reduced pressure to produce a cationic polymer-modified PLGA nanoparticle suspension. In this distillation step, if the organic solvent is distilled off under reduced pressure for a long period of time at a temperature above the glass transition point of the biocompatible polymer, the rigidity of the biocompatible polymer constituting the nanoparticles decreases and the fluidity increases, causing the nanoparticles to fuse together, significantly reducing the pressure filtration characteristics in the filtration sterilization step following the distillation step. Therefore, the distillation step must be carried out as quickly as possible at a low temperature. Specifically, it is preferable to carry out the distillation step at 45°C or below within 30 hours.

[0017] The cationic polymer-modified PLGA nanoparticles thus prepared have an average particle size of about 1 nm to 990 nm, about 10 nm to 700 nm, about 30 nm to 500 nm, about 30 nm to 500 nm, about 50 nm to 400 nm, or about 240 nm. The particle size can be measured, for example, by small-angle X-ray scattering, nanoparticle tracking analysis, etc.

[0018] (Binding of Capture Probe) A capture probe is bound to the coating layer of the cationic polymer-modified PLGA nanoparticle. The capture probe contains an oligonucleotide. The oligonucleotide contains a random sequence, such as a random primer, or a sequence specific to a target nucleotide strand.

[0019] The cationic polymer-modified PLGA nanoparticles can be prepared by adding oligonucleotides to a final concentration of about 0.001% to 0.05% to the cationic polymer-modified PLGA nanoparticle suspension obtained by the above evaporation step, freeze-drying the mixture, and then powdering it.

[0020] As the random primer, for example, random primers (for example, hexamer, heptamer, octamer, nonamer) manufactured by Promega Corporation can be used.

[0021] The target nucleotide chain-specific sequence is not limited as long as it can hybridize with the nucleotide chain to be detected (target nucleotide chain). The number of nucleotides in the primer having the target nucleotide chain-specific sequence is, for example, about 5 to 100 nucleotides, preferably about 16 to 60 nucleotides. Unlike the recovery method of Non-Patent Document 1, the use of a target nucleotide chain-specific sequence can increase the recovery amount of a specific nucleotide chain.

[0022] The target nucleotide strand may include an RNA strand and a DNA strand. The nucleotide strand may also include an oligonucleotide and a polynucleotide. The RNA strand may include mRNA, non-translated RNA, rRNA, tRNA, small RNA, microRNA, etc. The DNA strand may include genomic DNA, mitochondrial DNA, etc.

[0023] The target nucleotide chain is not limited as long as it is a nucleotide chain contained in the liquid sample. The target nucleotide chain may be a nucleotide chain derived from the subject from which the liquid sample is collected, or may be a nucleotide chain derived from a heterologous organism present in the subject's body as an infectious disease or as a normal flora. The heterologous organism may include, for example, bacteria, fungi, viruses, parasites, spirochetes, chlamydia, rickettsia, mycoplasma, etc.

[0024] For example, when the target nucleotide chain is miRNA, the candidate miRNAs for tuberculosis diagnostic markers are hsa-miR-423-3p, hsa-miR-451a, hsa-miR-532-3p, hsa-miR-203a-3p, hsa-miR-424-5p, hsa-miR-375-3p, hsa-miR-503-3p, hsa-miR-210-3p, hsa-miR-887-3p, Examples include hsa-miR-95-3p, hsa-miR-324-5p; and other miRNAs (hsa-miR-107, hsa-miR-181a-5p, hsa-miR-29b-3p, hsa-miR-92b-3p, hsa-let-7a-5p, hsa-let7f-5p, hsa-miR-30c-5p, hsa-miR-423-5p, etc.).

[0025] The capture probe is bound to the coating layer of the cationic polymer-modified PLGA nanoparticles via electrostatic binding. The surface of the cationic polymer-modified PLGA nanoparticles is positively charged, allowing negatively charged substances such as nucleic acids to be electrostatically bound.

[0026] Specifically, oligonucleotides and, for example, mannitol are added to a suspension of cationic polymer-modified PLGA nanoparticles, and the mixture is freeze-dried at, for example, about −45° C. to form a powder, thereby obtaining capture probe-bound cationic polymer-modified PLGA nanoparticles.

[0027] 2. Collection Membrane Filter for Collecting Nucleotide Chains One embodiment relates to a collection membrane filter for collecting nucleotide chains (hereinafter also simply referred to as a "collection membrane filter").

[0028] A membrane filter is a membrane with pores. The pore size is preferably about 0.1 μm to 0.5 μm. The material of the membrane filter is not particularly limited, but examples include nitrocellulose, glass, polyethersulfone, polycarbonate, cellulose acetate, polytetrafluoroethylene (PTFE), and polyvinylidene fluoride (PVDF). A material resistant to organic solvents, such as PVDF, is preferred.

[0029] The capture probe-bound cationic polymer-modified PLGA nanoparticles can be supported on a membrane filter by the following method.

[0030] For example, a suspension containing approximately 0.01 to 20 mg / ml of capture probe-bound cationic polymer-modified PLGA nanoparticles is prepared. The solvent used to prepare the suspension is preferably a hydrophilic solvent such as water or buffer. A membrane filter is placed in a suction filtration device. When the membrane filter is a hydrophobic PVDF membrane or the like, it is preferable to first permeate the membrane filter with methanol, followed by swelling with the hydrophilic solvent. The suspension is applied to the membrane filter and suctioned, thereby supporting the capture probe-bound cationic polymer-modified PLGA nanoparticles on the membrane filter and removing the hydrophilic solvent, thereby preparing a collection membrane filter.

[0031] 3. Collection Membrane Filter Unit for Collecting Nucleotide Chains One embodiment relates to a collection membrane filter unit 1 (hereinafter also simply referred to as "filter unit 1") for collecting nucleotide chains.

[0032] The structure of the filter unit 1 will be described using Figures 2(A) and (B). The filter unit 1 can employ a structure such as that described in JP-A-55-031480. The filter unit 1 includes a filter holder 13 incorporating the collection membrane filter 15 prepared in Section 2 above, an inlet tube 11b, and an outlet tube 12b. Reference numeral 11a in Figure 2(A) denotes an inlet, and reference numeral 12a denotes an outlet. The inlet 11a is connected to an injector (e.g., an injection syringe) for injecting a liquid sample. The connection is achieved by inserting the tip of the outlet side of the injector (the end where the injection needle is usually attached, in the case of an injection syringe) into the inlet 11a of the filter unit 1. Furthermore, to prevent the filter unit 1 from being dislodged by the pressure generated when injecting a liquid sample, the inlet 11a of the filter unit 1 preferably has a female luer lock structure. The filter holder 13 is preferably made of plastic so that it can be used disposably.

[0033] The flow of the injected liquid sample is indicated by arrows in Figure 2(A). Next, the structure inside the filter holder 13 will be explained using Figure 2(B). Figure 2(B) is a vertical cross-sectional view of the filter unit 1 at the midline.

[0034] The filter holder 13 is disk-shaped and includes a two-layer structure consisting of an upper filter holder 13a and a lower filter holder 13b. The filter holder 13 includes a collection membrane filter 15 and a clamping ring 13c for sealing the upper filter holder 13a and lower filter holder 13b. An injection tube 11b is connected to the center of the upper filter holder 13a, and the interior of the injection tube 11b has a tubular structure, forming the injection lumen 11c. An exhaust tube 12b is connected to the center of the lower filter holder 13b, and the interior of the exhaust tube 12b has a tubular structure, forming the exhaust lumen 12c. The upper filter holder 13a and lower filter holder 13b are designed to form a disk-shaped space 14 on the upper filter holder 13a side when the collection membrane filter 15 is sandwiched between them and clamped by the clamping ring 13c. This space 14 fills with liquid when liquid is injected and serves as a pressure chamber. The space 14 is formed by the inside of a collar 20, which extends circularly downward from the upper filter holder 13a. The upper part of the filter holder lower part 13b has a recessed structure that fits onto the outer edge of the collar 20. In addition, a spider web-like groove is cut into the bottom surface of the recessed structure from the opening of the discharge pipe 12b, and is designed to ensure a flow path for the liquid that has passed through the collection membrane filter 15.

[0035] Furthermore, an annular outward flange 17 is formed on the lower side surface of the filter folder upper part 13a. Furthermore, an annular outward flange 18 that is complementary to flange 17 is formed on the upper side surface of the filter folder lower part 13b. When the filter folder upper part 13a and the filter folder lower part 13b are overlapped, the tightening ring 13c is designed to fit into the overlapping portion of the flange 17 and the flange 18.

[0036] The collection membrane filter 15 is circular and is positioned so that it does not fit between the flanges 17 and 18, but fits over the fitting portion between the collar 20 and the recessed structure of the lower part 13b of the filter holder, and is fixed by being sandwiched between the collar 20 and the bottom surface of the recessed structure of the lower part 13b of the filter holder.

[0037] The liquid injected from the inlet 11a passes through the injection pipe 11b to fill the space 14, and due to the pressure of the injector that injected the liquid, passes through the collection membrane filter 15 and is discharged from the outlet 12a through the discharge pipe 12b.

[0038] When the liquid sample passes through the collection membrane filter 15, the nucleotide chains in the liquid sample are captured by the capture probe-bound cationic polymer-modified PLGA nanoparticles.

[0039] The clamping ring 13c is removable, and by removing the clamping ring 13c, the collection membrane filter 15 that has filtered the liquid sample can be collected.

[0040] The size of the collection membrane filter 15 is approximately 10 mm to 35 mm in diameter. Accordingly, the diameter of the filter holder 13 is also approximately 13 mm to 40 mm. Furthermore, the height from the inlet 11 a to the outlet 12 a is approximately 20 mm to 30 mm.

[0041] 4. Kit for collecting nucleotide chains from a liquid sample One embodiment relates to a kit for collecting nucleotide chains from a liquid sample. The kit includes the filter unit 1 described in 3 above and an injector. The injector may be a syringe or the like. Preferably, the injector can hold at least 10 mL of the liquid sample.

[0042] 5. Collection of Nucleotide Chains in a Liquid Sample Using Capture Probe-Bound Cationic Polymer-Modified PLGA Nanoparticles One embodiment relates to a method for collecting nucleotide chains in a liquid sample (hereinafter, sometimes simply referred to as a "collection method").

[0043] The collection method includes contacting a liquid sample with capture probe-bound cationic polymer-modified PLGA nanoparticles, and recovering nucleotide chains from the capture probe-bound cationic polymer-modified PLGA nanoparticles that have come into contact with the liquid sample.

[0044] The nucleotide chain is as explained in 1 above.

[0045] The liquid sample may be urine, saliva, serum, plasma, pleural effusion, ascites, cerebrospinal fluid, synovial fluid, puncture fluid, other puncture fluids, sputum, throat swab, etc., collected from a subject. Urine, saliva, serum, and plasma are preferred. Highly viscous throat swab fluids may be obtained by rinsing a cotton swab used to wipe the throat in sterile saline, and the rinsing solution may be used as the sample. Furthermore, impurities may be removed from the liquid sample by centrifugation, filtration, or the like. Furthermore, the liquid sample may be mixed with a cell lysate or a sample lysate, and the resulting mixture may be used to collect nucleotide chains.

[0046] Subjects may include humans, dogs, cats, rabbits, mice, rats, monkeys, cows, horses, sheep, goats, and the like.

[0047] The capture method can include the following two embodiments. (1) First Embodiment The first embodiment is a method in which the capture probe-bound cationic polymer-modified PLGA nanoparticles described in 1. above are directly added to a liquid sample, thereby bringing the liquid sample into contact with the capture probe-bound cationic polymer-modified PLGA nanoparticles, and then recovering the capture probe-bound cationic polymer-modified PLGA nanoparticles after contact. An overview of the capture method is shown in Figure 3(A). If there are nucleotide chains in the liquid sample that hybridize to the capture probe, they will be captured by the capture probe.

[0048] The liquid sample is brought into contact with the capture probe-bound cationic polymer-modified PLGA nanoparticles by mixing the liquid sample with the capture probe-bound cationic polymer-modified PLGA nanoparticles. The amount of the capture probe-bound cationic polymer-modified PLGA nanoparticles to be added to the liquid sample is about 0.1 mg to 10 mg, preferably about 0.5 mg to 5 mg, and more preferably about 0.7 mg to 3 mg per mL of the liquid sample.

[0049] The capture probe-bound cationic polymer-modified PLGA nanoparticles can be recovered, for example, by centrifuging at approximately 40,000 to 50,000 × g and approximately 0 to 10°C for approximately 20 to 40 minutes to precipitate the capture probe-bound cationic polymer-modified PLGA nanoparticles that have come into contact with the liquid sample, and then removing the supernatant.

[0050] (2) Second Embodiment The second embodiment is a method using the filter unit 1 described in 3 above. An overview is shown in Figure 3(B). A liquid sample is placed in a syringe, and the filter unit 1 is placed at the tip of the syringe.

[0051] The liquid sample is extruded from the injector, and the filtrate that passes through the collection membrane filter 15 is discarded from the outlet 12a of the filter unit 1. The liquid sample comes into contact with the capture probe-bound cationic polymer-modified PLGA nanoparticles supported on the collection membrane filter 15, and any nucleotide chains that hybridize to the capture probes are captured by the capture probes. The clamping ring 13c is removed, and the collection membrane filter 15 is recovered.

[0052] 6. Recovery of Nucleotide Chains from Capture Probe-Bound Cationic Polymer-Modified PLGA Nanoparticles In the case of the first embodiment described in 5 above, nucleotide chains can be recovered from capture probe-bound cationic polymer-modified PLGA nanoparticles that have come into contact with a liquid sample by adding a phenol / chloroform mixture to the capture probe-bound cationic polymer-modified PLGA nanoparticles, performing phenol / chloroform extraction, and then performing ethanol precipitation.

[0053] In the case of the second embodiment described in 5. above, the recovered collection membrane filter 15 is immersed in acetone or toluene to dissolve the PLGA nanoparticles and elute the nucleic acids from the membrane filter. A phenol / chloroform mixture is added to the eluate, followed by phenol / chloroform extraction and subsequent ethanol precipitation. Nucleic acids can be recovered by elution (including membrane filter dissolution) and extraction from the PLGA nanoparticles supported on the membrane filter using nucleic acid extraction reagents or nucleic acid extraction columns provided with commercially available kits.

[0054] The present invention will be described in more detail below with reference to examples, but the present invention should not be construed as being limited to these examples.

[0055] 1. Preparation of capture probe-conjugated chitosan-modified PLGA nanoparticles Chitosan-modified PLGA nanoparticles were prepared by ESD using a PLGA substrate (PLGA-7520; lactic acid:glycolic acid polymerization ratio = 3:1, average molecular weight 20,000 Da) as follows.

[0056] 200 mg of PLGA was dissolved in 13 mL of its good solvent, acetone, to prepare a polymer solution. 4 mL of ethanol was added and mixed to prepare a mixed good solvent. Next, 525 mg of 2 wt% chitosan (KIMICA Chitosan, KIMICA) aqueous solution was added to 5 g of 2 wt% polyvinyl alcohol (PVA: Gohsenol EG-05, Nippon Synthetic Chemical Industry) aqueous solution to prepare a poor solvent for PLGA. The mixed good solvent was added dropwise at a constant rate (20 mL / min) to the poor solvent while stirring at 400 rpm at 40 °C. A suspension of PLGA nanoparticles was obtained by diffusion of the good solvent into the poor solvent. After distilling off the acetone and ethanol under reduced pressure, random primer (Promega: Hexamer) was added to the resulting nanoparticle suspension to a final concentration of 0.0056%. The suspension was then lyophilized at -45 °C to obtain a powder of random primer-conjugated chitosan-modified PLGA nanoparticles.

[0057] 2. Recovery of urinary small RNA and miRNA using random primer-conjugated chitosan-modified PLGA nanoparticles. Approximately 2 ml of purified water was added to 10 mg of powdered random primer-conjugated chitosan-modified PLGA nanoparticles (hereinafter simply referred to as "PLGA nanoparticles") and uniformly suspended by sonication. 2 ml of the PLGA nanoparticle suspension was added to 10 mL of urine and mixed. The amount of PLGA nanoparticles added per 10 mL of urine was 10 mg. The mixture was then centrifuged at 48,000 × g, 4°C, for 30 minutes to precipitate the PLGA nanoparticles, and the supernatant was removed.

[0058] A mixture of Qiagen's Qiazol (trademark) / chloroform (5:1) was added to the precipitate, followed by phenol / chloroform extraction and ethanol precipitation to recover the RNA in the urine.

[0059] 3. Small RNA and miRNA Recovery Amounts RNA recovery amounts were measured using capillary electrophoresis. Capillary electrophoresis was performed using an Agilent 2100 Bioanalyzer Electrophoresis System, and RNA amounts were quantified using the 2100 Expert Software.

[0060] Figure 4 shows the recovery amounts of small RNA and miRNA in urine. (A) shows the recovery amounts of small RNA and miRNA in the control without PLGA addition. (B) shows the recovery amounts of small RNA and miRNA recovered directly from urine without using chitosan-modified PLGA nanoparticles without capture probes attached. (C) shows the recovery amounts of small RNA and miRNA recovered using Urine Conditioning Buffer™ (UCB). (D) shows the recovery amounts of small RNA and miRNA recovered directly from urine using capture probe-attached chitosan-modified PLGA nanoparticles.

[0061] The amount of recovered RNA, expressed as small RNA / miRNA (pg / μL), was 404 / 333 for the control, 360 / 276 for PLGA empty, 4,781 / 4,661 for UCB, and 4,220 / 4,059 for capture probe-conjugated chitosan-modified PLGA nanoparticles (PLGA-Random primer). Small RNA and miRNA were recovered in amounts similar to those recovered with UCB using capture probe-conjugated chitosan-modified PLGA nanoparticles.

[0062] 4. Chitosan-modified PLGA nanoparticles loading test on filters. Using a vacuum filtration system, chitosan-modified PLGA nanoparticles were loaded onto a 0.45 μm pore size nitrocellulose filter [HARG (CAT. NO. HABG04700)] and a 0.22 μm pore size PVDF filter (Dilapore GV). Each filter was placed in the filter compartment of the vacuum filtration system. First, water was added to the upper chamber of the vacuum filtration system, and the water was sucked through the water flow, allowing the water to fall into the lower chamber. Next, a suspension of chitosan-modified PLGA nanoparticles (22.4 mg of chitosan-modified PLGA nanoparticles per 2.2752 g of suspension) was added to the upper chamber of the vacuum filtration system and sucked through the water flow. Water was again added to the upper chamber of the vacuum filtration system and sucked through the water flow. After filtration, the filters were recovered and frozen. The frozen filters were fixed, dehydrated, and evaporated, and the filter surfaces were observed using a scanning electron microscope.

[0063] The results are shown in Figure 5. Figure 5(A) is a scanning electron microscope image of chitosan-modified PLGA nanoparticles supported on a nitrocellulose filter. Figure 5(B) is a scanning electron microscope image of chitosan-modified PLGA nanoparticles supported on a PVDF filter. In both cases, roughly spherical chitosan-modified PLGA nanoparticles were supported on the filters.

[0064] 5. Collection of nucleotide chains from body fluid samples using PLGA-loaded filters The random primer-loaded chitosan-modified PLGA nanoparticles prepared according to the method described in 1. above were loaded onto a filter by the method described in 4. above (hereinafter referred to as "PLGA-loaded filters"), and the nucleotide chain collection ability was evaluated using the filter. However, the amount of random primer loaded onto the chitosan-modified PLGA nanoparticles was half that of 2. above.

[0065] (1) Total RNA Collection. The following five samples were prepared for total RNA. RNA Sample 1: 1000 μL of saliva was mixed with 4x the supernatant volume of Lysis Buffer RAV1 (hereafter referred to as "lysis buffer") from the NucleoSpin™ RNA Virus Kit (Machley-Nagel) and incubated at 70°C for 5 minutes. After incubation, the solution was centrifuged at 11,000 g for 1 minute. PBS was added to the supernatant, and the resulting solution was brought to 10 mL and passed through a PLGA-coated filter. After removing as much liquid as possible from the PLGA-coated filter using an empty syringe, RNA was extracted using the Trizol method. Specifically, 1 mL of Trizol (Thermofisher) was passed through the PLGA-coated filter using a syringe. Total RNA was extracted from the Trizol that had passed through the PLGA-coated filter according to the manufacturer's protocol. During the RNA elution step, 25 μL of distilled water was added to recover total RNA. RNA Sample 2: 300 μL of serum was mixed with a fourfold volume of lysis buffer and incubated at 70°C for 5 minutes. PBS was added to the mixture, which was then diluted to 10 mL and passed through a PLGA-coated filter. Total RNA was then recovered as described for RNA Sample 1. RNA Sample 3: Urine was first pretreated by centrifuging the urine at 2,000 g for 20 minutes at room temperature and filtering the supernatant through a 0.45 μm pore size filter. 15 mL of pretreated urine was then passed through the PLGA-coated filter. After removing as much liquid as possible from the PLGA-coated filter with an empty syringe, total RNA was extracted using the miRNeasy mini Kit (QIAGEN™). Specifically, 1 mL of Qiazol (Qiagen) was passed through the PLGA-coated filter using a syringe, and total RNA was extracted from the Qiazol (Qiagen) that had passed through the PLGA-coated filter according to the manufacturer's protocol. In the RNA elution step, 25 μL of distilled water was added to recover total RNA. RNA sample 4: As a control, total RNA was extracted from saliva by the Trizol method.Specifically, 750 μL of Trizol LS (Thermofisher) was added to 250 μL of saliva, and total RNA was extracted according to the manufacturer's protocol. During the total RNA elution step, 25 μL of distilled water was added to recover total RNA. RNA sample 5: As a control, total RNA was extracted from serum using the Trizol method. Specifically, 900 μL of Trizol LS (Thermofisher) was added to 300 μL of serum, and total RNA was extracted according to the manufacturer's protocol. During the total RNA elution step, 25 μL of distilled water was added to recover total RNA.

[0066] (2) Collection of Total DNA The following three samples were prepared for total DNA. DNA Sample 1: 1000 μL of saliva (heat-treated at 95°C for 5 minutes and then rapidly cooled) was centrifuged, and total RNA was collected from the supernatant in the same manner as for RNA Sample 1 above. Total DNA was collected from the organic layer after total RNA collection. The amount of DNA collected was measured using a NanoDrop and found to be 6.1 ng / μL. DNA Sample 2: 300 μL of serum (heat-treated at 95°C for 5 minutes and then rapidly cooled) was centrifuged, and total RNA was collected from the supernatant in the same manner as for RNA Sample 2 above. Total DNA was collected from the organic layer after total RNA collection. The amount of DNA collected was measured using a NanoDrop and found to be 23.6 ng / μL. DNA sample 3: 15 mL of urine (heat-treated at 95°C for 5 minutes and then rapidly cooled) was centrifuged, and the supernatant was passed through a PLGA-coated filter. 1 mL of Trizol was then passed through the PLGA-coated filter using a syringe, and total DNA was recovered from the organic layer of Trizol that had passed through the PLGA-coated filter. The amount of recovered DNA was measured using a NanoDrop and found to be 4.6 ng / μL.

[0067] (3) cDNA synthesis and PCR. For RNA samples, cDNA was synthesized by reverse transcription (ReverTra Ace™, TOYOBO Co.) using 12 μL of extracted RNA as a template. PCR (Ex Taq premier™, Takara Bio.) was performed using 1 μL of the synthesized cDNA solution as a template to confirm whether RNA was successfully collected and extracted from each sample. PCR was performed using primers that amplify partial regions of the 18s rRNA gene, beta actin gene, and mitochondrial CO1 gene. PCR was also performed on DNA samples using primers that amplify partial regions of the 18s rRNA gene, beta actin gene, and mitochondrial CO1 gene. PCR was performed using primers that amplify partial regions of the 18s rRNA gene, beta actin gene, and mitochondrial CO1 gene.

[0068] (4) Results The amplification results for each RNA sample are shown in Figures 6(A) to 6(C). Figure 6(A) shows the results of RT-PCR detection of 18s rRNA in nucleotide strands repaired using a PLGA-supported filter or Trizol. Figure 6(B) shows the results of RT-PCR detection of beta actin mRNA in nucleotide strands repaired using a PLGA-supported filter or Trizol. Figure 6(C) shows the results of RT-PCR detection of mitochondrial CO1 mRNA in nucleotide strands repaired using a PLGA-supported filter or Trizol. In Figures 6(A) to 6(C), lane 1 shows the amplification product obtained by amplifying saliva using total RNA recovered through a PLGA-supported filter as a template. Lane 2 shows the amplification product obtained by amplifying serum using total RNA recovered through a PLGA-supported filter as a template. Lane 3 shows the amplification product obtained by amplifying urine using total RNA recovered through a PLGA-supported filter as a template. Lane 4 shows the amplification product obtained by amplifying saliva as a sample using total RNA recovered with Trizol as a template. Lane 5 shows the amplification product obtained by amplifying serum as a sample using total RNA recovered with Trizol as a template. M indicates a molecular weight marker, and C indicates a negative control in which distilled water was used instead of the template.

[0069] Amplification products were confirmed in all lanes, demonstrating that RNA can be recovered using the PLGA-loaded filter.

[0070] The amplification results for each DNA sample are shown in Figures 7(A) to 7(C). Figure 7(A) shows the results of PCR detection of the 18s rRNA gene in the nucleotide strand repaired using a PLGA-loaded filter. Figure 7(B) shows the results of PCR detection of the beta actin gene in the nucleotide strand repaired using a PLGA-loaded filter. Figure 7(C) shows the results of PCR detection of the mitochondrial CO1 gene in the nucleotide strand repaired using a PLGA-loaded filter. In Figures 7(A) to 7(C), lane 1 shows the amplification product amplified using total DNA collected from serum as a template. Lane 2 shows the amplification product amplified using total DNA collected from saliva as a template. Lane 3 shows the amplification product amplified using total DNA collected from urine as a template. M indicates a molecular weight marker, and C indicates a negative control in which distilled water was used instead of the template.

[0071] Amplification products were confirmed in all lanes, demonstrating that DNA can be recovered using PLGA-loaded filters. This also demonstrated that not only genomic DNA but also mitochondrial DNA can be recovered.

[0072] 6. Application of PLGA-Coated Filters to Clinical Samples (1) Dengue Virus Detection Serum samples were collected from three Laotian dengue fever patients, and each sample was labeled LaoDen A, LaoDen B, and LaoDen C. Each serum sample was pretreated by centrifuging at 3,000 g for 15 minutes and filtering the supernatant through a 0.45 μm pore size filter. 250 μL of each pretreated serum sample was mixed with four volumes of lysis buffer and incubated at 70°C for 5 minutes. Each mixture was then passed through the PLGA-coated filter used in step 5 above. Subsequently, 1 mL of QIAzol Lysis Reagent (QIAGEN™) was passed through the PLGA-coated filter, and the lysate that passed through the PLGA-coated filter was collected. Total RNA, including miRNAs, was extracted from the collected lysate using the miRNeasy mini kit according to the manufacturer's protocol. cDNA was synthesized using the extracted total RNA as a template and reverse transcriptase (ReverTra Ace™, TOYOBO Co.). Dengue virus RNA was amplified by quantitative PCR using the synthesized cDNA as a template. Dengue virus detection primer set used was Dengue Universal (Dus and Duc described in the Dengue Virus Infection Diagnostic Manual: National Institute for Health Risk Management, https: / / id-info.jihs.go.jp / diseases / ta / dengue / 020 / denguelabomanual.pdf). The Ct values ​​obtained by quantitative PCR were 21.56 (LaoDen A), 26.95 (LaoDen B), and 26.98 (LaoDen C), respectively.

[0073] Dengue virus-derived RNA was detected by RT-PCR. Human β-actin cDNA was amplified using the hβ-actin primer set as an internal standard. Electrophoresis of the PCR products obtained by RT-PCR is shown in Figure 8. Dengue virus was detected in three dengue patients.

[0074] (2) Detection of EB virus: Approximately 90% of Japanese people are infected with EB virus through saliva during childhood, but most cases are asymptomatic. Therefore, we detected the EB virus genome using saliva samples from healthy individuals.

[0075] Saliva was collected from two healthy subjects, and the samples from each subject were labeled sample 1 and sample 2.

[0076] Four volumes of lysis buffer were added to 1 mL of each sample, mixed, and incubated at 70°C for 5 minutes. After incubation, the mixture was filtered through a 0.45 μm pore size filter, and the filtrate was passed through the PLGA-coated filter used in step 5 above. Next, 1 mL of ISOGENOME (NIPPON GENE CO., LTD.) was passed through the PLGA-coated filter, and the lysate that passed through the PLGA-coated filter was recovered. DNA was extracted from the recovered lysate according to the ISOGENOME protocol. EBV-derived DNA was amplified by quantitative PCR using the extracted DNA as a template. The primer set for EBV detection used primers that amplify bases 94829 to 94889 (60 bases) of the EBV genomic DNA. The Ct values ​​by quantitative PCR were 32.05 (sample 1) and 31.07 (sample 2), respectively.

[0077] EB virus-derived DNA was detected by PCR. Human beta-actin DNA was amplified using the h beta actin primer set as an internal standard. The results of electrophoresis of the PCR products obtained by PCR are shown in Figure 9. EB virus was detected in two subjects.

[0078] 7. Collection of miRNAs using randomly primed PLGA-coated filters. Three urine samples from healthy humans were first pretreated by centrifuging at 2,000 g for 20 minutes at room temperature and filtering the supernatant through a 0.45 μm pore size filter. 15 mL of pretreated urine was passed through the PLGA-coated filter to capture urinary nucleic acids (including miRNAs and small RNAs). 2.25 mL of Qiazol was passed through the nucleic acid-coated PLGA-coated filter to elute the nucleic acids along with the PLGA. Total RNA was extracted and purified from the eluate using the miRNeasy Mini Kit. Total RNA was eluted from each sample column with 25 μL of distilled water. Reverse transcription (total volume: 10 μL) was performed using 7 μL of the eluate as template. The reverse transcription product was diluted 15-fold and subjected to quantitative PCR. For quantitative PCR, primers were used to detect hsa-miR-423-3p, hsa-miR-532-3p, and hsa-miR-451a, which are candidate miRNAs for tuberculosis diagnostic markers; and hsa-miR-107, hsa-miR-181a-5p, hsa-miR-29b-3p, hsa-miR-324-5p, hsa-miR-92b-3p, hsa-let-7a-5p, and hsa-let7f-5p, which are miRNAs reported to be associated with various other diseases.

[0079] Representative results are shown in Figure 10. As shown in the amplification curve (Figure 10(A)) and melting curve (dissociation curve) (Figure 10(B)), specific amplification was confirmed with all primers. Therefore, it was demonstrated that various miRNAs can be collected from human urine samples using PLGA-loaded filters bound with random primers.

[0080] 8. Collection of miRNA using PLGA-coated filters with miRNA-specific primers and comparison with other methods

[0081] (1) Collection of miRNA using PLGA-loaded filters with bound random primers The method described in Section 7 above was followed. Hereinafter, the PLGA-loaded filters with bound random primers will be referred to as "PLAG RP filters."

[0082] (2) Collection of miRNAs using PLGA-coated filters with miRNA-specific primers. Instead of the random primers bound to the PLGA-coated filters prepared in Section 4 above, specific miRNAs were collected using PLGA-coated filters bound with specific primers for the miRNAs hsa-miR-30C-5p and hsa-miR-423-5p. These PLGA-coated filters are designated "PLGA_30C-5p" and "PLGA_423-5p." Nucleic acids were collected from urine samples using each filter, extracted, and purified using the same method as in Section 7 above. Nucleic acids were eluted from the kit's column with 25 μL of distilled water for each sample. Reverse transcription was performed as in Section 7 above, and the reverse transcription product was diluted 15-fold and subjected to quantitative PCR.

[0083] (3) Extraction of total RNA using Urine Conditioning Buffer. A pellet containing nucleic acids was extracted from 15 mL of urine using Urine Conditioning Buffer (Zymo Research; hereafter referred to as "UCB"), a conventional method for extracting nucleic acid components from urine. The method followed the manufacturer's protocol. The extracted pellet was dissolved in 2.25 mL of Qiazol, and total RNA was extracted and purified using the miRNeasy mini Kit as described in Section 7 above. Total RNA from each sample was eluted from the kit's column with 25 μL of distilled water. Reverse transcription was performed as described in Section 7 above, and the reverse transcription product was diluted 15-fold and subjected to quantitative PCR.

[0084] (4) Quantitative PCR The diluted reverse transcription products obtained in steps 8. (1) to (3) above were amplified by quantitative PCR using primers specific for hsa-miR-30C-5p and hsa-miR-423-5p, respectively. The amplification results for hsa-miR-30C-5p are shown in Figure 11. Figure 11(A) shows the amplification curve, and Figure 11(B) shows the Ct value. In Figure 11, PLAG RP Filter 3-1 and PLAG RP Filter 3-2 represent the results for different samples. PLGA_30C-5p-1 and PLGA_30C-5p-2 represent the results for different samples. UCB indicates the amplification results for nucleic acids extracted using UCB. No template indicates the negative control. Figure 12 shows the amplification results for hsa-miR-423-5p. Figure 12(A) shows the amplification curve, and Figure 12(B) shows the Ct value. PLAG RP filter 3-1 and PLAG RP filter 3-2 are the results of different specimens. PLGA_423-5p-1 and PLGA_423-5p-2 are the results of different specimens. UCB indicates the results of amplification of nucleic acid extracted with UCB. No template indicates a negative control.

[0085] Similar to the UCB technique for nucleic acid recovery from samples, target nucleotides were detected using both randomly primer-conjugated PLGA filters and those conjugated with primers specific to the target nucleotide chain. This demonstrates that target nucleotides can be captured using both randomly primer-conjugated and target nucleotide chain-specific primer-conjugated PLGA filters. While specific recovery of target nucleotides is difficult with UCB, PLGA conjugated with primers specific to the target nucleotide chain allows for targeted recovery. Furthermore, randomly primer-conjugated or target nucleotide chain-specific PLGA filters have the advantage of lower running costs per sample than UCB, making them useful.

Claims

1. A method for capturing nucleotide chains from a liquid sample collected from a subject, comprising: bringing the liquid sample into contact with capture probe-bound cationic polymer-modified PLGA nanoparticles; and recovering the nucleotide chains from the capture probe-bound cationic polymer-modified PLGA nanoparticles that have come into contact with the liquid sample, wherein the capture probe-bound cationic polymer-modified PLGA nanoparticles have a nanoparticle core containing PLGA, a coating layer containing a cationic polymer that covers the surface of the core, and a capture probe bound to the coating layer, and the capture probe is an oligonucleotide.

2. The method of claim 1, wherein the cationic polymer is chitosan.

3. The method of claim 1, wherein the oligonucleotide comprises a random sequence or a target nucleotide strand-specific sequence.

4. A collection membrane filter for collecting nucleotide chains from a liquid sample, the collection membrane filter carrying capture probe-bound cationic polymer-modified PLGA nanoparticles, the capture probe-bound cationic polymer-modified PLGA nanoparticles having a nanoparticle core containing PLGA, a coating layer containing a cationic polymer covering the surface of the core, and a capture probe bound to the coating layer, the capture probe being an oligonucleotide.

5. A collection membrane filter unit for collecting nucleotide chains from a liquid sample, comprising: a filter holder incorporating the collection membrane filter according to claim 4; an inlet pipe; and a discharge pipe.

6. A kit for capturing nucleotide chains from a liquid sample, comprising: the capturing membrane filter unit according to claim 5; and an injector for injecting the liquid sample into the capturing membrane filter unit.

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