CN106645720A - Beta-glucuronidase detection reagent, reaction pad, preparation method of reaction pad and kit - Google Patents
Beta-glucuronidase detection reagent, reaction pad, preparation method of reaction pad and kit Download PDFInfo
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- CN106645720A CN106645720A CN201610881477.4A CN201610881477A CN106645720A CN 106645720 A CN106645720 A CN 106645720A CN 201610881477 A CN201610881477 A CN 201610881477A CN 106645720 A CN106645720 A CN 106645720A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a beta-glucuronidase detection reagent, a reaction pad, a preparation method of the reaction pad and a kit. The beta-glucuronidase detection reagent is prepared from polyvinylpyrrolidone, 5-bromo-4-chloro-3-indole-beta-D-glucuronide and new-born calf serum. The preparation method of the reaction pad comprises steps as follows: 1), polyvinylpyrrolidone and 5-bromo-4-chloro-3-indole-beta-D-glucuronide are dissolved in a phosphate buffer solution, and a soak solution A is obtained; 2), the new-born calf serum is dissolved in the phosphate buffer solution, and a soak solution B is obtained; 3), a carrier is put in the soak solution A and is sufficiently soaked and dried, and a soak solution A carrier is obtained; 4), the soak solution A carrier is put in the soak solution B and is sufficiently soaked and dried, and the reaction pad is obtained. According to the detection reagent, the stability of activity of beta-glucuronidase is specifically enhanced through addition of the new-born calf serum, so that the detection is more accurate.
Description
Technical field
The present invention relates to aerobic bacteria property vaginitis detection reagent field, and in particular to a kind of beta-glucuronidase enzyme detection
Reagent, reacting pad, its preparation method and kit.
Background technology
Vaginitis is the inflammation of connective tissue under vaginal mucosa and mucous membrane, is the common disease of Out-patient Clinic of Department of Gynecology.Normal health
Women, due to anatomy and biochemical characteristics, intrusion of the vagina to pathogen has natural defense function, when preventing naturally for vagina
Imperial function is destroyed, then pathogen is easy to invade, and causes colpitis.Common vaginitis has bacterial vaginitis (BV/
AV), colpomycosis (VVC) and trichomonas vaginitis (TV) three major types.Wherein, women Bacterial leaf steak can be divided into two
Kind, one kind is caused by anaerobic bacteria and facultative anaerobic bacteria, clinically claims bacterial vaginosis BV (BV), another kind to be caused by aerobic bacteria,
Clinically claim aerobic bacteria property vaginitis (AV).The pathogen of two kinds of bacterial vaginitis is different, clinical symptoms, therapeutic scheme and knot
Office also differs, and distinguishes both Bacterial leaf steaks, significant in the diagnosis and treatment of vagina infection.BV refers to a class
Normal flora (produces H in genesiology2O2Bacillus acidi lactici) reduce, instead Gardnerella, anaerobic bacteria, bend vibrios amount reproduction,
Intravaginal ecological balanced system changes the class disease for causing.AV is reduced by Vaginal lactobacilli, and aerobic bacteria infection causes
Colpitis.Common causative is streptococcus, staphylococcus and EHEC.Normal vagina is with Bacillus acidi lactici as advantage
The normal microflora of bacterium, vagina pH ＜ 4.5.And when aerobic bacteria property vaginitis is suffered from, Vaginal lactobacilli is reduced, and aerobic bacteria
Breeding causes congested vaginal wall mucous membrane, oedema and produces purulent secretion.Clinical manifestation is with vagina abnormal secretion, intercourse pain
It is main.Bacterium preformation enzyme is a series of relevant with the growth metabolism enzyme that bacterium synthesizes during growth and breeding, these enzymes
The substrate of various biochemical reactions can be decomposed, and can for a long time retain its activity in aerobic environment.Different genera it is thin
Bacterium, the species of its preformation enzyme is different.B races streptococcus and EHEC metabolism can produce β glucuronidases.By detection
β glucuronidases to diagnose vagina in aerobic bacteria Bacterial community be an effective, feasible method.But currently used for detection
The detection reagent of β glucuronidases its accuracy has been short of.
The content of the invention
In order to overcome the deficiencies in the prior art, first purpose of the present invention is to provide a kind of beta-glucuronidase enzyme
Detection reagent, the detection reagent specifically strengthens the stability of beta-glucuronidase enzymatic activity, inspection by addition NBCS
It is more accurate to survey.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：A kind of beta-glucuronidase enzyme detection
Reagent, including the chloro- 3- indoles-β of the bromo- 4- of polyvinylpyrrolidone, 5--D-Glucose aldehydic acid glycosides salt and NBCS and phosphoric acid
Salt buffer.
Preferably, the composition in the beta-glucuronidase enzyme detection reagent includes by weight：
1～10 part of polyvinylpyrrolidone；
The chloro- 3- indoles-β of the bromo- 4- of 5- -2.5～25 parts of D-Glucose aldehydic acid glycosides salt；
Wherein, the percent by volume that NBCS accounts for beta-glucuronidase enzyme detection reagent is 1～10%；
The molar concentration of phosphate buffer is 50～150mmol/L.
Preferably, the composition in the beta-glucuronidase enzyme detection reagent includes by weight：
2 parts of polyvinylpyrrolidone；
The chloro- 3- indoles-β of the bromo- 4- of 5- -5 parts of D-Glucose aldehydic acid glycosides salt；
Wherein, the percent by volume that NBCS accounts for beta-glucuronidase enzyme detection reagent is 1%；
The molar concentration of phosphate buffer is 100mmol/L.
Preferably, the polyvinylpyrrolidone is polyvinylpyrrolidone-K90.
Preferably, the chloro- 3- indoles-β-D-Glucose aldehydic acid glycosides salt of the bromo- 4- of the 5- be the chloro- 3- indoles-β of the bromo- 4- of 5--
D-Glucose aldehydic acid glycosides sodium.
Preferably, the dilution of the sample detected using the beta-glucuronidase enzyme detection reagent is physiology
Salt solution.
Second object of the present invention is to provide for a kind of reacting pad, and the reacting pad is convenient to use, and accuracy is high.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：A kind of reacting pad, including carrier and such as
The composition of upper described beta-glucuronidase enzyme detection reagent；The composition coating of the beta-glucuronidase enzyme detection reagent
On carrier；The reacting pad is used to detect beta-glucuronidase enzyme.
Preferably, the dilution of the sample detected using the reacting pad is physiological saline.
Third object of the present invention is to provide for a kind of preparation method of reacting pad, and by the preparation method inspection is combined
The composition of test agent obtains the high reacting pad of a kind of sensitivity height, accuracy.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：A kind of system of reacting pad as above
Preparation Method, comprises the following steps：
1) soak A is prepared：Polyvinylpyrrolidone and the chloro- 3- indoles-β of the bromo- 4- of 5--D-Glucose aldehydic acid glycosides salt is molten
In phosphate buffer, soak A is obtained；
2) soak B is prepared：NBCS is substantially soluble in phosphate buffer, soak B is obtained；
3) soak A process：Carrier is placed in soak A after fully immersion and is taken out, be then dried, soaked
Liquid A carriers；
4) soak B process：Soak A carriers are placed in soak B after fully immersion and are taken out, be then dried,
Obtain reacting pad.
Preferably, step 1) and 2) in, the pH of the phosphate buffer is 6.8～7.5；More preferably, the phosphorus
The pH of phthalate buffer is 6.8.
Preferably, step 1) and 2) in, the phosphate buffer be sodium phosphate buffer.
Preferably, step 3) in, the carrier is placed in soak A fully 10～20s of immersion；Step 4) in, it is described
Carrier is placed in soak B fully 10～20s of immersion.
Preferably, step 3) in, the temperature of the drying is 20～70 DEG C, and the time is 30～40min；Step 4) in,
The temperature of the drying is 20～70 DEG C, and the time is 30～40min.
Preferably, the carrier is filter paper.
Fourth object of the present invention is to provide for a kind of kit, and the kit is used to detect beta-glucuronidase
Enzyme.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：A kind of kit, including it is as described above
Reacting pad；The kit is used to detect beta-glucuronidase enzyme.
The design principle of the present invention is as follows：
When the glucuronidase activity in vaginal fluid is abnormal, the bromo- 4- of glucuronidase hydrolysis 5- are chloro-
3- indoles-β-D-Glucose aldehydic acid glycosides salt, indyl is presented blueness, depth and the β-grape alditol of colour generation under conditions of aerobic
The activity of neuraminidase is directly proportional；NBCS auxiliary strengthens the stability of beta-glucuronidase enzymatic activity so that detection knot
Fruit is accurately.
Compared to existing technology, the beneficial effects of the present invention is：
1st, detection reagent of the invention specifically strengthens the steady of beta-glucuronidase enzymatic activity by addition NBCS
Qualitative, it is more accurate to detect；
2nd, reacting pad of the invention can use physiological saline as the dilution of detection sample, and use condition is simple and convenient,
And the detection reacting pad of condition identical other bacterial classification preformation enzymes is may be used in combination while detecting to sample, it is in hgher efficiency；
3rd, reacting pad preparation method of the invention is to greatest extent coated on the composition of detection reagent in reacting pad, obtains
Reacting pad sensitivity it is high, accuracy is good.
Description of the drawings
Fig. 1 is the colour developing result schematic diagram of embodiment 8.
Specific embodiment
Below, with reference to accompanying drawing and specific embodiment, the present invention is described further：
A kind of beta-glucuronidase enzyme detection reagent, by weight including following component：
1～10 part of polyvinylpyrrolidone-K90；
The chloro- 3- indoles-β of the bromo- 4- of 5- -2.5～25 parts of D-Glucose aldehydic acid glycosides sodium；
Wherein, the percent by volume that NBCS accounts for beta-glucuronidase enzyme detection reagent is 1～10%；
The molar concentration of phosphate buffer is 50～150mmol/L；
Polyvinylpyrrolidone (PVP)-K90, at 25 DEG C, percentage by volume is 5% aqueous solution (aq), and its viscosity is
39.5～45.8mPas.
Beta-glucuronidase enzyme detection reagent is obtained by following preparation method：
By polyvinylpyrrolidone, the chloro- 3- indoles-β of the bromo- 4- of 5--D-Glucose aldehydic acid glycosides sodium and NBCS successively
In being dissolved in the phosphate buffer (PBS) that pH is 6.8～7.5, beta-glucuronidase enzyme detection reagent is obtained.
A kind of reacting pad, including the composition of carrier and beta-glucuronidase enzyme detection reagent as above；The β-
The composition of glucuronidase detection reagent is coated on carrier；The reacting pad is used to detect beta-glucuronidase enzyme.
When carrying out sample detection using the reacting pad, the dilution of sample is physiological saline.
The preparation method of the reacting pad is comprised the following steps：
1) get the raw materials ready：Prepare polyvinylpyrrolidone-K90, the chloro- 3- indoles-β-D-Glucoses of the bromo- 4- of 5- by above weight portion
Aldehydic acid glycosides salt and NBCS；
2) phosphate buffer (PBS) is prepared：Prepare the phosphate buffer that pH is 6.8～7.5 stand-by；
The phosphate buffer is NaH2PO4And Na2HPO4The aqueous solution；
3) soak A is prepared：Polyvinylpyrrolidone and the chloro- 3- indoles-β of the bromo- 4- of 5--D-Glucose aldehydic acid glycosides sodium is molten
In phosphate buffer, soak A is obtained；
4) soak B is prepared：NBCS is substantially soluble in phosphate buffer, soak B is obtained；
5) soak A process：Filter paper is placed in soak A after fully 10～20s of immersion and is taken out, be then 20 in temperature
～70 DEG C, the time is to be dried under conditions of 30～40min, obtains soak A filter paper；
6) soak B process：Soak A filter paper is placed in soak B after fully 10～20s of immersion and is taken out, Ran Hou
Temperature is 20～70 DEG C, and the time is to be dried under conditions of 30～40min, obtains reacting pad.
More specifically, the filter paper can be Whatman glass fiber filter papers, the fiber filter paper of GF/A Whatman
Grade3 or common quantitative filter papers.
Application response pad reagent preparation box：Reacting pad is placed in the reacting hole of kit, is then sealed reacting hole, obtained
To kit；Kit is placed in 2～8 DEG C and preserves；The kit is used to detect beta-glucuronidase enzyme.
Embodiment 1～3
Embodiment 1～3 discloses beta-glucuronidase enzyme detection reagent, and by table 1 three groups of material compositions are prepared respectively, presses
Table 2 prepares the concentration sodium phosphate buffer different with pH；, for embodiment 1, for embodiment 2, C groups are for embodiment for B groups for A groups
3。
By polyvinylpyrrolidone, the chloro- 3- indoles-β of the bromo- 4- of 5--D-Glucose aldehydic acid glycosides sodium and NBCS successively
In being dissolved in 100mL sodium phosphate buffers, beta-glucuronidase enzyme detection reagent is obtained.
The composition and content of the detection reagent of table 1
The preparation parameter of the sodium phosphate buffer of table 2
Detection reagent to obtaining in embodiment 1～3 is detected：Different β-the glucose of concentration of enzymatic activity is prepared respectively
Aldehyde neuraminidase standard solution, beta-glucuronidase enzyme standard solution using physiological saline as solvent, detection embodiment 1～
3 sensitivity.
Respectively each 1mL of beta-glucuronidase enzyme detection reagent of Example 1～3, is then added dropwise respectively standard items molten
Liquid 35uL, 8～10min of water-bath under conditions of temperature is for 37 ± 2 DEG C, the color change then observed in solution, solution colour developing
Represent positive for light blue, blue or bluish violet, represent negative without colour developing；As a result it is as shown in table 3：
The testing result of the embodiment 1～3 of table 3
+ represent positive ,-represent negative.
The detectable beta-glucuronidase enzyme of embodiment 1～3, and when beta-glucuronidase enzymatic activity is relatively low still
It is able to detect that beta-glucuronidase enzyme；Wherein when concentration of enzymatic activity is 3.75U/mL, embodiment 1 still can detect to be lacked
The beta-glucuronidase enzyme of amount, the sensitivity highest of the detection reagent of embodiment 1.
Embodiment 4～6
Embodiment 4～6 discloses a kind of reacting pad and preparation method thereof：
1) got the raw materials ready by table 1, by table 2 sodium phosphate buffer is prepared；, for embodiment 4, B groups are for embodiment 5, C for A groups
Group is used for embodiment 6；
2) soak A is prepared：Polyvinylpyrrolidone and the chloro- 3- indoles-β of the bromo- 4- of 5--D-Glucose aldehydic acid glycosides sodium is molten
In 100mL sodium phosphate buffers, soak A is obtained；
3) soak B is prepared：NBCS is substantially soluble in 100mL sodium phosphate buffers, soak B is obtained；
4) soak A process：Filter paper is placed in soak A after fully 10～20s of immersion and is taken out, be then 40 in temperature
DEG C, the time is to be dried under conditions of 30～40min, obtains soak A filter paper；
5) soak B process：Soak A filter paper is placed in soak B after fully 10～20s of immersion and is taken out, Ran Hou
Temperature is 40 DEG C, and the time is to be dried under conditions of 30～40min, obtains reacting pad.
Reacting pad to obtaining in embodiment 4～6 is detected：The different β of concentration of enzymatic activity-grape alditol is prepared respectively
Neuraminidase standard solution, beta-glucuronidase enzyme standard solution detects embodiment 4～6 using physiological saline as solvent
Sensitivity；Standard solution 35uL is added dropwise respectively in the reacting pad of embodiment 4～6, under conditions of temperature is for 37 ± 2 DEG C
8～10min of water-bath, the then color change in observing response pad, reacting pad colour developing represents positive for light blue, blue or bluish violet
Property, represent negative without colour developing；As a result it is as shown in table 5：
The testing result of the embodiment 4～6 of table 4
+ represent positive ,-represent negative.
The reacting pad of embodiment 4～6 can detect beta-glucuronidase enzyme, and when beta-glucuronidase enzymatic activity compared with
Remain able to detect beta-glucuronidase enzyme when low；Wherein when concentration of enzymatic activity is 3.75U/mL, embodiment 4 still can be examined
Measure a small amount of beta-glucuronidase enzyme, the reacting pad sensitivity highest of embodiment 4.
Embodiment 7
In example 4, soak A and soak B are adopted and sodium phosphate buffer molar concentration identical Tris-
Hcl buffer solutions, absolute methanol or physiological saline substitute sodium phosphate buffer, and adopt and identical β of embodiment 4～6-glucose
Aldehyde neuraminidase standard solution is detected that testing result is as shown in table 6：
Table 5 substitutes the testing result of the reacting pad obtained by sodium phosphate buffer using different solvents
As a result show：Using the sensitivity highest of phosphate buffer, still can when concentration of enzymatic activity is 3.75U/mL
Enough detections are obtained.
Embodiment 8
The reacting pad that embodiment 4 is obtained is placed in different preservation conditions by the present embodiment, detects its stability, is adopted after process
Detected that the colour developing result of reacting pad is as shown in Figure 1 with the identical detection method of embodiment 4～6, it is illustrated that in it is blank right
According to for physiological saline, as a result as shown in table 6：
The reacting pad sensitivity technique result processed under the different condition of table 6
Testing result shows no matter the reacting pad that with the addition of NBCS is processed under 37 DEG C of placements or cryogenic conditions,
Higher stability is shown, and sensitivity is still padded than being not added with the reaction of NBCS, shows NBCS
The stability of reacting pad is improve in the middle of the present invention.
For a person skilled in the art, technical scheme that can be as described above and design, make other each
Plant corresponding change and deform, and all these changes and deforms the protection model that should all belong to the claims in the present invention
Within enclosing.
Claims (14)
1. a kind of beta-glucuronidase enzyme detection reagent, it is characterised in that：Including polyvinylpyrrolidone, the chloro- 3- of the bromo- 4- of 5-
Indoles-β-D-Glucose aldehydic acid glycosides salt, NBCS and phosphate buffer.
2. beta-glucuronidase enzyme detection reagent as claimed in claim 1, it is characterised in that：The beta-glucuronidase
Composition in enzyme detection reagent includes by weight：
1～10 part of polyvinylpyrrolidone；
The chloro- 3- indoles-β of the bromo- 4- of 5- -2.5～25 parts of D-Glucose aldehydic acid glycosides salt；
Wherein, the percent by volume that NBCS accounts for beta-glucuronidase enzyme detection reagent is 1～10%；
The molar concentration of phosphate buffer is 50～150mmol/L.
3. beta-glucuronidase enzyme detection reagent as claimed in claim 1, it is characterised in that：The polyvinylpyrrolidone
For polyvinylpyrrolidone-K90.
4. beta-glucuronidase enzyme detection reagent as claimed in claim 1, it is characterised in that：The chloro- 3- Yin of the bromo- 4- of the 5-
Diindyl-β-D-Glucose aldehydic acid glycosides salt is the chloro- 3- indoles-β of the bromo- 4- of 5--D-Glucose aldehydic acid glycosides sodium.
5. beta-glucuronidase enzyme detection reagent as claimed in claim 1, it is characterised in that：Using the β-grape alditol
The dilution of the sample that neuraminidase detection reagent is detected is physiological saline.
6. a kind of reacting pad, it is characterised in that：Beta-glucuronidase enzyme including carrier and as described in claim 1-5 is arbitrary
The composition of detection reagent；The composition of the beta-glucuronidase enzyme detection reagent is coated on carrier；The reacting pad is used for
Detection beta-glucuronidase enzyme.
7. reacting pad as claimed in claim 6, it is characterised in that：The dilution of the sample detected using the reacting pad
For physiological saline.
8. a kind of preparation method of reacting pad as claimed in claim 6, it is characterised in that comprise the following steps：
1) soak A is prepared：Polyvinylpyrrolidone and the chloro- 3- indoles-β of the bromo- 4- of 5--D-Glucose aldehydic acid glycosides salt are dissolved in into phosphorus
In phthalate buffer, soak A is obtained；
2) soak B is prepared：NBCS is substantially soluble in phosphate buffer, soak B is obtained；
3) soak A process：Carrier is placed in soak A after fully immersion and is taken out, be then dried, obtain soak A
Carrier；
4) soak B process：Soak A carriers are placed in soak B after fully immersion and are taken out, be then dried, obtained
Reacting pad.
9. the preparation method of reacting pad as claimed in claim 8, it is characterised in that：Step 1) and 2) in, the phosphate delay
The pH for rushing liquid is 6.8～7.5.
10. the preparation method of reacting pad as claimed in claim 9, it is characterised in that：Step 1) and 2) in, the phosphate delay
The pH for rushing liquid is 6.8.
The preparation method of 11. reacting pads as claimed in claim 8, it is characterised in that：Step 1) and 2) in, the phosphate delay
Liquid is rushed for sodium phosphate buffer.
The preparation method of 12. reacting pads as claimed in claim 8, it is characterised in that：Step 3) in, the carrier is placed in immersion
Fully 10～20s of immersion in liquid A；Step 4) in, the carrier is placed in soak B fully 10～20s of immersion.
The preparation method of 13. reacting pads as claimed in claim 8, it is characterised in that：Step 3) in, the temperature of the drying is
20～70 DEG C, the time is 30～40min；Step 4) in, the temperature of the drying is 20～70 DEG C, and the time is 30～40min.
A kind of 14. kits, it is characterised in that：Including reacting pad as claimed in claim 6；The kit be used for detect β-
Glucuronidase.
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| CN201610881477.4A CN106645720A (en) | 2016-09-30 | 2016-09-30 | Beta-glucuronidase detection reagent, reaction pad, preparation method of reaction pad and kit |
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| CN114235789A (en) * | 2021-11-29 | 2022-03-25 | 北京华晟源医疗科技有限公司 | Preparation method of beta-glucuronidase detection paper |
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Application publication date: 20170510 |
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