Diagnostic method and reagent kit for MIDD

Abstract

The present invention relates to a method and a reagent kit for diagnosing MIDD. Specifically, a method for diagnosing MIDD is provided, which includes (1) detecting urinary lactate and (2) comparing the detected lactate with a control level. The method of the present invention is simple and rapid, provides highly accurate results, and allows for the immediate detection of MIDD patients.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from Chinese Patent Application No. 202211084715.0 filed on September 6, 2022, the contents of which are incorporated herein by reference in their entirety. [Technical Field]

[0002] The present invention belongs to the field of biotechnology, and specifically relates to a method and a reagent kit for diagnosing MIDD. [Background technology]

[0003] Diabetes mellitus (DDM) is a chronic disease caused by abnormalities in the body's glucose metabolism and is typically characterized by hyperglycemia. According to data from the International Diabetes Federation, as of 2019, the number of diabetes patients among the population aged 20 to 79 reached approximately 463 million, of which China had the highest number of diabetes patients in the world, at approximately 116.4 million. Diabetes is currently incurable, and patients require regular blood glucose monitoring, with some even requiring lifelong insulin injections or hypoglycemic medication. Therefore, diabetes has become one of the major diseases affecting the quality of life of the Chinese population. Diabetes can be classified based on its etiology into T1DM, T2DM, gestational diabetes, and specific diabetes. Among these, T2DM is the predominant type of diabetes in China, accounting for 53% of the total. Diabetes is a disease caused by an abnormality in the glucose metabolism regulation mechanism in the human body, and lactate is the end product of the glycolytic pathway in the human body. Currently, many literatures have reported that diabetic patients have abnormal lactate metabolism, but the relationship between these two has not yet been clearly elucidated. Summary of the Invention

[0004] Based on the deficiencies of the existing technology, a first aspect of the present invention provides a reagent for detecting lactate in a urine sample and its use in diagnosing MIDD. A first aspect of the present invention provides the use of a reagent for detecting lactate in a urine-derived sample, and an optional lactate standard, in the manufacture of a reagent kit for use in the diagnosis of MIDD (mitochondrial m.3243A>G mutation diabetes) or for the identification of MIDD in diabetic patients. In one or more embodiments, the lactate detection reagent comprises reagents necessary for detecting lactate using one or more methods selected from a chromatographic method, a titration method, a colorimetric method, an enzymatic analysis method, and an optical probe method. In one or more embodiments, the lactate detection reagents include a urine treatment reagent, such as 3NPH_HCl, EDC, and / or a buffer, which makes the lactate in the sample suitable for subsequent detection. In one or more embodiments, the lactate detection reagent comprises a reagent used for converting, concentrating, separating, or identifying lactate, and preferably comprises one or more selected from the group consisting of an antibody, lactate dehydrogenase, lactate oxidase, lactate binding protein, or functional variants thereof. In one or more embodiments, the reagent for detecting lactate comprises one or more selected from formic acid, acetonitrile, and isopropanol. In one or more embodiments, the functional variant of a lactate binding protein comprises a lactate optical probe.

[0005] In one or more embodiments, the lactate optical probe is selected from Laconic, Green Lindoblum, GEM-IL, eLACCO1.1, and LiLac. In one or more embodiments, the lactate optical probe comprises a lactate binding protein and an optically active polypeptide fused together, wherein the optically active polypeptide is located within the sequence of the lactate binding protein, at the N-terminus, or at the C-terminus. In one or more embodiments, the lactate optical probe is as described in any of the embodiments of CN202011516287.5. In one or more embodiments, the lactate level in a sample from a MIDD patient is significantly elevated compared to a control level, which is derived from the lactate level in a corresponding sample from a non-MIDD patient or a healthy individual. In one or more embodiments, the control level is 117±23 mμM or greater.

[0006] In a second aspect, the present invention provides a reagent kit for detecting lactic acid in urine, which comprises a lactic acid detection reagent and a urine treatment reagent. In one or more embodiments, the reagent for detecting lactate is as described in the first aspect of the present specification. In one or more embodiments, the urine treatment reagent includes, but is not limited to, 3NPH_HCl, EDC, and a buffer. In one or more embodiments, the buffer is a phosphate buffer or Tris, such as HEPES, PBS, or the like. In one or more embodiments, the reagent kit further comprises a lactate standard. In one or more embodiments, the lactate detection reagent includes a reagent used for converting, concentrating, separating, or identifying lactate, and preferably includes one or more of an antibody, lactate dehydrogenase, lactate oxidase, lactate binding protein or a functional variant thereof, formic acid, acetonitrile, and isopropanol.

[0007] The present invention further provides a reagent kit for detecting lactate in urine, comprising a lactate optical probe according to any embodiment of the first aspect of the present specification and a buffer solution. In one or more embodiments, the buffer is a phosphate buffer or Tris, such as HEPES, PBS, or the like. In one or more embodiments, the reagent kit further comprises a reagent for detecting the mitochondrial m.3243A>G mutation, a reagent for detecting blood glucose in a subject, and / or a reagent for detecting urinary glucose in a subject.

[0008] The present invention also provides a method for diagnosing MIDD or identifying MIDD in a diabetic patient, comprising (1) detecting urinary lactate and (2) comparing with a control level, wherein the urinary lactate level in a MIDD patient is higher than the control level. In one or more embodiments, the step of detecting lactate detects lactate using one or more methods selected from a chromatographic method, a titration method, a colorimetric method, an enzymatic analysis method, and an optical probe method. In one or more embodiments, the optical probe method includes mixing a urine sample with a lactate optical probe, measuring the fluorescence intensity, and determining the lactate content based on the fluorescence intensity. In one or more embodiments, the urinary lactate level of MIDD patients is significantly elevated compared to a control level, which is derived from the lactate level in a corresponding sample from a non-MIDD patient or a healthy individual. Preferably, the control level is 117±23 μM or greater. In one or more embodiments, the lactate optical probe comprises a lactate binding protein fused with an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the lactate binding protein, at the N-terminus, or at the C-terminus. Preferably, the lactate optical probe is one described in any of the embodiments of PCT / CN2020 / 137900. [Brief explanation of the drawings]

[0009] The present invention will be specifically described below with reference to the drawings and examples. [Figure 1] The clinical characteristics of healthy subjects, LADA, T2DM, and MIDD are shown herein. [Figure 2] 1 is a flow chart of a probe-based method for real-time detection of clinical samples. [Figure 3(A)] Serum test results for probe FiLa-H showing serum lactate levels. [Figure 3(B)] This shows the results of a serum test using the probe FiLa-H, and is a comparison of the detection results between UHPLC-MS and the probe FiLa-H. [Figure 4(A)] 10 is a serum test result for probe FiLa showing serum lactate levels. [Figure 4(B)] This shows the results of a serum test using the probe FiLa, and is a comparison of the detection results between UHPLC-MS and the probe FiLa. [Figure 5(A)] 10 is a urine test result for probe FiLa-H showing urinary lactate levels. [Figure 5(B)] FIG. 1 shows the results of a urine test using the probe FiLa-H, and is a comparison of the detection results between UHPLC-MS and the probe FiLa-H. [Figure 6] FIG. 1 compares lactate levels in three diseased samples and healthy samples. [Figure 7] 1 is a comparison of lactate levels in three disease samples. DETAILED DESCRIPTION OF THE INVENTION

[0010] As used herein, when referring to a numerical value or range, the term "about" means that the numerical value or range is within 20%, within 10%, and within 5% of the stated numerical value or range. As used herein, the terms "comprise", "comprise" and their equivalents include "contain" and "consist of", for example, a composition "comprising" X means that the composition may consist of X alone, or may include other substances, e.g., X+Y. Because there is no significant difference in the age of onset or clinical symptoms, patients with MIDD (a specific subtype of diabetes caused by the mitochondrial m.3243A>G mutation) are often misdiagnosed as T1DM or T2DM. We analyzed changes in lactate levels in serum and urine samples from patients with adult latent autoimmune diabetes (LADA) (a subtype of T1DM), T2DM (type 2 diabetes), and MIDD (a specific subtype of diabetes caused by the mitochondrial m.3243A>G mutation). The present invention found that elevated urinary lactate levels are closely associated with MIDD. Therefore, lactate can be used as a potential screening biomarker for this disease. Reassessing these clinical symptoms of diabetes (abnormally elevated urinary lactate and blood glucose) can facilitate rapid screening for MIDD. Thus, the present invention provides a method for diagnosing MIDD or identifying MIDD in diabetic patients, comprising (1) detecting urinary lactate and (2) comparing with a control level, wherein the urinary lactate level in MIDD patients is higher than the control level, and in particular, the urinary lactate level in MIDD patients is significantly elevated compared to the control level. As used herein, the term "control level" refers to a reference urinary lactate level that serves as a diagnostic standard. Such a level can be obtained by comparing a sample from a MIDD subject with a sample from a healthy subject or a non-MIDD subject. Furthermore, the control level can be a level from a healthy subject or a non-MIDD subject. The control level can be obtained from a single subject or a group of at least two or more individuals. Those skilled in the art can select an appropriate reference level depending on the desired sensitivity and specificity. In an exemplary embodiment, the control level is a urinary lactate level of 117±23 μM or higher.

[0011] In an exemplary embodiment, lactate is detected using chromatography and probe methods. However, those skilled in the art will understand that other lactate detection methods in this area can also be used in the present invention. For example, titration, colorimetry, enzymatic analysis, optical probe methods, etc. Other lactate detection methods are within the knowledge of those skilled in the art. Those skilled in the art will also be familiar with the lactate detection procedures and reagents used in these methods. Illustrative examples of the above-mentioned methods include chromatographic methods (e.g., HPLC), titration methods (Biomedical Chromatography 2012, 26(11):1408-1415; Food Chemistry 2012, 135(3):1078-1082), colorimetric methods, hydrogen peroxide detection methods, enzymatic cycling methods (Cell Stem Cell 2019, 25(6):754-767; Annals of Epidemiology 2013, 23(12):791-796; Analyst 1972, 97(151):142-145), enzyme-linked electrochemical analysis (Biochemistry Biophysics Reports 2016, 5:35-54), and nuclear magnetic resonance technology NMR (Nature Chemical Biology 2016, 12(11): 937-943), and liquid chromatography-mass spectrometry (LC-MS) (The Journal of Clinical Investigation 2021,131(2): e136055; Nature 2017, 551(7678):115-118).

[0012] Lactic acid detection Colorimetric method: Lactate dehydrogenase (LDH) is used to convert lactate to pyruvate, and oxidized coenzymes (e.g., NAD+) are converted to H +LDH accepts NAD+ and is reduced to a reduced coenzyme (e.g., NADH). The absorbance of NADH is measured at a wavelength of 340 nm, and the lactate content in the sample is calculated based on this. In the reaction system, the reaction catalyzed by LDH is reversible, so adding a hydrazine compound to the buffer solution to form a stable complex with pyruvate causes the reaction to proceed in one direction. Reagents required for colorimetric methods include, but are not limited to, lactate dehydrogenase (LDH), NAD+, a buffer solution (e.g., Tris), and hydrazine hydrate. Hydrogen peroxide detection method: Lactate oxidase is used to convert lactate to pyruvate, simultaneously generating hydrogen peroxide. Lactate is then quantitatively detected by measuring the hydrogen peroxide level. For example, lactate analyzers use amperometric and photometric detection, or peroxidase converts hydrogen peroxide to other detectable compounds (e.g., quinoneimine, 3-aminophthalic acid ester, etc.). Reagents required include, but are not limited to, lactate oxidase, peroxidase, and buffer solutions.

[0013] Chromatographic methods: Lactic acid can be detected by several colorimetric methods, including HPLC, LC-MS, and UHPLC-MS. For example, the lactate detection procedure using UHPLC-MS includes the following steps: Mix a sample or standard solution with 3NPH_HCl solution and EDC solution sequentially. The mixture is frozen and then centrifuged. The supernatant is collected for quantitative analysis. The supernatant is injected into a UHPLC system, separated with water, and eluted with formic acid and acetonitrile / isopropanol using a gradient elution. The paired ions used for lactate quantification are 224 / 137. Optical Probe Method: The present invention relates to a method for the real-time detection of lactate in a clinical sample based on a genetically encoded optical probe, the method comprising contacting an optionally diluted urine sample with a lactate optical probe and quantifying lactate by detecting a change in fluorescence of the lactate optical probe. As used herein, a lactate optical probe refers to a polypeptide probe that quantitatively detects lactate levels by utilizing changes in optical properties. Such probes typically contain a protein that recognizes lactate (a lactate-binding protein) and a protein whose optical properties change in response to the binding of the protein to lactate (i.e., an optically active polypeptide, such as a fluorescent protein). Typically, a lactate optical probe contains one or more lactate-binding proteins and one or more optically active polypeptides, wherein the one or more optically active polypeptides are located within the sequence of the one or more lactate-binding proteins, or at the N-terminus or C-terminus. A wide variety of optical probes that can be used to detect lactate are known in the art, see, for example, PCT / CN2020 / 137900, Laconic (PLos one 2013, 8(2), e57712), Green Lindoblum (Science Reports 2020, 10, 19562), GEM-IL (Cell Reports Methods 2021, 1(7), 100092), eLACCO1.1 (Nature Communications 2021, 12, 7058), and LiLac (Nature Communications 2022, 13, 2919). Those skilled in the art should understand that other lactate optical probes, not limited to those used in the examples, can also be used to detect lactate levels in the present invention.

[0014] An exemplary lactate optical probe for use in the present invention is the optical probe described in PCT / CN2020 / 137900, the contents of which are incorporated herein by reference in their entirety. The lactate optical probe comprises a lactate binding protein or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or a functional variant thereof is located within the sequence of the lactate binding protein or a functional variant thereof, at the N-terminus or C-terminus. In one or more embodiments, the lactate binding protein has the sequence set forth in SEQ ID NO:1 or a functional fragment thereof, or a variant thereof having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, and retains lactate binding function. In preferred embodiments, the lactate binding protein is set forth in amino acids 80-258 of SEQ ID NO:1, or a variant thereof having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. In one embodiment, the optically active polypeptide is a fluorescent protein or a functional fragment or variant thereof that retains its fluorescence response function. In one embodiment, the fluorescent protein is selected from yellow fluorescent protein, green fluorescent protein, blue fluorescent protein, or apple red fluorescent protein (e.g., cpYFP, ​​cpGFP, cpBFP, and cpmApple shown in SEQ ID NOs: 2-5 of PCT / CN2020 / 137900), and shown in SEQ ID NOs: 4-7 in the Sequence Listing of the present application, respectively. In one embodiment, the lactate optical probe further comprises one or more linkers linked to the side chains of the optically active polypeptide. The linkers of the present invention may be of any length and any amino acid sequence. In one embodiment, the side chains of the optically active polypeptide comprise a linker of 5 or fewer amino acids, for example, a linker of 0, 1, 2, 3, or 4 amino acids. In one embodiment, the linker in the side chain of the optically active polypeptide comprises amino acid Y. In one embodiment, linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the lactate optical probe is represented as follows: first portion B1 of lactate binding protein - Y - optically active polypeptide A - second portion B2 of lactate binding protein. In one embodiment, the lactate optical probe does not comprise a linker.

[0015] In one embodiment, the optically active polypeptide is selected from one or more of the following regions of lactate binding protein: 93 / 94, 93 / 95, 93 / 96, 93 / 97, 94 / 95, 94 / 96, 94 / 97, 95 / 96, 95 / 97, 96 / 97, 119 / 120, 119 / 121, 120 / 121, 137 / 138, 137 / 139, 137 / 140, 137 / 141, 138 / 139, 138 / 140, 138 / 141, 139 / 140, 139 / 141, 140 / 141, 158 / 159, 158 / 160, 158 / 161, 159 / 160, 159 / 161 1, 160 / 161, 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191, 190 / 191, 208 / 209, 208 / 210, 209 / 210, 230 / 231, 230 / 232 and / or 231 / 232, where the numbers correspond to the full length of the lactate binding protein. Preferably, the optically active polypeptide is located at one or more sites selected from the following of the lactate binding protein: 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191 or 190 / 191. In one or more embodiments, the B1-A-B2 lactate optical probe of the present invention may be a probe in which cpYFP is located at the 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191, or 190 / 191 site of a lactate binding protein or a functional fragment thereof.In an exemplary embodiment, the B1-A-B2 lactate optical probe may be a probe in which cpYFP is located at positions 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 187 / 189, 189 / 191, and 190 / 191 of a lactate binding protein or a functional fragment thereof. In one or more embodiments, the functional fragment of the lactate binding protein is set forth in positions 80 to 258 of SEQ ID NO:1.

[0016] The lactate binding protein in the lactate optical probe may have one or more mutations. Lactate optical probes containing mutated lactate binding proteins may have a higher or lower response to lactate than their non-mutated counterparts, and either may be used to detect lactate. Preferably, lactate optical probes may be used to detect lactate when their response to lactate (see PCT / CN2020 / 137900) is greater than 1.2-fold or less than 0.8-fold that of the control fluorescent protein. In one embodiment, the mutations are located at positions 185, 189, and / or 190 of the lactate binding protein or functional fragment thereof. Exemplarily, in one or more embodiments, the mutations are selected from the group consisting of P189R and P190D, P189R and P190A, P189R and P190I, P189R and P190Q, P189R and P190N, P189D and P190D, P189D and P190E, P189D and P190V, P189D and P190L, P189D and P190F, P189D and P190I, P189 D and P190Q, P189D and P190N, P189D and P190G, P189D and P190Y, P189D and P190W, P189E and P190R, P189E and P190A, P189E and P190V, P189E and P190Q, P189A and P190L, P189A and P190F, P189A and P190M, P189A, P189A and P190N, P189A and P190G, P189A and P190H, P189A and P190T, P189V and P190D, P189V and P190E, P189V and P190A, P189V, P189V and P190N, P189V and P190H, P189V and P190Y, P189L and P190V, P189L and P190F, P189L and P190M, P189L and P190G, P189L and and P190H, P189F and P190D, P189F and P190L, P189F and P190F, P189F and P190I, P189F and P190N, P189F and P190H, P189F and P190Y, P189F and P190K, P189F and P190T, P189F and P190W, P189I and P190R, P189I and P190D, P189I and P190A,P189I and P190V, P189I and P190M, P189I and P190Q, P189I and P190G, P189I and P190Y, P189I and P190S, P189I and P190T, P189M and P190R, P189M and P190D, P189M and P190E, P189M and P190F, P189M and P190G, P189M and P190S, P189M and P190W, P189C and P190D, P189C and P190E, P189C and P190F, P189C and P190I, P189C and P190 M, P189C and P190C, P189C, P189C and P190H, P189C and P190Y, P189C and P190S, P189C and P190W, P190L, P190F, P190I, P190Q, P190N, P190K, P190T, P189Q and P190E, P189Q and P190A, P189Q and P190V, P189Q and P190M, P189Q and P190C, P189Q and P190Q, P189Q and P190H, P189Q and P190S, P189N and P190R, P189N and P190D, P 189N and P190L, P189N and P190F, P189N and P190C, P189N, P189N and P190N, P189N and P190G, P189N and P190H, P189N and P190Y, P189N and P190T, P189G and P190V, P189G and P190F, P189G and P190M, P189G and P190C, P189G and P190G, P189G and P190H, P189G and P190K, P189G and P190W, P189H and P190R, P189H and P190D, P189H and P190E, P189H and P190L, P189H and P190S, P189Y and P190R, P189Y and P190L, P189Y and P190N, P189Y and P190H, P189Y and P190S, P189Y and P190T, P189K and P190D, P189K and P190E, P189K and P190V, P189K and P190L, P189K and P190F, P189K and P190I, P189K and P190M, P189K, P189K and P190Q, P189K and P190N, P189K and P190Y,P189K and P190K, P189K and P190T, P189S and P190E, P189S and P190A, P189S and P190L, P189S and P190F, P189S and P190M, P189S and P190C, P189S, P189S and P190Q, 189S and P190Y, P189S and P190K, P189S and P190S, P189T and P190R, P189T and P190D, P189T and P190M, P189T and P190C, P189T, P189T and P190Q, P189T and P190N, P189T and P190H, P189T and P190Y, P189T and P190K, P189T and P190W, P189W and P190A, P189W and P190V, P189W and P190F, P189W, P189W and P190Q, P189W and P190H, P189W and P190S, P189W and P190T, P189W and P190W.

[0017] In some specific embodiments, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K, preferably, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, or M185K. In some specific embodiments, the mutations are P189R and P190A, P189D and P190D, P189D and P190E, P189D and P190Q, P189D and P190Y, P189A and P190N, P189A and P190G, P189V and P190H, P189F and P190I, P189F and P190N, P189F and P190K ... 9I and P190D, P189I and P190A, P189I and P190V, P189I and P190M, P189M and P190R, P189M and P190E, P189M and P190F, P189M and P190G, P189M and P190S, P189C and P190E, P190Q, P189Q and P190M, P189Q and P190C, P189N and P190N , P189G and P190F, P189H and P190L, P189H and P190S, P189Y and P190L, P189K and P190V, P189K and P190T, P189S and P190A, P189S and P190M, P189S and P190Q, P189S and P190K, P189S and P190S, P189T and P190D, P189W and P190A, P P189W and P190T, P189C and P190D, P189C and P190Y, P189N and P190Y, P189R and P190I, P189M and P190D, P189H and P190R, P189N, P189F and P190D, P189F and P190H, P189N and P190F, P189C and P190F, P189H and P190D, or P189S.In some specific embodiments, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K, preferably, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, or M185K.

[0018] In some specific embodiments, the mutations include P189S, P189C and P190D, P189C and P190Y, P189N and P190Y, P189R and P190I, P189M and P190D, P189H and P190R, P189N, P189F and P190D, P189F and P190H, P189N and P190F, P189C and P190F, or P189H and P190D. In one or more embodiments, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K, preferably, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, or M185K. In some embodiments, the mutations include (1) P189C and P190D, P189M and P190D, P189F and P190D, or P189H and P190D, and optionally (2) M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K.

[0019] In some embodiments, the mutations are (1) P189C and P190D, P189M and P190D, or P189H and P190D, and optionally (2) M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, ... or the mutations include (1) P189F and P190D, and optionally (2) M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K.

[0020] In an exemplary embodiment, the lactate optical probe comprises a cpYFP insertion at the 185 / 189 position of a functional fragment of lactate binding protein, and one or more mutations selected from the following: P189R and P190D, P189R and P190A, P189R and P190I, P189R and P190Q, P189R and P190N, P189D and P190D, P189D and P190E, P189D and P190V, P189D and P190L, P189D and P190F, P189D and P190I, P189D and P190Q, P189 D and P190N, P189D and P190G, P189D and P190Y, P189D and P190W, P189E and P190R, P189E and P190A, P189E and P190V, P189E and P190Q, P189A and P190L, P189A and P190F, P189A and P190M, P189A, P189A and P190N, P189A and P190G, P189A and P190H, P189A and P190T, P189V and P190D, P189V and P190E, P189V and P190A, P189V, P189V and P 190N, P189V and P190H, P189V and P190Y, P189L and P190V, P189L and P190F, P189L and P190M, P189L and P190G, P189L and P190H, P189F and P190D, P189F and P190L, P189F and P190F, P189F and P190I, P189F and P190N, P189F and P190H, P189F and P190Y, P189F and P190K, P189F and P190T, P189F and P190W, P189I and P190R, P189I and P19 0D, P189I and P190A, P189I and P190V, P189I and P190M, P189I and P190Q, P189I and P190G, P189I and P190Y, P189I and P190S, P189I and P190T, P189M and P190R, P189M and P190D, P189M and P190E, P189M and P190F, P189M and P190G, P189M and P190S, P189M and P190W, P189C and P190D, P189C and P190E, P189C and P190F, P189C and P190I,P189C and P190M, P189C and P190C, P189C, P189C and P190H, P189C and P190Y, P189C and P190S, P189C and P190W, P190L, P190F, P190I, P190Q, P190N, P190K, P190T, P189Q and P190E, P189Q and P190A, P189Q and P190V, P189Q and P190M, P189Q and P190C, P189Q and P190Q, P189Q and P190H, P189Q and P190S, P189N and P190R, P189 N and P190D, P189N and P190L, P189N and P190F, P189N and P190C, P189N, P189N and P190N, P189N and P190G, P189N and P190H, P189N and P190Y, P189N and P190T, P189G and P190V, P189G and P190F, P189G and P190M, P189G and P190C, P189G and P190G, P189G and P190H, P189G and P190K, P189G and P190W, P189H and P190R, P189H and P190D, P189H and P190E, P189H and P190L, P189H and P190S, P189Y and P190R, P189Y and P190L, P189Y and P190N, P189Y and P190H, P189Y and P190S, P189Y and P190T, P189K and P190D, P189K and P190E, P189K and P190V, P189K and P190L, P189K and P190F, P189K and P190I, P189K and P190M, P189K, P189K and P190Q, P189K and P190N, P189K and P1 90Y, P189K and P190K, P189K and P190T, P189S and P190E, P189S and P190A, P189S and P190L, P189S and P190F, P189S and P190M, P189S and P190C, P189S, P189S and P190Q, 189S and P190Y, P189S and P190K, P189S and P190S, P189T and P190R, P189T and P190D, P189T and P190M, P189T and P190C, P189T, P189T and P190Q, P189T and P190N,The probe may have P189T and P190H, P189T and P190Y, P189T and P190K, P189T and P190W, P189W and P190A, P189W and P190V, P189W and P190F, P189W, P189W and P190Q, P189W and P190H, P189W and P190S, P189W and P190T, or P189W and P190W. In a further embodiment, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E or M185K, preferably the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R or M185K.

[0021] In an exemplary embodiment, the lactate optical probe may be a probe having cpYFP inserted at the 185 / 189 position of a functional fragment of a lactate binding protein and one or more mutations selected from the following: P189S, P189C and P190D, P189C and P190Y, P189N and P190Y, P189R and P190I, P189M and P190D, P189H and P190R, P189N, P189F and P190D, P189F and P190H, P189N and P190F, P189C and P190F, or P189H and P190D. In an exemplary embodiment, the functional fragment of the lactate binding protein is residues 80 to 258 of SEQ ID NO:1, and the mutations are P189N, P189S, P189C and P190F, P189N and P190F, P189N and P190Y, P189H and P190R, P189R and P190I, P189F and P190H, P189C and P190Y, P189C and P190D, P189M and P190D, P189H and P190D, or P189F and P190D. In a further embodiment, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E or M185K, preferably the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R or M185K.

[0022] In some specific embodiments, the lactate optical probe may be a probe having a mutation in which cpYFP is inserted at the 185 / 189 position of a functional fragment of a lactate binding protein, the functional fragment of the lactate binding protein being a sequence of residues 80 to 258 of SEQ ID NO:1, and the mutations include (1) P189C and P190D, P189M and P190D, P189F and P190D, or P189H and P190D, and optionally (2) M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K. Preferably, the mutations are (1) P189CoyobiP190D, P189M and P190D, or P189H and P190D, and optionally (2) M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T. , M185R, M185E, or M185K, or the mutations include (1) P189F and P190D, and optionally (2) M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, or M185K. Preferably, the lactate optical probe is a probe in which cpYFP is inserted at the 185 / 189 position of a functional fragment of a lactate binding protein and has P189F and P190D mutations, or a probe in which cpYFP is inserted at the 185 / 189 position of a functional fragment of a lactate binding protein and has M185L, P189H, and P190D mutations. In specific embodiments, the lactate optical probe comprises a lactate optical probe having a sequence set forth in SEQ ID Nos: 6-30, 34-40 of PCT / CN2020 / 137900. In exemplary embodiments, the lactate optical probe comprises or consists of the amino acid sequence SEQ ID NO: 2 or 3 (SEQ ID No: 30 or 35 of PCT / CN2020 / 137900) and variants thereof. In one embodiment, the lactate optical probe comprises a sequence having at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence SEQ ID No: 2 or 3.

[0023] The lactate optical probe can be fused to another functional polypeptide, for example, the functional polypeptide is located at the N-terminus and / or C-terminus of the lactate optical probe. In some embodiments, the functional polypeptide includes a purification tag or an immunoblot tag. A linker may be present between the optical probe and the other functional polypeptide. As used herein, the terms "functional variant," "derivative," and "analog" refer to a protein that essentially retains a biological function or activity homologous to that of the original polypeptide or protein (e.g., lactate-binding protein or fluorescent protein). A functional variant, derivative, or analog of a polypeptide or protein (e.g., lactate-binding protein or fluorescent protein) of the present invention may be (i) a protein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) have been substituted, and the substituted amino acid residues may or may not be encoded by the genetic code; (ii) a protein having a substitution at one or more amino acid residues; (iii) a protein formed by fusing a mature protein to another compound (e.g., a compound that extends the half-life of the protein, such as polyethylene glycol); or (iv) a protein formed by fusing an additional amino acid sequence to the protein sequence (e.g., a secretory sequence, a sequence for purifying the protein, or a protein-constituting sequence, or a fusion protein formed with an antigenic IgG fragment). As taught herein, these functional variants, derivatives, and analogs are well known to those skilled in the art. The analogs may differ from the original polypeptide or protein by differences in amino acid sequence, by modifications that do not affect sequence, or by both. These proteins include naturally occurring or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutations induced by radiation or exposure to mutagenic agents, or by site-directed mutagenesis or other known molecular biology techniques. In some embodiments, the lactate detection process includes creating a lactate standard curve, which is a common technique in the art and is obtained by measuring lactate standards of different known concentrations and correlating the concentrations with the measurement results or calculated values ​​based on the measurement results.

[0024] In the lactate analysis based on the optical probe, the optical probe is brought into contact with lactate in the sample, and then appropriate excitation and emission wavelengths are selected depending on the fluorescent protein used, and the fluorescence intensity is measured, and quantitative analysis is performed based on the lactate standard curve. In an exemplary embodiment, for fluorescent proteins with a single excitation wavelength (e.g., cpBFP and cpmApple), the data processing process includes: F=F Sample -F BLK F represents the actual fluorescence intensity in a single channel, and F Sample represents the fluorescence intensity of the probe-expressed sample, and F BLK represents the fluorescence intensity of a sample that does not express the probe. A standard curve is created using the fluorescence intensities of the standards, and the amount of lactate in the sample is quantitatively analyzed based on the standard curve. In an exemplary embodiment, the excitation wavelength for cpBFP is 360 nm (BP 10 nm) and the emission wavelength is 450 nm (BP 10 nm), while the excitation wavelength for cpmApple is 540 nm (BP 25 nm) and the emission wavelength is 590 nm (BP 20 nm). For fluorescent proteins with multiple excitation wavelengths (e.g., cpYFP and cpGFP), the data processing process includes: F=F Sample -F BLK R=F 励起波長1 / F 励起波長2 F represents the actual fluorescence intensity in a single channel, and F Sample represents the fluorescence intensity of the probe-expressed sample, and F BLK represents the fluorescence intensity of the sample that does not express the probe. F 励起波長1 represents the fluorescence intensity emitted at the emission wavelength (528 nm for cpYFP or cpGFP) after the probe is excited at the first excitation wavelength (485 nm BP 20 nm for cpYFP), and F 励起波長2represents the fluorescence intensity emitted at the emission wavelength (528 nm for cpYFP or cpGFP) after the probe is excited at the second excitation wavelength (420 nm BP 20 nm for cpYFP). The first and second excitation wavelengths are determined based on the optical spectral characteristics of the fluorescent protein contained in the probe.

[0025] R (Ratio) represents the fluorescence ratio of the probes. The filter bandwidth BP (band pass) indicates the total range on either side of the median, for example, 485 BP 20 nm means the range of 475-495 nm. [Lac] = Kd(RR min ) / (R max -R) [Lac] represents the lactate level, Kd represents the dissociation constant of the probe, and R min and R max where σ represents the fluorescence ratio of the probe protein without or after the addition of saturating concentrations of lactate, respectively, and R represents the fluorescence ratio of the sample. Typically, before detecting lactate, samples are pretreated to remove substances that may affect the measurement. Such pretreatment methods can be adjusted depending on the specific detection method. Those skilled in the art are familiar with such pretreatment processes and the necessary reagents. For example, in an analytical method using UHPLC-MS, as described in Xie et al., 2021, sample pretreatment involves sequentially mixing the sample (e.g., blood or urine) with a 3NPH_HCl solution and an EDC solution, then freezing it at -20°C, and collecting the supernatant for analysis. Furthermore, when detecting lactate in a sample using an optical probe, the sample (e.g., blood or urine) is first diluted with a buffer solution (e.g., HEPES), then mixed with the optical probe, and the fluorescence intensity is measured. Additionally, the present invention further provides a reagent kit for detecting lactate in urine, the kit comprising a lactate detection reagent and an optional urine treatment reagent. As used herein, the terms "lactate detection substance," "detection reagent," and "lactate detection reagent" are interchangeable and refer to a substance that targets lactate and can directly or indirectly measure its presence and / or concentration. To facilitate detection, the detection reagents of the present invention can be labeled with a detectable label, including, but not limited to, radioisotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (e.g., biotin or half-antibodies), and the like.

[0026] In some embodiments, the lactate detection reagent includes a reagent used for converting, concentrating, separating, or identifying lactate. As described above, those skilled in the art will understand that any lactate detection method, including chromatographic methods and probe methods, can be used in the present invention. For example, titration methods, colorimetric methods, enzymatic analysis methods, optical probe methods, etc. Those skilled in the art are also familiar with the lactate detection procedures and reagents used in these methods. Thus, by way of example, the lactate detection reagents described herein can be used in a variety of assays, including chromatographic methods (e.g., HPLC), titration methods (Biomedical Chromatography 2012, 26(11):1408-1415; Food Chemistry 2012, 135(3):1078-1082), colorimetric methods, hydrogen peroxide detection methods, enzymatic cycling methods (Cell Stem Cell 2019, 25(6): 754-767; Annals of Epidemiology 2013, 23(12): 791-796; Analyst 1972, 97(151):142-145), enzyme-linked electrochemical analysis (Biochemistry Biophysics Reports 2016, 5: 35-54), nuclear magnetic resonance technology (NMR) (Nature Chemical Biology 2016, 12(11):937-943) and liquid chromatography-mass spectrometry (LC-MS) (The Journal of Clinical Investigation 2021, 131(2):e136055; Nature 2017, 551(7678):115-118). Reagents used in the above methods include, but are not limited to, antibodies, lactate dehydrogenase, lactate oxidase, lactate-binding protein, or functional variants thereof. In an exemplary embodiment, the lactate detection reagent may further be one or more selected from 3NPH_HCl, EDC, formic acid, acetonitrile, and isopropanol. After using the urine treatment reagent, the lactate in the sample may become more suitable for subsequent detection. Such pretreatment methods can be adjusted depending on the specific detection method. Those skilled in the art are familiar with such pretreatment processes and the necessary reagents. For example, as described in Xie et al., 2021, an analytical method using UHPLC-MS involves sample pretreatment by sequentially mixing the sample (e.g., blood or urine) with a 3NPH_HCl solution and an EDC solution, then freezing it at -20°C, and collecting the supernatant for analysis. Furthermore, when detecting lactate in a sample using an optical probe, the sample (e.g., blood or urine) is first diluted with a buffer solution (e.g., HEPES) and then mixed with the optical probe, and the fluorescence intensity is measured. In one or more embodiments, the urine treatment reagent includes, but is not limited to, 3NPH_HCl, EDC, and a buffer solution. The reagent kit may further include a buffer solution. The role of the buffer solution is to provide a stable buffer environment for the reaction involved in lactate detection. Those skilled in the art can select an appropriate buffer solution based on experience, and examples include phosphate buffer solutions (HEPES, PBS) and Tris. The reagent kit may further include reagents necessary for other auxiliary detections that may be relevant to the diagnosis of MIDD, such as reagents for detecting the mitochondrial m.3243A>G mutation (e.g., PCR primers), reagents for detecting blood glucose in a subject (e.g., glucose oxidase), and reagents for detecting urinary sugar in a subject (e.g., glucose oxidase), all of which are within the knowledge of those skilled in the art.

[0027] The present invention further provides a use of the lactate detection reagent in the manufacture of a reagent kit for use in detecting MIDD or identifying MIDD in diabetic patients. Concentrations, contents, percentages, and other numerical values ​​may be expressed in range format herein. While range formats have been adopted for convenience and brevity, they should be understood to include the numerical values ​​expressly stated as the upper and lower limits of the range, and to include, as appropriate, all individual numerical values ​​or subranges contained within the range. The present invention has the following advantages: Probe-based body fluid sample measurement does not require time-consuming sample preparation (i.e., pretreatment or purification), making it extremely fast and convenient. Typically, the entire process from sample pipetting to measurement takes about 1 minute for one sample, and processing 96 samples in an automated microplate reader typically requires about 3 minutes. These advantages make this invention a promising technology for metabolic diagnostics and screening.

[0028] Some embodiments of the present invention: 1. Use of a reagent for detecting lactic acid in a sample of urine origin, or a reagent for detecting lactic acid in a sample of urine origin and a lactic acid standard, in the manufacture of a reagent kit for use in the diagnosis of MIDD or for the identification of MIDD in diabetic patients. 2. In the use described in Item 1, the lactate detection reagent is characterized by including a reagent necessary for detecting lactate using one or more methods selected from a chromatography method, a titration method, a colorimetric method, an enzyme analysis method, and an optical probe method. 3. In the use according to item 1 or 2, the reagent for detecting lactate comprises a reagent used for converting, concentrating, separating, or identifying lactate; Preferably, the lactate detection reagent is characterized by containing one or more substances selected from an antibody, lactate dehydrogenase, lactate oxidase, lactate binding protein or a functional variant thereof, formic acid, acetonitrile, and isopropanol. 4. In the use according to item 3, the functional variant of the lactate binding protein comprises a lactate optical probe; Preferably, the lactate optical probe comprises a lactate binding protein and an optically active polypeptide; More preferably, said optical probe has the following characteristics: the lactate binding protein comprises the sequence set forth in SEQ ID NO: 1 or a functional fragment thereof; the lactate binding protein has a mutation at the following sites: (1) P189 and / or P190, and optionally (2) M185; The optically active polypeptide is selected from one or more sites of the lactate binding protein, namely, 93 / 94, 93 / 95, 93 / 96, 93 / 97, 94 / 95, 94 / 96, 94 / 97, 95 / 96, 95 / 97, 96 / 97, 119 / 120, 119 / 121, 120 / 121, 137 / 138, 137 / 139, 137 / 140, 137 / 141, 138 / 139, 138 / 140, 138 / 141, 139 / 140, 139 / 141, 140 / 141, 158 / 159, 158 / 160, 158 / 161, 159 / 16 Located in 0, 159 / 161, 160 / 161, 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191, 190 / 191, 208 / 209, 208 / 210, 209 / 210, 230 / 231, 230 / 232 or 231 / 232 The present invention is characterized by having one or more selected from the following:

[0029] 5. A reagent kit for detecting lactic acid in urine, the reagent kit comprising a lactic acid detection reagent and a urine treatment reagent; Preferably, the lactate detection reagent comprises a reagent necessary for detecting lactate using one or more methods selected from a chromatography method, a titration method, a colorimetric method, an enzymatic analysis method, and an optical probe method; More preferably, the reagent kit includes a reagent for detecting lactate that is used for converting, concentrating, separating, or identifying lactate. 6. The reagent kit according to Item 5, wherein the lactate detection reagent comprises one or more selected from the group consisting of an antibody, lactate dehydrogenase, lactate oxidase, a lactate binding protein or a functional variant thereof, 3NPH_HCl, EDC, formic acid, acetonitrile, and isopropanol; The urine treatment reagent contains 3NPH_HCl, EDC, and a buffer solution, and preferably the buffer solution is a phosphate buffer solution or Tris. 7. A reagent kit for detecting lactate in urine, comprising a lactate optical probe and a buffer solution, wherein the lactate optical probe comprises a lactate binding protein and an optically active polypeptide; Preferably, the optically active polypeptide is located within the sequence of the lactate binding protein, at the N-terminus or C-terminus, Preferably, the buffer is a phosphate buffer or Tris, such as HEPES or PBS. 8. In the reagent kit according to item 7, the optical probe has the following characteristics: the lactate binding protein comprises the sequence set forth in SEQ ID NO: 1 or a functional fragment thereof; the lactate binding protein has a mutation at the following sites: (1) P189 and / or P190, and optionally (2) M185; The optically active polypeptide is selected from one or more sites of the lactate binding protein, namely, 93 / 94, 93 / 95, 93 / 96, 93 / 97, 94 / 95, 94 / 96, 94 / 97, 95 / 96, 95 / 97, 96 / 97, 119 / 120, 119 / 121, 120 / 121, 137 / 138, 137 / 139, 137 / 140, 137 / 141, 138 / 139, 138 / 140, 138 / 141, 139 / 140, 139 / 141, 140 / 141, 158 / 159, 158 / 160, 158 / 161, 159 / 16 Located in 0, 159 / 161, 160 / 161, 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191, 190 / 191, 208 / 209, 208 / 210, 209 / 210, 230 / 231, 230 / 232 or 231 / 232 The present invention is characterized by having one or more selected from the following:

[0030] 9. The reagent kit according to any one of Items 5 to 8, further comprising a reagent for detecting the mitochondrial m.3243A>G mutation, a reagent for detecting blood glucose in a subject, and / or a reagent for detecting urinary glucose in a subject. 10. A method for diagnosing MIDD or identifying MIDD in a diabetic patient, comprising (1) detecting urinary lactate and (2) comparing with a control level, wherein the urinary lactate level in a MIDD patient is higher than the control level. The present invention will be further described below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and do not limit the scope of the present invention. The methods and reagents used in the examples are conventional methods and reagents in the art unless otherwise specified. [Example]

[0031] Detection method: 1. Inducible Protein Expression and Purification The probe plasmid was transformed into the BL21(DE3) or JM109(DE3) expression strain, and a single colony was cultured in a test tube. The next day, the cultured bacteria were inoculated into an Erlenmeyer flask at a ratio of 1:100, and the OD 600 When the RI reaches 0.4 to 0.6, IPTG is added to a final concentration of 1 mM, and the mixture is cultured at 18°C ​​to induce expression of the target protein. (1) Harvesting of bacterial cells: After 24 to 48 hours of protein expression, bacterial cells containing the expressed protein are harvested using a centrifuge at 4,000 rpm for 10 minutes and resuspended in buffer A. (2) Ultrasonic disruption: The ultrasonic disruption device is programmed to emit ultrasonic waves for 1 second and stop for 3 seconds, with a cycle time of 300 seconds, a Φ15 probe, and an output of 55%. (3) Centrifugation: After treating the bacterial cells with ultrasound until they become transparent, the cells are centrifuged at 9600 rpm at 4°C for 30 minutes, the supernatant is collected, and the precipitate is discarded. (4) Column pretreatment: A homemade nickel ion affinity chromatography column is flushed with 5 column volumes (CV) of deionized water and equilibrated with 5 column volumes (CV) of buffer A. (5) Protein column loading: The supernatant after disruption and centrifugation is loaded onto a pretreated nickel column. (6) Washing: Contaminating proteins are removed using a washing buffer containing 50 mM imidazole. (7) Elution: The target protein is eluted from the nickel column using an elution buffer containing 300 mM imidazole. (8) Nickel column treatment: After use, the nickel column is washed with 5 column volumes (CV) of buffer B (a buffer containing 500 mM imidazole), and then treated with 5 column volumes (CV) of deionized water. Finally, the column packing is immersed in a 20% ethanol solution for storage. Buffer A: 20 mM phosphate, 0.5 M NaCl, 10 mM imidazole. Buffer B: 20 ​​mM phosphate, 0.5 M NaCl, 500 mM imidazole. After purification on the nickel column, the protein is dissolved in the elution buffer. However, because the solution needs to be exchanged when evaluating the protein's properties or storing it, the resulting protein must be desalted. First, the desalting column must be pretreated by washing with 5 column volumes (CV) of deionized water, followed by treatment with 5 column volumes (CV) of desalting buffer. Next, the protein to be desalted is loaded onto the desalting column, the protein is collected, and finally, the desalting column is treated with 10 column volumes (CV) of deionized water.

[0032] 2. Clinical Sample Collection Serum samples from diabetic patients and healthy controls were provided by the Sixth Affiliated People's Hospital of Shanghai Jiao Tong University and Shanghai Oriental Hospital. Sample collection and subsequent experiments were approved by the institutional review boards, and informed consent was obtained from all participants. 3. Serum and urine testing methods For the fluorescent protein probe-based lactate assay, serum samples were diluted 50-fold or 100-fold with HEPES buffer, and urine samples were diluted 20-fold. Measurements were performed using a 96-well black microplate to set up different lactate standard gradients. 50 μL of diluted sample was added to 50 μL of 0.8 μM probe protein solution. Alternatively, 0.5 μL of serum sample or 2.5 μL of urine sample was directly mixed with 100 μL of probe protein (0.4 μM) using an Echo 650 sonic pipetting system and a BioTek MultifloFX automated pipetting device. The fluorescence intensity was measured immediately using a Synergy Neo2 multifunction plate reader with filters set to excitation wavelengths of 485 BP 20 nm or 420 BP 10 nm and emission wavelengths of 528 BP 20 nm. Quantitative analysis was performed based on the lactate standard curve.

[0033] Fluorescence detection data processing (using FiLa probe as an example) F=F Sample -FBLK R (Ratio)=F 485 / F 420 F represents the actual fluorescence intensity in a single channel, and F Sample represents the fluorescence intensity of the probe-expressed sample, and F BLK represents the fluorescence intensity of the sample that does not express the probe. F 485 represents the fluorescence intensity when the fluorescent protein sample is excited at 485 nm and emitted at 528 nm, and F 420 Ratio represents the fluorescence intensity when a fluorescent protein sample is excited at 420 nm and emitted at 528 nm. FiLa indicates the fluorescence intensity ratio of the probes. The bandwidth BP (band pass) of a filter indicates the total range on either side of the median, so 485 BP 20 nm means 475-495 nm. [Lac] = Kd(R-Rmin) / Rmax-R) [Lac] represents the lactate level, Kd represents the dissociation constant of the FiLa probe, and R min and R max represents the fluorescence ratio of the probe protein without the addition of lactate or after the addition of a saturating concentration of lactate, respectively, and R represents the fluorescence ratio of urine or serum. 4. UHPLC-MS Analysis of Serum and Urinary Lactate Lactate in serum and urine was measured using UHPLC (Agilent 1290) and a triple quadrupole mass spectrometer (Agilent 6460C) (Analytical Chemistry 93, 5709-5717). A 5 μL aliquot of serum or urine sample or standard solution was mixed sequentially with 25 μL of 160 mM 3NPH-HCl solution and 25 μL of 120 mM EDC solution. The mixture was frozen at -20°C for 20 minutes, then centrifuged, and the supernatant was used for quantitative analysis. A 5 μL sample was injected onto an Agilent 1290 UHPLC system, separated with water, and gradient eluted with 0.1% formic acid and acetonitrile / isopropanol (7:3, v / v). Mass spectrometry was performed using negative ESI mode with multiple reaction monitoring (MRM). The paired ions used for quantification of lactate were 224 / 137. [Example]

[0034] Serum samples were measured from patients with adult latent autoimmune diabetes mellitus (LADA) (a subtype of T1DM), T2DM, and MIDD (a special subtype of diabetes caused by the mitochondrial m.3243A>G mutation) provided by affiliated hospitals. Fifteen patients were selected for each disease type. To eliminate metabolic differences due to indicators such as age, sex, height, and weight, in this example, patient samples were matched to healthy control samples based on indicators such as age, sex, and BMI of the diabetic patients. Their clinical characteristics are shown in Tables 1-4 and Figure 1. [Table 1] [Table 2] [Table 3] [Table 4] First, we established a probe-based instantaneous detection method for clinical samples, with a measurement time of less than 1 minute, and measurement is possible with 0.5 μL of serum or 2.5 μL of urine in a 96-well plate. The detection flow is shown in Figure 2. [Example]

[0035] Random serum tests using the probe FiLa-H (SEQ ID NO:2) showed that serum lactate levels were highest in the T2DM group (3.33 ± 0.25 mM), followed by the m.3243A>G mutation group (2.80 ± 0.23 mM), LADA group (2.67 ± 0.25 mM), and control group (2.10 ± 0.20 mM). To verify the accuracy of lactate detection using the probe method, this study compared the results with those of the probe method using ultra-high performance liquid chromatography / mass spectrometry (UHPLC-MS). Compared with HPLC-MS technology, UHPLC-MS has higher resolution, sensitivity, and detection efficiency. In this study, correlation analysis and Bland-Altman analysis were used to evaluate the measurement results of both methods. The Pearson correlation coefficient (r) value of the correlation analysis reached 0.961. Furthermore, Bland-Altman analysis showed that most of the analytical results were within the 95% limits of agreement, indicating high agreement and reliability of the measurement results from both methods (see Figure 3(A) and Figure 3(B)). Furthermore, as shown in Figures 4(A) and (4B), similar measurements were performed using the FiLa probe (SEQ ID NO:3) and other probes described in the Examples of PCT / CN2020 / 137900, and similar results were obtained, confirming high correlation and consistency. [Example]

[0036] As shown in Figure 5(A), random urine analysis using the FiLa-H probe revealed that the lactate level in the m.3243A>G mutation group was 344±73 μM, which was significantly higher than that in the other three groups. However, no significant difference was observed between the LADA, T2DM, and control groups (127±25 μM vs. 111±21 μM vs. 117±23 μM, p>0.5). Furthermore, measurements were performed using the UHPLC-MS method, and correlation analysis and Bland-Altman analysis were performed to evaluate the measurement results of both methods. As a result, it was found that the measurement results using the probe were consistent with the measurement results of UHPLC-MS (shown in Figure 5(B)). [Example]

[0037] The serum lactate concentration and urinary lactate concentration of the subject samples were measured using the probe FiLa-H, and the results are shown in Tables 1 to 4. ROC analysis showed that urinary lactate showed adequate discriminatory ability when comparing MIDD and healthy controls, with an area under the curve (AUC) of 0.867. However, when comparing LADA and healthy controls, no significant discriminatory ability was observed for urinary lactate (0.502, failure score), nor was it observed in the comparison between T2DM and healthy controls (0.511, failure score) (Figure 6). Therefore, elevated urinary lactate levels are a significant clinical parameter in MIDD (m.3243A>G) patients, a novel finding that has not been reported previously. Furthermore, because there is no significant discriminatory relationship between the age of onset and clinical symptoms, patients with MIDD are often misdiagnosed as T1DM or T2DM. As shown in Figure 7, the urinary lactate levels of MIDD patients were significantly different from those of T1DM and T2DM. Specifically, significant differences were observed between MIDD and LADA (urinary lactate: 0.804, serum lactate: 0.520) and T2DM (urinary lactate: 0.853, serum lactate: 0.684). Therefore, a urinary lactate feature score is useful for distinguishing MIDD from LADA and T2DM. These results suggest that elevated urinary lactate is closely associated with MIDD patients and can be used as a potential screening biomarker for the disease. Reassessing these clinical symptoms of diabetes (abnormally elevated urinary lactate and blood glucose) can facilitate rapid screening for MIDD.

Claims

1. Use of a reagent for detecting lactic acid in a urine-derived sample, or a reagent for detecting lactic acid in a urine-derived sample and a lactic acid standard, in the manufacture of a reagent kit for use in diagnosing MIDD or for identifying MIDD in diabetic patients.

2. 2. The use according to claim 1, wherein the lactate detection reagent comprises a reagent necessary for detecting lactate using one or more methods selected from a chromatography method, a titration method, a colorimetric method, an enzymatic analysis method, and an optical probe method.

3. In the use according to claim 1 or 2, the reagent for detecting lactate comprises a reagent used for converting, concentrating, separating or identifying lactate, Preferably, the lactate detection reagent comprises one or more selected from an antibody, lactate dehydrogenase, lactate oxidase, lactate binding protein or a functional variant thereof, formic acid, acetonitrile, and isopropanol.

4. 4. The method of claim 3, wherein the functional variant of the lactate binding protein comprises a lactate optical probe; Preferably, the lactate optical probe comprises a lactate binding protein and an optically active polypeptide; More preferably, said optical probe has the following characteristics: The lactate binding protein comprises the sequence set forth in SEQ ID NO: 1 or a functional fragment thereof; the lactate binding protein has mutations at the following sites: (1) P189 and / or P190, and optionally (2) M185; The optically active polypeptide is selected from one or more sites of the lactate binding protein, namely, 93 / 94, 93 / 95, 93 / 96, 93 / 97, 94 / 95, 94 / 96, 94 / 97, 95 / 96, 95 / 97, 96 / 97, 119 / 120, 119 / 121, 120 / 121, 137 / 138, 137 / 139, 137 / 140, 137 / 141, 138 / 139, 138 / 140, 138 / 141, 139 / 140, 139 / 141, 140 / 141, 158 / 159, 158 / 160, 158 / 161, 159 / 16 Located at 0, 159 / 161, 160 / 161, 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191, 190 / 191, 208 / 209, 208 / 210, 209 / 210, 230 / 231, 230 / 232 or 231 / 232 A use characterized by having one or more selected from the following.

5. A reagent kit for detecting lactic acid in urine, the reagent kit comprising a lactic acid detection reagent and a urine treatment reagent; Preferably, the lactate detection reagent comprises a reagent necessary for detecting lactate using one or more methods selected from a chromatography method, a titration method, a colorimetric method, an enzymatic analysis method, and an optical probe method; More preferably, the reagent kit includes a reagent for detecting lactate that is used for converting, concentrating, separating, or identifying lactate.

6. 6. The reagent kit according to claim 5, wherein the lactate detection reagent comprises one or more selected from the group consisting of an antibody, lactate dehydrogenase, lactate oxidase, a lactate binding protein or a functional variant thereof, formic acid, acetonitrile, and isopropanol; The urine treatment reagent contains 3 NPH_HCl, EDC, and a buffer solution, and preferably the buffer solution is a phosphate buffer solution or Tris.

7. A reagent kit for detecting lactate in urine, comprising a lactate optical probe and a buffer solution, wherein the lactate optical probe comprises a lactate binding protein and an optically active polypeptide; Preferably, the optically active polypeptide is located within the sequence of the lactate binding protein, at the N-terminus or C-terminus, Preferably, the buffer solution is a phosphate buffer or Tris, for example, HEPES, PBS, etc. A reagent kit for detecting lactic acid in urine.

8. 8. The reagent kit according to claim 7, wherein the optical probe has the following characteristics: The lactate binding protein comprises the sequence set forth in SEQ ID NO: 1 or a functional fragment thereof; the lactate binding protein has mutations at the following sites: (1) P189 and / or P190, and optionally (2) M185; The optically active polypeptide is selected from one or more sites of the lactate binding protein, namely, 93 / 94, 93 / 95, 93 / 96, 93 / 97, 94 / 95, 94 / 96, 94 / 97, 95 / 96, 95 / 97, 96 / 97, 119 / 120, 119 / 121, 120 / 121, 137 / 138, 137 / 139, 137 / 140, 137 / 141, 138 / 139, 138 / 140, 138 / 141, 139 / 140, 139 / 141, 140 / 141, 158 / 159, 158 / 160, 158 / 161, 159 / 16 Located at 0, 159 / 161, 160 / 161, 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191, 190 / 191, 208 / 209, 208 / 210, 209 / 210, 230 / 231, 230 / 232 or 231 / 232 A reagent kit comprising one or more selected from the following:

9. The reagent kit according to any one of claims 5 to 8, further comprising a reagent for detecting the mitochondrial m.3243A>G mutation, a reagent for detecting blood glucose in a subject, and / or a reagent for detecting urinary glucose in a subject.

10. A method for diagnosing MIDD or identifying MIDD in a diabetic patient, comprising: (1) detecting urinary lactate; and (2) comparing with a control level, wherein the urinary lactate level in a MIDD patient is higher than the control level.

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