CN115901659B - Kit and method for detecting urinary calculus forming coefficient - Google Patents
Kit and method for detecting urinary calculus forming coefficient Download PDFInfo
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Abstract
The invention relates to the technical field of urine calculus forming coefficient detection, in particular to a urinary calculus forming coefficient detection kit and a detection method, wherein the kit comprises an STD reagent and a Test reagent, the STD reagent comprises oxalic acid solution with the concentration of 40mM, oxalic acid solution with the concentration of 160mM and calcium chloride solution with the concentration of 100mM, and the Test reagent comprises buffer solution with the pH value of 7.4 and a sterile needle filter with the concentration of 0.22 mu m; the core of the kit is that a certain amount of oxalic acid and a sufficient amount of calcium chloride are added to activate calcium oxalate in urine to form at a limited time and a limited temperature. After the crystallization is collected, the crystallization is fully dissolved, the absorbance of the solution is detected by a spectrophotometer, the lithogenic coefficient of urine is calculated through a standard curve, the integral balance state of lithogenic ions and lithostatic ions in urine can be reflected, the lithogenic coefficient is taken as a result, and the lithogenic state and risk of a urine sample can be intuitively reflected.
Description
Technical Field
The invention relates to the technical field of urinary calculus forming coefficient detection, in particular to a urinary calculus forming coefficient detection kit and a urinary calculus forming coefficient detection method.
Background
Urinary or kidney stones are a widely recognized public health problem throughout the world. The overall prevalence of the disease varies across the world: asia 1-5%, europe 5-9%, north america 13%, sauter 20%. According to the study of the literature, if the patients with urinary system stone are not provided with effective preventive treatment after the primary treatment, the recurrence rate of the urinary system stone for 3 years can reach 50 percent. Urinary tract stone types are classified according to the main mineral components. Most commonly calcium oxalate stones account for 75% of all stone types, with other stone types including magnesium ammonium phosphate, uric acid ammonium, calcium phosphate, uric acid, and cystine.
The mechanism of kidney stone formation is quite complex and is not fully understood at present. It is accepted in the academia that the primary driving force for kidney stone formation is supersaturation of urine, the saturation being the state in which the lithogenic ions in solution are in equilibrium with their respective salts. Increased excretion of urolith forming ions (also known as stone promoters), such as calcium, oxalic acid and phosphate, and decreased urine volume and stone inhibitors, such as citrate, potassium and magnesium, will increase free ion activity, which can lead to supersaturation of urine, leading to crystal formation and growth. Many abnormalities in urine, including low urine volume, high oxalic acid urine, high calcium urine, low uric acid urine, hyperuricemia, and low magnesium urine, are risk factors for CaOx stone formation.
Currently, there are some methods focused on assessing the lithogenic potential in urine, all of which are more focused on urine saturation. The Boen Risk Index (BRI) focused on the in vitro calcium oxalate crystallization behavior of natural synthetic urine samples to establish the CaOx crystallization risk index of the unprepared natural urine. The method requires slowly heating the urine sample to 37 ℃, evaluating the initial concentration of free calcium by an ion selective electrode (RAPIDLab 855 blood gas analyzer), and gradually adding ammonium oxalate solution (40 mmoL/L, 0.5mL per step, 0.75mL per minute) to initiate the formation of CaOx particles in 200mL aliquots of the urine sample. The start of calcium oxalate crystallization and the amount of oxalic acid (Ox 2) required to induce the process were measured using a 1101M photometer with a 585nm filter. The measured values are recorded as BRI values. The method is one of the main methods for measuring the saturation of urine at present, but has low sensitivity, requires a special high-value instrument to observe the time and state of initial crystal formation in the urine, has complex operation steps and is not easy to observe and quantify. In 1998, sriboonlue et al found that oxalic acid could co-precipitate with excess calcium ions and ethanol as calcium oxalate precipitate at pH 5. This is called an indirect method of measuring oxalic acid in urine. However, this method cannot directly detect oxalic acid or react to the saturation state of urine, so that the present method is less applied. In 1997 Grases et al used the principle that unprotected stone surfaces remained in contact with urine and that the supersaturated material contained thereon crystallized sooner or later. Thus, they used this feature to assess the risk of urine CaOx formation. However, the observation of the crystallization is not easy to control, the time required for the experiment is long, and the experiment is not easy to be used for clinical operation.
Disclosure of Invention
The invention aims to provide a detection kit and a detection method for urinary calculi Dan Jishu (UCI), which have a larger detection range, can be used for measuring the lithogenic potential in urine, have higher sensitivity and specificity, and have the advantages of simple and quick operation.
In order to achieve the above purpose, the invention adopts the following technical scheme: a urinary calculus formation factor detection kit comprising:
a Standard (STD) reagent comprising the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 160mM and calcium chloride solution;
a Test article (Test) reagent comprising the following components: buffer at pH 7.4 and a 0.22 μm sterile needle filter.
Further: the concentration of the calcium chloride is 100mM/L.
Further: the concentration of the buffer solution is 1M/L, and the diameter of the filter is 20mm.
Further: the buffer is phosphate buffered saline (TBS).
The detection principle of the kit of the invention is as follows:
urinary lithogenic index is calculated by measuring the crystals produced by urine over a defined period of time
Oxalic acid+CaCO 3 CaOx (crystal) +CO 2 +H 2 O
Further: standard (STD) reagents also include: oxalic acid with a concentration of 60mM, oxalic acid with a concentration of 80mM or/and grass with a concentration of 120 mM.
As a preferred embodiment of the present invention, the kit may further comprise oxalic acid having a concentration of 60mM, oxalic acid having a concentration of 80mM and oxalic acid having a concentration of 120 mM. It will be apparent to those skilled in the art that oxalic acid solutions having concentrations of 60mM, 80mM and 120mM can be formulated from 40mM and 160mM oxalic acid solutions during actual use.
The calcium carbonate solution in the Standard (STD) reagent is used for preparing the detection sample, and is directly used without dilution.
The buffer solution in the Test reagent is phosphate buffer salt solution with pH value of 7.4, and is used for washing formed crystals, removing impurities and improving the accuracy of the detection result.
A 0.22 μm sterile spike filter was used to filter macromolecules and impurities in urine, typically twice per urine sample.
The method for detecting the Dan Chengdan coefficient of the urinary system by using the kit comprises the following specific steps:
(1) Preparing a standard substance and a detection sample;
the standard substance is as follows: 950 mu L of deionized water and 50 mu L of standard oxalic acid liquid with different concentrations are added into a glass test tube, and the standard oxalic acid liquid is fully and uniformly mixed, wherein the concentration of the final oxalic acid standard liquid is as follows: 1mM (90 mg), 1.5mM (135 mg), 2mM (180 mg), 3mM (270 mg) and 4mM (360 mg);
preparing a detection sample: taking 2-3mL of urine, filtering with a sterile syringe needle filter of 0.22 μm for 2 times, adding 950 μL+50 μL oxalic acid standard solution with concentration of 40mM into a glass test tube, and fully and uniformly mixing;
(2) Adding 1mL of 100mM calcium chloride solution into a standard substance and a detection sample test tube, uniformly mixing, and standing in a 37 ℃ incubator for reaction 1 h;
(3) Taking out the test tube, centrifuging at 4000rpm for 15min, removing supernatant, collecting crystals, washing the crystals once with phosphoric acid buffer (TBS), centrifuging again at 4000rpm for 10min, removing supernatant, air drying the crystals, and dissolving the crystals with anhydrous hydrochloric acid;
(4) Detecting absorbance of the solution by a spectrophotometer at a wavelength of 215 nm;
(5) The OD value of the standard solution is used for calculating a standard curve, the oxalic acid amount is expressed in mg/L, and urine is obtained by the following formula: OD (optical density) (urine) = OD (test sample) - OD (1 mM oxalic acid) Obtaining OD value of urine, and lithogenic coefficient (UCI) =OD of urine (urine) Standard curve coefficient, results are expressed in mg/L;
(6) The urinary lithogenic factor x 24 hour urine volume, shown in mg content per day.
The urinary calculus forming coefficient can reflect the balance state of Dan Yinzi (mainly oxalic acid) related to calcium ions in urine, can reflect the saturation state of urine, can evaluate the risk of urine forming calculus, and has a certain auxiliary effect on early screening and postoperative follow-up of urinary calculus.
The beneficial technical effects of the invention are as follows:
(1) The core of the kit is that a certain amount of oxalic acid and a sufficient amount of calcium chloride are added to activate calcium oxalate in urine to form in a limited time and a limited temperature (in a 37 ℃ incubator). After the crystallization is collected, the crystallization is fully dissolved, the absorbance of the solution at the OD215nm is detected by a spectrophotometer, and the lithogenic coefficient of urine is calculated through a standard curve.
(2) The detection method has the advantages that the whole chemical reaction process is nontoxic and harmless, the reaction product is free from residue and pollution, and the reagent required by detection does not contain any fluorescent agent; the spectrophotometer and the incubator are laboratory routine experimental instruments, and besides, no special experimental instrument is needed, the experimental steps are simple, the operation is easy, batch detection can be realized, and the spectrometer and the incubator are suitable for clinical and laboratory popularization.
(3) The existing related methods for detecting the urine saturation are all aimed at describing the oxalic acid amount required at the beginning of crystallization in urine, and as a result, the whole saturation and the balance state of the urine cannot be represented. The method can reflect the overall balance state of the lithogenic ions and the lithogenic ions in the urine, and can intuitively reflect the lithogenic state and the risk of the urine sample by taking the lithogenic coefficient as a result.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a flow chart of a method for detecting a kit of the present invention;
FIG. 2 shows UCI value standard curves at different wavelengths;
FIG. 3 UCI value calibration curve (OD 215) test one;
FIG. 4 UCI value calibration curve (OD 215) test two;
FIG. 5 UCI value calibration curve (OD 215) test three;
FIG. 6 ROC analysis (UCI concentration);
FIG. 7ROC analysis (UCI content).
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The urinary calculus forming coefficient detection kit comprises an STD reagent and a Test reagent:
the STD reagent comprises the following components: oxalic acid solution with the concentration of 40mM, oxalic acid solution with the concentration of 160mM and calcium chloride solution, wherein the concentration of calcium chloride is 100mM/L;
the Test reagent comprises the following components: phosphate buffer at pH 7.4 and a 0.22 μm sterile needle filter with a diameter of 20mm.
Example 2
The urinary calculus forming coefficient detection kit comprises an STD reagent and a Test reagent:
the STD reagent comprises the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 60mM, oxalic acid solution with concentration of 160mM and calcium chloride solution, wherein the concentration of calcium chloride is 100mM/L;
the Test reagent comprises the following components: phosphate buffer at pH 7.4 and a 0.22 μm sterile needle filter with a diameter of 20mm.
Example 3
The urinary calculus forming coefficient detection kit comprises an STD reagent and a Test reagent:
the STD reagent comprises the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 80mM, oxalic acid solution with concentration of 160mM and calcium chloride solution, wherein the concentration of calcium chloride is 100mM/L;
the Test reagent comprises the following components: phosphate buffer at pH 7.4 and a 0.22 μm sterile needle filter with a diameter of 20mm.
Example 4
The urinary calculus forming coefficient detection kit comprises an STD reagent and a Test reagent:
the STD reagent comprises the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 120mM, oxalic acid solution with concentration of 160mM and calcium chloride solution, wherein the concentration of calcium chloride is 100mM/L;
the Test reagent comprises the following components: phosphate buffer at pH 7.4 and a 0.22 μm sterile needle filter with a diameter of 20mm.
Example 5
The urinary calculus forming coefficient detection kit comprises an STD reagent and a Test reagent:
the STD reagent comprises the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 60mM, oxalic acid solution with concentration of 120mM, oxalic acid solution with concentration of 160mM and calcium chloride solution, wherein the concentration of calcium chloride is 100mM/L;
the Test reagent comprises the following components: phosphate buffer at pH 7.4 and a 0.22 μm sterile needle filter with a diameter of 20mm.
Example 6
The urinary calculus forming coefficient detection kit comprises an STD reagent and a Test reagent:
the STD reagent comprises the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 60mM, oxalic acid solution with concentration of 80mM, oxalic acid solution with concentration of 120mM, oxalic acid solution with concentration of 160mM and calcium chloride solution, wherein the concentration of calcium chloride is 100mM/L;
the Test reagent comprises the following components: phosphate buffer at pH 7.4 and a 0.22 μm sterile needle filter with a diameter of 20mm.
Detection method example 1
The detection steps are shown in fig. 1, and the specific operation is as follows:
(1) Standard and test sample preparation
Preparing a standard substance: 950 mu L of deionized water and 50 mu L of standard oxalic acid liquid with different concentrations are added into a glass test tube, and the standard oxalic acid liquid is fully and uniformly mixed, wherein the concentration of the final oxalic acid standard liquid is as follows: 1mM (90 mg), 1.5mM (135 mg), 2mM (180 mg), 3mM (270 mg) and 4mM (360 mg);
adding 1mL of 100mM calcium chloride solution into each standard test tube, uniformly mixing, and standing in a 37 ℃ incubator for reaction 1 h;
taking out the test tube, centrifuging at 4000rpm for 15min, removing supernatant, air drying the crystal, and dissolving the crystal with anhydrous hydrochloric acid;
detecting absorbance (OD value) of the dissolved solution at 215nm wavelength by using a spectrophotometer;
the OD value of the standard solution was used to calculate a standard curve, expressed in mg/L.
(2) As shown in fig. 2: and detecting the absorbance of crystals and a standard curve under different wavelengths, and finding that the UCI value can reach the optimal effect at OD 215.
(3) As shown in fig. 3-5: the UCI value standard curve was repeatedly measured and found to be relatively stable at OD215, with R2 being greater than 0.99.
Detection method example 2
(1) A sample detection method;
preparing a detection sample: taking 2-3mL of urine, filtering with a sterile syringe needle filter of 0.22 μm for 2 times, adding 950 μL+50 μL40mM oxalic acid standard solution into a glass test tube, and fully and uniformly mixing;
adding 1mL of 100mM calcium chloride solution into a detection sample test tube, uniformly mixing, and then placing in a 37 ℃ incubator for standing reaction 1 h;
taking out the test tube, centrifuging at 4000rpm for 15min, removing the supernatant, collecting crystals, washing the crystals once with TBS buffer, centrifuging again at 4000rpm for 10min, removing the supernatant, airing the crystals, and dissolving the crystals with anhydrous hydrochloric acid;
detecting absorbance of the solution by a spectrophotometer at a wavelength of 215 nm;
urine first passes the formula: OD (optical density) (urine) = OD (test sample) - OD (1 mM oxalic acid) Obtaining the OD value of urine;
lithogenic coefficient of Urine (UCI) =od (urine) Standard curve coefficient x 24 hours urine volume, results are expressed in mg/day;
(2) Clinical testing
200 persons are randomly selected from healthy people and lithiasis patients, and urine is used for detecting the lithiasis coefficient. After balancing the baseline data (age, sex), groups 71 (healthy people) and 72 (lithiasis patients), respectively, the results (table 1) suggest that there is a significant difference in UCI values (concentration and content) for healthy people and lithiasis patients.
Table 1: clinical trial test data
As shown in the ROC analysis results of FIGS. 6-7, the method can well distinguish urine of healthy people and lithiasis patients, and the AUC values are more than 90%. Wherein if the content is used as an index, the AUC value and 95% CI are over 90%, which indicates that the method meets the clinical use standard.
The segmentation value (Cut Off value) of UCI detection is calculated by the result of ROC analysis. As shown in table 2: when UCI was 165 mg/day, sensitivity was 83.33%, specificity was 97.18%, and accuracy was 90.21%. Therefore, the detection method and the detection result of the kit all accord with clinical use standards.
Table 2 UCI split value calculation
Detection method example 3
Illustrated in one case:
(1) The patient was first instructed to collect 24 hours of urine, then the total amount of 24 hours of urine was measured, and 10 ml was left after slightly mixing.
(2) Urine 1ml was taken with a 2 ml syringe and filtered twice through a 0.22 μm sterile syringe needle filter.
(3) The filtered urine 950. Mu.L was added to a test tube, and 50. Mu.L of 40mM oxalic acid standard solution (STD kit) was added thereto, followed by mixing well to prepare a test sample.
(4) To the test tube of the test sample, 1ml of 100mM calcium chloride solution (STD kit) was added, and after mixing, the mixture was allowed to stand in an incubator (37 ℃) for reaction for 1 hour.
(5) Taking out the test tube, centrifuging at 4000rpm for 15min, removing the supernatant, collecting crystals, washing the crystals once with TBS buffer, centrifuging again at 4000rpm for 10min, removing the supernatant, air drying the crystals, and dissolving the crystals with anhydrous hydrochloric acid.
(6) The absorbance (OD value) of the solution was measured at a wavelength of 215nm by a spectrophotometer, and the result of the measurement was 1.17.
(7) OD value of urine = OD value of test article OD-1mM standard, calculated using the standard curve of fig. 4, the OD value of urine is 1.17-0.54 = 0.63
(8) Lithogenic coefficient of urine (UCI value) =od (urine) The standard curve coefficient is multiplied by 90mg/L multiplied by 24 hours urine volume (L/day), and the result is shown in mg/day. This urineThe UCI value of the fluid=0.63/0.006×2.23 (L/day) = 234.15 mg/day, greater than the split value 165 mg/day, so this urine has a higher risk of stone formation.
Finally, what should be said is: the above embodiments are only for illustrating the technical aspects of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention, which is intended to be encompassed by the claims.
Claims (6)
1. Application of urinary calculus forming coefficient detection kit in urinary calculus forming coefficient detection, which is characterized in that the kit comprises:
a standard reagent comprising the following components: oxalic acid solution with concentration of 40mM, oxalic acid solution with concentration of 160mM and calcium chloride solution;
a test article reagent comprising the following components: buffer with pH value of 7.4 and sterile needle filter with 0.22 μm;
the application detection step comprises the following steps:
(1) Preparing a standard substance and a detection sample;
preparing a detection sample: taking 2-3mL of urine, filtering with a sterile syringe needle filter of 0.22 μm for 2 times, adding 950 μL+50 μL oxalic acid standard solution with concentration of 40mM into a glass test tube, and fully and uniformly mixing;
(2) Adding 1mL of 100mM calcium chloride solution into a standard substance and a detection sample test tube, uniformly mixing, and standing in a 37 ℃ incubator for reaction 1 h;
(3) Taking out the test tube, centrifuging at 4000rpm for 15min, removing the supernatant, collecting crystals, washing the crystals once with phosphoric acid buffer, centrifuging again at 4000rpm for 10min, removing the supernatant, airing the crystals, and dissolving the crystals with anhydrous hydrochloric acid;
(4) Detecting absorbance of the solution by a spectrophotometer at a wavelength of 215 nm;
(5) OD value of standard solution forCalculating a standard curve, wherein the oxalic acid amount is expressed in mg/L, and urine is obtained by the following formula: OD (optical density) (urine) = OD (test sample) - OD (1 mM oxalic acid) Obtaining the OD value of urine, the lithogenic coefficient of urine=OD (urine) Standard curve coefficient, results are expressed in mg/L;
(6) The urinary lithogenic factor x 24 hour urine volume, shown in mg content per day.
2. The use according to claim 1, characterized in that: the standard reagent further comprises: oxalic acid solution with concentration of 60mM, oxalic acid solution with concentration of 80mM or/and oxalic acid solution with concentration of 120 mM.
3. The use according to claim 1, wherein the concentration of the calcium chloride solution is 100mM/L.
4. The use according to claim 1, wherein the concentration of the buffer is 1M/L and the diameter of the filter is 20mm.
5. The use according to claim 4, wherein the buffer is phosphate buffered saline.
6. The use according to claim 1, wherein in step (1), the standard is: 950 mu L of deionized water and 50 mu L of standard oxalic acid liquid with different concentrations are added into a glass test tube, and the standard oxalic acid liquid is fully and uniformly mixed, wherein the concentration of the final oxalic acid standard liquid is as follows: 1mM, 1.5mM, 2mM, 3mM and 4mM.
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| 草酸钙尿结石患者的成因分析;曹秋生;胡燕霞;黄士杰;黄存嫦;;中国现代医学杂志(11);第1359-1361页 * |
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