Lactose intolerance detection kit and method of use thereof

Abstract

The present invention discloses a lactose intolerance detection kit and its use method, and relates to the field of biomedical technology. The kit includes liquid A and liquid B, and liquid A is made by mixing phosphate (PB) buffer, sodium azide, sucrose, bovine serum albumin, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and Triton X-100; liquid B is made by mixing Tris buffer, sodium chloride, potassium ferrocyanide, L-tris (2-carboxyethyl) phosphine hydrochloride, dodecyl polyethylene glycol ether, disodium ethylenediaminetetraacetate, o-nitrobenzene-β-D-pyranogalactoside (ONPG) and sodium azide. Feces is placed in liquid A and mixed, and mixed with liquid B to prepare a sample solution, and the absorbance of the sample solution and the absorbance at 15min are immediately read, and the reaction degree is calculated to obtain β-D-galactosidase activity. The kit of the present invention has the advantages of high sensitivity and high accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a lactose intolerance detection kit and a method for using the kit. Background Art

[0002] Milk and dairy products are very important nutrients in human life. They can provide nutrition for human growth and development, and also play an important role in bone development. Lactose is the main carbohydrate in dairy products, but it cannot be directly absorbed by the human body. After lactose enters the human body, it is broken down into glucose and galactose by the action of lactase in the small intestine. Glucose and galactose are both monosaccharides that can be directly absorbed by the human body. After the baby is weaned, the secretion of lactase gradually decreases with age, and eventually some people will develop symptoms of "lactose intolerance". "Lactose intolerance" refers to a series of symptoms such as diarrhea and bloating caused by the inability to digest and absorb lactose in the intestines after consuming milk or breast milk due to the lack of enzymes in the intestines that break down lactose.

[0003] There are four methods for diagnosing lactose intolerance:

[0004] 1. Breath test: In patients with lactase deficiency, the lactose that is not absorbed by the small intestine enters the colon and is acted upon by bacteria in the colon to generate gases such as H2, CO2 and CH4. These gases diffuse into the blood and are then excreted from the lungs. Detecting the content of the above gases in the exhaled gas is helpful in the diagnosis of lactase deficiency.

[0005] 2. Determination of fecal reducing sugar and pH value: The sugar ingested by breastfeeding infants is mainly lactose. Lactose that is not decomposed in the intestine is excreted with feces. At the same time, the feces contain organic acids (acetic acid, lactic acid, propionic acid, butyric acid, etc.) produced by lactose metabolism, which reduces the pH value of feces. Therefore, the content of reducing sugar and pH value in feces can reflect the decomposition of lactose. The lead acetate method is used to detect the content of reducing sugar in feces. If reducing sugar ≥ (++) is positive, and the fecal pH value is <5.5, it indicates lactase deficiency.

[0006] 3. Lactose tolerance test: Lactose tolerance test is a traditional detection method. Blood is drawn at different time points after oral lactose ingestion to test blood sugar levels. If the blood sugar level is <200 mg / L, it is positive, indicating lactase deficiency.

[0007] 4. Jejunal biopsy to determine lactase activity and pathological histological observation: This method is currently the most direct and reliable method for diagnosing lactase deficiency. If the lactase / sucrase ratio is ≤0.3, or the absolute value of lactase is <5IU / g protein or <1.5IU / g intestinal tissue, lactase deficiency can be diagnosed. At the same time, pathological histological examination is performed. Primary lactase deficiency only has decreased lactase activity, but no intestinal mucosal lesions; secondary lactase deficiency is often accompanied by intestinal mucosal lesions. It can also distinguish between single or multiple sugar malabsorption.

[0008] However, there are the following disadvantages: 1. Breath test: 4-8 samples are required, and the sampling process lasts for 2-4 hours. The sampling process is cumbersome and time-consuming; it requires high cooperation from the subjects, and young children (<3 years old) cannot perform it; about 2% of the normal population does not produce H 2 , the test cannot be performed; diabetic patients affect the test results; it is greatly affected by diet and is prone to false positives. 2. Stool reducing sugar determination: because the sugars ingested by older children and adults, in addition to lactose, also include sucrose, maltose, galactose, fructose, glucose, etc., which are all reducing sugars. At this time, a positive lead acetate method can only indicate malabsorption of sugars, and the diagnosis of lactase deficiency must be confirmed in combination with dietary history. 3. Lactose tolerance test: multiple blood draws are required, which is difficult for children to accept, with a high false positive rate and poor specificity. 4. Jejunal biopsy to determine the activity of lactase and pathological histological observation: It is an invasive examination and is difficult to use as a routine diagnostic method.

[0009] At present, there is an urgent need for a lactose intolerance detection kit with a simple detection method and high detection accuracy. By detecting the activity of β-D-galactosidase in feces, it can confirm whether the patient has lactose intolerance and provide a basis for the auxiliary diagnosis of lactose intolerance. Summary of the invention

[0010] In view of the above-mentioned defects of the prior art, the present invention provides a lactose intolerance detection kit and a method of using the same to solve the problems raised by the above-mentioned background technology.

[0011] In order to achieve the above object, the present invention provides the following technical solutions:

[0012] A lactose intolerance detection kit comprises liquid A and liquid B, wherein the liquid A is composed of phosphate (PB) buffer, sodium azide, sucrose, bovine serum albumin, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and Triton X-100; and the liquid B is composed of Tris buffer, sodium chloride, potassium ferrocyanide, tris(2-carboxyethyl)phosphine hydrochloride, dodecyl polyethylene glycol ether, disodium ethylenediaminetetraacetate, o-nitrophenyl-β-D-galactopyranoside and sodium azide.

[0013] Preferably, the A solution is prepared by uniformly mixing 50-200 mM phosphate buffer, 0.5-2 g / L sodium azide, 10-50 g / L sucrose, 10-50 g / L bovine serum albumin, 0.05-0.20 g / L tris(2-carboxyethyl)phosphine hydrochloride and 1-5 g / L Triton X-100.

[0014] Preferably, the concentration of sucrose in the solution A is 30 g / L, the concentration of bovine serum albumin is 25 g / L, and the concentration of tris(2-carboxyethyl)phosphine hydrochloride is 0.1 g / L.

[0015] Preferably, the phosphate buffer in liquid A can be replaced by PIPES / MOPSO / MES / MOPS / HEPES, pH 7.0-8.0; sodium azide can be replaced by PC300 / KroVin series / IZU / FAD / sodium deoxyacetate, sucrose can be replaced by trehalose / dextran / sorbitol / mannitol / xylitol to enhance the stability of the enzyme; bovine serum albumin can be replaced by human serum albumin / calf serum / casein / glycine / proline to enhance the stability of the enzyme; TCEP can be replaced by β-mercaptoethanol or dithiothreitol to increase the stability of the enzyme structure; Triton X-100 can be replaced by Tween 20 / Tween 80 / Brij-35 / polyoxyethylene alkyl ether as a surfactant to aid solubilization, so that the enzyme in the feces can be better dissolved in liquid A.

[0016] Preferably, the B solution is prepared by uniformly mixing 50-200 mM Tris buffer, 0.5-3% sodium chloride, 0.05-0.1 g / L potassium ferrocyanide, 0.05-0.20 g / L tris(2-carboxyethyl)phosphine hydrochloride, 1-5 g / L dodecyl polyethylene glycol ether, 0.5-2 g / L disodium ethylenediaminetetraacetate (EDTA-2Na), 1-3 g / L o-nitrophenyl-β-D-pyranogalactoside and 0.5-2 g / L sodium azide.

[0017] Preferably, the concentration of potassium ferrocyanide in the solution B is 0.08 g / L, and the concentration of tris(2-carboxyethyl)phosphine hydrochloride is 0.1 g / L.

[0018] Preferably, the Tris buffer in solution B can be replaced by TAPS / AMPSO / CHES / CAPSO / AMP / boric acid-borax buffer, TCEP can be replaced by β-mercaptoethanol or dithiothreitol to increase the stability of the enzyme structure; EDTA-2Na can be replaced by EDTA-2K to chelate the competitively bound metal ions and enzyme inhibitors in the reaction system, o-nitrophenyl-β-D-pyranogalactoside (ONPG) can be replaced by chloronitrophenyl N-acetyl-β-D-aminogalactoside (CNP-GAL) / 4-nitrophenyl-β-D-pyranoside (PNPG) as a substrate; sodium azide can be replaced by PC300 / KroVin series / IZU / FAD / sodium deoxyacetate.

[0019] Preferably, the test sample of the lactose intolerance test kit is feces.

[0020] Preferably, the lactose intolerance detection kit comprises the following steps:

[0021] S1. Place the stool sample in liquid A and shake for 8 to 15 seconds;

[0022] S2, taking the liquid A and liquid B prepared in step S1 and mixing them evenly to prepare a sample solution, and immediately reading the absorbance using an ultraviolet spectrophotometer or other optical measuring instrument, which is recorded as A1; wherein the liquid A and the liquid B are mixed in a volume ratio of 1:20;

[0023] S3. Read the absorbance of the sample solution after 15 min and record it as A2;

[0024] S4. Calculate the reaction degree = A2-A1, substitute the reaction degree into the standard curve for calculation, and obtain the β-D-galactosidase activity in the sample; wherein the formula is β-D-galactosidase activity = (standard enzyme activity × sample reaction degree) / standard reaction degree;

[0025] S5. Determine whether lactose intolerance exists based on the β-D-galactosidase activity obtained in step S4.

[0026] Preferably, the β-D-galactosidase activity of a lactose-tolerant sample is generally known to be 10 U / L, and if it is lower than this, the sample is judged to be lactose-intolerant.

[0027] Preferably, in step S1, the mass volume ratio of feces to liquid A is (10-30) μg:1 μL.

[0028] Preferably, the main technical principle is: o-nitrophenyl-β-D-pyranogalactoside (hereinafter referred to as ONPG) can be hydrolyzed by β-D-galactosidase into galactose and yellow o-nitrophenol (ONP), and o-nitrophenol has a maximum absorption peak at a wavelength of 420nm.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The kit of the present invention has the advantages of high sensitivity and high accuracy. The detection object is feces, which is non-invasive detection and has a high degree of acceptance by patients. In the method of the present invention, sodium azide, sucrose, bovine serum albumin, potassium ferrocyanide and TCEP are added to enhance enzyme stability, avoid interference of various sugars, hemoglobin, pigments, reducing substances and the like in the sample on the test results, the test results are highly accurate, specific, and sampling can be performed at any time, which is convenient. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0032] Example 1

[0033] A lactose intolerance detection kit comprises a liquid A and a liquid B, wherein the liquid A is prepared by uniformly mixing 50 mM phosphate buffer, 0.5 g / L sodium azide, 10 g / L sucrose, 10 g / L bovine serum albumin, 0.05 g / L tris(2-carboxyethyl)phosphine hydrochloride and 1 g / L Triton X-100.

[0034] The B solution is prepared by uniformly mixing 50 mM Tris buffer, 0.5% sodium chloride, 0.05 g / L potassium ferrocyanide, 0.05 g / L tris(2-carboxyethyl)phosphine hydrochloride, 1 g / L dodecyl polyethylene glycol ether, 0.5 g / L disodium ethylenediaminetetraacetate, 1 g / L o-nitrobenzene-β-D-pyranogalactoside and 0.5 g / L sodium azide.

[0035] A method for using a lactose intolerance detection kit comprises the following steps:

[0036] S1. Place the feces sample in liquid A and shake for 8 seconds, where the mass volume ratio of feces to liquid A is 10 μg:1 μL;

[0037] S2, taking the liquid A and liquid B of step S1 and mixing them evenly to prepare a sample solution, and immediately reading the absorbance with a UV spectrophotometer, which is recorded as A1; wherein the liquid A and the liquid B are mixed in a volume ratio of 1:20;

[0038] S3. After reacting at 37°C for 15 min, read the absorbance of the sample solution and record it as A2;

[0039] S4. Calculate the reaction degree = A2-A1, substitute the reaction degree into the standard curve for calculation, and obtain the β-D-galactosidase activity in the sample; wherein the formula is β-D-galactosidase activity = (standard enzyme activity × sample reaction degree) / standard reaction degree;

[0040] S5. Determine whether lactose intolerance exists based on the β-D-galactosidase activity obtained in step S4.

[0041] The lactose intolerance test results have been tested and found to be 100% accurate.

[0042] Example 2

[0043] A lactose intolerance detection kit comprises a liquid A and a liquid B, wherein the liquid A is prepared by uniformly mixing 200 mM phosphate buffer, 2 g / L sodium azide, 50 g / L sucrose, 50 g / L bovine serum albumin, 0.20 g / L tris(2-carboxyethyl)phosphine hydrochloride and 5 g / L Triton X-100.

[0044] The B solution is prepared by uniformly mixing 200 mM Tris buffer, 3% sodium chloride, 0.1 g / L potassium ferrocyanide, 0.20 g / L tris(2-carboxyethyl)phosphine hydrochloride, 5 g / L dodecyl polyethylene glycol ether, 2 g / L disodium ethylenediaminetetraacetate, 3 g / L o-nitrophenyl-β-D-pyranogalactoside and 2 g / L sodium azide.

[0045] A method for using a lactose intolerance detection kit comprises the following steps:

[0046] S1. Place the feces sample in liquid A and shake for 15 seconds, where the mass volume ratio of feces to liquid A is 30 μg:1 μL;

[0047] S2, taking the liquid A and liquid B of step S1 and mixing them evenly to prepare a sample solution, and immediately reading the absorbance with a UV spectrophotometer, which is recorded as A1; wherein the liquid A and the liquid B are mixed in a volume ratio of 1:20;

[0048] S3. After reacting at 37°C for 15 min, read the absorbance of the sample solution and record it as A2;

[0049] S4. Calculate the reaction degree = A2-A1, substitute the reaction degree into the standard curve for calculation, and obtain the β-D-galactosidase activity in the sample; wherein the formula is β-D-galactosidase activity = (standard enzyme activity × sample reaction degree) / standard reaction degree;

[0050] S5. Determine whether lactose intolerance exists based on the β-D-galactosidase activity obtained in step S4.

[0051] The lactose intolerance test results have been tested and found to be 100% accurate.

[0052] Example 3

[0053] A lactose intolerance detection kit comprises a liquid A and a liquid B, wherein the liquid A is prepared by uniformly mixing 100 mM phosphate buffer, 1 g / L sodium azide, 30 g / L sucrose, 25 g / L bovine serum albumin, 0.1 g / L tris(2-carboxyethyl)phosphine hydrochloride and 2 g / L Triton X-100.

[0054] The B solution is prepared by uniformly mixing 100 mM Tris buffer, 1% sodium chloride, 0.08 g / L potassium ferrocyanide, 0.10 g / L tris(2-carboxyethyl)phosphine hydrochloride, 3 g / L dodecyl polyethylene glycol ether, 1 g / L disodium ethylenediaminetetraacetate, 2 g / L o-nitrophenyl-β-D-pyranogalactoside and 1 g / L sodium azide.

[0055] A method for using a lactose intolerance detection kit comprises the following steps:

[0056] S1. Place the feces sample in liquid A and shake for 10 seconds, where the mass volume ratio of feces to liquid A is 15 μg:1 μL;

[0057] S2, taking the liquid A and liquid B of step S1 and mixing them evenly to prepare a sample solution, and immediately reading the absorbance with a UV spectrophotometer, which is recorded as A1; wherein the liquid A and the liquid B are mixed in a volume ratio of 1:20;

[0058] S3. After reacting at 37°C for 15 min, read the absorbance of the sample solution and record it as A2;

[0059] S4. Calculate the reaction degree = A2-A1, substitute the reaction degree into the standard curve for calculation, and obtain the β-D-galactosidase activity in the sample; wherein the formula is β-D-galactosidase activity = (standard enzyme activity × sample reaction degree) / standard reaction degree;

[0060] S5. Determine whether lactose intolerance exists based on the β-D-galactosidase activity obtained in step S4.

[0061] The lactose intolerance test results have been tested and found to be 100% accurate.

[0062] Comparative Example 1

[0063] The difference between this comparative example and Example 3 is that solution A does not contain sucrose, bovine serum albumin and TCEP, and the other components and steps remain unchanged.

[0064] A lactose intolerance detection kit comprises liquid A and liquid B, wherein the liquid A is prepared by uniformly mixing 100 mM phosphate buffer, 1 g / L sodium azide and 2 g / L Triton X-100.

[0065] The B solution is prepared by uniformly mixing 100 mM Tris buffer, 1% sodium chloride, 0.08 g / L potassium ferrocyanide, 0.10 g / L tris(2-carboxyethyl)phosphine hydrochloride, 3 g / L dodecyl polyethylene glycol ether, 1 g / L disodium ethylenediaminetetraacetate, 2 g / L o-nitrophenyl-β-D-pyranogalactoside and 1 g / L sodium azide.

[0066] Comparative Example 2

[0067] The difference between this comparative example and Example 3 is that solution B does not contain potassium ferrocyanide and TCEP, and the other components and steps remain unchanged.

[0068] A lactose intolerance detection kit comprises a liquid A and a liquid B, wherein the liquid A is prepared by uniformly mixing 100 mM phosphate buffer, 1 g / L sodium azide, 30 g / L sucrose, 25 g / L bovine serum albumin, 0.1 g / L tris(2-carboxyethyl)phosphine hydrochloride and 2 g / L Triton X-100.

[0069] The B solution is prepared by uniformly mixing 100 mM Tris buffer, 1% sodium chloride, 3 g / L dodecyl polyethylene glycol ether, 1 g / L disodium ethylenediaminetetraacetate, 2 g / L o-nitrobenzene-β-D-pyranogalactoside and 1 g / L sodium azide.

[0070] Comparative Example 3

[0071] The difference between this comparative example and Example 3 is that the o-nitrophenyl-β-D-pyranogalactoside in solution B is replaced by an N,N-dimethylformamide solution of 5-bromo-4-chloro-3-indole-β-D-galactoside, and the other components and steps remain unchanged.

[0072] A lactose intolerance detection kit comprises a liquid A and a liquid B, wherein the liquid A is prepared by uniformly mixing 100 mM phosphate buffer, 1 g / L sodium azide, 30 g / L sucrose, 25 g / L bovine serum albumin, 0.1 g / L tris(2-carboxyethyl)phosphine hydrochloride and 2 g / L Triton X-100.

[0073] The B solution is prepared by uniformly mixing 100 mM Tris buffer, 1% sodium chloride, 0.08 g / L potassium ferrocyanide, 0.10 g / L tris(2-carboxyethyl)phosphine hydrochloride, 3 g / L dodecyl polyethylene glycol ether, 1 g / L disodium ethylenediaminetetraacetate, 2 g / L 5-bromo-4-chloro-3-indole-β-D-galactoside N,N-dimethylformamide solution and 1 g / L sodium azide.

[0074] Experimental Example 1 Stability of β-D-galactosidase

[0075] 1.1 Experimental Materials and Methods: The lactose intolerance test kit prepared in Example 3 and Comparative Example 1 was used to test the stability of β-D-galactosidase after the A solution + enzyme samples were placed for 0 days, 14 days, 29 days and 1 year. The initial sample enzyme activities were 1100U / L, 400U / L and 200U / L.

[0076] 1.2 Test Results

[0077] As shown in Table 1:

[0078] Table 1 Enzyme activity

[0079]

[0080]

[0081] As can be seen from Table 1, after the A solution + enzyme sample was placed for one year, the enzyme activity decrease rates of Comparative Example 1 were: A solution + enzyme (1100U / L): 90.40%, A solution + enzyme (400U / L): 90.63% and A solution + enzyme (200U / L): 96.53%; the enzyme activity decrease rates of Example 3 were: A solution + enzyme (1100U / L): 8.0%, A solution + enzyme (400U / L): 17.02% and A solution + enzyme (200U / L): 29.93%, indicating that the addition of sucrose, bovine serum albumin and TCEP to A solution is beneficial to the stability of the enzyme.

[0082] Experimental Example 2 Accuracy

[0083] 2.1 Experimental Materials and Methods: The lactose intolerance detection kit prepared in Example 3 and Comparative Example 2 was used to test the accuracy of β-D-galactosidase in the A solution + enzyme / (feces) sample. A human feces sample was divided into 20 portions as a control, and the initial sample enzyme activity was 400 U / L.

[0084] 2.2 Test Results

[0085] Table 2 is the test results of Comparative Example 2, and Table 3 is the test results of Experimental Example 3:

[0086] Table 2 Enzyme activity of samples in comparative example 2

[0087]

[0088]

[0089] Table 3 Enzyme activity of samples in Example 3

[0090]

[0091]

[0092] It can be seen from Tables 2 and 3 that the average enzyme activity of Comparative Example 2 (No. 1 to 20) is 58.41 U / L, and the average enzyme activity of Example 3 (No. 1 to 20) is 283.95 U / L. Compared with the standard, Example 3 has higher accuracy, indicating that potassium ferrocyanide and TCEP in liquid B can eliminate interference from reducing substances, hemoglobin, etc. in stool, which is beneficial to improve the accuracy of the lactose intolerance test kit.

[0093] Experimental Example 3 Specificity

[0094] The lactose intolerance detection kits prepared in Example 3 and Comparative Example 3 were used to detect the activity of β-D-galactosidase in the feces of patients with known lactose intolerance. The results showed that the kit of Comparative Example 3 failed to detect that the patient had lactose intolerance symptoms. It can be seen that replacing o-nitrophenyl-β-D-pyranogalactoside in Solution B of the present application with an N,N-dimethylformamide solution of 5-bromo-4-chloro-3-indole-β-D-galactoside failed to test the known effect (lactose intolerance symptoms), indicating that the present application as a whole technical solution, β-D-galactosidase has specificity for the test substrate.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A lactose intolerance detection kit, characterized in that: The kit comprises liquid A and liquid B, wherein the liquid A is prepared by uniformly mixing 50-200 mM phosphate buffer, 0.5-2 g / L sodium azide, 10-50 g / L sucrose, 10-50 g / L bovine serum albumin, 0.05-0.20 g / L tris(2-carboxyethyl)phosphine hydrochloride and 1-5 g / L Triton X-100; and the liquid B is prepared by uniformly mixing 50-200 mM Tris buffer, 0.5-3% sodium chloride, 0.05-0.1 g / L potassium ferrocyanide, 0.05-0.20 g / L tris(2-carboxyethyl)phosphine hydrochloride, 1-5 g / L dodecyl polyethylene glycol ether, 0.5-2 g / L disodium ethylenediaminetetraacetate, 1-3 g / L o-nitrophenyl-β-D-pyranogalactoside and 0.5-2 g / L sodium azide. The test sample of the lactose intolerance test kit is feces.

2. The lactose intolerance detection kit according to claim 1, characterized in that: The concentration of sucrose in the solution A is 30 g / L, the concentration of bovine serum albumin is 25 g / L, and the concentration of tris(2-carboxyethyl)phosphine hydrochloride is 0.1 g / L.

3. The lactose intolerance detection kit according to claim 1, characterized in that: The concentration of potassium ferrocyanide in the solution B is 0.08 g / L, and the concentration of tris(2-carboxyethyl)phosphine hydrochloride is 0.1 g / L.

4. A method for using a lactose intolerance detection kit, characterized in that: The method of using the lactose intolerance detection kit according to any one of claims 1 to 3 to perform detection comprises the following steps: S1. Place the stool sample in liquid A and shake for 8 to 15 seconds; S2, taking the liquid A and liquid B of step S1 and mixing them evenly to prepare a sample solution, and immediately reading the absorbance, which is recorded as A1; wherein the liquid A and the liquid B are mixed in a volume ratio of 1:20; S3. Read the absorbance of the sample solution after 15 min and record it as A2; S4. Calculate the sample reactivity = A2-A1, substitute the sample reactivity into the formula to calculate, and obtain the β-D-galactosidase activity in the sample; wherein the formula is β-D-galactosidase activity = (enzyme activity of standard product × sample reactivity) / standard reactivity; S5. Determine whether lactose intolerance exists based on the β-D-galactosidase activity obtained in step S4.

5. The method for using the lactose intolerance detection kit according to claim 4, characterized in that: In step S1, the mass volume ratio of feces to liquid A is (10-30) μg:1 μL.

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