CN102288559B - Method and kit for detecting amylase - Google Patents
Method and kit for detecting amylase Download PDFInfo
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- CN102288559B CN102288559B CN201110153417.8A CN201110153417A CN102288559B CN 102288559 B CN102288559 B CN 102288559B CN 201110153417 A CN201110153417 A CN 201110153417A CN 102288559 B CN102288559 B CN 102288559B
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- 238000000034 method Methods 0.000 title claims abstract description 32
- 102000013142 Amylases Human genes 0.000 title abstract description 31
- 108010065511 Amylases Proteins 0.000 title abstract description 31
- 239000004382 Amylase Substances 0.000 title abstract description 10
- 235000019418 amylase Nutrition 0.000 title abstract description 10
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims abstract description 63
- 238000001514 detection method Methods 0.000 claims abstract description 37
- 229920002472 Starch Polymers 0.000 claims abstract description 33
- 239000008107 starch Substances 0.000 claims abstract description 33
- 235000019698 starch Nutrition 0.000 claims abstract description 30
- JLKDVMWYMMLWTI-UHFFFAOYSA-M potassium iodate Chemical compound [K+].[O-]I(=O)=O JLKDVMWYMMLWTI-UHFFFAOYSA-M 0.000 claims abstract description 21
- 239000001230 potassium iodate Substances 0.000 claims abstract description 21
- 229940093930 potassium iodate Drugs 0.000 claims abstract description 21
- 235000006666 potassium iodate Nutrition 0.000 claims abstract description 21
- 238000002835 absorbance Methods 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims abstract description 11
- 239000000203 mixture Substances 0.000 claims abstract description 9
- 239000007853 buffer solution Substances 0.000 claims abstract description 6
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 6
- 230000002478 diastatic effect Effects 0.000 claims description 25
- 238000012360 testing method Methods 0.000 claims description 21
- 239000002253 acid Substances 0.000 claims description 15
- 239000008363 phosphate buffer Substances 0.000 claims description 12
- 238000013016 damping Methods 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims description 6
- 235000010234 sodium benzoate Nutrition 0.000 claims description 6
- 239000004299 sodium benzoate Substances 0.000 claims description 6
- 239000008057 potassium phosphate buffer Substances 0.000 claims description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 4
- 239000001257 hydrogen Substances 0.000 claims 4
- 229910052739 hydrogen Inorganic materials 0.000 claims 4
- 238000006243 chemical reaction Methods 0.000 abstract description 13
- 210000002966 serum Anatomy 0.000 abstract description 13
- 210000002700 urine Anatomy 0.000 abstract description 13
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 abstract description 10
- 229910052740 iodine Inorganic materials 0.000 abstract description 10
- 239000011630 iodine Substances 0.000 abstract description 10
- 239000011259 mixed solution Substances 0.000 abstract description 3
- 230000002378 acidificating effect Effects 0.000 abstract 3
- 150000001875 compounds Chemical class 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- 238000000859 sublimation Methods 0.000 abstract 1
- 230000008022 sublimation Effects 0.000 abstract 1
- 239000003153 chemical reaction reagent Substances 0.000 description 31
- 229940111205 diastase Drugs 0.000 description 20
- 239000000758 substrate Substances 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 230000007062 hydrolysis Effects 0.000 description 6
- 238000006460 hydrolysis reaction Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 229920001353 Dextrin Polymers 0.000 description 2
- 239000004375 Dextrin Substances 0.000 description 2
- 229920001503 Glucan Polymers 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 235000019425 dextrin Nutrition 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000005360 mashing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229940005654 nitrite ion Drugs 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 229960004839 potassium iodide Drugs 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 210000003079 salivary gland Anatomy 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 238000004879 turbidimetry Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 208000016222 Pancreatic disease Diseases 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- YVZLYNHKJASIHA-UHFFFAOYSA-L [Na+].[K+].OP(O)([O-])=O.OP(O)([O-])=O Chemical compound [Na+].[K+].OP(O)([O-])=O.OP(O)([O-])=O YVZLYNHKJASIHA-UHFFFAOYSA-L 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
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- 230000002421 anti-septic effect Effects 0.000 description 1
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- 210000001124 body fluid Anatomy 0.000 description 1
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- 239000000872 buffer Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- CSVGEMRSDNSWRF-UHFFFAOYSA-L disodium;dihydrogen phosphate Chemical compound [Na+].[Na+].OP(O)([O-])=O.OP(O)([O-])=O CSVGEMRSDNSWRF-UHFFFAOYSA-L 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000004204 exocrine pancreatic function Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000024924 glomerular filtration Effects 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- TWNIBLMWSKIRAT-VFUOTHLCSA-N levoglucosan Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@H]2CO[C@@H]1O2 TWNIBLMWSKIRAT-VFUOTHLCSA-N 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- 239000007981 phosphate-citrate buffer Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 229940042633 potassium 8 mmol Drugs 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- FYKDNWHPKQOZOT-UHFFFAOYSA-M sodium;dihydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].OP(O)([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O FYKDNWHPKQOZOT-UHFFFAOYSA-M 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of medical detection and discloses a method and a kit for detecting amylase. The method for detecting the amylase provided by the invention comprises the following steps of: in a buffer solution with a pH value of 7.2, mixing a sample to be detected and soluble starch to obtain a mixed solution; mixing potassium iodide and acidic potassium iodate and mixing the mixture of the potassium iodide and the acidic potassium iodate with the obtained mixed solution; detecting absorbance in a 630nm wavelength; and calculating the concentration of the amylase in the sample to be detected according to the absorbance of a non-enzymatic blank tube which is subjected to the same treatment. In the method, the potassium iodide and the potassium iodate generate iodine under the acidic condition, and the iodine is combined with the unreacted starch to form a blue compound, so influence on reaction color development due to reduction of components caused by easiness in sublimation and volatilization of an iodine solution which is directly used is avoided, and the accuracy of a detection result is improved. The kit is good in stability, long in preservation time, wide in application range and convenient for popularization and can be applied to determination of the content of the amylase in serum or urine by hospitals, health departments and medical biological scientific research institutes at all levels.
Description
Technical field
The present invention relates to medical test determination techniques field, relate to specifically a kind of diastatic method of detection and kit.
Background technology
Diastase (α-Amylase) is called for short AMS or AMY, and a class can be by the enzyme of starch molecule hydrolysis, and general action is in α-1 such as soluble starch, amylose, glycogen, 4-glucosan, hydrolyzing alpha-Isosorbide-5-Nitrae-glycosidic bond, produces glucosan, maltose and glucose molecule.Diastase is rich content in pancreas and salivary gland, and amylopsin enters alimentary canal by pancreas with activated state, is the enzyme of most important hydrolysis carbohydrates, with the same AMS that all belongs to of diastase of salivary gland secretion.Diastase molecular weight is little, can pass through glomerular filtration, with urine, discharges.When suffering from pancreatic disease or have exocrine pancreatic function obstacle, can cause that amylase activity raises or reduces, therefore, the diastase of measuring in serum and urine is significant to pancreatitic diagnosis.The fluctuation of amylase in urine level is larger, so preferably serum amylase detects, or both measure simultaneously.
In serum and urine, the assay method of amylase activity has a variety ofly, mainly contains viscosimetry, turbidimetry, mashing system, iodine-starch colourimetry, enzyme rate method and dyestuff method for releasing etc.Wherein, viscosimetry and turbidimetry, because influence factor is more, lack specificity and sensitivity, are eliminated.Although mashing system can directly be measured enzymatic activity, complicated operation is not suitable for routine clinical inspection.At present widely used is iodimetric titration, enzyme rate method and dyestuff method for releasing.Wherein, because having, cost is low, detection does not need expensive instrument, stable reagent, easy and simple to handle, reliable results for iodine-starch colourimetry, is used widely clinically.
In iodine-starch colorimetric method for determining serum and urine, diastatic reaction principle is α-Isosorbide-5-Nitrae hydrolysis of glycoside bond in the AMS catalysis starch molecule in serum (slurry) and urine, produces glucose, maltose and contains α-1, the dextrin of 6 glycosidic bond side chains.Under the quantitative condition of substrate, after reaction, add iodine liquid and the starch not being hydrolyzed to be combined into blue complex, its blue depth with without the blank tube comparison of enzymatic reaction, amylase activity is larger, remaining starch is fewer, and reactant liquor colour developing is more shallow, and final product is placed under ultraviolet/visible light analyser or under semi-automatic biochemical analyzer, detection is in absorbance size at predominant wavelength 630nm, thus diastatic content in measuring and calculating sample.
Yet said method developer iodine liquid easily distils, volatilization causes composition to reduce the thin out reaction solution that affects, and causes testing result accuracy to reduce.
Summary of the invention
In view of this, the object of the invention is to provide the kit of the detection diastase enzyme that a kind of stability is high, accuracy is high.
For realizing object of the present invention, the present invention adopts following technical scheme:
Detect a diastatic method, comprising:
Step 1, in the damping fluid of pH7.2, testing sample and soluble starch are mixed to get mixed liquor;
Step 2, potassium iodide mix with acid Potassiumiodate, and the mixed liquor obtaining with step 1 reacts, and under 630nm wavelength, detects absorbance, according to the absorbance of the blank tube without enzymatic through above-mentioned same treatment, calculate diastatic concentration in testing sample.
α-Isosorbide-5-Nitrae hydrolysis of glycoside bond in AMS catalysis starch molecule in serum (slurry) and urine, produces glucose, maltose and contains α-1, the dextrin of 6 glycosidic bond side chains.Under the quantitative condition of substrate, after reaction, add iodine liquid and the starch not being hydrolyzed to be combined into blue complex, its blue depth with without the blank tube comparison of enzymatic reaction, detect and be in absorbance size at predominant wavelength 630nm, thus diastatic content in measuring and calculating sample.Detection method of the present invention is usingd potassium iodide and acid potassium iodide as nitrite ion, potassium iodide and Potassiumiodate generate iodine under acid condition, be combined with unreacted starch and form blue complex, avoid directly using the volatilization that easily distils of iodine liquid to cause composition to reduce the thin out reaction solution that affects, improve testing result accuracy.
Preferably, the mol ratio of described Potassiumiodate and potassium iodide is 1: 5～6.
Preferably, the potassium iodate solution that acid Potassiumiodate is 8～12mmol/LpH2.0～3.0.
For the good combination of enzyme-to-substrate, enzyme and substrate all need damping fluid that the suitable environment that dissociates is provided.The ionic strength of damping fluid also affects the activity of enzyme, and ionic strength is too high, electrolyte interferases and Binding Capacity, and enzymatic activity will progressively decline, also may kinase activity but ionic strength is too low.The ionic strength that general selection is more approaching with the body fluid of physiological environment.Therefore detection method of the present invention is in order to provide a suitable enzymolysis environment, and testing sample mixes in the damping fluid of pH7.2 with soluble starch.The described damping fluid at pH7.2 comprises: sodium hydrogen phosphate-citrate buffer solution, Tris-hydrochloride buffer and phosphate buffer.As preferably, described in detection method of the present invention, the buffer solution of pH7.2 is the phosphate buffer of 50～100mmol/L pH7.2, comprises sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution and sodium hydrogen phosphate-potassium phosphate buffer.
Preferably, described soluble starch concentration is 960mg/L.
The present invention also provides a kind of detection diastatic kit, comprises 960mg/L soluble starch, the phosphate buffer of 50～100mmol/L pH7.2, the potassium iodate solution of 8～10mmol/LpH2.0～3.0 and 30～50mmol/L potassium iodide.
Preferably, kit of the present invention also comprises the Sodium Benzoate of 50～70mmol/L, and Sodium Benzoate is as antiseptic guarantee reagent stability.
The diastatic kit of detection provided by the invention can be double reagent, the phosphate buffer of soluble starch and pH7.2 and potassium iodide are made to the first reagent, acid Potassiumiodate is made to the second reagent, when utilizing this kit to detect diastase, by the first reagent and the second reagent mix, thereby detect the diastase in testing sample.The diastatic kit of detection provided by the invention can also be many reagent, the phosphate buffer of soluble starch and pH7.2 is made to the first reagent, acid Potassiumiodate is made to the second reagent, potassium iodide is made the 3rd reagent, while detecting diastase, by the first reagent and the second reagent and the 3rd reagent mix, detect the diastase in testing sample.Because soluble starch aqueous stability is poor, therefore kit of the present invention is made powdered reagent by the reagent of soluble-containing starch, before using, dissolves.
From above-mentioned technical scheme, can find out, detection method of the present invention is usingd potassium iodide and acid potassium iodide as nitrite ion, potassium iodide and Potassiumiodate generate iodine under acid condition, be combined with unreacted starch and form blue complex, avoid directly using the volatilization that easily distils of iodine liquid to cause composition to reduce the thin out reaction solution that affects, improve testing result accuracy.Test shows, detection method of the present invention has high accuracy and precision, same sample is carried out to repeatability to be detected, standard rate (coefficient of variation) between each result is 1.92%, be less than 3% of standard, and the range of linearity is wide, in serum, diastase can be surveyed the range of linearity and can reach 800U/L, and in urine, diastase can be surveyed the range of linearity and can reach 1600U/L.Kit good stability of the present invention, long shelf-life, applied widely, be convenient to promote the use of, can be applicable to situation of all-level hospitals, sanitary precaution department and medical biotechnology R&D institution and measure diastase content in serum or urine.
Embodiment
The embodiment of the invention discloses a kind of diastatic method of detection and kit.Those skilled in the art can use for reference content herein, suitably improve technological parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Product of the present invention and method are described by preferred embodiment, and related personnel obviously can change method as herein described or suitably change and combination within not departing from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
In order further to understand the present invention, below in conjunction with embodiment, the present invention is described in detail.
Embodiment 1: the diastatic kit of detection of the present invention.
The diastatic kit of detection of the present invention is many reagent.
The first reagent is powdered reagent, comprises phosphate buffer and the 60mmol/L Sodium Benzoate of 960mg/L soluble starch, 50mmol/LpH7.2;
The second reagent is 30mmol/L potassium iodide;
The potassium iodate solution that the 3rd reagent is 8mmol/LpH2.0.
Embodiment 2: the diastatic kit of detection of the present invention.
The diastatic kit of detection of the present invention is many reagent.
The first reagent is powdered reagent, comprises phosphate buffer and the 50mmol/L Sodium Benzoate of 960mg/L soluble starch, 20mmol/LpH7.2;
The second reagent is 50mmol/L potassium iodide;
The potassium iodate solution that the 3rd reagent is 12mmol/LpH3.0.
Embodiment 3: the diastatic kit of detection of the present invention.
The diastatic kit of detection of the present invention is double reagent.
The first reagent is powdered reagent, comprises phosphate buffer, 48mmol/L potassium iodide and the 70mmol/L Sodium Benzoate of 960mg/L soluble starch, 100mmol/LpH7.2;
The second reagent is the potassium iodate solution of 8mmol/LpH2.5.
Embodiment 4: detect diastatic method.
Set 37 ℃ of semi-automatic biochemical analyzer temperature of reaction, reaction method is end-point method, measures predominant wavelength 630nm, and the Direction of Reaction is negative reaction.Getting testing sample mixes with 960mg/L soluble starch, hatch 360s, then add the acid Potassiumiodate mixed solution of 40mmol/L potassium iodide and 8mmol/LpH2.0, mix and be placed on semi-automatic biochemical analyzer, detect and record the absorbance under 630nm wavelength.Blank tube without enzymatic is identical with above-mentioned processing.According to computing formula C
sample=(A
blank-A
sample)/A
blank* (C * V)/(t * V
sample) calculate diastatic concentration in testing sample, wherein, C
samplefor sample to be tested concentration; A
samplefor sample absorbance; A
blankfor blank tube absorbance; C is soluble starch concentration; V is soluble starch volume; T is reaction time (min); V
samplefor testing sample volume.Because every liter of example reaction 1min hydrolysis 4mg starch of international unit regulation is 1 international unit (U/L), therefore in order to be that international unit need to be according to formula C by gained diastase concentration conversion
sample (international unit)=C
sample* (1 * 1000)/4, wherein 1 reaction time (min) for international unit regulation; 1000 are converted into the factor of L for mL; 4 is every liter of sample hydrolysis amount of starch that international unit is stipulated.
Embodiment 5: detection method analysis of the accuracy of the present invention
Get 20 routine clinical samples, respectively according to method and Gal-G described in embodiment 4
2-CNP substrate method, utilizes CB171 semi-automatic biochemical analyzer to detect the diastase content in sample, the results are shown in Table 1.
Table 1 sample diastase content detection result
From table 1 result, with existing Gal-G
2-CNP substrate method is compared, and detection method testing result of the present invention is close, and related coefficient is 0.995, and detection method testing result of the present invention accurately and reliably.
Embodiment 6: detection method repeatability of the present invention detects
Using conventional serum sample as testing sample, according to method described in embodiment 4, utilize CB171 semi-automatic biochemical analyzer to detect the diastase content in testing sample, with existing Gal-G
2the comparison of-CNP substrate method method, the results are shown in Table 2.
Table 2 sample repeatability testing result
| Detection method of the present invention | Gal-G 2-CNP substrate method |
| 124 | 127 |
| 121 | 121 |
| 122 | 124 |
| 118 | 119 |
| 124 | 124 |
| 120 | 122 |
| 121 | 121 |
| 117 | 120 |
| 119 | 125 |
| 121 | 122 |
| CV％＝1.92％ | CV％＝2.01％ |
Result from table 2, adopt detection method of the present invention to detect diastatic coefficient of variation CV=1.92%, be less than 5%, meet < < external diagnosis reagent General Requirement > >, and be less than existing Gal-G
2-CNP substrate method the coefficient of variation, shows that detection method of the present invention is reproducible.
Embodiment 7: the detection of the method for the invention linearity
Get the high value serum that diastase concentration approaches 800U/L, be diluted to 5 different concentration gradients, theoretical concentration value is followed successively by 50,100,200,400,800U/L, according to method described in embodiment 4, utilize CB171 semi-automatic biochemical analyzer to detect the diastase in testing sample, each concentration determination 2 times, averages, according to formula, calculate correlation coefficient r value, the results are shown in Table 3.
Get diastase concentration and approach the high value urine of 1600U/L, be diluted to 5 different concentration gradients, theoretical concentration value is followed successively by 100,200,400,800,1600U/L, according to method described in embodiment 4, utilize CB171 semi-automatic biochemical analyzer to detect the diastase in testing sample, each concentration determination 2 times, averages, according to formula, calculate correlation coefficient r value, the results are shown in Table 4.
Table 3 blood serum sample result of linear detection
Table 4 urine sample result of linear detection
From the result of table 3 and table 4, in detection method serum of the present invention, the diastatic range of linearity can reach 800U/L, and in urine, the diastatic range of linearity can reach 1600U/L, shows that the detection method range of linearity of the present invention is wide, applied widely.
The explanation of above embodiment is just for helping to understand method of the present invention and core concept thereof.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of the claims in the present invention.
Claims (5)
1. detect a diastatic method, it is characterized in that, comprising:
Step 1, in the damping fluid of pH7.2, testing sample and soluble starch are mixed to get mixed liquor;
Step 2, potassium iodide mix with acid Potassiumiodate, and the mixed liquor obtaining with step 1 reacts, and under 630nm wavelength, detects absorbance, according to the absorbance of the blank tube without enzymatic through above-mentioned same treatment, calculate diastatic concentration in testing sample;
Wherein, described soluble starch concentration is 960mg/L;
The buffer solution of described pH7.2 is the phosphate buffer of 50～100mmol/L pH7.2;
The phosphate buffer of described 50～100mmol/L pH7.2 is Lin acid hydrogen Er Na – phosphate sodium dihydrogen buffer solution or Lin acid hydrogen Er Na – potassium phosphate buffer.
2. detection method according to claim 1, is characterized in that, the mol ratio of described Potassiumiodate and potassium iodide is 1:4～6.
3. detection method according to claim 1, is characterized in that the potassium iodate solution that described acid Potassiumiodate is 8～12mmol/LpH2.0～3.0.
4. detect a diastatic kit, it is characterized in that, consist of: 960mg/L soluble starch, the phosphate buffer of 50～100mmol/L pH7.2, the Potassiumiodate of 8～12mmol/LpH2.0～3.0 and 30～50mmol/L potassium iodide;
The phosphate buffer of described 50～100mmol/L pH7.2 is Lin acid hydrogen Er Na – phosphate sodium dihydrogen buffer solution or Lin acid hydrogen Er Na – potassium phosphate buffer.
5. kit according to claim 4, is characterized in that, also comprises the Sodium Benzoate of 50～70mmol/L.
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|---|---|---|---|
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103472015B (en) * | 2013-09-17 | 2016-02-17 | 四川农业大学 | Based on iodine-starch color development system dynamic absorbance quantitative analysis method |
| CN103789400B (en) * | 2014-02-28 | 2015-12-02 | 多多药业有限公司 | A kind of detection method of beta-amylase |
| EP3463058B1 (en) | 2016-05-31 | 2023-11-15 | Indian Institute of Technology, Guwahati | A transmittance based system/kit for point-of-care quantification of biomarkers sample and use thereof |
| CN109001189B (en) * | 2017-06-06 | 2021-04-27 | 中粮营养健康研究院有限公司 | Method for detecting amylase content range in sugar product prepared from sugarcane and application of method |
| CN107978220A (en) * | 2017-11-07 | 2018-05-01 | 阜阳师范学院 | A kind of intelligence egg shelf-life indicating label |
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| CN111157465A (en) * | 2019-12-30 | 2020-05-15 | 武汉艾迪康医学检验所有限公司 | α -amylase detection method |
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| CN101100652A (en) * | 2006-07-03 | 2008-01-09 | 河南农业大学 | Swine alimentary canal rumen lactobacillus with transferred amylase gene and application thereof |
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