Preparation method of high-temperature storage urine galactose kit

Abstract

The invention discloses a preparation method of a high-temperature storage urine galactose kit. The preparation method comprises the following steps: preparing a reaction solution and a substrate solution based on microcrystalline cellulose, agarose and a surfactant; placing the reaction solution and the substrate solution on a support carrier in a solid phase manner to prepare a dry chemical reaction reagent pad and a substrate pad for detecting galactourine; the high-temperature galactose storage kit is prepared on the basis of a dry chemical reaction reagent pad and a substrate pad for detecting galactose. The invention also discloses a kit capable of storing galactose at high temperature, which is prepared by the preparation method. The kit comprises a reaction device, a urine purifying and sampling device and a calibration reagent, a reaction hole and a calibration hole are formed in the reaction device, and visual windows are arranged on the reaction hole and the calibration hole; a reaction reagent is cured in the reaction hole and the calibration hole; the urine purifying and sampling device is used for purifying tested raw urine; an activated carbon adsorbent is arranged in the urine purification device; the calibration reagent is used for being dropwise added into the calibration hole during urine galactose detection, and after color development, the calibration reagent is used for being compared with color development of the reaction hole.

Description

Technical Field

[0001] The invention relates to the technical field of protection of urine galactose detection enzyme reagents, and in particular to a method for preparing a urine galactose kit for high-temperature storage. Background Art

[0002] Lactase is a hydrolase secreted by the brush border of epithelial cells on the surface of the small intestinal villi, which can hydrolyze lactose into glucose and galactose. When lactase is deficient, the body cannot absorb lactose normally, resulting in lactose malabsorption. In severe cases, lactose malabsorption can cause lactose intolerance symptoms with clinical manifestations of abdominal distension, diarrhea, and abdominal pain. There are many methods for lactase determination. Direct detection of jejunal mucosal lactase is the most direct and reliable, but it is an invasive test. The hydrogen breath test is convenient, accurate, sensitive, and non-invasive, but it requires special instruments, is expensive, takes a long time to operate, and requires good patient compliance. Lactose barium meal X-ray examinations carry radiation risks.

[0003] The urine galactose determination method has the characteristics of simple operation, low price, high specificity and sensitivity, and is suitable for testing in medical institutions at all levels. The principle of this method is that under the action of galactose oxidase, galactose generates hexadiol and hydrogen peroxide, the latter of which enters the trinder color reaction, and the subject can be judged whether it is lactose intolerant by comparing the depth of the sample and the standard color. However, although the kit prepared based on this method is simple to operate and does not require professional training, it is not currently suitable for special environments and remote mountainous areas and other harsh environments, because the current urine galactose kit requires high storage conditions, and cold chain transportation and low temperature storage are required at ultra-normal temperatures, which will greatly increase the cost of using the kit. Among them, the enzyme involved in the urine galactose method is easily inactivated at room temperature, which is one of the important reasons. Therefore, the prior art has a technical demand to solve the problem of the stability of the enzyme in the urine galactose method detection reagent at high temperature (40℃-45℃), and to realize the high temperature (40℃-45℃) storage and transportation of the urine galactose detection kit. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method of a urine galactose kit for high temperature storage and a urine galactose kit capable of high temperature storage, thereby maintaining the stability of the enzyme in the urine galactose method reagent to achieve the purpose of a urine galactose detection kit for self-testing during high temperature (40°C-45°C) storage and transportation.

[0005] The first aspect of the present invention is to provide a method for preparing a high-temperature storage urine galactose kit, which is used to provide high-temperature storage protection for the detection enzyme reagent of the urine galactose kit, comprising: S1, preparation of reaction solution and substrate solution based on microcrystalline cellulose, agarose and surfactant; S2, solidifying the reaction solution and the substrate solution on a support carrier to prepare a dry chemical reaction reagent pad and a substrate pad for detecting urine galactose; S3, preparing the high temperature storage urine galactose kit based on the dry chemical reaction reagent pad and substrate pad for detecting urine galactose.

[0006] Preferably, the step of preparing the reaction solution in S1 comprises: (1) Prepare microcrystalline cellulose buffer R1; (2) Using the microcrystalline cellulose buffer R1 as solvent, a reaction solution R2 is prepared.

[0007] Preferably, the step S1 of preparing the substrate solution comprises: (1) Prepare microcrystalline cellulose buffer R1; (2) Using the microcrystalline cellulose buffer R1 as solvent, a substrate solution R3 is prepared.

[0008] Preferably, the steps of preparing the microcrystalline cellulose buffer R1 include: (a) preparing a phosphate buffer solution having a pH of 7.5 to 7.8 as a stock solution; (b) dissolving microcrystalline cellulose, agarose and a surfactant in the mother solution to prepare the microcrystalline cellulose buffer R1.

[0009] Preferably, the microcrystalline cellulose is one or more of methylcellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose and carboxytetramethyl cellulose; the agarose is one or more of ordinary agarose, low melting point agarose, low electroosmotic agarose, high strength agarose, Seakem HE agarose, Seakem HEEO agarose, SeaKem ME agarose, SeaKem HGT agarose, SeaKem HGT (P) agarose and IsoGel agarose; the phosphate buffer with a pH of 7.5-7.8 is 50-200 mmol / L, a PBK (KH2PO4 / K2HPO4) buffer with a pH of 7.5-7.8 or a PBS (Na2HPO4 / KH2PO4) buffer with a pH of 7.5-7.8.

[0010] Preferably, the step of preparing the substrate solution R3 using the microcrystalline cellulose buffer R1 as a solvent comprises: The galactosidase substrate is added into the microcrystalline cellulose buffer R1 and fully dissolved to form a substrate solution R3, wherein the formed substrate solution R3 contains 0.1-0.5 g / mL of the galactosidase substrate; the galactosidase substrate is one or more of O-nitro-β-D-pyranoside (ONPG), 5-bromo-4-chloro-3-indole-β-D-pyranoside (x-gal), chlorophenol red-β-D-pyranoside (CPRG), PNPG and IPTG.

[0011] Preferably, the step S2 of preparing a dry chemical reaction reagent pad for detecting urine galactose comprises: (1) solidifying the reaction solution R2 on a first supporting carrier; (2) subjecting the reaction solution R2 in solid phase on the first supporting carrier to variable temperature drying under a first condition; wherein the first condition includes a gas condition, a variable temperature range, and a drying time; in the first condition, the gas condition is a dry inert gas condition; the variable temperature range is 15-35°C, and the drying time is 5 hours.

[0012] Preferably, the step S2 of preparing the substrate pad comprises: (1) solidifying the substrate solution R3 on a second support carrier; (2) subjecting the substrate solution R3 in solid phase on the second support carrier to variable temperature drying under second conditions; wherein the second conditions include gas conditions, variable temperature range and drying time; in the second conditions, the gas conditions are dry inert gas conditions; the variable temperature range is 15-35°C, and the drying time is 5 hours.

[0013] Preferably, the first supporting carrier and the second supporting carrier are one or more of hydroxypropyl methylcellulose and methylcellulose.

[0014] The second aspect of the present invention is to provide a high temperature storage urine galactose kit prepared by the preparation method of the high temperature storage urine galactose kit of the first aspect, comprising: Reaction apparatus, urine purification and dosing apparatus, and calibration reagents; The reaction device is provided with a reaction hole and a calibration hole, and the reaction hole and the calibration hole are both provided with a visualization window; the reaction reagent is solidified in the reaction hole and the calibration hole, and the reaction reagent is obtained by drying the reaction solution R2 in an inert gas at 15°C-35°C for 5 hours; The urine purification and sample addition device is used to purify the original urine of the subject; an activated carbon adsorbent is arranged in the urine purification device; the body of the urine purification and sample addition device is a rectangular structure and is made of an inert material; The calibration reagent is used to be added dropwise to the calibration well when performing urine galactose detection, and is used to compare the color development with that of the reaction well after color development; The reaction reagents include a buffer solution, an enzyme label solution and a substrate solution R3; The buffer is a PBK (KH2PO4 / K2HPO4) buffer with a pH of 7.5-7.8 and a 50-200 mmol / L buffer. The buffer contains 3% g / V microcrystalline cellulose, 3-5% g / V agarose and 0.1-2% g / V surfactant. The microcrystalline cellulose includes one or more of methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose and carboxytetramethyl cellulose. The agarose is one or more of ordinary agarose, low melting point agarose, low electroosmotic agarose, high strength agarose, Seakem HE agarose, Seakem HEEO agarose, SeaKem ME agarose, SeaKem HGT agarose, SeaKem HGT (P) agarose and IsoGel agarose. The enzyme labeling solution is prepared by using the microcrystalline cellulose buffer R1 as a solvent, adding galactose oxidase, peroxidase and a color developer; the galactose oxidase is one or more of galactose hydrolyzing oxidase and β-galactosidase; the peroxidase is one or more of horseradish peroxidase, superoxide dismutase (SOD), peroxidase (GPX), aldehyde oxidase activity (GR), hydroxyperoxidase (GAT) and lactoperoxidase; the color developer is one or more of 4-aminoantipyrine and 3-methyl-2-thiazolinonehydrazone hydrochloride hydrate (MBTH); The substrate solution R3 uses the microcrystalline cellulose buffer R1 as a solvent, and a galactosidase substrate is added to fully dissolve the substrate solution R3, wherein the substrate solution R3 contains 0.1 g / mL of a galactosidase substrate, and the substrate solution R3 contains one or more of O-nitro-β-D-pyranoside (ONPG), 5-bromo-4-chloro-3-indole-β-D-pyranoside (x-gal), chlorophenol red-β-D-pyranoside (CPRG), PNPG, and IPTG; The calibration reagent is a 1.0mmol / L D-galactose standard solution, which is prepared by adding D-galactose and sodium chloride into purified water and dissolving them; the standard reagent includes the following components: 0.15-0.2g / L of 1.0mmol / L D-galactose standard solution and 0.8-1.0g / L of sodium chloride are added into purified water and dissolved; the solvent of the standard reagent is an artificial urine base solution; the artificial urine base solution includes: 1-3g of disodium hydrogen phosphate, 0.75-1g of potassium dihydrogen phosphate, 1g of urea 0-12g, creatinine 1.0-1.5g, glucose 10-15g, thimerosal 0-1.5g, disodium hydrogen phosphate 2-3g, potassium dihydrogen phosphate 0.5-0.75g, sodium chloride 7-10g, urea 7-10g, creatinine 0.7-1.0g, glucose 15-20g, thimerosal 0.5-1g, bilirubin 0.2-1.0g, distilled water 500ml, and use NaOH to adjust the standard reagent to a pH of 5.7.

[0015] A third aspect of the present invention is to provide an electronic device, comprising a processor and a memory, wherein the memory stores a plurality of instructions, and the processor is used to read the instructions and execute the method described in the first aspect.

[0016] A fourth aspect of the present invention is to provide a computer-readable storage medium, wherein the computer-readable storage medium stores a plurality of instructions, and the plurality of instructions can be read by a processor to execute the method described in the first aspect.

[0017] Beneficial effects of the method and system of the present invention: (1) The difference between the present kit and the normal temperature storage kit is that the components in the reaction solution are changed: sericin is used to stabilize the enzyme in the normal temperature kit; microcrystalline cellulose is added to the buffer solution for high temperature (40°C-45°C) storage and transportation of the present invention to dissolve the enzyme and immobilize the enzyme, thereby increasing the upper limit of the storage temperature by 50%; the peroxidase in the normal temperature kit is superoxide dismutase (SOD) or hydroxyperoxidase (GAT), which is replaced by horseradish peroxidase in the present invention.

[0018] (2) Technology upgrade (embedding technology): After the enzyme is solidified, microcrystalline cellulose solution is added to the small holes to achieve enzyme embedding technology to keep it active. (The crystal form of microcrystalline cellulose does not change after ultrasonic activation treatment, and the grain size remains basically unchanged, but the ultrasonic treatment partially destroys the hydrogen bonds of cellulose, reduces the crystallinity, loosens the structure, increases the water retention value and specific surface area of ​​cellulose, and significantly improves the accessibility and reaction activity of cellulose to reagents.) (3) High temperature storage is possible without covering the reaction wells and calibration wells with aluminum foil. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the related technologies, the drawings required for use in the specific embodiments or the related technical descriptions will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 A flow chart of a method for preparing a high-temperature urine galactose storage kit according to an embodiment of the present invention; Figure 2 A schematic diagram of an electronic device provided according to an embodiment of the present invention. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0023] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. Embodiment 1

[0024] like Figure 1 As shown, the first aspect of the present invention provided in this embodiment is to provide a method for preparing a high-temperature storage urine galactose kit, which is used to provide high-temperature storage protection for the detection enzyme reagent of the urine galactose kit, comprising: S1, preparation of reaction solution and substrate solution based on microcrystalline cellulose, agarose and surfactant; S2, solidifying the reaction solution and substrate solution on a support carrier to prepare a dry chemical reaction reagent pad and substrate pad for detecting urine galactose.

[0025] S3, preparing the high temperature storage urine galactose kit based on the dry chemical reaction reagent pad and substrate pad for detecting urine galactose.

[0026] As a preferred embodiment, the preparation of the reaction solution in S1 comprises: (1) Prepare microcrystalline cellulose buffer R1; (2) Using the microcrystalline cellulose buffer R1 as solvent, a reaction solution R2 is prepared.

[0027] As a preferred embodiment, the step S1 of preparing the substrate solution comprises: (1) Prepare microcrystalline cellulose buffer R1; In this embodiment, the steps of preparing the microcrystalline cellulose buffer R1 include: (a) preparing a phosphate buffer solution having a pH of 7.5 to 7.8 as a stock solution; (b) dissolving microcrystalline cellulose, agarose and a surfactant in the mother solution to prepare the microcrystalline cellulose buffer R1.

[0028] As a preferred embodiment, the microcrystalline cellulose is one or more of methylcellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose and carboxytetramethyl cellulose; the agarose is one or more of ordinary agarose, low melting point agarose, low electroosmotic agarose, high strength agarose, Seakem HE agarose, Seakem HEEO agarose, SeaKem ME agarose, SeaKemHGT agarose, SeaKem HGT (P) agarose and IsoGel agarose; the phosphate buffer with a pH of 7.5-7.8 is 50-200 mmol / L, a PBK (KH2PO4 / K2HPO4) buffer with a pH of 7.5-7.8 or a PBS (Na2HPO4 / KH2PO4) buffer with a pH of 7.5-7.8.

[0029] (2) Using the microcrystalline cellulose buffer R1 as solvent, a substrate solution R3 is prepared.

[0030] In this embodiment, the preparation of substrate solution R3 using the microcrystalline cellulose buffer R1 as solvent includes: The galactosidase substrate is added into the microcrystalline cellulose buffer R1 and fully dissolved to form a substrate solution R3, wherein the formed substrate solution R3 contains 0.1-0.5 g / mL of the galactosidase substrate; the galactosidase substrate is one or more of O-nitro-β-D-pyranoside (ONPG), 5-bromo-4-chloro-3-indole-β-D-pyranoside (x-gal), chlorophenol red-β-D-pyranoside (CPRG), PNPG and IPTG.

[0031] As a preferred embodiment, the S2 preparation of a dry chemical reaction reagent pad for detecting urine galactose comprises: (1) solidifying the reaction solution R2 on a first supporting carrier; (2) Drying the reaction solution R2 in solid phase on the first support carrier under a first condition by variable temperature; wherein the first condition includes a gas condition, a variable temperature range, and a drying time.

[0032] As a preferred embodiment, in the first condition, the gas condition is a dry inert gas condition; the variable temperature range is 15-35° C., and the drying time is 5 hours. Of course, those skilled in the art should know that the variable temperature range and the drying time can be adjusted according to specific preparation conditions, all within the scope of protection of the present invention.

[0033] As a preferred embodiment, the step S2 of preparing the substrate pad comprises: (1) solidifying the substrate solution R3 on a second support carrier; (2) Drying the substrate solution R3 in solid phase on the second support carrier under second conditions; wherein the second conditions include gas conditions, temperature range and drying time.

[0034] As a preferred embodiment, in the second condition, the gas condition is a dry inert gas condition; the variable temperature range is 15-35° C., and the drying time is 5 hours. Of course, those skilled in the art should know that the variable temperature range and the drying time can be adjusted according to specific preparation conditions, all within the scope of protection of the present invention.

[0035] As a preferred embodiment, the first supporting carrier and the second supporting carrier are both one or more of hydroxypropyl methylcellulose and methylcellulose. Embodiment 2

[0036] This embodiment provides a high-temperature storage urine galactose kit prepared by the preparation method of the high-temperature storage urine galactose kit of the first embodiment, comprising: Reaction apparatus, urine purification and dosing apparatus, and calibration reagents; As a preferred embodiment, the reaction device is provided with a reaction hole and a calibration hole, and the reaction hole and the calibration hole are both provided with a visualization window; the reaction reagent is solidified in the reaction hole and the calibration hole, and the reaction reagent is obtained by drying the reaction solution R2 in an inert gas at 15°C-35°C for 5 hours; The urine purification and sample addition device is used to purify the original urine of the subject; an activated carbon adsorbent is arranged in the urine purification device; The calibration reagent is used to be added dropwise to the calibration well when performing urine galactose detection, and is used to compare the color development with that of the reaction well after color development; The reaction reagents include a buffer solution, an enzyme label solution and a substrate solution R3.

[0037] As a preferred embodiment, the buffer is a PBK (KH2PO4 / K2HPO4) buffer with a concentration of 50-200 mmol / L and a pH of 7.5-7.8.

[0038] In this embodiment, 0.68 g KH2PO4 and 0.871 g K2HPO4 were weighed by an electronic balance and dissolved in deionized water to prepare a 50 mmol / LPBK buffer solution (the reagent sources: KH2PO4 was manufactured by Sigma, CAS: 7778-77-0, K2HPO4 was manufactured by Sigma, CAS: 7758-11-4), and 3 g of activated microcrystalline cellulose was weighed by an electronic balance and dispersed in DMSO and 50 mmol / LPBK buffer was added to prepare a 3% g / V Solution (the sources of the reagents: microcrystalline cellulose is manufactured by Sigma, CAS: 9004-34-6; DMSO is manufactured by Sigma, CAS: 67-68-5), 3g of Seakem HEEO agarose (manufactured by Sigma, CAS: 9012-36-6) and 0.1% g / V surfactant Tween 80 (manufactured by Sigma, CAS: 9005-65-5) are weighed with an electronic balance to prepare a buffer support solution with PBK buffer, and the pH is adjusted to 7.5.

[0039] Microcrystalline cellulose properties (1) Chemical stability: MCC is stable in nature and can withstand high temperature, high humidity, and strong light exposure; (2) Chemical reaction performance: Microcrystalline cellulose and cellulose show some similar characteristics in chemical reaction characteristics. In addition, the surface is porous and the structure is loose, so reagents can easily penetrate during chemical reactions, which greatly improves the uniformity of the reaction and produces products with excellent properties. Compared with general dissolving pulp, although microcrystalline cellulose has a higher degree of crystallinity, it has higher reaction performance during the carboxymethylation, acetylation, and esterification processes, and changes in mechanical properties, flammability, and especially solubility.

[0040] As a preferred embodiment, the buffer contains 3% g / V microcrystalline cellulose, 3-5% g / V agarose and 0.1-2% g / V surfactant; the microcrystalline cellulose includes: one or more of methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose and carboxytetramethyl cellulose; the microcrystalline cellulose MCC is stable in nature, resistant to high temperature, high humidity, strong light irradiation, porous surface, loose structure, and reagents can easily penetrate during chemical reactions, thereby greatly improving the uniformity of the reaction; the microcrystalline cellulose is prepared by acid method, alkali method, oxidation method, bioenzyme method, etc.

[0041] In this embodiment, microcrystalline cellulose is preferably prepared according to the following method: hydrochloric acid is added to 10% cotton pulp for acidification, and after reaching the limit degree of polymerization, the finished product can be obtained through neutralization, dehydration, drying and other operation steps. The process flow is as follows: weigh a certain amount of cotton pulp, cut the coarse powder, dissolve it in water, and prepare 40% starch milk; add 5% hydrochloric acid solution for acidification and heat it to 90°C. After 1 hour, the reaction is completed; after washing with water twice, dehydration, drying and other operation steps are carried out until the water content of the material reaches less than 10%, the material is discharged, crushed, and the finished product is obtained. After passing the inspection, it is ready for use. Activation process: weigh a certain amount of microcrystalline cellulose and add it to 100ml deionized water, treat it with an ultrasonic cell crusher, the ultrasonic power is 750W, and the temperature of the treatment system is controlled below 40°C by an ice water bath, and the treatment is carried out for 90 minutes. The treated microcrystalline cellulose is fully washed with deionized water, filtered, vacuum dried, and ground for use.

[0042] The agarose is one or more of ordinary agarose, low melting point agarose, low electroosmotic agarose, high strength agarose, Seakem HE agarose, Seakem HEEO agarose, SeaKem ME agarose, SeaKem HGT agarose, SeaKem HGT (P) agarose and IsoGel agarose.

[0043] As a preferred embodiment, the enzyme-labeled solution is prepared by using the microcrystalline cellulose buffer R1 as a solvent, adding galactose oxidase, peroxidase and a color developer; the galactose oxidase is one or more of lactose oxidase and β-galactosidase; the peroxidase is one or more of horseradish peroxidase, superoxide dismutase (SOD), peroxidase (GPX), aldehyde oxidase activity (GR), hydroxyperoxidase (GAT) and lactoperoxidase; the color developer is one or more of 4-aminoantipyrine and 3-methyl-2-thiazolinonehydrazone hydrochloride hydrate (MBTH).

[0044] In this embodiment, 0.012g of galactose oxidase (manufactured by Sigma, CAS: 9028-79-9), 0.2-0.3g of horseradish peroxidase (manufactured by Sigma, CAS: 9003-99-0), and 2g of 4-aminoantipyrine were weighed on an electronic balance. The buffer support was used as a solvent, galactose oxidase was added, and after fully dissolving, 4-aminoantipyrine was added. After fully dissolving, horseradish peroxidase was added and fully dissolved. The final concentration of the solution was 50U / L galactose oxidase, 1% 4-aminoantipyrine (manufactured by Sigma, CAS: 1675-58-8) and 50U / L peroxidase. (manufactured by Sigma, product number 1.16216) 10g of galactosidase substrate (manufactured by Sigma, product number 10105031001) is weighed on an electronic balance, and the galactosidase substrate is fully dissolved to prepare a reaction solvent, and the final solution contains 60U / L of galactosidase substrate. 3g of activated microcrystalline cellulose is weighed on an electronic balance and added to deionized water heated to 90°C, and stirred while adding to prepare a 3% g / V solution as an embedding solution (the source of the reagent: microcrystalline cellulose is manufactured by Sigma, CAS: 9004-34-6). The body of the urine purification sample addition device is a rectangular structure, the body material is an inert material PE, and is provided with a reaction hole and a calibration hole, and the size of the reaction hole and the calibration hole is Φ4mm. The reaction hole and the calibration hole are visualized. Add 20ul of reaction solvent to the reaction wells and calibration wells (stirring with a glass rod while adding) to solidify, and dry in an oven with inert gas at 56°C for 30 minutes until completely dry. Similarly, add 20ul of 3% g / V microcrystalline cellulose embedding solution to the reaction wells and calibration wells (stirring with a glass rod while adding) to solidify, and dry in an oven with inert gas at 56°C for 30 minutes until completely dry, and store at room temperature.

[0045] As a preferred embodiment, the substrate solution R3 uses the microcrystalline cellulose buffer R1 as a solvent, and the galactosidase substrate is added to fully dissolve it. The substrate solution R3 contains 0.1 g / mL of galactosidase substrate, and the substrate solution R3 contains 0.1 g / mL of O-nitro-β-D-pyranoside (ONPG), 5-bromo-4-chloro-3-indole-β-D-pyranoside (x-gal), chlorophenol red-β-D-pyranoside (CPRG), PNPG, and IPTG. One or more.

[0046] As a preferred embodiment, the urine purification and sample addition device body is a rectangular structure, and the material is an inert material; the manufacturing method is the purification device manufacturing method described in patent CN200910079299.3.

[0047] As a preferred embodiment, the calibration reagent is a 1.0mmol / L D-galactose standard solution, which is prepared by adding D-galactose and sodium chloride to purified water and dissolving them; the standard reagent includes the following components: 1.0mmol / L D-galactose standard solution 0.15-0.2g / L, sodium chloride 0.8-1.0g / L added to purified water and dissolved; the solvent of the standard reagent is artificial urine base liquid; the artificial urine base liquid includes: disodium hydrogen phosphate 1-3g, potassium dihydrogen phosphate 0.75-1g, urea 1 0-12g, creatinine 1.0-1.5g, glucose 10-15g, thimerosal 0-1.5g, disodium hydrogen phosphate 2-3g, potassium dihydrogen phosphate 0.5-0.75g, sodium chloride 7-10g, urea 7-10g, creatinine 0.7-1.0g, glucose 15-20g, thimerosal 0.5-1g, bilirubin 0.2-1.0g, distilled water 500ml, and use NaOH to adjust the standard reagent to a pH of 5.7.

[0048] In this embodiment, 2g of disodium hydrogen phosphate and 1g of potassium dihydrogen phosphate were weighed on an electronic balance and added to 500 ml of deionized water to prepare a buffer solution. 1g of disodium hydrogen phosphate, 0.75g of potassium dihydrogen phosphate, 10g of urea, 1.0g of creatinine, 10g of glucose, 0.5g of thimerosal, 7g of urea, 0.7g of creatinine, 0.5-1g of thimerosal, and 0.2g of bilirubin were weighed on an electronic balance and added to the buffer solution and stirred to dissolve. 500ml of urine base solution with a pH of 5.7 was prepared with NaOH; 0.1g of D-galactose and 0.4g of sodium chloride were weighed and added to the urine base solution to dissolve to prepare a 0.2g / L D-galactose calibration reagent.

[0049] Application examples: According to the subject's weight: calculated as 10 ml of fresh milk per kilogram of body weight (485 ml per person per time for adults). The subject empties the urine, drinks the fresh milk, and does not eat or drink water within 2 hours after drinking the milk. The subject drains the urine in the first hour after drinking the milk, and collects the urine in the second hour after drinking the milk for testing. The day before the test, the subject must not eat foods or drugs rich in VC. Please test in time after collecting urine. Samples that cannot be measured in time should be stored at 2°C~8°C, but the measurement should be completed within 8 hours. Take out the kit from, tear open the aluminum foil packaging bag, and take out the reaction device. Gently peel off the aluminum foil strip on the reaction well. Put the urine purification device in the kit (manufactured as described in the purification device manufacturing in patent CN200420088097.8).

[0050] Remove the sealing cap, use a pipette to draw 2 ml of urine, and inject it into the purification device. Cover the sealing cap of the purification device and shake the purification device for about 20 times.

[0051] Remove the sealing cap and connect it to the syringe, gently push the syringe, discard the first few drops of purified urine, and drop two drops of purified urine into the reaction well on the reaction device; then drop two drops of galactose standard solution into the calibration well of the reaction device. After the reaction device is placed at room temperature for 20 minutes, immediately observe the color depth of the reaction well and the calibration well to read the results. The reaction well and the calibration well show red. If the color of the reaction well is darker than that of the calibration well, it means that the concentration of galactosidase in the urine of the test subject is greater than 1.0mmol / L. If the color of the reaction well is lighter than that of the calibration well, it means that the concentration of galactosidase in the urine of the test subject is less than 1.0mmol / L.

[0052] Example Kit stability test tests the stability of the enzyme in the urine galactose test reagent at super normal temperature (40°C-45°C). The prepared 13 batches of urine galactose test kits (10 tests in each batch were tested by 10 testers) were tested at 45°C in the first month, second month, third month, fourth month, fifth month, sixth month, seventh month, eighth month, ninth month, tenth month, eleventh month, twelfth month, and thirteenth month in parallel with the newly configured kit to verify the stability and accuracy of the reagents.

[0053] Test method: Add two drops of galactose standard solution (1mmol / L) to the reaction well and calibration well, and observe the color change. The color of the reaction well and the calibration well is consistent, and the color of the three parallel reaction devices is also consistent.

[0054] The present invention also provides a memory storing a plurality of instructions, wherein the instructions are used to implement the method as in the first embodiment.

[0055] like Figure 2As shown, the present invention also provides an electronic device, including a processor 301 and a memory 302 connected to the processor 301, the memory 302 stores a plurality of instructions, and the instructions can be loaded and executed by the processor so that the processor can execute the method as in the first embodiment.

[0056] Through the description of the above implementation methods, those skilled in the art can clearly understand that the above embodiments can be implemented by software, or by means of software plus necessary general hardware platforms. Based on such understanding, the technical solutions of the above embodiments can be embodied in the form of software products, which can be stored in a non-volatile storage medium (which can be a CD-ROM, a USB flash drive, a mobile hard disk, etc.), including several instructions for enabling a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a high-temperature storage urine galactose kit, which is used to provide high-temperature storage protection for the detection enzyme reagent of the urine galactose kit, characterized in that: include: S1, preparation of reaction solution and substrate solution based on microcrystalline cellulose, agarose and surfactant; S2, solidifying the reaction solution and the substrate solution on a support carrier to prepare a dry chemical reaction reagent pad and a substrate pad for detecting urine galactose; S3, preparing the high temperature storage urine galactose kit based on the dry chemical reaction reagent pad and substrate pad for detecting urine galactose.

2. The method for preparing a high temperature storage urine galactose kit according to claim 1, characterized in that: The preparation of the reaction solution in S1 comprises: (1) Prepare microcrystalline cellulose buffer R1; (2) Using the microcrystalline cellulose buffer R1 as solvent, a reaction solution R2 is prepared.

3. The method for preparing a high temperature storage urine galactose kit according to claim 2, characterized in that: The step S1 of preparing the substrate solution comprises: (1) Prepare microcrystalline cellulose buffer R1; (2) Using the microcrystalline cellulose buffer R1 as solvent, a substrate solution R3 is prepared.

4. The method for preparing a high temperature storage urine galactose kit according to claim 3, characterized in that: The steps of preparing the microcrystalline cellulose buffer R1 include: (a) preparing a phosphate buffer solution having a pH of 7.5 to 7.8 as a stock solution; (b) dissolving microcrystalline cellulose, agarose and a surfactant in the mother solution to prepare the microcrystalline cellulose buffer R1.

5. The method for preparing a high temperature storage urine galactose kit according to claim 4, characterized in that: The microcrystalline cellulose is one or more of methylcellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose and carboxytetramethyl cellulose; the agarose is one or more of ordinary agarose, low melting point agarose, low electroosmotic agarose, high strength agarose, Seakem HE agarose, Seakem HEEO agarose, SeaKem ME agarose, SeaKem HGT agarose, SeaKem HGT (P) agarose and IsoGel agarose; the phosphate buffer with a pH of 7.5-7.8 is 50-200 mmol / L, a PBK (KH2PO4 / K2HPO4) buffer with a pH of 7.5-7.8 or a PBS (Na2HPO4 / KH2PO4) buffer with a pH of 7.5-7.

8.

6. The method for preparing a high temperature storage urine galactose kit according to claim 5, characterized in that: The method of preparing the substrate solution R3 using the microcrystalline cellulose buffer R1 as a solvent comprises: The galactosidase substrate is added into the microcrystalline cellulose buffer R1 and fully dissolved to form a substrate solution R3, wherein the formed substrate solution R3 contains 0.1-0.5 g / mL of the galactosidase substrate; the galactosidase substrate is one or more of O-nitro-β-D-pyranoside (ONPG), 5-bromo-4-chloro-3-indole-β-D-pyranoside (x-gal), chlorophenol red-β-D-pyranoside (CPRG), PNPG and IPTG.

7. The method for preparing a high temperature storage urine galactose kit according to claim 6, characterized in that: The S2 preparation of a dry chemical reaction reagent pad for detecting urine galactose comprises: (1) solidifying the reaction solution R2 on a first supporting carrier; (2) subjecting the reaction solution R2 in solid phase on the first supporting carrier to variable temperature drying under a first condition; wherein the first condition includes a gas condition, a variable temperature range, and a drying time; in the first condition, the gas condition is a dry inert gas condition; the variable temperature range is 15-35°C, and the drying time is 5 hours.

8. The method for preparing a high temperature storage urine galactose kit according to claim 7, characterized in that: The step S2 of preparing the substrate pad comprises: (1) solidifying the substrate solution R3 on a second support carrier; (2) subjecting the substrate solution R3 in solid phase on the second support carrier to variable temperature drying under second conditions; wherein the second conditions include gas conditions, variable temperature range and drying time; in the second conditions, the gas conditions are dry inert gas conditions; the variable temperature range is 15-35°C, and the drying time is 5 hours.

9. The method for preparing a high temperature storage urine galactose kit according to claim 8, characterized in that: The first supporting carrier and the second supporting carrier are both one or more of hydroxypropyl methylcellulose and methylcellulose.

10. A high temperature storage urine galactose kit prepared by the preparation method of the high temperature storage urine galactose kit according to any one of claims 1 to 9, characterized in that: include: Reaction apparatus, urine purification and dosing apparatus, and calibration reagents; The reaction device is provided with a reaction hole and a calibration hole, and the reaction hole and the calibration hole are both provided with a visualization window; the reaction reagent is solidified in the reaction hole and the calibration hole, and the reaction reagent is obtained by drying the reaction solution R2 in an inert gas at 15°C-35°C for 5 hours; The urine purification and sample addition device is used to purify the original urine of the subject; an activated carbon adsorbent is arranged in the urine purification device; the body of the urine purification and sample addition device is a rectangular structure and is made of an inert material; The calibration reagent is used to be added dropwise to the calibration well when performing urine galactose detection, and is used to compare the color development with that of the reaction well after color development; The reaction reagents include a buffer solution, an enzyme label solution and a substrate solution R3; The buffer is a PBK (KH2PO4 / K2HPO4) buffer with a pH of 7.5-7.8 and a 50-200 mmol / L buffer. The buffer contains 3% g / V microcrystalline cellulose, 3-5% g / V agarose and 0.1-2% g / V surfactant. The microcrystalline cellulose includes one or more of methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose and carboxytetramethyl cellulose. The agarose is one or more of ordinary agarose, low melting point agarose, low electroosmotic agarose, high strength agarose, Seakem HE agarose, Seakem HEEO agarose, SeaKem ME agarose, SeaKem HGT agarose, SeaKem HGT (P) agarose and IsoGel agarose. The enzyme labeling solution is prepared by using the microcrystalline cellulose buffer R1 as a solvent, adding galactose oxidase, peroxidase and a color developer; the galactose oxidase is one or more of galactose hydrolyzing oxidase and β-galactosidase; the peroxidase is one or more of horseradish peroxidase, superoxide dismutase (SOD), peroxidase (GPX), aldehyde oxidase activity (GR), hydroxyperoxidase (GAT) and lactoperoxidase; the color developer is one or more of 4-aminoantipyrine and 3-methyl-2-thiazolinonehydrazone hydrochloride hydrate (MBTH); The substrate solution R3 uses the microcrystalline cellulose buffer R1 as a solvent, and a galactosidase substrate is added to fully dissolve the substrate solution R3, wherein the substrate solution R3 contains 0.1 g / mL of a galactosidase substrate, and the substrate solution R3 contains one or more of O-nitro-β-D-pyranoside (ONPG), 5-bromo-4-chloro-3-indole-β-D-pyranoside (x-gal), chlorophenol red-β-D-pyranoside (CPRG), PNPG, and IPTG; The calibration reagent is a 1.0mmol / L D-galactose standard solution, which is prepared by adding D-galactose and sodium chloride into purified water and dissolving them; the standard reagent includes the following components: 0.15-0.2g / L of 1.0mmol / L D-galactose standard solution and 0.8-1.0g / L of sodium chloride are added into purified water and dissolved; the solvent of the standard reagent is an artificial urine base solution; the artificial urine base solution includes: 1-3g of disodium hydrogen phosphate, 0.75-1g of potassium dihydrogen phosphate, 1g of urea 0-12g, creatinine 1.0-1.5g, glucose 10-15g, thimerosal 0-1.5g, disodium hydrogen phosphate 2-3g, potassium dihydrogen phosphate 0.5-0.75g, sodium chloride 7-10g, urea 7-10g, creatinine 0.7-1.0g, glucose 15-20g, thimerosal 0.5-1g, bilirubin 0.2-1.0g, distilled water 500ml, and use NaOH to adjust the standard reagent to a pH of 5.7.

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