CN102323230B - Seminal fructose concentration detection kit and application - Google Patents
Seminal fructose concentration detection kit and application Download PDFInfo
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- CN102323230B CN102323230B CN 201110233674 CN201110233674A CN102323230B CN 102323230 B CN102323230 B CN 102323230B CN 201110233674 CN201110233674 CN 201110233674 CN 201110233674 A CN201110233674 A CN 201110233674A CN 102323230 B CN102323230 B CN 102323230B
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Abstract
The invention discloses a seminal fructose concentration quantitative detection kit which comprises a R1 reagent, a R2 reagent, and fructose standard liquid, wherein the R1 reagent comprises 150-300 micromol/L of buffer with a pH of 4.5-5.0, 50-70 micromol/L of chromogenic reagents, 285-300 micromol/L of electron acceptors, 0.01-0.1% of preservatives; the R2 reagent comprises the following components: 150-300 micromol/L of buffer with a pH of 4.5-5.0, 2.5-6.0 U/ml of fructose dehydrogenase, 0.01-0.1% of preservatives; the fructose standard liquid comprises the following components: 150-300 micromol/L of buffer with a pH of 4.5-5.0, 20-60 mmol/L of fructose, 0.01-0.1% of preservatives. The invention also discloses an application of the seminal fructose concentration quantitative detection kit in the detection of seminal fructose concentration. Protein removal of the sample of the invention is unnecessary; a velocity method is adopted for the detection, and the use of concentrated hydrochloric acid is avoided; the reaction temperature is not needed to reach above 60 DEG C; an enzyme with specific catalysis for fructose is adopted, and thus the detection results are more accurate and specific; a full-automatic biochemical analyzer is used for the automatic large-scale detection, which greatly increases the detection efficiency.
Description
Technical field
The invention belongs to medical test determination techniques field, relate to a kind of in-vitro diagnosis kit and detection method thereof, specifically a kind of seminal plasma fructose concentration detection by quantitative kit and application.
Background technology
The fructose of seminal vesicle secretion is the main source of sperm energy, participates in the capacitation and the fertilization of sperm directly.In dependent diagnostic such as male sterility and research, the mensuration of fructose concentration is occupied very consequence always.The concentration of fructose can reflect the secreting function of seminal vesicle, congenital seminal vesicle lack as or when suffering from seminal vesiculitis, the content of fructose obviously reduces; When incomplete ejaculation or ejaculation overfrequency, the content of fructose also reduces.In addition, fructose is in conjunction with the detection of other index such as alpha-glucosidase etc., also can be used for differentiating the azoospermatism that azoospermatism due to the simple property obstruction of vas deferen and vas deferens, seminal vesicle depauperation cause.Studies show that, the secretion of the level affects seminal vesicle fructose of testosterone, hypoandrogenism can cause fructose content to reduce, so fructose content can also reflect the function of interstitial glands secretion testosterone indirectly.
At present, the chemical colorimetry of detection fructose mainly contains compound development process and enzyme process; The compound development process mainly contains indoles method and resorcinol method, the two reagent cost is lower, but all need use concentrated hydrochloric acid, high volatility, dangerous big, operator and environment are all had harm, and sample also needs that deproteinized is handled, incubation temperature is up to more than 60 ℃, causes clinical use very inconvenient; Enzyme process mainly contains three kinds of methods, wherein a kind of need are used hexokinase, glucose phosphate isomerase, glucose-6-phosphate dehydrogenase (G6PD), the detection sensitivity height, but the purity requirement to these enzymes is very high, any pollution can not be arranged, and the substrate specificity of hexokinase is not very high in addition, glucose, sorbose, maltose, galactose etc. all can disturb this reaction, cause testing result higher; Another relates to SDH, and reaction has only a step, but this enzyme can reversibly change into fructose with sorbose, and sample also needs pre-treatment step such as centrifugal and heating; The last related Fructose dehydrogenase of a kind of this law just, this zymolyte high specificity can be avoided steps such as centrifugal, the heating of sample or deproteinized simultaneously, and reaction velocity is fast, as a result the accuracy height.But have a bit, more than all methods be craft or semi-automatic operation, the application of sample program is many, and the agents useful for same amount is big, the reagent cost height, reagent and after the sample mixing regular hour, by spectrophotometer colorimetric estimation one by one, calculate the result according to formula then, same sample once can only detect one, expended a large amount of time and manpower, and the manual operation error is also bigger.
Now, the clinical biochemical check has all realized automated analysis basically.Full automatic biochemical apparatus is exactly with the sampling in the original process hand-manipulated, mixing, incubation, detection, calculating as a result, judgement, demonstration and all or part of automatic operation of steps such as print result and cleaning, because its measuring speed is fast, accuracy is high, the consumption amount of reagent is little, now obtained in hospitals at different levels, epidemic prevention station, family planning service station using widely, be used and improve routine biochemistry efficiency of test and income greatly.Regrettably, by the end of at present, no matter be abroad or domestic, detection for the seminal plasma fructose project, except early stage scientific payoffs, still do not have a kind of detection kit that can match with full automatic biochemical apparatus, commercial to come out, therefore, set up a kind of seminal plasma fructose concentration detection by quantitative kit and detection method of using that match with full automatic biochemical apparatus and just seem particularly urgent and important.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, fill up the vacancy on the market, a kind of seminal plasma fructose concentration detection by quantitative kit that matches with full automatic biochemical apparatus and use is provided.
Another object of the present invention is to provide the application of described seminal plasma fructose concentration detection by quantitative kit in detecting seminal plasma fructose concentration.
For solving the problems of the technologies described above, the present invention has adopted following technical scheme:
A kind of seminal plasma fructose concentration detection by quantitative kit comprises R1 reagent, R2 reagent and fructose titer, wherein,
1) R1 reagent comprises the component of following content:
PH4.5-5.0 damping fluid 150-300 μ mol/L;
Developer 50-70 μ mol/L;
Electron accepter 285-300 μ mol/L;
Antiseptic 0.01-0.1%;
2) R2 reagent comprises the component of following content:
PH4.5-5.0 damping fluid 150-300 μ mol/L;
Fructose dehydrogenase 2.5-6.0U/ml;
Antiseptic 0.01-0.1%;
3) the fructose titer comprises the component of following content:
PH4.5-5.0 damping fluid 150-300 μ mol/L;
Fructose 20-60mmol/L;
Antiseptic 0.01-0.1%;
Wherein: described damping fluid is McIlvaine ' s damping fluid, Walpole ' s damping fluid or the kaliumphosphate buffer of pH4.5-5.0; Described developer is tetrazolium bromide, iodonitrotetrazolium purple or 1-methoxyl-5-sylvan piperazine Methylsulfate salt; Described electron accepter is azophenlyene dimethyl ester sulfate or 1-methoxyl-5-sylvan piperazine Methylsulfate salt; Described antiseptic is any one in Sodium azide, gentamicin or the ProClin series antiseptic.
The application of seminal plasma fructose concentration detection by quantitative kit in detecting seminal plasma fructose concentration.
The application of seminal plasma fructose concentration detection by quantitative kit in detecting seminal plasma fructose concentration may further comprise the steps:
1) specimen preparation: fresh semen liquefaction back is by the centrifugal 10～20min of 3000～5000rpm, and the supernatant that is obtained promptly is;
2) the input basic parameter is as follows in the function software of full automatic biochemical apparatus:
Test item: Fru;
Sample reagent ratio: 1: 30;
Reagent ratio: R1: R2=1: 4;
Predominant wavelength: 560-590nm;
Detection method: rate method;
Calibrating method: single-point calibration or two-point calibration;
The Direction of Reaction: rise;
Reaction time: 180-270 second;
Detection time: 90-120 second;
3) scale operation: calibrate with the fructose titer, instrument can draw the relational expression between a fructose concentration and the absorbance rate of change according to the concentration and the per minute absorbance rate of change thereof of fructose titer:
Single-point calibration: Y=AX; Or two-point calibration: Y=AX+B,
Wherein, the constant A of single-point calibration or the constant A of two-point calibration, B provide after calibration finishes automatically, and Y represents the concentration of fructose titer, and X represents the per minute absorbance rate of change of fructose titer;
4) pattern detection:
With 1) in the sample and R1 reagent, the R2 reagent that prepare be placed on the relevant position of full automatic biochemical apparatus, after the detection information of input sample, begin to detect; After the end of test (EOT), the concentration that concentration that instrument draws according to calibration and the relational expression between the absorbance rate of change calculate fructose in the sample automatically.
A kind of seminal plasma fructose concentration detection by quantitative kit provided by the invention comprises R1 reagent, R2 reagent and fructose titer; Described R1 reagent is the buffer solution that contains the pH4.5-5.0 of developer, electron accepter and antiseptic; Described R2 reagent is the buffer solution that contains the pH4.5-5.0 of Fructose dehydrogenase and antiseptic; Described fructose titer is the pH4.5-5.0 buffer solution that contains fructose and antiseptic.
In the described R1 reagent, the damping fluid of pH4.5-5.0 is used to reaction that the proper acidic reaction conditions is provided; Described developer be tetrazolium bromide (3-(and 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT), content is 50-70 μ mol/L, and this scope is and effective binding capacity of fructose reaction, works to generate coloring matter in reaction; In addition, developer can also be the iodonitrotetrazolium purple (Iodonitrotetrazolium purple, INT), the potassium ferricyanide (Potassium ferricynate); Electron accepter be azophenlyene dimethyl ester sulfate (Phenazine Methosulfate, PMS), content is 285-300 μ mol/L, this scope is the effective electron transmission capacity of Fructose dehydrogenase, as the electron accepter in the Fructose dehydrogenase catalytic reaction; In addition, electron accepter can also be 1-methoxyl-5-sylvan piperazine Methylsulfate salt (1-Methoxy-5-methylphenazinium methyl sulfate, 1-methoxyPMS).
In the described R2 reagent, Fructose dehydrogenase (Fructose Dehydrogenase, FDH) content is 2.5-6.0U/ml, when being lower than the lower limit 2.5U/ml of Fructose dehydrogenase, the rate of change of per minute absorbance is very little, the micro fluctuation of absorbance rate of change promptly can cause result's very cataclysm, consider the optimality that reagent cost and reagent play a role, when being limited to 6.0U/ml on the selection Fructose dehydrogenase, in the 2.5-6.0U/ml scope, the concentration of Fructose dehydrogenase is high more, and reaction velocity is fast more, and the rate of change of per minute absorbance is also big more;
Described antiseptic is a Sodium azide, and content is 0.01-0.1%, and this scope can be to not reacted any spinoff, the term of validity that can prolong kit in effective sterilization; Can also be other have in the gentamicin of antisepsis and the ProClin series antiseptic any one etc., but cannot be thimerosal, it has certain inhibiting effect to Fructose dehydrogenase.
Above-mentioned detection method, in the described step 1), prepared refining sample can detect in separating the same day, also can preserve more than 2 weeks at least, but should avoid multigelation in frozen standby below-20 ℃; Described step 2) in, sample reagent should be taken into account the minimum application of sample amount of instrument than deciding according to the parameter of full automatic biochemical apparatus, the reactant liquor volume scope of cuvette etc.; In the described step 3), the calibration pattern can be selected voluntarily, can select the single-point calibration for use, and promptly list is calibrated with a fructose titer; Also can select two-point calibration for use, promptly use distilled water as a concentration point, the fructose titer is calibrated as the another one concentration point; In the described step 4), the excessive concentration of fructose in sample when exceeding the range of linearity of kit, is used and is resurveyed after physiological saline dilutes, and the result multiply by extension rate and gets final product;
Detection method of the present invention is a rate method, and described R1 reagent, R2 reagent are the McIlvaine ' s buffer solution that contains the PH4.5-5.0 of developer MTT, electron accepter PMS, Fructose dehydrogenase FDH and antiseptic, detect principle to be,
Fructose in the Fructose dehydrogenase catalysis sample, under the acting in conjunction of MTT and PMS, reaction generates coloured complex MTT Jia Za, this compound has maximum absorption band at 560-590nm wavelength place, by monitoring its per minute absorbance rate of change, and then detect the fructose concentration in the sample at 560-590nm wavelength place.
Compared with prior art, Tu Chu technique effect is:
1, this kit adopts rate method to detect the concentration of fructose in the refining sample, only adopt 2 kinds of liquid reagents, compare with the end-point method of manual inspection, the kind of agents useful for same and consumption all obviously reduce, avoided having the use of strong corrosion and volatile concentrated hydrochloric acid, temperature of reaction does not need to reach more than 60 ℃ yet again;
2, adopt this kit and detection method, do not need deproteinized again after sample prepares, also do not need pre-dilution, no longer need to do separately blank pipe, standard pipe in the testing process, simplified operation steps, reduced personal error; In addition, compare with existing hexokinase method detection kit, Fructose dehydrogenase makes testing result more accurately with special to the selectivity catalytic action of substrate fructose;
3, the present invention has realized from manual or semi-automatic transformation to full-automatic detection method, filled up the vacancy on the market, adopt full automatic biochemical apparatus to carry out the detection of robotization, scale, discharged great amount of manpower, saved a large amount of consumptive materials, to the accurate control and the monitoring of reaction time, course of reaction, significantly improved the accuracy and the reliability of testing result;
4; employing the present invention can be when detecting same this fructose concentration of increment; the refining γ that can also match-L-glutamyl transpeptidase with full automatic biochemical apparatus; alpha-glucosidase; the fructose kit is united use; and detect these projects simultaneously; after detecting end; can print all results that this institute of same increment surveys project with the form of report; can satisfy the relevant section office of hospital's male sterility; human sperm bank; mechanisms such as family planning research institute are to semen quality; the needs of sperm screening; and for auxiliary diagnosis of male sterility etc.; improve efficient and income that the seminal fluid routine biochemistry is analyzed greatly, be very suitable for conventional sense and popularization.
Embodiment:
According to following embodiment, can better understand the present invention.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, process conditions and result thereof only are used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1
A kind of seminal plasma fructose concentration detection by quantitative kit in the present embodiment comprises R1 reagent, R2 reagent and fructose titer, respectively by following composition and consumption preparation:
1) R1 reagent:
PH4.5McIlvaine ' s damping fluid 150 μ mol/L;
Tetrazolium bromide (MTT) 54 μ mol/L;
Azophenlyene dimethyl ester sulfate (PMS) 294 μ mol/L;
Sodium azide 0.1%;
2) R2 reagent:
PH4.5McIlvaine ' s damping fluid 150 μ mol/L;
Fructose dehydrogenase (FDH) 2.5U/ml;
Sodium azide 0.08%;
3) fructose titer:
PH4.5McIlvaine ' s damping fluid 150 μ mol/L;
Fructose 20mmol/L;
Gentamicin 0.05%;
Mentioned reagent is divided the bottle of packing into after all dissolving, and makes liquid double reagent and fructose titer, keeps in Dark Place standby in 2-8 ℃.
Embodiment 2
A kind of seminal plasma fructose concentration detection by quantitative kit in the present embodiment comprises R1 reagent, R2 reagent and fructose titer, respectively by following composition and consumption preparation:
1) R1 reagent:
PH4.8McIlvaine ' s damping fluid 200 μ mol/L;
The purple 60 μ mol/L of iodonitrotetrazolium;
1-methoxyl-5-sylvan piperazine Methylsulfate salt 285 μ mol/L;
Gentamicin 0.1%;
2) R2 reagent:
PH4.8McIlvaine ' s damping fluid 200 μ mol/L;
Fructose dehydrogenase 4.0U/ml;
Sodium azide 0.05%;
3) fructose titer:
PH4.8McIlvaine ' s damping fluid 200 μ mol/L;
Fructose 40mmol/L;
ProClin150 0.02％；
Mentioned reagent is divided the bottle of packing into after all dissolving, and makes liquid double reagent and fructose titer, keeps in Dark Place standby in 2-8 ℃.
Embodiment 3
A kind of seminal plasma fructose concentration detection by quantitative kit in the present embodiment comprises R1 reagent, R2 reagent and fructose titer, respectively by following composition and consumption preparation:
1) R1 reagent:
PH5.0Walpole ' s damping fluid 300 μ mol/L;
The purple 70 μ mol/L of iodonitrotetrazolium;
1-methoxyl-5-sylvan piperazine Methylsulfate salt 300 μ mol/L;
ProClin150 0.05％；
2) R2 reagent:
PH5.0Walpole ' s damping fluid 300 μ mol/L;
Fructose dehydrogenase (FDH) 6.0U/ml;
ProClin300 0.02％；
3) fructose titer:
PH5.0McIlvaine ' s damping fluid 300 μ mol/L;
Fructose 60mmol/L;
Sodium azide 0.1%;
Mentioned reagent is divided the bottle of packing into after all dissolving, and makes liquid double reagent and fructose titer, keeps in Dark Place standby in 2-8 ℃.
Embodiment 4
Utilize the method for fructose concentration in the kit detection refining among the embodiment 1, the concrete operations step is as follows:
1) specimen preparation:
Fresh semen liquefaction back is by the centrifugal 10min of 5000rpm, and the supernatant that is obtained promptly is;
2) the input basic parameter is as follows in the function software of Olympus AU400 full automatic biochemical apparatus:
Test item: Fru;
Sample size: 10 μ l;
R1 reagent and R2 reagent dosage ratio are 4: 1; R1 amount of reagent: 240 μ l; R2 amount of reagent: 60 μ l;
Detection method: rate method;
Calibration pattern: AB (single-point calibration);
Predominant wavelength: 570nm;
The Direction of Reaction: rise;
Main reading point 1:20;
Main reading point 2:25;
3) scale operation:
Calibrate with 20mmol/L fructose titer, instrument can draw the relational expression Y=AX between a fructose concentration and the per minute absorbance rate of change according to the rate of change of the concentration and the per minute absorbance of fructose titer; Wherein, constant A provides after calibration finishes automatically, and Y represents the concentration of fructose titer, and X represents the per minute absorbance rate of change of fructose titer;
For example, calibrate with 20mmol/L fructose titer, the per minute absorbance rate of change that records is 0.0068, then by calibration relation formula Y=AX, instrument calculates A=2941.18 automatically, also promptly, fructose concentration that is drawn by scale operation and the relational expression between the per minute absorbance rate of change are Y=2941.18X;
4) pattern detection:
With 1) in the sample and R1 reagent, the R2 reagent that prepare be placed on the relevant position of full automatic biochemical apparatus, after the detection information of input sample, begin to detect.After the end of test (EOT), instrument calculates the concentration of fructose in the sample automatically according to the calibration relation formula, i.e. fructose concentration Y (mmol/L)=2941.18X, and wherein, the per minute absorbance rate of change of X representative sample is measured automatically on full automatic biochemical apparatus and is provided.
For example, the per minute absorbance rate of change that full automatic biochemical apparatus records the fructose in certain part of refining is 0.0096, then fructose concentration Y=2941.18 * 0.0096=28.24mmol/L in this part refining
Embodiment 5
Utilize the method for fructose concentration in the kit detection refining among the embodiment 2, the concrete operations step is as follows:
1) specimen preparation:
Fresh semen liquefaction back is by the centrifugal 20min of 3000rpm, and the supernatant that is obtained promptly is;
2) the input basic parameter is as follows in the function software of Olympus AU640 full automatic biochemical apparatus:
Test item: Fru;
Sample size: 8 μ l;
R1 reagent and R2 reagent dosage ratio are 4: 1; R1 amount of reagent: 192 μ l; R2 amount of reagent: 48 μ l;
Detection method: rate method;
Calibration pattern: AA (two-point calibration);
Predominant wavelength: 570nm;
The Direction of Reaction: rise;
Main reading point 1:18;
Main reading point 2:24;
3) scale operation:
Calibrate with distilled water 0mmol/L and 40mmol/L fructose titer, instrument can draw the relational expression Y=AX+B between a fructose concentration and the per minute absorbance rate of change according to concentration and absorbance separately; Wherein, constant A, B provide after calibration finishes automatically, and Y represents the concentration of fructose in the fructose titer, and X represents the per minute absorbance rate of change of fructose titer.
For example, after carrying out scale operation with distilled water and fructose titer, the per minute absorbance rate of change of measuring is respectively 0.0003 and 0.0098, then by calibration relation formula Y=AX+B, instrument calculates A=4210.5 automatically, B=-1.2632, also, fructose concentration that is drawn by scale operation and the relational expression between the per minute absorbance rate of change are Y=4210.5X-1.2632.
4) pattern detection:
With 1) in the sample and R1 reagent, the R2 reagent that prepare be placed on the relevant position of full automatic biochemical apparatus, after the detection information of input sample, begin to detect.After the end of test (EOT), instrument calculates the concentration of fructose in the sample automatically according to the calibration relation formula, i.e. fructose concentration Y (mmol/L)=4210.5X-1.2632, and wherein, X is the per minute absorbance rate of change of sample, measures automatically to provide on full automatic biochemical apparatus;
For example, after certain part of refining sample diluted 2 times with physiological saline, the per minute absorbance rate of change of the fructose that full automatic biochemical apparatus records was 0.0105, then concentration Y=2 * (4210.5 * the 0.0105-1.2632)=85.9mmol/L of fructose in this part refining.
Embodiment 6
Utilize the method for fructose concentration in the kit detection refining among the embodiment 3, the concrete operations step is as follows:
1) specimen preparation:
Fresh semen liquefaction back is by the centrifugal 15min of 4000rpm, and the supernatant that is obtained promptly is;
2) the input basic parameter is as follows in the function software of Laura Faith-1000 full automatic biochemical apparatus:
Test item: Fru;
Sample size: 11 μ l;
R1 reagent and R2 reagent dosage ratio are 4: 1; R1 amount of reagent: 264 μ l; R2 amount of reagent: 66 μ l;
Detection method: rate method;
Calibration pattern: single-point calibration;
Predominant wavelength: 578nm;
The Direction of Reaction: rise;
Main reading point 1:25;
Main reading point 2:30;
3) scale operation:
Calibrate with 60mmol/L fructose titer, instrument can draw the relational expression Y=AX between a fructose concentration and the per minute absorbance rate of change according to the rate of change of the concentration and the per minute absorbance of fructose titer; Wherein, constant A provides after calibration finishes automatically, and Y represents the concentration of fructose titer, and X represents the per minute absorbance rate of change of fructose titer;
For example, calibrate with 60mmol/L fructose titer, the per minute absorbance rate of change of measuring is 0.0155, then by calibration relation formula Y=AX, instrument calculates A=3870.97 automatically, also promptly, fructose concentration that is drawn by scale operation and the relational expression between the per minute absorbance rate of change are Y=3870.97X;
4) pattern detection:
With 1) in the sample and R1 reagent, the R2 reagent that prepare be placed on the relevant position of full automatic biochemical apparatus, after the detection information of input sample, begin to detect.After the end of test (EOT), instrument calculates the concentration of fructose in the sample automatically according to the calibration relation formula, i.e. fructose concentration Y (mmol/L)=3870.97X, and wherein, X is the per minute absorbance rate of change of sample, measures automatically to provide on full automatic biochemical apparatus.
For example, the per minute absorbance rate of change that full automatic biochemical apparatus records the fructose in certain part of refining is 0.0184, then fructose concentration Y=3870.97 * 0.0184=71.23mmol/L in this part refining
Embodiment 7
In on the basis of embodiment 1 and embodiment 4, criticizing and the detection (being the reappearance of the testing result of kit of the present invention) of betweenrun precision
Select each 1 part in low, the high mixing refining sample that is worth of fructose concentration, prepare 5 batches detection kit, each batch kit is surveyed same concentration refining sample 8 times, have 40 data, the result of each sample replication 8 times calculates withinrun precision from 5 batches, calculate betweenrun precision from all 40 results, the result is that CV% represents with the percentage of the coefficient of variation all.Wherein, CV%=standard deviation/mean value * 100%, this coefficient is more little, shows that same this duplicate detection of increment result's dispersion degree is more little, and promptly the reappearance of testing result is good more.The result and the result of manual or semi-automatic detection relatively see Table 1, and the method good reproducibility that uses this kit to detect seminal plasma fructose concentration is described.
Table 1 is precision result manual, semi-automatic and full-automatic detection compare
| The manual inspection method | Method for semi-automatically detecting | The full-automatic detection method of the present invention | |
| Withinrun precision | CV％≤15％ | CV％≤15％ | CV％≤8％ |
| Betweenrun precision | CV％≤15％ | CV％≤15％ | CV％≤9％ |
Embodiment 8
Detection (being the concentration range that kit of the present invention can accurately be measured) in the basic enterprising line linearity scope of embodiment 1 and embodiment 4.
Get high concentration fructose titer (80mmol/L), be prepared into the sample of 11 isoconcentration gradients by table 2, each concentration samples duplicate detection is averaged absorbance 4 times, according to experimental result, is X-axis with the fructose final concentration, with the mean light absorbency is Y-axis, carry out straight-line regression, find fructose concentration in the 0-72mmol/L scope, the correlativity between fructose concentration and the absorbance is good, coefficient R 〉=0.990 is the concentration range that kit of the present invention can accurately be measured.
Show the sample preparation of 2:11 isoconcentration gradient level
| Catalogue number(Cat.No.) | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| Physiological saline (ml) | 1.0 | 0.9 | 0.8 | 0.7 | 0.6 | 0.5 | 0.4 | 0.3 | 0.2 | 0.1 | 0.0 |
| 80mmol/L fructose (ml) | 0.0 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 | 0.6 | 0.7 | 0.8 | 0.9 | 1.0 |
| Fructose final concentration (mmol/L) | 0.0 | 8.0 | 16.0 | 24.0 | 32.0 | 40.0 | 48.0 | 56.0 | 64.0 | 72.0 | 80.0 |
Embodiment 9
On the basis of embodiment 1 and embodiment 4, verify accuracy by recovery test.
Choose the low value and each 1 part of high value mixing sample of fructose concentration, with the low value mixing sample high value mixing sample is carried out the different proportion dilution, the low value sample accounts for 75%, 50%, 25% respectively, every this replication of increment 4 times calculates average recovery rate respectively, and its average recovery rate is expressed as the recovery 1, the recovery 2 and the recovery 3 respectively, wherein, the recovery=measured result/notional result * 100%, the recovery are more near 100%, and the accuracy of expression testing result is high more.Gained result and result manual, semi-automatic detection relatively see Table 3, show the method accuracy height that uses this kit to detect seminal plasma fructose concentration.
Table 3 is recovery result manual, semi-automatic and full-automatic detection compare
| The manual inspection method | Method for semi-automatically detecting | The full-automatic detection method of the present invention | |
| The recovery 1 | 68％ | 72％ | 93％ |
| The recovery 2 | 65％ | 74％ | 98％ |
| The recovery 3 | 69％ | 74％ | 101％ |
| Average recovery rate | 67％ | 73％ | 97％ |
Embodiment 10
On the basis of embodiment 1 and embodiment 4, carry out the detection of reagent blank absorbance rate of change
Change the sample in the fructose reaction system into distilled water and detect, other testing conditions is constant, and measured result is reagent blank absorbance rate of change (Δ A/min), is one of quality index of this kit.Repeated detection result shows, Δ A/min≤0.002, and when exceeding this value, prompting reagent may out of date or misoperation.
Kit and detection method in embodiment 2 of the present invention and embodiment 5, embodiment 3 and embodiment 6 and described other concentration range, the detection method of the rate of change of its precision, accuracy, the range of linearity and reagent blank absorbance and step and embodiment 1 and embodiment's 4 is similar, does not repeat them here.
Claims (2)
1. a seminal plasma fructose concentration detection by quantitative kit is characterized in that, form by R1 reagent, R2 reagent and fructose titer, wherein,
1) R1 reagent comprises the component of following content:
2) R2 reagent comprises the component of following content:
PH4.5-5.0 damping fluid 150-300 μ mol/L;
Fructose dehydrogenase 2.5-6.0U/ml;
Antiseptic 0.01-0.1%;
3) the fructose titer comprises the component of following content:
PH4.5-5.0 damping fluid 150-300 μ mol/L;
Fructose 20-60mmol/L;
Antiseptic 0.01-0.1%;
Wherein: described damping fluid is McIlvaine's damping fluid, Walpole's damping fluid or the kaliumphosphate buffer of pH4.5-5.0;
Described developer is tetrazolium bromide, iodonitrotetrazolium purple or 1-methoxyl-5-sylvan piperazine Methylsulfate salt;
Described electron accepter is azophenlyene dimethyl ester sulfate or 1-methoxyl-5-sylvan piperazine Methylsulfate salt;
Described antiseptic is any one in Sodium azide, gentamicin or the ProClin series antiseptic.
2. the application of the described seminal plasma fructose concentration of claim 1 detection by quantitative kit in detecting seminal plasma fructose concentration is characterized in that may further comprise the steps:
1) specimen preparation: fresh semen liquefaction back is by the centrifugal 10～20min of 3000～5000rpm, and the supernatant that is obtained promptly makes sample;
2) the input basic parameter is as follows in the function software of Olympus AU400 or Olympus AU640 or Laura Faith-1000 full automatic biochemical apparatus:
Test item: Fru;
Sample reagent ratio: 1:30;
Reagent ratio: R1:R2=1:4;
Predominant wavelength: 560-590nm;
Detection method: rate method;
Calibrating method: single-point calibration or two-point calibration;
The Direction of Reaction: rise;
Reaction time: 180-270 second;
Detection time: 90-120 second;
3) scale operation: calibrate with the fructose titer, instrument can draw the relational expression between fructose concentration and the absorbance rate of change according to the concentration and the per minute absorbance rate of change thereof of fructose titer:
Single-point calibration: Y=A ＇ X; Or two-point calibration: Y=AX+B,
Wherein, the constant A ＇ of single-point calibration or the constant A of two-point calibration, B provide after calibration finishes automatically, and Y represents the concentration of fructose titer, and X represents the per minute absorbance rate of change of fructose titer;
4) sample detection:
With 1) in the sample and R1 reagent, the R2 reagent that prepare be placed on the relevant position of described full automatic biochemical apparatus, after the detection information of input sample, begin to detect; After the end of test (EOT), the concentration that concentration that described full automatic biochemical apparatus draws according to calibration and the relational expression between the absorbance rate of change calculate fructose in the sample automatically.
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