CN103667421A - Seminal plasma fructose detection kit - Google Patents
Seminal plasma fructose detection kit Download PDFInfo
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- CN103667421A CN103667421A CN201310653263.8A CN201310653263A CN103667421A CN 103667421 A CN103667421 A CN 103667421A CN 201310653263 A CN201310653263 A CN 201310653263A CN 103667421 A CN103667421 A CN 103667421A
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Abstract
The invention discloses a seminal plasma fructose detection kit, relates to a biochemistry kit, and belongs to the medical field. The seminal plasma fructose detection kit includes a reagent A and a reagent B; the reagent A includes hexokinase of 15-30 KU/L, glucose phosphate isomerase of 10-30 KU/L, NADP+ (oxidized form of nicotinamide adenine dinucleotide phosphate) of 1.2 mmol/L, a buffer solution of 70 mmol/L and a preservative of 0.95 g/L; and the reagent B includes glucose-6-phosphate dehydrogenase of 10-30 KU/L, ATP (adenosine triphosphate) of 15-30 mmol/L and a preservative of 0.95 g/L. The beneficial effects of the seminal plasma fructose detection kit reside in that the seminal plasma fructose detection kit is suitable for an automatic biochemical analyzer to make seminal plasma fructose detection convenient and accurate, reduces operating errors, shortens the test time, realizes the simultaneous determination of bulk specimens, and is suitable for large-scale promotion of use.
Description
Technical field
The present invention relates to a kind of biological chemical reagent box, belong to medical field, specifically a kind of detection seminal plasma fructose test kit.
Background technology
In recent years, along with people progressively improve the requirement of prenatal and postnatal care, semen analysis also becomes the important detection means of clinical conventional evaluation male fertility, and male sex's sexual accessory gland function is more and more subject to people's attention the impact of male reproductive function.Some biochemical markers in refining can reflect gonad function, and wherein seminal plasma fructose, from seminal vesicle fluid, is secreted by seminal vesicle, are the energy derives of sperm motility.The secretion of the level affects seminal vesicle fructose of testosterone, hypoandrogenism can cause fructose content to reduce, so fructose content can reflect the function of interstitial glands Testosterone Secretion indirectly.
Seminal plasma fructose can be used as the index of seminal vesicle secreting function.Seminal plasma fructose is measured the azoospermia that the azoospermia can be used for differentiating due to pure obstruction of vas deferen and vas deferens, seminal vesicle dysplasia cause.Seminal plasma fructose is measured judgement male fertility significant.Research is found, the case that Seminal fructose concentration reduces, and sperm survival rate is often lower, and the motility of sperm obviously weakens.Fructose content is obviously relevant to sperm concentration, and sperm concentration is higher, and fructose rate of decomposition is higher, therefore fructose content reduces with the increase of sperm concentration.
Given this, the detection of seminal plasma fructose just seems particularly important.At present, China part andrology has launched the detection of seminal plasma fructose in laboratory, and the method that tradition is used has gas chromatography, indoles method, Resorcinol Method, enzyme process.Above detection method is specifically described below: gas chromatography principle is to utilize fructose and the physicochemical difference of other each components in refining, when moving phase process stationary phase, due to the difference of each component in two alternate absorption, parsing or other avidity, the translational speed of each component is also different, and make fructose obtain separation from other components, and make the concentrated and enrichment of fructose, according to peak height or the area of color atlas, calculate its content.It is high that gas chromatography has tolerance range, and specificity is good, to the less-in-demand feature of sample, but due to this method to plant and instrument require highly, be difficult at clinical expansion, often using it as the reference method of measuring fructose.
The seminal plasma fructose quantitative detecting method that WHO recommends is indoles method, and its principle is that fructose and indoles generate coloured complex in strong acid, heating environment, and this coloring matter has maximum absorption band at 470nm wavelength, and its absorbancy size is directly proportional to fructose content.Applicable instrument is that microplate reader is compared automatic clinical chemistry analyzer complex operation, is unfavorable for promoting; Atopic is not ideal enough; Under concentrated hydrochloric acid environment, react, because concentrated hydrochloric acid is volatile, easily cause reaction poor repeatability, when especially Samples detection amount is large, due to the prolongation of operating time, this phenomenon is more obvious; Biological safety is poor, the easy contaminate environment of strong acid and perishable instrument.
The principle that Resorcinol Method is measured seminal plasma fructose is to utilize fructose to be incubated with Resorcinol in sour environment, generates a kind of red-purple mixture, according to the depth of its color, obtains fructose content.Applicable instrument is semi-automatic biochemical analyzer, and operating process requirement water bath heat preservation is compared with automatic clinical chemistry analyzer complex operation, is unfavorable for promoting; Resorcinol ethanol was at room temperature placed for a long time can affect color developing effect, should not store for a long time, needs fresh preparation; Under concentrated hydrochloric acid environment, react, because concentrated hydrochloric acid is volatile, easily cause reaction poor repeatability, when especially Samples detection amount is large, due to the prolongation of operating time, this phenomenon is more obvious; Biological safety is poor, the easy contaminate environment of strong acid and perishable instrument.
The principle of enzymatic assays seminal plasma fructose is that hexokinase decomposition fructose is fructose-6-phosphate, and the latter can allosteric be G-6-P, glucose-6-phosphate dehydrogenase (G6PD) catalysis G-6-P and NAD
+generate NADH, by the absorbance of the monitoring 340nm wavelength NADH of place, and eliminate in sample glucose on the impact of result after, can detect the concentration of fructose in sample.But the applicable instrument of this technology be microplate reader compared with automatic clinical chemistry analyzer complex operation, be unfavorable for promoting; Refining sample needs Deproteinization to measure reagent before measuring be lyophilized powder, needs to dissolve complex steps before mensuration.
Summary of the invention
The present invention proposes the seminal plasma fructose test kit after a kind of improvement, this reagent has sample to be processed without Deproteinization, and reagent is liquid reagent, have advantages of without before using and dissolve, make advantage simple to operate, that accuracy is high, reproducible, immunity from interference is strong.
For achieving the above object, a kind of detection seminal plasma fructose test kit of the present invention, described test kit comprises reagent A and reagent B, each component of described reagent A and concentration range thereof: Hexokinase 1 5KU/L, glucose phosphate isomerase 30KU/L, NADP
+1.2mmol/L, damping fluid 70mmol/L, sanitas 0.95g/L; Above-mentioned each component adds respectively purified water to mix; Each component of reagent B and concentration range thereof: glucose-6-phosphate dehydrogenase (G6PD) 10-30KU/L, ATP15-30mmol/L, sanitas 0.95g/L; Above-mentioned each component adds respectively purified water to mix.
Described damping fluid is a kind of in phosphate buffered saline buffer, tris buffer, PIPES damping fluid, HEPES damping fluid, TAPS damping fluid, glycine buffer or borate buffer.
The pH value of described damping fluid is 6.1-7.5.
Described sanitas is a kind of in sodium azide, ethyl mercury Sulfothiorine, P-hydroxybenzoic acid, ethyl p-hydroxybenzoate or phenol wide-spectrum bactericide.
The pH of described sanitas is 7.0-9.0.
A kind of detection seminal plasma fructose reagent of the present invention, it is as follows that it realizes technical scheme principle: it is fructose-6-phosphate that hexokinase is decomposed fructose, and the latter can allosteric be G-6-P, glucose-6-phosphate dehydrogenase (G6PD) catalysis G-6-P and NAD
+generate NADH, the fructose content in sample is directly proportional to the changing value of the absorbancy of enzymatic reaction product, can calculate the concentration of fructose by measuring the absorbance of the 340nm wavelength NADPH of place rising.
Hexokinase, glucose phosphate isomerase, NADP in described reagent A
+after mixing with PIPES damping fluid, sodium azide (pH7.0), liquid reagent can be stablized to 12 months, without redissolution.
In described reagent B, after glucose-6-phosphate dehydrogenase (G6PD), ATP and sanitas mixing, liquid reagent also can be stablized to 12 months, without redissolution.
A kind of method that seminal plasma fructose test kit detects Seminal fructose concentration that detects of the present invention, the method comprises the steps:
Yi ﹑ sets detect parameters according to reaction principle and instrument performance: described detecting instrument is automatic clinical chemistry analyzer, and its parameter is: item types is end-point method; Predominant wavelength is 340nm; Commplementary wave length: 670nm ± 10nm; Project Unit is g/L; Temperature of reaction is 37 ℃; Reaction times is 5-6min;
After Er ﹑ adds 12 μ l distilled water and adds 240 μ l reagent A to mix again in blank tube, at constant temperature 1min, then add reagent B60 μ l, after mixing, at 37 ℃ of constant temperature 5-6min, 340nm wavelength is sentenced blank tube zeroing and is measured absorbancy, reading result; In standard pipe, add 12 μ l fructose standard substance, then add 240 μ l reagent A to mix in 37 ℃ of constant temperature 1min, then add reagent B60 μ l, after mixing, at 37 ℃ of constant temperature 5-6min, 340nm wavelength is sentenced blank tube zeroing and is measured absorbancy, reading result; Prepare refining sample, the refining gathering is mixed with the volume ratio of 1:4 with distilled water, the sample after fully mixing is at 15-25 ℃ of stable 8h or 2-8 ℃ of stable 24h; In sample hose, add refining sample 12 μ l, then add 240 μ l reagent A to mix in 37 ℃ of constant temperature 1min, then add reagent B60 μ l, after mixing, at 37 ℃ of constant temperature 5min, 340nm wavelength is sentenced blank tube zeroing and is measured absorbance A, reading result.
The described method of preparing refining sample is, the refining gathering is mixed with the volume ratio of 1:4 with distilled water, and the sample after fully mixing is at 15-25 ℃ of stable 8h or 2-8 ℃ of stable 24h.
Described detecting instrument is automatic clinical chemistry analyzer, and its parameter is: item types is end-point method; Predominant wavelength is 340nm; Commplementary wave length: 670nm ± 10nm; Project Unit is g/L; Temperature of reaction is 37 ℃; Reaction times is 6min.
Described a kind of detection seminal plasma fructose test kit, its storage and stability are strong, 2～8 ℃ of airtight storages, can stablize 12 months.
Described a kind of detection seminal plasma fructose test kit, this measuring and calculation method is:
Once the ejaculate seminal fluid cumulative volume of the ejaculation of quantity of fructose=fructose concentration * once
In formula: Au-mensuration absorbance
Ac-standard absorbance value
Cc-normal concentration value
A kind of detection seminal plasma fructose test kit of the present invention, its beneficial effect is: the reagent comprising in seminal plasma fructose mensuration test kit of the present invention is without redissolution, and reagent can be stablized 12 months, is applicable to automatic clinical chemistry analyzer, seminal plasma fructose measurement result is had to specificity good, accuracy rate is high, and immunity from interference is strong, simple to operate, and because mensuration process is full-automatic, reduce the error in operating process, shorten minute, storage and stability are strong; Can in medical institutions, promote the use of.
Embodiment
Below in conjunction with the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only one of them embodiment of the present invention, rather than whole embodiment.Embodiment based in the present invention, those of ordinary skills, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
1. prepare reagent A: each component of described reagent A and concentration range thereof: Hexokinase 1 5KU/L, glucose phosphate isomerase 30KU/L, NADP
+1.2mmol/L, phosphate buffered saline buffer 70mmol/L, sodium azide 0.95g/L; Above-mentioned each component adds respectively purified water to mix; The pH of described phosphate buffered saline buffer is 6.1, and the pH of sodium azide is 7.0;
2. prepare reagent B: each component of reagent B and concentration range thereof: glucose-6-phosphate dehydrogenase (G6PD) 10KU/L, ATP15mmol/L, ethyl mercury sodium thiosulfate 0.95g/L; Above-mentioned each component adds respectively purified water to mix; The pH of described ethyl mercury sodium thiosulfate is 7.0.
Embodiment 2
1. prepare reagent A: each component of described reagent A and concentration range thereof: Hexokinase 1 5KU/L, glucose phosphate isomerase 30KU/L, NADP
+1.2mmol/L, tris buffer 70mmol/L, P-hydroxybenzoic acid 0.95g/L; Above-mentioned each component adds respectively purified water to mix; The pH of described tris buffer is 7.0, and the pH of P-hydroxybenzoic acid is 8.0;
2. prepare reagent B: each component of reagent B and concentration range thereof: glucose-6-phosphate dehydrogenase (G6PD) 20KU/L, ATP25mmol/L, phenol 0.95g/L; Above-mentioned each component adds respectively purified water to mix; The pH of described phenol is 8.5.
Embodiment 3
1. prepare reagent A: each component of described reagent A and concentration range thereof: Hexokinase 1 5KU/L, glucose phosphate isomerase 30KU/L, NADP
+1.2mmol/L, glycine buffer 70mmol/L, ethyl p-hydroxybenzoate 0.95g/L; Above-mentioned each component adds respectively purified water to mix; The pH of described glycine buffer is 7.5, and the pH of ethyl p-hydroxybenzoate is 9.0;
2. prepare reagent B: each component of reagent B and concentration range thereof: glucose-6-phosphate dehydrogenase (G6PD) 30KU/L, ATP30mmol/L, ethyl p-hydroxybenzoate 0.95g/L; Above-mentioned each component adds respectively purified water to mix; The pH of described ethyl p-hydroxybenzoate is 9.0.
Embodiment 4
With a kind of in embodiment 1, detect the method that reagent that seminal plasma fructose test kit comprises detects Seminal fructose concentration, the concrete steps of the method are as follows:
1. set detecting instrument parameter: described detecting instrument is automatic clinical chemistry analyzer, and its parameter is: item types is end-point method; Predominant wavelength is 340nm; Commplementary wave length: 670nm ± 10nm; Project Unit is g/L; Temperature of reaction is 37 ℃; Reaction times is 5min;
Table 1: detecting instrument parameter
| Detection method | End-point method |
| Predominant wavelength | 340nm |
| Commplementary wave length | 670nm±10nm |
| Temperature of reaction | 37℃ |
| Reaction times | 5min |
2. after adding 12 μ l distilled water add 240 μ l reagent A to mix again in blank tube, at 37 ℃ of constant temperature 1min, then add reagent B60 μ l, after mixing, at 37 ℃ of constant temperature 5min, 340nm wavelength place records absorbancy 0.0591; In standard pipe, add 12 μ l fructose standard substance, then add 240 μ l reagent A to mix in 37 ℃ of constant temperature 1min, then add reagent B60 μ l, after mixing, at 37 ℃ of constant temperature 5min, 340nm wavelength place records absorbancy 1.034; Prepare refining sample, the refining gathering is mixed with the volume ratio of 1:4 with distilled water, sample after fully mixing is at 15 ℃ of stable 8h, in sample hose, add refining sample 12 μ l, add again 240 μ l reagent A to mix in 37 ℃ of constant temperature 1min, then add reagent B60 μ l, after mixing, at 37 ℃ of constant temperature 5min, 340nm wavelength place records absorbancy 0.2100.
Table 2: reaction conditions
This measuring and calculation method is:
Once the ejaculate seminal fluid cumulative volume of the ejaculation of quantity of fructose=fructose concentration * once
In formula: Au-mensuration absorbance
Ac-standard absorbance value
Cc-normal concentration value
Record numerical value normal people refining term of reference >8.33mmol/L; Overproof explanation sample fructose exceeds standard, and may suffer from the disease of refining aspect.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (9)
1. detect a seminal plasma fructose test kit, it is characterized in that: described test kit comprises reagent A and reagent B each component of described reagent A and concentration range thereof: Hexokinase 1 5KU/L, glucose phosphate isomerase 30KU/L, NADP
+1.2mmol/L, damping fluid 70mmol/L, sanitas 0.95g/L; Above-mentioned each component adds respectively purified water to mix; Each component of reagent B and concentration range thereof: glucose-6-phosphate dehydrogenase (G6PD) 10-30KU/L, ATP15-30mmol/L, sanitas 0.95g/L; Above-mentioned each component adds respectively purified water to mix.
2. a kind of detection seminal plasma fructose test kit as claimed in claim 1, is characterized in that: described damping fluid is a kind of in phosphate buffered saline buffer, tris buffer, PIPES damping fluid, HEPES damping fluid, TAPS damping fluid, glycine buffer or borate buffer.
3. a kind of detection seminal plasma fructose test kit as claimed in claim 1, is characterized in that: the pH value of described damping fluid is 6.1-7.5.
4. a kind of detection seminal plasma fructose test kit as claimed in claim 1, is characterized in that: described sanitas is a kind of in sodium azide, ethyl mercury Sulfothiorine, P-hydroxybenzoic acid, ethyl p-hydroxybenzoate or phenol wide-spectrum bactericide.
5. a kind of detection seminal plasma fructose test kit as claimed in claim 1, is characterized in that: the pH of described sanitas is 7.0-9.0.
6. a kind of testing method that detects seminal plasma fructose reagent as claimed in claim 1, it is characterized in that: in sample cup, add 12 μ l products to be tested, after adding again 240 μ l reagent A to mix at constant temperature 1min, then add reagent B60 μ l, after mixing at 37 ℃ of constant temperature 5-6min, 340nm wavelength is sentenced blank zeroing and is measured absorbancy, reading result.
7. a kind of testing method that detects seminal plasma fructose reagent as claimed in claim 6, it is characterized in that: described product to be tested is refining sample, its preparation method is: the refining gathering is mixed with the volume ratio of 1:4 with distilled water, and the sample after fully mixing is at 15-25 ℃ of stable 8h.
8. a kind of testing method that detects seminal plasma fructose reagent as claimed in claim 6, it is characterized in that: described product to be tested is refining sample, its preparation method is: the refining gathering is mixed with the volume ratio of 1:4 with distilled water, and the sample after fully mixing is at 2-8 ℃ of stable 24h.
9. a kind of testing method that detects seminal plasma fructose reagent as claimed in claim 6, is characterized in that: described detection method is: end-point method; It detects predominant wavelength is 340nm; Detect commplementary wave length: 670nm ± 10nm; Temperature of reaction is 37 ℃; Reaction times is 6min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020006940A1 (en) * | 2018-07-03 | 2020-01-09 | 中国农业科学院北京畜牧兽医研究所 | Method and kit thereof for quantitatively detecting lactulose in liquid milk by using microplate reader enzymatic method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102061332A (en) * | 2010-11-05 | 2011-05-18 | 深圳市博锐德生物科技有限公司 | Quantitative fructose assay kit and application thereof as well as quantitative seminal plasma fructose assay method |
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- 2013-12-06 CN CN201310653263.8A patent/CN103667421A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102061332A (en) * | 2010-11-05 | 2011-05-18 | 深圳市博锐德生物科技有限公司 | Quantitative fructose assay kit and application thereof as well as quantitative seminal plasma fructose assay method |
Non-Patent Citations (4)
| Title |
|---|
| KUNST, A., DRAEGER, B. AND ZIEGENHOR,J.: "《Methods of Enzymatic Analysis》", 31 December 1984 * |
| 刘瑜，何林: "男性不育实验诊断几种常用项目检测方法学及试剂盒简介", 《中国男科学杂志》 * |
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| 陈正炎: "《临床生物化学和生物化学检验实验指导》", 31 August 2002 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020006940A1 (en) * | 2018-07-03 | 2020-01-09 | 中国农业科学院北京畜牧兽医研究所 | Method and kit thereof for quantitatively detecting lactulose in liquid milk by using microplate reader enzymatic method |
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Application publication date: 20140326 |