Microsphere reagent for nucleic acid amplification

Abstract

The invention provides a nucleic acid amplification reagent, the nucleic acid amplification reagent comprises an enzyme and a necessary reagent required by nucleic acid amplification, the enzyme is wrapped by a shell formed by a thermoplastic polymer material to form microspheres, so that the enzyme is isolated from the necessary reagent for nucleic acid amplification, the nucleic acid amplification can be completed only by a single-tube reagent, and the operation is simple and convenient. Independent split charging of amplification components is not needed, and errors caused by manual operation are reduced. According to the method, the enzyme is separated from magnesium ions, and in the nucleic acid amplification process, along with the increase of the amplification temperature, the high polymer material wrapping the enzyme is cracked to release the enzyme, so that the nucleic acid amplification is started, and in addition, the sensitivity of the nucleic acid amplification is also improved.

Description

[0001] The present application claims priority to Chinese prior application No. 2024109754347, filed on July 19, 2024, and U.S. provisional application No. 63 / 677,237, filed on July 30, 2024, the contents of which are incorporated herein in their entirety. TECHNICAL FIELD

[0002] The present application belongs to the technical field of molecular biology detection, and in particular relates to a preparation method and application of a reagent for nucleic acid amplification. BACKGROUND

[0003] The following background description is only an introduction to some background knowledge and does not constitute any limitation to the present application.

[0004] Nucleic acid amplification or testing is a commonly used modern inspection method. By designing primers and through the action of enzymes, the nucleic acid in the sample can be exponentially increased. This method can detect 1 copy of nucleic acid material in the sample, and is a method with high test sensitivity and high test accuracy. The reagent required for nucleic acid amplification usually includes salt ions such as magnesium ions and primers or energy-providing substances, and also includes enzymes such as polymerase. Generally, the enzyme exists alone and cannot be mixed with magnesium ions. Generally, contact can activate the activity of the enzyme. If the enzyme is in contact with magnesium for a long time without amplification, the activity of the enzyme will be reduced.

[0005] A traditional method to solve this problem is to let magnesium ions and enzymes exist in different solutions and be separately packaged. When amplification is needed, the two are mixed to start nucleic acid amplification. Another way is to use freeze-dried form, such as magnesium ions and enzymes are prepared as freeze-dried powders to reduce the contact between magnesium and enzymes. However, after freeze-drying, although the contact between magnesium and enzymes can be reduced, nucleic acid amplification cannot be performed until the freeze-dried reagent forms a solution. In some cases where immediate testing is required, it is challenging to form a solution from freeze-dried reagents in a short period of time. Moreover, additional reagents need to be added during the freeze-drying process, which may affect the efficiency of nucleic acid amplification in the subsequent process.

[0006] In view of the technical problems of the above-mentioned traditional products, the present application provides an improved method to solve the deficiencies of the existing traditional technology. SUMMARY

[0007] The present application provides a nucleic acid amplification reagent, which includes primers, probes, and other nucleic acid amplification reagents, such as magnesium ions, and enzymes. The enzymes are encapsulated in microspheres made of thermoplastic polymer materials, so that the contact between the enzymes and the magnesium ions is reduced. At the same time, such reagents can be divided into a tube. In some ways, the enzymes for nucleic acid amplification and other nucleic acid amplification reagents are separately included in microspheres, and then mixed when used, and then broken by heating to release the nucleic acid amplification reagents. The enzyme for nucleic acid amplification is a polymerase. The nucleic acid amplification reagent includes magnesium ions, or other energy substances, or buffer solutions.

[0008] In another aspect, the present application provides an enzyme, especially an enzyme used in nucleic acid amplification, which is encapsulated in a thermoplastic polymer material. In this way, the enzyme is stored in a shell formed by the polymer material, so that the enzyme is relatively independent in space, facilitating the storage of the enzyme and ensuring the activity of the enzyme. In some ways, the polymer material has a microporous structure, which allows the encapsulated enzyme to be vacuum freeze-dried, and water molecules escape from the shell through the micropores to prepare a freeze-dried enzyme with very low water content, thereby facilitating the storage of the enzyme.

[0009] In some ways, when PCR is used for nucleic acid amplification, the melting or melting temperature of the selected thermoplastic polymer material is less than 100°C, at which temperature the thermoplastic polymer material can melt into a liquid state to encapsulate the nucleic acid amplification reagent, and when the temperature is lower than the melting temperature, the polymer material solidifies to form particles.

[0010] In some ways, the melting temperature of the selected thermoplastic polymer material is below 100°C, such as below 80°C, 60°C, or 50°C, and vice versa. In the process of encapsulation, it is appropriate to select a material with a melting temperature below 100°C, for example, between 40-100°C, for example, less than 100°C, less than 90°C, less than 80°C, less than 70°C, less than 60°C, or less than 50°C, to encapsulate the nucleic acid amplification reagent. Above the above temperatures, the polymer material changes from a solid state to a liquid state and becomes flowable, and then after the encapsulation is completed, the polymer material changes from a liquid state to a solid state when it encounters a lower temperature, thereby forming microspheres. In the use of nucleic acid amplification, such as PCR amplification, when a high temperature is encountered, the included reagents, such as polymerase or nucleic acid amplification reagents, are released to contact each other to activate the activity of the enzyme, thereby allowing the amplification of the target nucleic acid.

[0011] In some embodiments, the thermoplastic polymer material is one or more of polycaprolactone (PCL); polyvinylpyrrolidone (PVP); ethylene-vinyl acetate copolymer (EVA); partially thermoplastic polyurethane (TPU); polyisobutylene (PIB); partially styrene block copolymer (e.g., SIS, SEBS); polyvinyl butyral (PVB); polybutylene succinate (PBS); partially polyhydroxyalkanoate (PHA); random polypropylene (APP, Tg: - 20 °C), petroleum resin (C5 / C9); processing temperature: 70-90 °C; low temperature wax modified polymer: example: Fischer-Tropsch wax (melting point: 60-100 °C) + polyethylene blend.

[0012] In some embodiments, when the encapsulated reagent is a polymerase, a polyester-based polymer material is used, such as PCL, PBS, or PHB, etc. When the encapsulated reagent is a nucleic acid amplification reagent, a polyolefin-based polymer material is used, such as LMW-PE; high VA content EVA; polyisobutylene (PIB), etc.

[0013] In some aspects, the encapsulating material is a liposome material, the liposome comprising one or more ionizable lipids, one or more non-ionizable lipids, one or more sterol-based lipids, and / or one or more PEG-modified lipids; examples of suitable lipids can include, for example, phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Non-limiting examples of ionizable lipids can include, but are not limited to, C12-200, MC3, DLinDMA, DLin-MC3-DMA, DLinkC2DMA, cKK-E12, ICE (imidazolyl), HGT5000, HGT5001, OF-02, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, SM-102, ALC-0315, HGT4003, and JK-102-CA, etc., or combinations thereof. Non-limiting examples of non-ionizable lipids can include, but are not limited to, ceramides, cerebrosides, cerebrosides, diacylglycerols, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol sodium salt (DPPG), 1,2-distearyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), sphingomyelin, or combinations thereof. In some embodiments, the PEG-modified lipids can be poly(ethylene)glycol chains of PEG length of 1000-5000 Da covalently linked to a lipid with an alkyl chain of C6-C20 length. Non-limiting examples of PEG-modified lipids can include, but are not limited to, DMG-PEG 1000 , DMG-PEG 1300 , DMG-PEG1500 DMG-PEG 1800 DMG-PEG 2000 DMG-PEG 2200 DMG-PEG 2500 DMG-PEG 2700 DMG-PEG 3000 DMG-PEG 3200 DMG-PEG 3500 DMG-PEG 3700 DMG-PEG 4000 DMG-PEG 4200 DMG-PEG 4500 DMG-PEG 4700 DMG-PEG 5000 ALC-0159, M-DTDAM-2000, C8-PEG, DOGPEG, ceramide PEG, and DSPE-PEG, or a combination thereof.

[0014] Advantages

[0015] With the above system and method, the nucleic acid amplification reagent is convenient to store, and a single tube reagent is used, so that the amplification reagent does not need to be separately packaged, the operation is more simple and convenient, and the sensitivity of the test can be improved. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a curve chart of the microsphere-encapsulated enzyme provided in the embodiments of the present application, which is placed for 30 days at room temperature and placed for 0 days at room temperature, using a QuantStudio 5 real-time PCR instrument. TM 5 real-time PCR instrument.

[0017] Figure 2 is a curve chart of the microsphere-encapsulated enzyme provided in the embodiments of the present application, which is placed for 30 days at room temperature and a commercially available enzyme reagent, using a QuantStudio 5 real-time PCR instrument. TM 5 real-time PCR instrument.

[0018] Figure 3 is a microsphere morphology chart of the microsphere-encapsulated enzyme provided in the embodiments of the present application, which is placed for 12 months at room temperature in an 8-union PCR reaction tube containing an appropriate amount of pure water.

[0019] DETAILED DESCRIPTION

[0020] The structures related to the present application or the technical terms used are further described below, and if not specifically indicated, are understood and interpreted according to the general terms in the art.

[0021] High molecular material forming microspheres

[0022] The reagent encapsulation method of this invention primarily utilizes the fact that the reagent is in a liquid state during processing, but becomes a fixed granular state after encapsulation. This facilitates the storage and protection of the reagent encapsulated within the microspheres. The desired outcome is that the reagent is liquid during processing, solidifies upon cooling after encapsulation, and then, upon release, reverts to a liquid state at a certain temperature, allowing the release of the encapsulated reagent. Meeting this requirement essentially involves selecting thermoplastic polymer materials, as this process typically involves a physical change (melting into a flowable liquid upon heating, then solidifying upon cooling) and the process is reversible. As the temperature rises, the polymer melts into a liquid state, facilitating encapsulation; as the temperature drops, it solidifies, forming the encapsulated reagent; and when the temperature rises again, the polymer melts and releases the encapsulated reagent. In this invention, "melting" and "dissolving" are interchangeable and refer to the same thing. In some methods, when the encapsulated reagent is a nucleic acid amplification reagent, such as the various reagents listed in Tables 8-9 of this invention, these reagents are basically not degraded or denatured at high temperatures. Materials with high melting temperatures can be used for encapsulation. However, when using these materials for nucleic acid amplification, the highest amplification temperature is 95-100℃. Therefore, at temperatures around 95-100℃, the reagent can transition from solid to liquid. Of course, temperatures can be lower, such as below 80℃, 60℃, or 50℃. Conversely, during processing, materials with melting temperatures below 100℃ are more suitable, such as polymers with melting temperatures between 40-100℃, or less than 100℃, 90℃, 80℃, 70℃, 60℃, or 50℃, to encapsulate the nucleic acid amplification reagent. Then, after being cooled to a low temperature, such as below the melting temperature, the reagent solidifies into microparticles. When used, the temperature is raised again to the melting temperature of the polymer, releasing the encapsulated reagent to participate in nucleic acid amplification. It's understandable that when polymerases are included, the polymerases used for nucleic acid amplification (PCR) are extracted from strains of bacteria found in relatively hot springs, and can withstand temperatures of 95-100℃ without denaturation. Of course, if other enzymes are encapsulated, such as recombinant enzymes for isothermal amplification, the thermoplastic polymer material can be chosen with a melting temperature lower than the protein denaturation temperature. This way, as the temperature rises, the thermoplastic polymer material changes from a solid to a liquid, but the included proteases retain their activity.

[0023] That is, this has a direct relationship with the choice of the reagent to be encapsulated, when the encapsulation does not contain nucleic acid amplification reagents such as protein polymerase, the following high molecular materials can be selected as the encapsulated reagent. For example, common thermoplastic materials, commodity thermoplastics: polyethylene (PE) type, such as low-density polyethylene (LDPE): melting point about 105-115℃; linear low-density polyethylene (LLDPE): melting point about 120-125℃; polyvinyl chloride (PVC): hard PVC (uPVC): glass transition temperature (Tg) about 80℃, processing temperature about 160-210℃. Soft PVC: plasticizer reduces the softening point, and the processing temperature is lower. Polystyrene (PS): general polystyrene (GPPS): glass transition temperature (Tg) about 100℃, processing temperature about 180-280℃ (no sharp melting point, softening flow). High-impact polystyrene (HIPS): similar to GPPS, but with higher impact strength. Acrylonitrile butadiene styrene (ABS): no obvious sharp melting point, glass transition temperature (Tg) about 105℃, processing temperature about 200-250℃ (softening flow). Polymethyl methacrylate (PMMA / Acrylic): glass transition temperature (Tg) about 105℃, processing temperature about 210-250℃ (softening flow). Thermoplastic elastomers (TPEs): such materials have rubber-like elasticity at room temperature, but can melt and flow and process like thermoplastics after heating. Styrene block copolymers (SBCs): such as SBS (styrene-butadiene-styrene), SEBS (hydrogenated SBS). Softening / melting temperature depends on the styrene block, usually flows in the range of 100-200℃. Thermoplastic polyurethane elastomers (TPU): wide range of melting temperatures, soft segment type determines low temperature performance, hard segment determines melting point, usually melts in the range of 120-220℃. Bio-based / biodegradable thermoplastics: polylactic acid (PLA): melting point about 150-160℃ (crystalline type with high L-isomer content). Polyhydroxyalkanoates (PHA): such as PHB (polyhydroxybutyrate), melting point about 170-180℃; polybutylene succinate (PBS): melting point about 115℃.

[0024] Of course, some of the above materials have melting points exceeding 100℃. You can choose to mix multiple metamaterials to lower the melting temperature, for example, by mixing them with materials with low melting temperatures, thereby reducing the melting temperature, which is the temperature required for processing.

[0025] In some cases, thermoplastic materials can also be selected, such as polycaprolactone (PCL): melting point approximately 60°C (very low). Polyvinylpyrrolidone (PVP K30), although it does not have a fixed melting point, has a low glass transition temperature (approximately 100°C) and can be water-processed, making it particularly suitable for heat-sensitive proteins.

[0026] In some preferred examples, thermoplastic high-molecular-weight materials with melting points below 100°C can be selected, such as polycaprolactone (PCL), melting point: approximately 60°C; low-melting-point polyethylene (PE), melting point: 80–100°C (such as ultra-low molecular weight polyethylene or certain copolymer-modified grades); for example, polyolefins; ethylene-vinyl acetate copolymer (EVA), melting point: 70–90°C.

[0027] (Grades with vinyl acetate content >28%); Partial thermoplastic polyurethane (TPU), melting point: 80–

[0028] 100℃ (grades with a high proportion of soft segments, such as polyether-based TPU); Polyisobutylene (PIB); melting point approximately 70℃, processing temperature: 90–120℃; Partial styrene block copolymers (such as SIS, SEBS); glass transition temperature (polystyrene block): 100℃, but overall flow temperature can be as low as 80–120℃; Polyvinyl butyral (PVB); glass transition temperature 60–70℃, processing temperature: 120–150℃.

[0029] Biodegradable thermoplastic materials can also be selected, such as modified polybutylene succinate (PBS); melting point: 90–100℃ (melting point lowered by copolymerization); partially polyhydroxyalkanoates (PHA); melting point: 80–160℃ (e.g., PHBV copolymers can be as low as ~100℃). Other similar thermoplastic materials can also be selected as the encapsulation material of this invention. For example, hot melt adhesive polymers; e.g., atactic polypropylene (APP, Tg: - 20℃), petroleum resin (C5 / C9); processing temperature: 70–90℃; low-temperature wax-modified polymer: e.g., Fischer-Tropsch wax (melting point: 60–100℃) + polyethylene blend.

[0030] When the material to be encapsulated is a protein such as an enzyme, a thermoplastic polymer such as polycaprolactone (PCL) can be used, for example, Capa 6500, Perstorp; low melting point EVA, 65-80°C; fatty acids; 50-70°C. For example, the following specific preparation process: PCL is heated to 70°C to melt, mixed with a protein suspension emulsion. By spray chilling or melt dropping forming, contact with cold air / liquid to solidify instantly. When the temperature is raised, the melting point is lowered so that the protein can be released and participate in the amplification of nucleic acids. For example, the reagents in Table 1 below can be used as the polymer material including enzymes of the present application, which can be purchased commercially.

[0031] Table 1: Manufacturers and molecular weights of thermoplastic polymers

[0032]

[0033] In some embodiments, a thermoplastic polymer with a melting temperature less than 100°C is selected.

[0034] Table 2: Polyolefins and manufacturers

[0035]

[0036]

[0037] Table 3: Polyesters and representative manufacturers

[0038]

[0039] Table 4: Styrenic elastomers and representative brands

[0040]

[0041]

[0042] In some embodiments, some bio-based / degradable materials are also included as the encapsulating material of the present application.

[0043] Table 5: Bio-based / degradable materials

[0044]

[0045] In some embodiments, some water-soluble polymer materials are also included as the encapsulating material of the present application.

[0046] Table 6: Water-soluble polymer materials

[0047]

[0048]

[0049] Table 7: Low temperature hot melt adhesive polymers

[0050] Material Processing temperature (°C) Properties Random polypropylene (APP) 70-85 Strong adhesion, fast curing Petroleum resin (C5 / C9) 80-95 Tackifier, used with EVA / PCL Microfluidic dripping Septon 4055 (SEBS) 60 °C injection, room temperature curing Spray cooling Capa TM 6500 (PCL) Melt: 70 °C → Curing: 5 °C

[0051] In some aspects, liposomes can also be used as the encapsulating material. The "melting temperature" of a liposome refers essentially to the transition temperature (Tm) of its phospholipid bilayer. At this temperature, the lipid membrane transitions from an ordered gel state to a disordered liquid crystal state, resulting in a sudden increase in membrane fluidity and permeability, thereby releasing the encapsulated drug or molecule. Thus, liposomes can also be used as the encapsulating material of the present application. The transition temperature of a liposome is generally between 30-60°C, at which temperature the encapsulated agent can be released, and in the specific preparation process, the liposome is dissolved in an organic solvent, and then nanoparticles are prepared by evaporating the organic solvent.

[0052] The lipid nanoparticle can include one or more ionizable lipids, one or more non-ionizable lipids, one or more sterol-based lipids, and / or one or more PEG-modified lipids. The liposome can include three or more different lipid components, one of the different components of lipids being a sterol-based lipid. In some embodiments, the sterol-based lipid is an imidazolyl cholesterol ester or "ICE" lipid (see WO 2011 / 068810, which is incorporated by reference herein). In some embodiments, the sterol-based lipid can constitute no more than 70% (e.g., no more than 65% and 60%) of the total lipids in the lipid nanoparticle (e.g., liposome). Examples of suitable lipids can include, for example, phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Non-limiting examples of ionizable lipids can include, but are not limited to, C12-200, MC3, DLinDMA, DLin-MC3-DMA, DLinkC2DMA, cKK-E12, ICE (imidazolyl), HGT5000, HGT5001, OF-02, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, SM-102, ALC-0315, HGT4003, and JK-102-CA, etc., or combinations thereof. Non-limiting examples of non-ionizable lipids can include, but are not limited to, ceramides, cerebrosides, sphingomyelin, diacylglycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol sodium salt (DPPG), 1,2-distearyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), sphingomyelin, or combinations thereof.In some embodiments, the PEG-modified lipid can be a poly(ethylene) glycol chain of 1000-5000 Da in length covalently linked to a lipid having an alkyl chain of C6-C20 length. Non-limiting examples of PEG-modified lipids can include, but are not limited to, DMG-PEG. 1000 DMG-PEG 1300 DMG-PEG 1500 DMG-PEG 1800 DMG-PEG 2000 DMG-PEG 2200 DMG-PEG 2500 DMG-PEG 2700 DMG-PEG 3000 DMG-PEG 3200 DMG-PEG 3500 DMG-PEG 3700 DMG-PEG 4000 DMG-PEG 4200 DMG-PEG 4500 DMG-PEG 4700 DMG-PEG 5000 ALC-0159, M-DTDAM-2000, C8-PEG, DOGPEG, Ceramide PEG, and DSPE-PEG, or combinations thereof.

[0053] Encapsulated reagent

[0054] In the present application, the reagents that can be encapsulated by the thermoplastic polymer to form the microspheres are generally the reagents necessary for nucleic acid amplification, so the reagents for nucleic acid amplification can be any principle of nucleic acid amplification, such as isothermal amplification, variable temperature amplification. In some ways, the most typical variable temperature amplification is PCR amplification reagents, such as the necessary reagents in the nucleic acid amplification system in Example 1 of the present application, such as (Tris-HCl PH = 9.0); dATP dGTP, dCTP, dTTP; ammonium sulfate; magnesium chloride; potassium chloride, and primers and probes needed to amplify the target fragment. And the polymerase for amplification, such as Taq enzyme, Taq DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, reverse transcription PCR (RT-PCR) specific enzyme, etc.

[0055] In some aspects, generally, the encapsulation of enzymes and other amplification reagents other than enzymes are included in two microspheres, respectively, for example, each microsphere includes an amount of 10 U, 5 U, 12 U of enzymes, so that when a PCR amplification is needed to be performed, it is indicated that one microsphere needs to be placed to meet the requirement. Of course, if liposomes are used to encapsulate enzymes and amplification reagents, the number of nanometers can be obtained by calculation. In some aspects, microdroplets can be used to form micrometer-sized particles, and each droplet can include 10 U of protein, and of course, several droplets can be used to form an amount of 10-20, 5-8, 4 U of enzymes. In the same way, nucleic acid amplification reagents can also be encapsulated in an amount of one nucleic acid amplification, and of course, it can be understood that one microsphere can include multiple target amplification reagents.

[0056] Preparation method

[0057] The most basic method of encapsulating nucleic acid amplification reagents with thermoplastic materials is to first let the thermoplastic material change from solid to liquid at the melting temperature, then mix with the included reagents to form liquid droplets, and then the droplets encounter low temperature (lower than the melting temperature) to change from liquid to solid, so that the encapsulated reagents are encapsulated in the microparticles formed by the high molecular material. Specifically, the following methods can be used, the most commonly used method is to use microchannels to prepare microspheres, and the encapsulation material is formed into a liquid at a certain temperature, and then the nucleic acid amplification reagents can be in an aqueous state, and then micrometer-sized droplets are formed by microfluidic method, and then the reagents are encapsulated and solidified at room temperature, thereby forming particles.

[0058] For example, spray cooling method is used to prepare microsphere particles, melt emulsification steps: (1) the thermoplastic material (such as PCL) is heated to 70-80°C to melt; (2) add water phase / freeze-dried powder containing protein, high-speed homogenization (10,000 rpm, 2 min) to form W / O emulsion, and add emulsifier (Span 80) to prevent phase separation; then the emulsion is atomized through a nozzle (particle size is controlled by nozzle aperture: 50-300 μm), and cold air is used for instant solidification, thereby forming microspheres, which can form 50-300 micrometer microspheres. Of course, it can be understood that the particle size can be millimeter level by adjusting the nozzle aperture, so that the tail flow diameter is millimeter level, for example, 1-3 millimeter microspheres, and the preferred particle size is between 2.0-2.35 millimeters. At the same time, the content of the included protein can be controlled by adjusting the parameters, for example, the enzyme activity unit U is set, and it is noted that the enzyme activity U is not directly related to the mass of the enzyme, and in the nucleic acid amplification of the present application, the activity of the polymerase is mainly used to achieve it, and generally the activity of the enzyme for a single target nucleic acid is 5 U, 10 U, 20 U, which is sufficient to complete, and when multiple target nucleic acids are tested at the same time, generally 10 U is also sufficient to complete, and less can be used.

[0059] The double emulsion-solvent evaporation method (water-soluble protein preferred) can also be selected, which is the preferred method for water-soluble proteins. The specific method is: the high molecular material such as PLGA, EC, PCL is dissolved in an organic solvent to form a solution, and then primary emulsification (W / O) is carried out: protein aqueous phase + high molecular material / dichloromethane solution → homogeneous W / O emulsion; double emulsification (W / O / W): pour the W / O emulsion into the water phase containing PVA emulsifier → stirring to form W / O / W double emulsion; evaporation and solidification: continue stirring for 6h to evaporate the organic solvent → centrifugal collection of microspheres. The diameter of the microspheres prepared in this way can be micron level, or millimeter level, for example, the diameter is 1-5 millimeters, 2-3 millimeters, etc., and the preferred particle size is between 2.0-2.35 millimeters.

[0060] Of course, in order to adapt to large-scale production, the melt extrusion-spheronization method can also be used; EVA, PCL, PEG-based copolymer can be selected as the wrapped high molecular material, first: melt mixing: melt blending of high molecular material + protein powder in a twin-screw extruder (temperature 70-80°C); extrusion granulation: hot melt is extruded into strips through a porous die → cutting into cylinders by rotating blades; hot spheronization: heated to above the Tg of the material in the fluidized bed → spheroidization under the action of surface tension. The diameter of the microspheres prepared in this way can be micron level, or millimeter level, for example, the diameter is 1-5 millimeters, 2-3 millimeters, etc., and the preferred particle size is between 2.0-2.35 millimeters.

[0061] Generation of droplets

[0062] In one embodiment, the present application provides a droplet generation device capable of generating thousands of droplets, and then dispersing the polymerase-containing droplets into thousands of individual droplets, each droplet containing polymerase, such as 5U, 10U, 100U of droplets. After the droplets are generated, the droplets are allowed to solidify at a temperature below the melting temperature of the high molecular material to form microspheres. In one embodiment, the droplet generation device is a microfluidic platform capable of generating and dispersing droplets of polymerase into a large number of individual droplets. In some ways, the droplet arrangement device of the present application includes a droplet generation device, and the droplet generation device is in communication with the arrangement device through a microchannel.

[0063] Those skilled in the art will readily appreciate that different types and forms of droplet generation devices can be used in the present application, as long as such devices are capable of generating droplets suitable for use in the present application. In one embodiment, the inlet provided by the droplet generator is used to introduce various liquids (such as oil or liquid polymer material, samples and reagents for conducting reactions) to the droplet generator. In one embodiment, the various liquids used to generate the droplets are provided to the droplet generator through the same inlet. In one embodiment, the various liquids used to generate the droplets are provided to the droplet generator through different inlets. Droplets with millimeter scale diameters can also be prepared using conventional droplet generation equipment and then solidified into microspheres of the present application.

[0064] The droplet generation device of the present application can be any structure or system that is capable of dividing a liquid into a plurality of droplets. In one embodiment, the droplet generation device includes, but is not limited to, flow focusing structures, cross-flow structures, co-flow structures, step emulsification, and microchannel emulsification. Some techniques for generating droplets are described by P. Zhu and L. Wang (2017), the contents of which are hereby incorporated by reference in their entirety as part of the present disclosure.

[0065] In one embodiment, the present droplet generator is a shear-based droplet generation device that uses shear force to pinch a stream into small droplets. In one embodiment, the shear-based droplet generation device includes, but is not limited to, those consisting of cross-flow structures, co-flow structures, and flow focusing structures.

[0066] In one embodiment, the present droplet generation device is a surface tension-based droplet generation device in which surface tension is the dominant driving force during droplet breakup. In one embodiment, the surface tension-based droplet generation device includes, but is not limited to, those consisting of T-junction structures, step emulsification, and microchannel emulsification structures.

[0067] In one embodiment, the present droplet generation device includes droplet generation structures as described in WO2016189383A1, the contents of which are hereby incorporated by reference in their entirety as part of the present disclosure. In one embodiment, methods capable of generating droplets can be used in the present application to generate droplets, including but not limited to high shear agitation, ultrasonic emulsification, high pressure homogenization, and membrane emulsification.

[0068] In one embodiment, the present droplet generation device includes flow focusing structures that can compress flow to enhance the focusing effect. In one embodiment, the flow focusing structure is a 2D planar flow focusing structure.

[0069] In one embodiment, the present droplet generation device includes cross-flow structures that allow the continuous and dispersed phases to intersect at an angle θ. In one embodiment, the present droplet generator includes T-junction, Y-junction, double-T-junction, K-junction, or V-junction structures.

[0070] In one embodiment, the present droplet generation device comprises a co-flow structure in which the dispersed stream is struck by a continuous phase of surrounding flow. In one embodiment, the co-flow structure is a 2D planar co-flow structure.

[0071] In one embodiment, the present droplet generation device comprises a step emulsification structure. In one embodiment, the present droplet generation device comprises a step emulsification structure in combination with a parallel or perpendicular T-structure.

[0072] In one embodiment, the present droplet generation device comprises a microchannel emulsification structure.

[0073] In one embodiment, the components or parts of the droplet generation structure responsible for generating the droplets have a hydrophobic surface. This can be achieved by chemical surface coating of the components or parts with hydrophobic groups. In one embodiment, a surfactant such as Span 80, Tween 20 or Abil EM90, PFPE-PEG-PFPE, perfluoropolyether-polyethyleneoxide-perfluoropolyether triblock copolymer) is added to the molten polymeric thermoplastic material or to the aqueous phase to prevent droplet coalescence or to prevent molecules such as enzymes, DNA or RNA from sticking to solid surfaces or water-oil interfaces.

[0074] In one embodiment, the generated droplets are emulsion droplets and are not limited to a specific type of emulsion. In one embodiment, the emulsion includes but is not limited to oil-in-water, water-in-oil and water-oil-water double emulsions.

[0075] In one embodiment, an oil (which can also be referred to as an oil phase) and a surfactant are used to generate the droplets. In one embodiment, the ratio of surfactant to oil is 1-5% (w / w). In one embodiment, the oil used to generate the droplets includes a thermoplastic polymer material as described above which is melted into a liquid state. In one embodiment, the surfactant used includes but is not limited to Span 80, Tween 20 / 80, ABIL EM 90 and phospholipids, PFPE-PEG-PFPE. Baret and Jean-Christophe (2012) describe surfactants used in droplet-based microfluidics, the contents of which are incorporated by reference into the present disclosure.

[0076] In one embodiment, the droplet generation device is capable of generating polymerase in water-in-oil droplets (droplet diameter 2-3 mm) at a frequency of about 1 Hz to about 20 Hz. In one embodiment, the frequency of generating the droplets is about 5 to 10 Hz. The size of the generated droplets is in the millimeter range.

[0077] In some embodiments, the present application provides a production device capable of producing gel microspheres, the device comprising a microfluidic channel, a first inlet for inputting a polymerase solution and a second inlet for inputting a gel solution, the first inlet and the second inlet being connected by the microfluidic channel, at the connection for contacting the gel solution with the cell solution and forming gel microspheres, each of the at least some of the gel microspheres comprising a fixed unit of polymerase.

[0078] The microfluidic channel herein comprises a plurality of microfluidic channels that are in fluid communication with each other. The gel microsphere structure with gel encapsulated or encapsulated droplets of polymerase, or oil phase encapsulated or encapsulated gel microspheres produced in the first microfluidic channel and / or the second microfluidic channel can be achieved by any of the structures in the prior art. For example, a cross-flow structure that allows the continuous phase and the dispersed phase to intersect at an angle θ. In one embodiment, the present droplet generator comprises a T-junction, a Y-junction, a double T-junction, a K-junction, or a V-junction.

[0079] Droplet properties

[0080] In one embodiment, the number, size (e.g., diameter), volume, and emulsification type of the droplets produced or used by the present application depend on the subsequent process or analysis required. In one embodiment, the number of droplets produced ranges from hundreds to millions.

[0081] In one embodiment, the size of the droplets produced ranges from about 5 microns to about 200 microns, 1-5 millimeters. In one embodiment of polymerase scattering, the size of the droplets produced ranges from about 10 microns to about 200 microns, or the individual droplets are between 1-5 millimeters. In one embodiment, the droplets produced have a uniform diameter. In one embodiment, the droplets produced have a uniform diameter with a coefficient of variation of less than 5%. In another embodiment, droplets of different diameters can be produced by adjusting the loading pressure. In some ways, each droplet contains 10 U of polymerase, for example, 10 U of polymerase in a solidified microsphere with a diameter of 2 millimeters.

[0082] The droplets produced by the present application are dropped into a solution below the melting temperature of the high molecular material in the subsequent preparation process, so as to be solidified to form microspheres, and the solidified microspheres have encapsulated reagents, such as polymerase, or reagents necessary for nucleic acid amplification.

[0083] For example, microspheres can be prepared using a microfluidic approach as follows. A microfluidic droplet generation method can also be used, and a microfluidic chip design can be used. The internal phase (aqueous phase) can include a protein (polymerase) buffer + 0.5% sodium alginate (sodium alginate can be omitted). The external phase (oil phase) can include molten PCL (80°C). The two phases can form monodisperse W / O droplets (50-200 μm, or 1-5 mm in diameter) in a flow focusing structure. The droplets can be cooled and solidified by dropping into a 4°C PBS solution to form PCL shell solidified microspheres. Of course, the aqueous phase can also include nucleic acid amplification reagents, such as the reagent formulations in Tables 8-9 of the Examples. The microfluidic droplet generation method can be performed using commercially available microfluidic droplet generation equipment. When droplets of 1-5 mm in diameter are desired, the microchannel can be configured to be 1X1 mm or larger, and a T-shaped structure or a flow focusing structure can be used. The flow rate of the oil phase can be controlled, and the flow rate of the oil phase can be reduced to reduce the shear force on the aqueous phase. The oil phase can be allowed sufficient time to form large droplets by surface tension or periodic perturbation. In the present application, the polymer material has a melting point of less than 100°C, and after being melted into a liquid phase, the surface tension is very large, sufficient to form millimeter-sized droplets, and each droplet can contain sufficient enzyme activity units for nucleic acid amplification, such as 10-15 U / droplet, or 10-15 / microsphere.

[0084] In one embodiment, the microfluidic channels are made of silicon, glass, plastic, and polydimethylsiloxane (PDMS). In one embodiment, the same type or configuration of microfluidic channels can be used in various components of the integrated droplet microfluidic system of the present application. In another embodiment, various types or configurations of microfluidic channels can be used in various components of the integrated droplet microfluidic system of the present application.

[0085] In one embodiment, the droplet generator of the present application includes two microfluidic channels for transporting oil and one or more microfluidic channels for transporting encapsulated reagents. In one embodiment, the actual configuration depends on the type of emulsion selected and the number of inlets required. In one embodiment, the outlet of the present application includes one microfluidic channel with a diameter of a few hundred microns, and the droplets exiting the outlet are directly dropped into a low-temperature PBS solution to form solidified microspheres.

[0086] Detection

[0087] Detection indicates testing for the presence or absence of a substance or material, such as, but not limited to, a chemical, an organic compound, an inorganic compound, a metabolite, a drug or a drug metabolite, an organic tissue or a metabolite of an organic tissue, a nucleic acid, a protein, or a polymer. In addition, detection indicates testing for the amount of a substance or material. Further, testing also indicates immunoassay, chemical assay, enzymatic assay, etc.

[0088] Sample

[0089] The sample of the detection device of the present application includes biological fluids (e.g. case fluids or clinical samples). The fluid sample or specimen can be derived from a solid or semi-solid sample, including excreta, biological tissue and food samples. The solid or semi-solid sample can be converted to a fluid sample by any suitable method, such as mixing, mashing, macerating, incubating, dissolving or digesting the solid sample with enzymatic action in a suitable solution (e.g. water, phosphate buffer or other buffer solution). A "biological sample" includes samples derived from animals, plants and food samples, such as including urine, saliva, blood and components thereof, spinal fluid, vaginal secretions, semen, fecal matter, sweat, secretions, tissues, organs, tumors, tissue and organ cultures, cell cultures and media derived from humans or animals. Preferably, the biological sample is urine, and more preferably, the biological sample is saliva. Food samples include food processing materials, end products, meat, cheese, wine, milk and drinking water. Plant samples include samples derived from any plant, plant tissue, plant cell cultures and media.

[0090] An "environmental sample" is derived from the environment (e.g. a fluid sample from a lake or other body of water, a sewage sample, a soil sample, ground water, sea water and effluent samples). Environmental samples can also include sewage or other waste water.

[0091] The sample here is a substance containing target nucleic acid, such as some viruses, bacteria, fungi, etc. can be extracted and amplified nucleic acid, such as new coronavirus, influenza virus, HPV virus, etc.

[0092] PCR nucleic acid amplification

[0093] PCR (Polymerase Chain Reaction) is a technology to realize the amplification of target sequences in the case of realizing temperature variation, see Table 10 for the way of amplification, when the temperature is at 95℃, the microspheres containing enzymes or nucleic acid reagents are melted to release the reagents to participate in the amplification of nucleic acid, this release is a slow release, which improves the utilization efficiency of enzymes, and also improves the sensitivity of amplification. PCR can be divided into conventional PCR, nested PCR, multiplex PCR, hot start PCR, real-time fluorescent quantitative PCR, digital PCR, DNA PCR, reverse transcription PCR, one-step RT-PCR, two-step RT-PCR, etc. All of them can use the reagents of the present application to amplify target nucleic acid. DETAILED DESCRIPTION

[0094] The detailed description of the present application is to further illustrate how the present application is implemented, and cannot form any limitation on the present application, the protection scope of the present application is limited by the claims.

[0095] Example 1

[0096] This example is a test comparison of the microspheres of the present application with commercially available enzyme reagents.

[0097] 1. Information of commercially available enzyme reagents

[0098] The commercially available enzyme used was Robustart Taq (antibody modified Taq enzyme) with the item number E16, purchased from Zhuhai Baoshui Biotechnology Co., Ltd. in a unit of 2000 U). Taq DNA polymerase Buffer (Taq DNA polymerase Buffer), 10X (KL-25-06840) was purchased from Shanghai Kanglang Biotechnology Co., Ltd. Example sub 1.1 : Preparation process of microspheres

[0099] The basic principle of the device for droplet generation is that the microchip of the droplet generator is made of polydimethylsiloxane (PDMS), silicon or plastic (polycarbonate, cyclic olefin copolymer (COC)). For the fabrication of a PDMS microchip, a mold is created by photolithography using SU-8 photoresist (Microchem) on a silicon dioxide substrate. The mold is used to fabricate a PDMS replica. The PDMS replica is bonded to a cover glass using plasma treatment to create the PDMS chip. The chip surface is treated with fluorosilane (Aquapel) to obtain hydrophobicity. Silicon chips are fabricated in a similar manner. A silicon wafer with patterned etching is bonded to a glass wafer with inlet and outlet ports drilled using an anodic bonding technique. The bonded silicon wafer is cut into individual chips. The silicon wafer surface is treated with fluorosilane (Aquapel) to obtain hydrophobicity.

[0100] The present application employs the equipment improved by Zhejiang Dapu Biological Company according to the public patent CN217450211U to produce the droplets of the present application, the microchannel is set to be 1 mm wide and 1.5 mm deep, and a T-shaped structure is used at the junction of the oil phase and the water phase to prepare the microspheres of the present application. The inner phase (water phase): buffer solution containing Taq enzyme + 0.5% sodium alginate + 0.1% Tween 80 (to reduce surface tension), the flow rate in the channel is 1 microliter / minute; the outer phase (oil phase): molten PCL (80°C), the flow rate is 40 microliters per minute; the two phases form monodisperse W / O droplets (particle size 2.25 microns) in the T-shaped structure; cooling and solidification: the droplets fall into 4°C PBS → PCL shell solidification to form microparticles. Each microparticle contains about 10 U of enzyme, and this method is to calculate the amount of enzyme and the number of droplets, and 2000 U of enzyme can be used to prepare about 200 microspheres, each containing about 10 U of enzyme, and of course 10-15 U of enzyme can also be prepared in each microsphere. The diameter of each microsphere is about 2.25 microns (2.0-2.25 microns). In order to control the size, a micro-imaging device can be used to take pictures of the size of the droplets at the outlet, and the relative flow rates of the two phases can be adjusted to control the size of the droplets. The above method is to prepare the microspheres of the present application by improving the traditional micropore method.

[0101] The above flow rate and size are fixed parameters selected by adjustment to prepare microspheres with an average size of about 2.25 microns. It can be understood that in the embodiments of the present application, the size of the droplets is affected by the size of the microchannel, the properties of the polymer material itself, and the melting temperature of the organic phase to become a flowable liquid phase, the water phase is generally a buffer solution containing enzyme, and auxiliary reagents are added to reduce the surface tension. The control of the relative flow rate of the two phases adopts pressure and the like, and these can be adjusted to obtain the target particle size through limited experiments according to the needs. Of course, it can be understood that any one of the methods for preparing microspheres in the present application can be used for preparation, and the preparation method is the existing traditional method, and the traditional method can be engineered to achieve, and will not be repeated again.

[0102] Example sub 1.2: Microspheres of nucleic acid amplification reagents (except polymerase)

[0103] The aqueous solution of the enzyme was replaced with a nucleic acid amplification reagent solution (see Table 8), such as a combination of each reagent in Table 8, and primers for amplifying the target sequence (Table 9) were also prepared in droplets according to the method described in Example 1.1, and the microdroplets were collected. After each droplet solidified, a nucleic acid amplification reagent was formed with an average particle size of 2.35 mm. If the amount of the nucleic acid amplification reagent was 25 μL, it was encapsulated with 25 μL of an aqueous solution of microspheres with a particle size of about 2.35 mm, and the number of microspheres was adjusted until the encapsulation was complete. The encapsulating polymer material can be selected at will, for example, a thermoplastic polymer material with a melting temperature below 100°C. Example sub 1.3: Effect of different encapsulation materials on the encapsulation of polymerase and amplification reagents.

[0104] According to the method of Example 1.1, different thermoplastic polymer materials were used for the oil phase to encapsulate the polymerase and the nucleic acid amplification reagent, respectively, and it was found that for the amplification effect, the amplification sensitivity, enzyme activity, and stability of the polyester-encapsulated polymerase were better than those of the polyolefin-encapsulated polymerase. However, for the amplification reagent, the opposite result was obtained, i.e., the amplification sensitivity and stability of the polyolefin-encapsulated nucleic acid amplification reagent (without polymerase) were better than those of the polyester-encapsulated nucleic acid amplification reagent. Therefore, in the preferred mode, the polyester is used to encapsulate the polymerase, and the polyolefin is used to encapsulate the nucleic acid amplification reagent. The specific test results are shown in Table 11.2.

[0105] 2. Preparation of the nucleic acid amplification system and the amplification method are shown in Table 8 below:

[0106] Table 8 Preparation of the nucleic acid amplification system (except for the enzyme, the total amount is 25 μL)

[0107]

[0108] According to the public information of WHO and the Chinese CDC, the primers and probes for the gene region of HPV18 type of human papillomavirus were selected, the genome sequence of human papillomavirus was downloaded from Virologic.org, GISAID, and the GenBank platform of the American NCBI, the alignment analysis was performed using BioEdit, the conserved region of the gene was selected, and the Primer Express 3.0 software was used to design specific primers and probes, as shown in Table 9 below. All the primers and probes used in this embodiment were synthesized by Shanghai Shuo Ying Biological Technology Co., Ltd.

[0109] Table 9 HPV18 type primer and probe sequences

[0110]

[0111] 3. Sample information

[0112] The test sample was a human papillomavirus (HPV) HPV18 type positive standard, which contained different copy numbers of templates.

[0113] 4. The PCR amplification program is set as shown in Table 10:

[0114] Table 10: PCR amplification program

[0115]

[0116] 5. Test results

[0117] The test results of the microspheres encapsulated enzyme (Example 1.1) of the present application and the commercially available enzyme, when the nucleic acid amplification reagent is according to Table 8-9 but not encapsulated. The microspheres encapsulated enzyme provided by the present application and the commercially available enzyme reagent (the enzyme content is equal, about 10 U) are tested using QuantStudio 5 real-time fluorescence PCR instrument, and the CT value statistical results are shown in Tables 11.1-11.2: TM 5. Test results

[0118] Table 11.1 CT value statistical results

[0119]

[0120]

[0121] From the above results, it can be seen that the polymerase encapsulated by the present application and the polymerase without encapsulation amplify different concentrations of templates, and it is found that the encapsulated enzyme of the present application has higher sensitivity than the enzyme without encapsulation under the same U, and the threshold value is exceeded under a relatively small number of cycles, and the result can appear faster. When the template concentration is very low, the present application still has CT value, indicating high sensitivity, while the traditional non-encapsulated enzyme has no CT value, indicating that the result cannot be tested and the sensitivity is low. The amplification result of the microspheres encapsulated enzyme provided by the present application is better than that of the commercially available enzyme reagent, especially in terms of sensitivity; this may be that the microspheres encapsulated enzyme prepared by the present application is continuously broken and releases the enzyme to participate in the nucleic acid amplification during the temperature change in the nucleic acid amplification process, thereby improving the sensitivity of the nucleic acid amplification, and the activity of the enzyme is used most efficiently to improve the detection sensitivity.

[0122] According to the method of Example 1.3, Table 11.2, the CT values of different microspheres encapsulated enzyme reagents are compared (the same method is used for nucleic acid amplification, and the enzyme is encapsulated by different encapsulation materials, the specific encapsulation process is as shown in Example 1.1, and the CT value of the enzyme content is 10 U, and the particle size is about 2.35 mm).

[0123]

[0124] The number of cycles to reach the threshold for amplification of low concentration templates was higher for PCL than for PBS and PHB, but no results were obtained for polyolefins at low concentrations. Although it is generally believed that polyolefins, such as high VA content EVA, are suitable for inclusion of enzymes, the polyolefins did not appear to be as effective as the polyesters as encapsulating materials when the enzymes were included and used to perform PCR nucleic acid amplification. Similarly, the group also tried to include the polymerase enzymes in some of the materials listed in Tables 4-7 and tested the performance of the polymerase enzymes encapsulated in these materials. The overall results were better than the PCR amplification results for the unencapsulated polymerase enzymes, but the polyesters were still better, especially in terms of sensitivity, than the polyolefins when compared side by side. Similarly, the group also tried to include the nucleic acid amplification reagents in some of the materials listed in Tables 4-7 and although the results were better than the PCR amplification results for the unencapsulated reagents, the polyolefins were still better, in terms of sensitivity, than the polyesters (data not shown).

[0125] Example 2

[0126] This example is to evaluate the enzyme activity of the microencapsulated enzymes of different sizes in the present application. 1. Three batches of microencapsulated enzymes of different sizes were prepared using the preparation method of Example 1.1 (encapsulating material is PBS), and the sizes of the microencapsulated enzymes were 1.7-1.85 mm, 1.85-2.0 mm, and 2.0-2.35 mm, respectively. 2. The enzyme activities of the three batches of microencapsulated enzymes of different sizes were tested (the mass of the enzymes was equal, but the sizes were different), and the results are shown in Table 12 below.

[0127] Table 12 Enzyme activity test

[0128]

[0129]

[0130] The enzyme activity test results of the three batches of microencapsulated enzymes of different sizes showed that the activity of the microencapsulated enzymes with a size of 2.0-2.35 mm was the highest. This indicates that the activity of the enzymes encapsulated in microspheres of different sizes is significantly related to the size of the microspheres, which may be due to the influence of the microenvironment on the enzymes in the microspheres, thereby affecting the enzyme activity. This directly affects the efficiency and sensitivity of PCR nucleic acid amplification.

[0131] Example 3

[0132] 1. Three batches of microencapsulated enzymes with a size of 2.0-2.35 mm were prepared (Example 1.1, encapsulating material is PHB).

[0133] 2. Test was performed using QuantStudio TM 5 Real-Time PCR instrument, amplification system was shown in Example 1, 1*10 6 copy / ml of template was added in each batch, 10 tests were performed in each batch. Precision test data statistics were shown in Table 13.

[0134] Table 13 Precision test data statistics

[0135]

[0136]

[0137] The above precision evaluation results show that the coefficient of variation CV values of the three batches of microspheres encapsulating enzymes provided by the application are less than 5%, and the precision performance test is qualified.

[0138] Example 4

[0139] This example is a test comparison between the microspheres (particle size of 2.0-2.35 millimeters) prepared in the application and the commercially available kit.

[0140] 1. Sample and commercially available kit information

[0141] The sample is a commercially available monkeypox virus nucleic acid detection reagent national reference (purchased from Jingliang Gene Technology (Shenzhen) Co., Ltd.). DNA extraction is performed by using a nucleic acid extraction kit (magnetic bead method), and the extraction kit is purchased from Jiangsu Shoushi Biological Technology Co., Ltd., with the item number: SDK60104.

[0142] The selected commercially available kit is a monkeypox virus nucleic acid detection kit (fluorescent PCR method), with the item number: YJC70115NW, purchased from Jiangsu Shoushi Biological Technology Co., Ltd. (the amount of enzyme used is 10U).

[0143] 2. Microsphere preparation

[0144] The amplification solution and polymerase enzyme in the monkeypox virus nucleic acid detection kit (fluorescent PCR method) with the item number YJC70115NW are prepared into microspheres (particle size of 2.0-2.35 millimeters) respectively, and the specific preparation method refers to the microfluidic preparation method in Example 1.1, wherein the polymerase is encapsulated by PCL, 10U of polymerase, and the nucleic acid amplification solution is encapsulated by polyisobutylene (PIB), Oppanol B10 (particle size of 2.0-2.35 millimeters).

[0145] 3. Test comparison

[0146] The microspheres provided by the application and the commercially available kit (YJC70115NW) are used for testing using QuantStudioTM 5 Real-time PCR instrument was used to test the positive reference of the monkeypox virus nucleic acid detection reagent national reference.

[0147] 4. Test results

[0148] The microspheres in the present application and the commercially available reagent were tested using QuantStudio TM 5 Real-time PCR instrument was used for testing, and the PCR amplification results are shown in Table 14, and the data statistics are shown in the following table:

[0149] Table 14 Comparison of test data statistics

[0150]

[0151] Through the above comparison test results, it is shown that the test performance of the prepared microspheres (amplification liquid and enzyme preparation into microspheres) of the present application is better than that of the commercially available reagent, and the sensitivity is better. When in the case of low concentration, the present application can still test the results, while the nucleic acid amplification liquid and polymerase not wrapped cannot be tested.

[0152] Example 5

[0153] This example is a test comparison of a prepared full reagent microsphere and a commercially available kit in the present application.

[0154] 1. Information of commercially available kit

[0155] The selected commercially available kit is Mycoplasma pneumoniae nucleic acid assay kit (fluorescence PCR method), item number: YJB20109N, purchased from Jiangsu Shoushi Biological Technology Co., Ltd. (enzyme concentration is 10 U).

[0156] 2. Preparation of full reagent microsphere

[0157] The PCR amplification components (amplification liquid, enzyme and detection liquid) of the Mycoplasma pneumoniae nucleic acid assay kit (fluorescence PCR method) with item number YJB20109N were prepared into microspheres; the specific preparation method refers to the microfluidic preparation method in Example 1.1, wherein the polymerase is wrapped with PCL, 10 U of polymerase (particle size is 2.0-2.35 millimeters), and the nucleic acid amplification and detection liquid is wrapped with polyisobutylene (PIB), Oppanol B10 (particle size is 2.0-2.35 millimeters).

[0158] 3. Test comparison

[0159] QuantStudio TM 5 Real-time PCR instrument was used to test the full reagent microsphere provided by the present application and the commercially available kit (YJC70115NW),

[0160] 4. Test results

[0161] The full reagent microspheres in the present application and the commercially available reagents are tested by using QuantStudio TM 5 real-time PCR instrument, and the data statistics are shown in the following table:

[0162] Table 15 Comparison of test data statistics

[0163]

[0164]

[0165] Through the above comparison test results, it is shown that the test performance of the full reagent microspheres of the present application is better than that of the commercially available reagents, and the sensitivity is better.

[0166] Example 6

[0167] This example is the stability evaluation of the microsphere-encapsulated enzyme in the present application

[0168] 1. The microsphere-encapsulated enzyme (particle size: 2.0-2.35 mm) in the present application is placed at room temperature for 30 days and the microsphere-encapsulated enzyme placed at room temperature for 0 days, and the two are tested by using QuantStudio TM 5 real-time PCR instrument, and the amplification system is shown in Example 1, and the PCR amplification results are shown in the following figure: Figure 1 The polymerase uses PCL as the encapsulating material.

[0169] 2. The microsphere-encapsulated enzyme in the present application is placed at room temperature for 12 months and the commercially available enzyme reagent (Robustart Taq (antibody modified Taq enzyme), item number: E16, purchased from Zhuhai Baorui Biological Technology Co., Ltd.), and the two are tested by using QuantStudio TM 5 real-time PCR instrument, and the amplification system is shown in Example 1, and the PCR amplification results are shown in the following figure: Figure 2 The above stability evaluation results show that the microsphere-encapsulated enzyme provided by the present application is stable in performance after being placed at room temperature for 12 months.

[0170] Example 7

[0171] This example is the water-soluble stability evaluation of the microsphere-encapsulated enzyme in the present application Figure 3 1. The microsphere-encapsulated enzyme (Example 1.1, encapsulated material: PCL, particle size: 2.0-2.35 mm) in the present application is respectively placed in 8 PCR reaction tubes, and then an appropriate amount of pure water is added into the reaction tubes, and the 8 PCR reaction tubes are placed at room temperature for 12 months. It is found by visual observation that the morphology of the microspheres does not change, as shown in the following figure:

[0172] 2. After 8-tube PCR reaction tube containing microspheres encapsulated enzyme was placed at room temperature for 12 months, the microspheres were gently taken out with tweezers, and the moisture on the surface of the microspheres was controlled.

[0173] 3. The microspheres encapsulated enzyme and the commercially available enzyme reagent (Robustart Taq (antibody modified Taq enzyme), product number: E16, purchased from Zhuhai Baosheng Biological Technology Co., Ltd.) were tested using QuantStudio TM 5 Real-time PCR instrument, the amplification system is shown in Example 1, and the CT value statistical results are shown in Table 16.

[0174] Table 16 CT value statistical results

[0175] Template concentration (copies / mL) Microsphere-encapsulated enzyme Commercially available enzyme 1 x 10 8 ]]> 20.38 21.32 1 x 10 7 ]] 24.23 25.26 1 x 10 6 ]]> 27.32 28.36 1 x 10 5 ]] 31.71 45.65

[0176] The above water stability evaluation results show that the microspheres encapsulated enzyme provided by the application is placed in an 8-tube PCR reaction tube containing an appropriate amount of pure water, and is placed at room temperature for 12 months, and the water stability is good. The amplification performance is good. Compared with the conventional enzyme without encapsulation, the activity of the enzyme is still good, and the activity of the enzyme without encapsulation may decrease, and the number of CT cycles increases, which indicates that the activity of the enzyme decreases.

[0177] Example 8

[0178] The present embodiment provides a preparation method of a freeze-dried reagent of a microsphere encapsulated enzyme.

[0179] Some high molecular materials have a microporous structure, and the microporous structure can allow the encapsulated enzyme to be vacuum freeze-dried, and the water molecules escape from the shell through the micropores, and the freeze-dried enzyme with extremely low water content is prepared, thereby facilitating the storage of the microsphere encapsulated enzyme and prolonging its shelf life.

[0180] Specifically includes the following steps:

[0181] 1. Prepare the microsphere encapsulated enzyme according to the method in Example 1, and the encapsulated material is PCL (particle size is 2.0-2.35 millimeters).

[0182] 2. The microsphere encapsulated enzyme particles are divided into 8-tube PCR reaction tubes.

[0183] 3. The 8-tube PCR reaction tube is placed in a vacuum freeze dryer for freeze-drying.

[0184] 4. Under the condition that the performance of the vacuum freeze dryer is normal, the freeze-drying program parameters are set as shown in Table 17.

[0185] Table 17 Freeze-drying program parameter setting

[0186]

[0187] 5. After the end of freeze-drying, the temperature and humidity of the environment where the freeze-drying machine is located are strictly controlled. After the temperature and humidity of the environment meet the requirements, the freeze-drying machine door is opened, and the 8 PCR reaction tube freeze-dried reagents are taken out.

[0188] 6. The microsphere-encapsulated enzyme particle freeze-dried reagent is sealed and packaged, and is subjected to accelerated stability at 37°C. After 60 days of accelerated stability, the performance is tested.

[0189] 7. Performance test of microsphere-encapsulated enzyme particle freeze-dried reagent.

[0190] The microsphere-encapsulated enzyme freeze-dried reagent and the commercially available enzyme reagent (Robustart Taq (antibody modified Taq enzyme), product number: E16, purchased from Zhuhai Baoshui Biotechnology Co., Ltd.) are tested using a QuantStudio TM 5 real-time PCR instrument, and the amplification system is as shown in Example 1. The CT value statistical results are shown in Table 17.

[0191] Table 17 CT value statistical results

[0192]

[0193]

[0194] The above results show that the microsphere-encapsulated enzyme freeze-dried reagent prepared in this embodiment has good test performance, and the test results can appear in a smaller number of amplification cycles.

[0195] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the application pertains, and are incorporated by reference. All patents and publications recited herein are expressly incorporated by reference herein in their entirety for all purposes to the same extent as if each were incorporated by reference individually. The present application may, however, be carried out in ways not specifically enumerated herein without departing from the essential scope and nature of the application. For example, the term "comprising" as used herein is used in the sense of "including", "containing", or "characterized by" and not in the more restrictive sense of "consisting only of". The term "a" or "an" as used herein means "one or more" and thus includes two or more unless the context clearly indicates otherwise. The term "another" as used herein means "at least a second or more" and thus includes two or more unless the context clearly indicates otherwise. The term "plurality" as used herein means "two or more" unless the context clearly indicates otherwise. The term "another" as used herein means "at least a second or more" and thus includes two or more unless the context clearly indicates otherwise. The term "plurality" as used herein means "two or more" unless the context clearly indicates otherwise. The term "comprising" as used herein is used in the sense of "including", "containing", or "characterized by" and not in the more restrictive sense of "consisting only of". The term "a" or "an" as used herein means "one or more" and thus includes two or more unless the context clearly indicates otherwise. The term "another" as used herein means "at least a second or more" and thus includes two or more unless the context clearly indicates otherwise. The term "plurality" as used herein means "two or more" unless the context clearly indicates otherwise. The term "comprising" as used herein is used in the sense of "including", "containing", or "characterized by" and not in the more restrictive sense of "consisting only of". The term "a" or "an" as used herein means "one or more" and thus includes two or more unless the context clearly indicates otherwise. The term "another" as used herein means "at least a second or more" and thus includes two or more unless the context clearly indicates otherwise. The term "plurality" as used herein means "two or more" unless the context clearly indicates otherwise.

Claims

1. A reagent for performing amplification of a target nucleic acid, said amplification reagent comprising: The enzyme and the necessary reagent for nucleic acid amplification, wherein the enzyme is encapsulated by a shell formed by a thermoplastic polymer to form a microsphere, so that the enzyme and the necessary reagent for nucleic acid amplification are isolated.

2. The agent of claim 1, wherein, The thermoplastic polymer can be changed from solid to liquid at or above the melting temperature, and can be changed from liquid to solid at a temperature lower than the melting temperature.

3. The agent of claim 2, wherein, The melting temperature of the polymer encapsulating the enzyme is below 100℃.

4. The agent of claim 3, wherein, The melting temperature is below 95-60℃.

5. The agent of claim 4, wherein, The enzyme is an enzyme for nucleic acid amplification, such as a nucleic acid polymerase, a cleavage enzyme, or an enzyme for gene editing.

6. The agent of claim 5, wherein, The necessary reagent for nucleic acid amplification is also encapsulated by a shell formed by a thermoplastic polymer to form a microsphere containing the necessary reagent for amplification.

7. The agent of claim 6, wherein, The polymer encapsulating the necessary reagent for nucleic acid amplification is a polyolefin polymer.

8. The agent of claim 7, wherein, The polyolefin polymer includes one or more of LMW-PE, high-VA-content EVA, and polyisobutylene (PIB).

9. The agent of claim 8, wherein, The polymer encapsulating the polymerase is a polyester polymer.

10. The agent of claim 9, wherein, The polyester polymer includes PCL, one or more of PBS or PHB.

11. The agent of claim 5, wherein, The particle size of the microsphere is 2.0- 2.35 mm.

12. The reagent of claim 1, wherein the thermoplastic polymer is a polymer selected from one or more of polyolefins, polyesters, polyurethanes, acrylics, and polysulfones.

13. The reagent of claim 12, wherein the polyolefins include polyethylene (PE) or polypropylene (PP).

14. The reagent of claim 13, wherein the polyesters include PET (polyethylene terephthalate).

15. The agent of claim 1, wherein, The thermoplastic polymer includes a styrene elastomer, and the styrene elastomer includes SIS block copolymer or SEBS hydrogenated elastomer.

16. The agent of claim 1, wherein, The thermoplastic polymer includes a biodegradable polymer, and the biodegradable polymer includes a low-molecular-weight polylactic acid (LOW-MW PLA), a polycaprolactone-lactide copolymer, or a modified starch thermoplastic.

17. The agent of claim 1, wherein, The thermoplastic polymer includes a water-soluble polymer, and the water-soluble polymer includes polyvinylpyrrolidone (PVP) or polyethylene oxide (PEO).

18. The agent of claim 1, wherein, The thermoplastic polymer is a liposome material.

19. The reagent of claim 1, wherein the necessary reagent for nucleic acid amplification includes Tris-HCl, KCl, MgCl2, and DNTP, and primer sequences for amplifying target nucleic acids.

20. The agent of claim 1, wherein, The nucleic acid amplification is PCR amplification.

Images