Preparation method of kit for detecting D-lactic acid, kit and detection method

Abstract

The embodiment of the invention belongs to the technical field of detection, and relates to a preparation method of a kit for detecting D-lactic acid, the kit and a detection method, and the preparation method of the kit for detecting D-lactic acid comprises the following steps: preparing a detection reagent solution; the detection reagent solution comprises a buffer solution with the concentration of 20 to 500 mmo l / L, a stabilizer with the concentration of 1 to 4000 mmo l / L, NAD < + > with the concentration of 1 to 6 mmo l / L and D-lactic dehydrogenase with the concentration of 1000 to 80000 U / L. The method is high in measuring speed and high in accuracy.

Description

Technical Field

[0001] The present application relates to the field of detection technology, and in particular to a method for preparing a kit for detecting D-lactic acid, the kit and a detection method. Background Art

[0002] Lactic acid (C3H603, 2-hydroxypropionic acid) is a natural metabolite of all organisms. In 1780, Swedish chemist Scheele discovered and isolated it from milk. Since the C2 carbon of lactic acid is asymmetric, it has two enantiomers, L-lactic acid and D-lactic acid, which have similar physical and chemical properties. Under normal physiological conditions in the human body, it usually exists in the form of lactate ions. The main metabolic site of L-lactic acid in the human body is the liver, where it is metabolized to pyruvate by L-lactate dehydrogenase (L-LDH). Due to the general lack of D-lactate dehydrogenase (D-LDH) in mammalian cells, it is generally believed that mammals cannot effectively metabolize D-lactic acid and can only excrete D-lactic acid in urine.

[0003] The main sources of D-lactic acid in the human body can be divided into endogenous and exogenous. Exogenous D-lactic acid comes from daily diet (especially fermented food). Studies have shown that consuming a large amount of yogurt may lead to an increase in the D-lactic acid content in plasma). Another way to obtain exogenous D-lactic acid is that D-lactic acid produced by intestinal microbial fermentation is absorbed by the human intestine. The main endogenous source of d-lactic acid is produced by the methylglyoxal (MG) pathway. MG is produced by a small part of fat, carbohydrate and protein metabolites. MG in the human body is mainly degraded by the glyoxalase system, which consists of glyoxalase I (S-lactylglutathione methylglyoxal lyase), glyoxalase II (S-2-hydroxyglutathione hydrolase) and reduced glutathione (GSH). Glyoxalase I catalyzes the coupling of MG and glutathione (GSH) to generate the intermediate SD-lactylglutathione, and glyoxalase II decomposes SD-lactylglutathione to generate GSH and D-lactic acid. Therefore, in mammals, the amount of D-lactic acid is considered a biological indicator of the amount of MG in the body. Lactic acid is widely distributed in the human body and is also present in many of the foods we eat.

[0004] In addition, it is known that the level of delactate in human tissues is closely associated with various disease conditions. It has been confirmed that changes in the concentration of D-lactic acid in the human body are associated with certain diseases to a certain extent. The current methods for measuring plasma and whole blood lactate can only detect the L-lactic acid isomer. However, in many research experiments and other situations, it is important to measure D-lactic acid. D-lactic acidosis (plasma D-lactic acid>3.0mmol / L, blood pH<7.35) is one of the complications of short bowel syndrome, secondary to diffuse small bowel disease or resection. Due to defective carbohydrate absorption, the substrate delivery to carbohydrate fermenting bacteria in the large intestine increases. These bacteria can produce D-lactic acid and L-lactic acid, which are absorbed. L-LDH in the body can metabolize L-lactic acid, but humans have a limited ability to metabolize D-lactic acid, resulting in metabolic acidosis. The level of D-lactic acid required to cause acidemia is more than 100 times the level of healthy people. D-lactic acidosis manifests as neurological dysfunction and encephalopathy. The L-lactic acid level in these people is usually normal. In addition, the plasma D-lactic acid level of people with DKA (diabetic ketoacidosis) was significantly higher than that of the healthy control group.

[0005] Moreover, in the food industry, D-lactic acid is a potentially useful quality indicator. For example: an increase in D-lactic acid concentration in beer may indicate bacterial contamination that can lead to undesirable changes in taste. D-lactic acid can also be a good indicator of the quality of wine, milk, meat and juice. Therefore, D-lactic acid content is of great significance in food analysis. Therefore, determining the concentration of D-lactic acid in biological samples is of great value.

[0006] Since the physical and chemical properties of the two isomers of D-lactic acid and L-lactic acid are similar, it is difficult to separate and determine the isomers of lactic acid in biological samples. In recent years, several analytical techniques have been developed and applied to the detection and separation of D- and L-lactic acid, including high performance liquid chromatography (HPLC) detection, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS) and capillary electrophoresis (CE). There are also enzymatic reactions using ultraviolet and fluorescence methods. Common D-lactic acid detection methods include:

[0007] (1) Enzymatic reaction is a relatively simple method for detecting D-lactic acid. Its basic principle is to detect D-lactic acid and NAD + Pyruvate NADH is generated under the catalysis of D-lactate dehydrogenase. The concentration of D-lactate in the sample can be determined by detecting the change in absorbance of reduced nicotinamide adenine dinucleotide (NADH) at a wavelength of 340nm.

[0008] (2) The high performance liquid chromatography (HPLC) detection method is to separate the two chiral molecules, D-lactic acid and L-lactic acid, by adding a chiral chromatographic column and utilizing the different interaction forces between the stationary phase filled in the chromatographic column and the chiral molecules.

[0009] (3) Capillary electrophoresis (CE) method: The principle is to add a chiral additive (CA) to the electrophoresis buffer. CA binds to the enantiomers through electrostatic attraction and non-covalent bonds such as hydrogen bonds to form reversible diastereomeric complexes with different stabilities. The chiral molecules are then separated in the chiral separator by electrophoresis.

[0010] Although HPLC, LC-MS or GC-MS methods show good sensitivity, accuracy and reproducibility for D-lactic acid determination, these chromatographic methods are time-consuming and require expensive instrumentation, making them unsuitable for widespread application. Summary of the invention

[0011] The purpose of the embodiments of the present application is to provide a preparation method of a kit for detecting D-lactic acid, a kit and a detection method, which have fast determination speed and high accuracy.

[0012] In order to solve the above technical problems, the present application provides a method for preparing a kit for detecting D-lactic acid, which adopts the following technical solution:

[0013] A method for preparing a kit for detecting D-lactic acid comprises the following steps:

[0014] preparing a detection reagent solution;

[0015] The detection reagent solution comprises 20-500 mmol / L of buffer, 1-4000 mmol / L of stabilizer, 1-6 mmol / L of NAD+ and 1000-80000 U / L of D-lactate dehydrogenase.

[0016] Furthermore, the stabilizer includes at least one of ammonium sulfate, glycerol, propylene glycol, ethylene glycol and a preservative.

[0017] Furthermore, the buffer comprises glycine-sodium hydroxide.

[0018] Furthermore, after the step of preparing the detection reagent solution, the method further comprises:

[0019] The detection reagent solution is dried, and after the drying process, each substance in the detection reagent solution becomes a dry powder state.

[0020] In order to solve the above technical problems, the present application also provides a kit for detecting D-lactic acid, which adopts the following technical solution:

[0021] A kit for detecting D-lactic acid, prepared using the preparation method of the kit for detecting D-lactic acid, the kit comprising a detection reagent solution;

[0022] The detection reagent solution comprises 20-500 mmol / L of buffer, 1-4000 mmol / L of stabilizer, 1-6 mmol / L of NAD+ and 1000-80000 U / L of D-lactate dehydrogenase.

[0023] Furthermore, when the kit is a single-dose kit, the detection reagent solution includes 100 mmol / L of the buffer, 500 mmol / L of the stabilizer, 3 mmol / L of the NAD+ and 12000 U / L of the D-lactate dehydrogenase.

[0024] Furthermore, when the kit is a two-dose kit, the detection reagent solution includes a first detection reagent and a second detection reagent, and the stabilizer includes a first stabilizer and a second stabilizer;

[0025] The first detection reagent includes 100 mmol / L of the buffer, 50 mmol / L of the first stabilizer and 3 mmol / L of NAD+;

[0026] The second detection reagent includes the 100 mmol / L buffer, 500 mmol / L of the second stabilizer and 12000 U / L of D-lactate dehydrogenase.

[0027] In order to solve the above technical problems, the present application also provides a method for detecting D-lactic acid for non-diagnostic purposes, which adopts the following technical solution:

[0028] A method for detecting D-lactic acid using the above-mentioned kit comprises the following steps:

[0029] The sample to be tested is uniformly mixed with the detection reagent solution to react to obtain a reactant solution;

[0030] The reactant solution is placed in a biochemical analyzer, and the speed or degree of the increase in the absorbance of the main wavelength is detected to calculate the concentration of D-lactic acid in the sample to be tested.

[0031] Furthermore, when the reagent in the kit is a single-dose kit, the volume ratio of the sample to be tested to the detection reagent solution is 1:25.

[0032] Further, when the reagents in the kit are two-dose kits, the detection reagent solution includes a first detection reagent and a second detection reagent;

[0033] The volume ratio of the sample to be tested, the first detection reagent and the second detection reagent is 2:20:5.

[0034] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0035] The present application proposes a method for measuring or continuously monitoring the change in absorbance of reduced nicotinamide adenine dinucleotide (NADH) at a wavelength of 340 nm using a D-lactic acid-specific NADH fluorescence method, thereby determining the concentration of D-lactic acid. The present application also provides a D-lactic acid determination kit for implementing the method. The reagent can not only be used to determine the concentration of D-lactic acid on an ultraviolet light analyzer, a visible light analyzer, a semi-automatic biochemical analyzer or a fully automatic biochemical analyzer, but also has a fast determination speed and high accuracy, so it can be effectively promoted and applied. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the scheme in the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments of the present application. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 is a flow chart of an embodiment of a method for detecting D-lactic acid according to the present application;

[0038] Figure 2 is a linear graph of the detection results of one embodiment of the kit for qualitatively detecting D-lactic acid in a serum sample of the present application;

[0039] Figure 3 It is a linear graph of the detection results of another embodiment of the kit for qualitatively detecting D-lactic acid in serum samples of the present application. DETAILED DESCRIPTION

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application; the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of the present application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0041] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0042] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0043] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0044] The principle of the method of this application is as follows:

[0045]

[0046] According to the above chemical formula, D-lactic acid and NAD can generate pyruvic acid and NADH under the action of D-lactate dehydrogenase.

[0047] Based on the above principle, the present application provides a method for preparing a kit for detecting D-lactic acid, comprising the following steps:

[0048] preparing a detection reagent solution;

[0049] The detection reagent solution comprises 20-500 mmol / L of buffer, 1-4000 mmol / L of stabilizer, 1-6 mmol / L of NAD+ and 1000-80000 U / L of D-lactate dehydrogenase.

[0050] As an option in the present application, the stabilizer includes at least one of ammonium sulfate (Ammonia Sulfate), glycerol (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and a preservative.

[0051] As an option in the present application, the buffer comprises glycine-sodium hydroxide.

[0052] As an option of the present application, after the step of preparing the detection reagent solution, the following step is further included:

[0053] The detection reagent solution is dried, and after the drying process, each substance in the detection reagent solution becomes a dry powder state.

[0054] The present application also provides a kit for detecting D-lactic acid, which is prepared using the preparation method of the kit for detecting D-lactic acid, and the kit includes a detection reagent solution; the detection reagent solution includes 20 to 500 mmol / L of buffer, 1 to 4000 mmol / L of stabilizer, 1 to 6 mmol / L of NAD+ and 1000 to 80000 U / L of D-lactate dehydrogenase. Specifically as shown in Table 1:

[0055] Table 1 Concentration range of each substance in the kit

[0056] substance concentration Buffer 20~500mmol / L Stabilizer 1~4000mmol / L NAD+ 1~6mmol / L D-Lactate dehydrogenase 1000~80000U / L

[0057] The present kit is specifically a kit for qualitatively detecting D-lactic acid in serum samples, and the sample applicable to the kit of the present application is serum.

[0058] The substances in the kit can be in the form of dry powder, which can be dissolved in water before use; or they can be prepared into liquid reagents, packaged in the kit, and used directly.

[0059] The kits of the present application include single-dose kits and double-dose kits.

[0060] As an option of the present application, when the kit is a single-dose kit, the detection reagent solution includes 100 mmol / L of the buffer, 500 mmol / L of the stabilizer, 3 mmol / L of NAD+ and 12000 U / L of D-lactate dehydrogenase. Specific details are shown in Table 2.

[0061] Table 2 Concentration of substances in single dose reagents

[0062] Substance (single dose reagent) concentration Buffer (Glycine-Sodium Hydroxide) 100mmol / L Stabilizer (glycerol or ammonium sulfate) 500mmol / L NAD+ 3mmol / L D-Lactate dehydrogenase 12000U / L

[0063] In addition, a single dose of reagent can be in solution state or dry powder state. The dry powder state is: after all the reagents are dissolved and prepared, they are divided into bottles and freeze-dried to make dry powder reagents; before use, pure water needs to be added and the reagents need to be reconstituted before use.

[0064] As an option of the present application, when the kit is a two-dose kit, the detection reagent solution includes a first detection reagent and a second detection reagent; the stabilizer includes a first stabilizer and a second stabilizer; the first detection reagent includes 100mmol / L of the buffer, 50mmol / L of the first stabilizer and 3mmol / L of the NAD+; the second detection reagent includes 100mmol / L of the buffer, 500mmol / L of the second stabilizer and 12000U / L of the D-lactate dehydrogenase. Among them, the first stabilizer includes glycerol, and the second stabilizer includes ammonium sulfate. As shown in Tables 3 and 4:

[0065] Table 3 Concentration of each substance in the first detection reagent

[0066] Substance (first test reagent of two-dose test kit) concentration Buffer (Glycine-Sodium Hydroxide) 100mmol / L First stabilizer (glycerol) 50mmol / L NAD+ 3mmol / L

[0067] Table 4 Concentration of each substance in the second detection reagent

[0068] Substance (Second Detection Reagent of Dual-Dose Test) concentration Buffer (Glycine-Sodium Hydroxide) 100mmol / L Second stabilizer (ammonium sulfate) 500mmol / L D-Lactate dehydrogenase 12000U / L

[0069] The two-dose reagent consists of a buffer, a stabilizer, NAD+, and D-lactate dehydrogenase. After all the reagents are dissolved and prepared, they are divided into bottles to make a liquid two-dose reagent that can be used directly.

[0070] The present application also includes coenzyme A and pyruvate dehydrogenase. In the case of double reagents, coenzyme A and pyruvate dehydrogenase are in the first detection reagent (or referred to as: reagent 1); D-lactate dehydrogenase is in the second detection reagent (or referred to as: reagent 2).

[0071] The present application also provides a method for detecting D-lactic acid, using the above-mentioned kit, comprising the following steps:

[0072] S1: uniformly mixing the sample to be tested with the detection reagent solution to react and obtain a reactant solution;

[0073] S2: placing the reactant solution in a biochemical analyzer, detecting the speed or degree of the increase in the absorbance at the main wavelength, and calculating the concentration of D-lactic acid in the sample to be tested.

[0074] As an option of the present application, when the reagents in the kit are single-dose kits, the volume ratio of the sample to be tested to the detection reagent solution is 1:25.

[0075] As an option of the present application, when the reagents in the kit are a two-dose kit, the detection reagent solution includes a first detection reagent and a second detection reagent;

[0076] The volume ratio of the sample to be tested, the first detection reagent and the second detection reagent is 2:20:5.

[0077] This application specifically includes the following embodiments:

[0078] Example 1

[0079] When the kit is a single-dose kit, the components are shown in the following table:

[0080] Table 5 The kit is a single-dose kit

[0081] Single dose reagent concentration Buffer (Glycine-Sodium Hydroxide) 100mmol / L Ammonium sulfate 500mmol / L NAD+ 3mmol / L D-Lactate dehydrogenase 12000U / L

[0082] When the detection reagent solution of the single-dose test kit is in a solution state, it can be used directly. When the detection reagent solution is dried to a dry powder state, it needs to be added with purified water first and then reconstituted before use.

[0083] The following settings are made on a biochemical analyzer (e.g., a fully automatic biochemical analyzer): temperature 37°C, reaction time 10 minutes, initial absorbance ≤ 0.1, test main wavelength 340 nm, test sub-wavelength 405 nm, volume ratio of the test sample (or referred to as: test D-lactic acid sample) to the test reagent solution is 1:25, reaction direction is positive reaction (i.e., ascending reaction), delay time is approximately 0 minutes, and detection time is approximately 5 minutes.

[0084] The sample to be tested is mixed with the detection reagent solution to react and obtain a reactant solution; the reactant solution is placed in a biochemical analyzer to detect the degree or speed of the absorbance increase at the main wavelength of 340nm, thereby calculating the concentration of D-lactic acid in the sample to be tested.

[0085] Example 2

[0086] When the kit is a two-dose kit, the components are shown in the following table:

[0087] Table 6 The kit is a double-dose kit

[0088] The first test reagent of the two-dose test kit concentration Glycine-Sodium Hydroxide 100mmol / L glycerin 50mmol / L NAD+ 3mmol / L Second test reagent for dual-dose test kit concentration Glycine-Sodium Hydroxide 100mmol / L Ammonium sulfate 500mmol / L D-Lactate dehydrogenase 12000U / L

[0089] The detection reagent solution is in a liquid state and can be used directly.

[0090] The following settings are made on a biochemical analyzer (e.g., a fully automatic biochemical analyzer): temperature 37° C., reaction time 10 minutes, initial absorbance ≤ 0.1, test main wavelength 340 nm, test sub-wavelength 405 nm, volume ratio of the test sample (or referred to as: test D-lactic acid sample), the first detection reagent and the second detection reagent is 2:20:5, reaction direction is positive reaction (i.e., ascending reaction), delay time is about 0 minutes, and detection time is about 5 minutes.

[0091] The sample to be tested is mixed with the detection reagent solution to react and obtain a reactant solution; the reactant solution is placed in a biochemical analyzer to detect the degree or speed of the absorbance increase at the main wavelength of 340nm, thereby calculating the concentration of D-lactic acid in the sample to be tested.

[0092] The present invention mixes the sample to be tested with the detection reagent solution in a certain volume ratio to cause a series of enzymatic reactions, and then places the reactants in an ultraviolet light analyzer, a visible light analyzer, a semi-automatic biochemical analyzer or a fully automatic biochemical analyzer. The NADH generated by the reaction is proportional to the D-lactic acid content in the sample. The D-lactic acid content can be measured by measuring the NADH generation rate (i.e., the degree or speed of the absorbance increase) at the main wavelength of 340nm.

[0093] The present application also provides the test data of Example 1 and Example 2, wherein the linearity results of the test kit are as shown in the following table and Figure 2 As shown, Figure 2 This is a linear graph of the detection results of an embodiment of the kit for qualitatively detecting D-lactic acid in serum samples of the present application, specifically a linear graph of the detection results of Example 1:

[0094] Table 7 Linearity results

[0095]

[0096] The above results show that Examples 1 and 2 have good linearity. Figure 1 As shown, the linear equation of Example 1 is y=1.0015x+15.008, R2=0.9998; Figure 2 The linear equation of Example 2 shown is y=1.0068x+16.094, R2=0.9998.

[0097] The reagents prepared in Example 1 and Example 2 and the purchased finished reagents were opened at 2-8°C for 15 days, and the high and low values ​​were tested. The results are shown in the following table:

[0098] Table 8 Opening stability

[0099]

[0100] The above results show that the bottle opening stability of Example 1 and Example 2 is good.

[0101] 20 clinical samples were tested, including 10 plasma samples from healthy people and 10 plasma samples from patients with short bowel syndrome. The test results are shown in the following table:

[0102] Table 9 Clinical comparison

[0103]

[0104] The above results were all low values ​​in the plasma of 10 healthy people, and high values ​​in the plasma of 10 patients with short bowel syndrome. The yin and yang judgments of clinical samples were basically good.

[0105] Beneficial effects of this application:

[0106] 1) The present application proposes a method for measuring or continuously monitoring the change in absorbance of reduced nicotinamide adenine dinucleotide (NADH) at a wavelength of 340 nm using a D-lactic acid-specific NADH fluorescence method to thereby determine the concentration of D-lactic acid.

[0107] 2) The present application also provides a D-lactic acid determination kit for implementing the method. The reagent can not only be used to determine the D-lactic acid concentration on an ultraviolet analyzer, a visible light analyzer, a semi-automatic biochemical analyzer or a fully automatic biochemical analyzer, but also has a fast determination speed and high accuracy, and can therefore be effectively promoted and applied.

[0108] The above description discloses the product, purpose, method of use and advantages of the present invention. It should be pointed out that the above embodiments are only for the purpose of clearly illustrating the examples given in this application and cannot be used to limit the claims of this patent. For those skilled in the art, certain improvements and substitutions can be made without departing from the spirit and principle of the present invention, which are all within the scope of protection required by the present invention.

[0109] It should be understood that, although the steps in the flowchart of the accompanying drawings are displayed in sequence as indicated by the arrows, these steps are not necessarily executed in sequence in the order indicated by the arrows. Unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least a part of the steps in the flowchart of the accompanying drawings may include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn or alternately with other steps or at least a part of the sub-steps or stages of other steps.

[0110] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A method for preparing a kit for detecting D-lactic acid, characterized in that: The following steps are involved: preparing a detection reagent solution; The detection reagent solution comprises 20-500 mmol / L of buffer, 1-4000 mmol / L of stabilizer, 1-6 mmol / L of NAD+ and 1000-80000 U / L of D-lactate dehydrogenase.

2. The method for preparing a kit for detecting D-lactic acid according to claim 1, characterized in that: The stabilizer includes at least one of ammonium sulfate, glycerin, propylene glycol, ethylene glycol and a preservative.

3. The method for preparing a kit for detecting D-lactic acid according to claim 1, characterized in that: The buffer comprises glycine-sodium hydroxide.

4. The method for preparing a kit for detecting D-lactic acid according to claim 1, characterized in that: After the step of preparing the detection reagent solution, the method further comprises: The detection reagent solution is dried, and after the drying process, each substance in the detection reagent solution becomes a dry powder state.

5. A kit for detecting D-lactic acid, prepared using the method for preparing a kit for detecting D-lactic acid according to any one of claims 1 to 4, characterized in that: The kit includes a detection reagent solution; The detection reagent solution comprises 20-500 mmol / L of buffer, 1-4000 mmol / L of stabilizer, 1-6 mmol / L of NAD+ and 1000-80000 U / L of D-lactate dehydrogenase.

6. The kit according to claim 5, characterized in that When the kit is a single-dose kit, the detection reagent solution includes 100 mmol / L of the buffer, 500 mmol / L of the stabilizer, 3 mmol / L of the NAD+ and 12000 U / L of the D-lactate dehydrogenase.

7. The kit according to claim 5, characterized in that When the kit is a two-dose kit, the detection reagent solution includes a first detection reagent and a second detection reagent, and the stabilizer includes a first stabilizer and a second stabilizer; The first detection reagent includes 100 mmol / L of the buffer, 50 mmol / L of the first stabilizer and 3 mmol / L of NAD+; The second detection reagent includes the 100 mmol / L buffer, 500 mmol / L of the second stabilizer and 12000 U / L of D-lactate dehydrogenase.

8. A method for detecting D-lactic acid for non-diagnostic purposes, using the kit according to any one of claims 5 to 7, characterized in that: The steps include: The sample to be tested is uniformly mixed with the detection reagent solution to react to obtain a reactant solution; The reactant solution is placed in a biochemical analyzer, and the speed or degree of the increase in the absorbance of the main wavelength is detected to calculate the concentration of D-lactic acid in the sample to be tested.

9. The method for detecting D-lactic acid according to claim 8, characterized in that: When the reagent in the kit is a single-dose kit, the volume ratio of the sample to be tested to the detection reagent solution is 1:

25.

10. The method for detecting D-lactic acid according to claim 8, characterized in that: When the reagents in the kit are a two-dose kit, the detection reagent solution includes a first detection reagent and a second detection reagent; The volume ratio of the sample to be tested, the first detection reagent and the second detection reagent is 2:20:5.

Images