CN104181155A - Kit and preparation method thereof - Google Patents
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- CN104181155A CN104181155A CN201410390094.8A CN201410390094A CN104181155A CN 104181155 A CN104181155 A CN 104181155A CN 201410390094 A CN201410390094 A CN 201410390094A CN 104181155 A CN104181155 A CN 104181155A
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Abstract
The invention discloses a kit for detecting sulfydryl metabolite in urine prompting the cervical lesion and a preparation method thereof. The kit comprises a reference agent, a testing agent and a color developing agent, wherein the reference agent is a mercuric chloride solution or a mercury sulfate solution, and the color developing agent is a phosphotungstic acid reagent; the kit is prepared in a sterility packaging manner. The remarkable increment of sulfydryl metabolite in female urine is proved to prompt the cervical lesion. The kit can detect the content of the sulfydryl metabolite at a preclinical phase and a clinical phase, the positive rate and accuracy for detecting the cervical cancer are high, the missed diagnosis can be avoided, a subject is prevented from being wounded and crossly infected, and instruments and equipment are not needed; the detection process is simple, the acceptance of the subject is high, the manpower and material resource can be greatly saved, workload of the examination personnel can be remarkably alleviated, the bottleneck that the screening number cannot reach the standard can be effectively broken through, and the kit is particularly suitable for surveying the cervical cancer in a wide range.
Description
Technical field
The present invention relates to biology and field of medical examination, be specifically related to a kind of for pointing out urine sulfydryl metabolite detection kit of cervical carcinoma pathology and preparation method thereof.
Background technology
Common screening methods of cervical cancer has HPV (Human infectious warts virus) to detect at present, and TCT (ThinPrep cytologic test), Pap smear detect and pathological diagnosis.Existing detection method reagent cost is high, and testing process is more complicated, and the cycle is long, can not adapt at present the needs of cervical cancer screening on a large scale.
Along with the development of biology techniques, researchist finds the remarkable rising of sulfydryl metabolite level in women's urine, the generation that can point out cervical carcinoma pathology.And can predict in advance preferably the generation of cervical carcinoma pathology by the detection of sulfydryl metabolite.This is because HPV (Human infectious warts virus) is considered to cause the main cause of disease of cervical carcinoma, the E6 proto-oncogene protein of HPV16 coding connects mediation by E6AP and is combined with cell protein P53, by ubiquitin approach in body, promote P53 to degrade again, cause the thiol molecule of P53 albumen in cervical cell or repressor protein a large amount of and irreversiblely come off and discharge from urine.Normal women does not have sulfydryl metabolin to discharge from urine, by the generation that in women's urine, sulfydryl metabolite horizontal detection can be pointed out cervical carcinoma pathology.
Summary of the invention
The present invention discloses a kind of kit and preparation method thereof according to the deficiencies in the prior art.The problem to be solved in the present invention is to provide a kind of for pointing out the urine sulfydryl metabolite detection kit of cervical carcinoma pathology, and the present invention also provides preparation method and application's detection method of mentioned reagent box.
The detection kit that first the present invention provides:
Kit is comprised of following reagent: one group of dummy, one group of test agent, two groups of developers;
Described dummy is mercuric chloride solution or mercuric sulfate solution, and concentration is 0.02mol-0.05mol/l; Described test agent is distilled water;
Described developer is phosphotungstic acid reagent and the buffer solution potpourri of 1:1 by volume; Wherein phosphotungstic acid reagent is comprised of the aqueous solution of following component concentration: sodium tungstate 40g-60g/l, 85% phosphoric acid 30ml-50ml/l, lithium sulfate 30g-50g/l; Described buffer solution is sodium acetate/acetic acid buffer solution or sodium dihydrogen phosphate/disodium hydrogen phosphate buffer solution, and buffer solution PH is PH5.4-PH5.8.
Above-mentioned every box kit comprises one group of 1ml dummy, one group of 1ml test agent and two groups of each 2ml developers, and each group reagent is all encapsulated in 5ml ampoule.
The present invention also provides the preparation method of mentioned reagent box, comprising:
(1) distilled water preparation aseptic packaging and testing agent;
(2) dummy preparation: take mercuric chloride or mercuric sulfate by concentration 0.02mol-0.05mol/l requirement, be dissolved in sterile packaged after distilled water;
(3) developer preparation:
A, the preparation of phosphotungstic acid reagent, take sodium tungstate 40g-60g, is dissolved in distilled water 300ml-500ml, add 85% phosphoric acid 30ml-50ml, reflux and boil 2 hours, be cooled to room temperature, adding distil water is settled to 1000ml, finally adds lithium sulfate 30g-50g, is placed in 2 ℃ of-8 ℃ of stored refrigerated standby;
B, buffer solution preparation: buffer solution is PH5.4-PH5.8, by pH value requirement preparation sodium acetate/acetic acid buffer solution, or sodium dihydrogen phosphate/disodium hydrogen phosphate buffer solution, be placed in 2 ℃ of-8 ℃ of stored refrigerated standby;
C, phosphotungstic acid reagent solution prepared by step a and step b and buffer solution by volume 1:1 mix, and are placed in 2 ℃ of-8 ℃ of stored refrigerated sterile packaged standby.
The present invention also provides the application detection method of mentioned reagent box:
Get respectively in the dummy and test agent that urina sanguinis sample 0.5ml splashes into a box kit, shake up, in standing a moment, add respectively one group of developer; Observe the change color of dummy solution and test agent solution, if two solution colours are unanimously judged to be feminine gender; If two solution colours are different, and the blueness that test agent color shows is more deeply judged to be the positive than dummy color.
The present invention utilizes in the sour environment of certain PH, and sulfydryl makes phosphotungstic acid be reduced into tungsten blue and is blue characteristic realization.Research is found: the material that phosphotungstic acid can be had a reductibility by sulfydryl etc. reduces, and the uric acid existing in urine under suitable PH condition, Vc etc. also can make phosphotungstic acid reduction, cause detecting the interference of sulfydryl metabolin.Kit of the present invention establishes dummy (chloride containing mercury or mercuric sulfate) and test agent (distilled water) is got rid of interference, because mercuric chloride or mercuric sulfate can be combined with sulfydryl before phosphotungstic acid adds, other reducing substances will be reduced into blueness by phosphotungstic acid as uric acid, Vc, as contained sulfydryl metabolite in sample, the sulfydryl metabolite of not being combined with mercuric chloride or mercuric sulfate can reduce phosphotungstic acid make blue show darker.Lithium sulfate in developer in redox reaction as the middle acceptor of H+.
Helpfulness of the present invention: the invention provides a kind of for pointing out urine sulfydryl metabolite detection kit of cervical carcinoma pathology and preparation method thereof, studies have shown that the remarkable rising of sulfydryl metabolite level in women's urine, can point out the generation of cervical carcinoma pathology, in urine, sulfydryl metabolite detection kit can detect the content of the cervical cancer patient urine sulfydryl metabolite of preclinical phase and clinical stage, clinical research proof kit of the present invention is high to cervical carcinoma Positive rate and accuracy, without failing to pinpoint a disease in diagnosis, person under inspection is without wound, without cross-infection, do not need instrument and equipment, testing process is easy, person under inspection's acceptance is high, can significantly save human and material resources and drop into, and significantly reduces census enumerator's workload, effectively breaks through examination number bottleneck not up to standard, is applicable to very much cervical cancer screening on a large scale, preparation method of the present invention is easy, and preparation cost is low, is applicable to the needs of extensive generaI investigation and examination.
Embodiment
Below by embodiment, the present invention is specifically described; the present embodiment is only for being further detailed invention; can not be interpreted as limiting the scope of the invention, some nonessential improvement that those skilled in the art makes according to the content of foregoing invention and adjustment also belong to the scope of protection of the invention.
Embodiment 1
Make 500 box kits
One, test agent is distilled water, prepares distilled water 500ml stand-by.
Two, dummy solution is mercuric chloride solution, the mercuric chloride solution 500ml of preparation 0.02mol/l.
Three, developer is phosphotungstic acid reagent and the buffer solution potpourri of 1:1 by volume.
1. phosphotungstic acid reagent, takes sodium tungstate 41g, is dissolved in distilled water 320ml, adds 85% phosphoric acid 35ml, refluxes and boils 2 hours, is cooled to room temperature, and adding distil water is settled to 1000ml; Add lithium sulfate 35g to dissolve, put 2 ℃ of-8 ℃ of stored refrigerated standby.
2. buffer solution PH5.4, by pH value requirement, preparation sodium acetate/acetic acid buffer solution 1000ml: 2mol/L sodium acetate solution 833.3ml is mixed with 2mol/L acetic acid solution 166.7ml, put 2 ℃ of-8 ℃ of stored refrigerated standby.
3. phosphotungstic acid reagent and the buffer solution mixing of 1:1 are by volume placed on to 2 ℃ of-8 ℃ of stored refrigerated standby.
The solution that above-mentioned " one " is prepared to " three ", presses respectively 1 1ml of dummy, 1 1ml of test agent pipe, and 2 2ml sterile packaged of developer, in 5ml ampoule, can be made kit totally 500 boxes.
Embodiment 2
Make 500 box kits
One, test agent is distilled water, prepares distilled water 500ml stand-by.
Two, dummy solution is mercuric chloride solution, the mercuric chloride solution 500ml of preparation 0.03mol/l.
Three, developer is phosphotungstic acid reagent and the buffer solution potpourri of 1:1 by volume.
1. phosphotungstic acid reagent, takes sodium tungstate 45g, is dissolved in distilled water 350ml, adds 85% phosphatase 24 0ml, refluxes to boil 2 hours, is cooled to room temperature, and adding distil water is settled to 1000ml, adds lithium sulfate 35g to dissolve, and puts 2 ℃ of-8 ℃ of stored refrigerated standby.
2. buffer solution PH5.7, by pH value requirement, preparation sodium dihydrogen phosphate/disodium hydrogen phosphate buffer solution 1000ml: 0.2mol/L sodium dihydrogen phosphate 935ml is mixed with 0.2mol/L disodium phosphate soln 65ml, put 2 ℃ of-8 ℃ of stored refrigerated standby.
3. phosphotungstic acid reagent and the buffer solution mixing of 1:1 are by volume placed on to 2 ℃ of-8 ℃ of stored refrigerated standby.
The solution that above-mentioned " one " is prepared to " three ", presses respectively 1 1ml of dummy, 1 1ml of test agent pipe, and 2 2ml sterile packaged of developer, in 5ml ampoule, can be made kit totally 500 boxes.
Embodiment 3
Make 1000 box kits
One, test agent is distilled water, prepares distilled water 1000ml stand-by.
Two, dummy solution is mercuric sulfate solution, the mercuric sulfate solution 1000ml of preparation 0.02mol/l.
Three, developer is phosphotungstic acid reagent and the buffer solution potpourri of 1:1 by volume.
1. phosphotungstic acid reagent, takes sodium tungstate 80g, is dissolved in distilled water 700ml, adds 85% phosphoric acid 80ml, refluxes to boil 2 hours, is cooled to room temperature, and adding distil water is settled to 2000ml, adds lithium sulfate 65g to dissolve, and puts 2 ℃ of-8 ℃ of stored refrigerated standby.
2. buffer solution PH5.8, by pH value requirement, preparation sodium dihydrogen phosphate/disodium hydrogen phosphate buffer solution 2000ml: 0.2mol/L sodium dihydrogen phosphate 1840ml is mixed with 0.2mol/L disodium phosphate soln 160ml, put 2 ℃ of-8 ℃ of stored refrigerated standby.
3. phosphotungstic acid reagent and the buffer solution mixing of 1:1 are by volume placed on to 2 ℃ of-8 ℃ of stored refrigerated standby.
The solution that above-mentioned " one " is prepared to " three ", presses respectively 1 1ml of dummy, 1 1ml of test agent pipe, and 2 2ml sterile packaged of developer, in 5ml ampoule, can be made kit totally 1000 boxes.
Embodiment 4
Test example
Get respectively in the dummy and test agent that urina sanguinis sample 0.5ml splashes into a box kit, shake up, in standing a moment, in dummy and test agent, add respectively one group of developer; Observe the change color of dummy solution and test agent solution, if two solution colours are unanimously judged to be feminine gender; If two solution colours are different, and the blueness that test agent color shows is more deeply judged to be the positive than dummy color.
To 1000 routine precancerous lesions of uterine cervixs, cervical carcinoma (early, middle and late phase) patient and uterine neck normal women and the non-malignant pathology patient of uterine neck, apply this kit and detect the sulfydryl metabolite in their urine, result: specificity 93.6%, other indexs see the following form.
| Project | Positive rate | Accuracy | Positive predictive value | Negative predictive value |
| Precancerous lesions of uterine cervix group | 36.8％ | 86.4％ | 50.3％ | 92.1％ |
| Early cervical carcinoma group | 67.5％ | 86.7％ | 81.5％ | 91.2％ |
| Advanced cervical cancer group | 82.5％ | 91.3％ | 89.4％ | 92.6％ |
Precancerous lesions of uterine cervix refer to cancer occur before the pathology that occurs of this position, and the pathology of cause cancer thus.
Early cervical carcinoma, i.e. carcinoma in situ, cancer knurl is confined to epithelium of cervix uteri and there is no local infiltration.
Advanced cervical cancer, cancer knurl has infiltrated or has shifted, and cancer knurl exceeds uterine neck.
Positive rate: refer in whole testing results, occur positive findings (cervical carcinoma or its precancerous lesion) percent.
Accuracy: the ratio that refers to test positive in ill (cervical carcinoma or its precancerous lesion) crowd.
Positive predictive value: refer in the positive situation of testing result the probability of ill (cervical carcinoma).
Negative predictive value: refer in the negative situation of testing result the probability of do not fall ill (cervical carcinoma).
Specificity: refer to the negative ratio of detection in normal population (other patients of Healthy People, non-cervical carcinoma).
From testing result, can find out, positive findings is judged to be precancerous lesions of uterine cervix or cervical cancer patient has higher accuracy and positive predictive value; Negative findings distinguishing benign and pernicious cervical lesions have very high specificity and negative predictive value.Therefore the examination that, this kit is applied to precancerous lesions of uterine cervix and cervical carcinoma has good clinical detection application value.
Claims (4)
1. a kit, is characterized in that being comprised of following reagent: one group of dummy, one group of test agent, two groups of developers;
Described dummy is mercuric chloride solution or mercuric sulfate solution, and concentration is 0.02mol-0.05mol/l; Described test agent is distilled water;
Described developer is phosphotungstic acid reagent and the buffer solution potpourri of 1:1 by volume; Wherein phosphotungstic acid reagent is comprised of the aqueous solution of following component concentration: sodium tungstate 40g-60g/l, 85% phosphoric acid 30ml-50ml/l, lithium sulfate 30g-50g/l; Described buffer solution is sodium acetate/acetic acid buffer solution or sodium dihydrogen phosphate/disodium hydrogen phosphate buffer solution, and buffer solution PH is PH5.4-PH5.8.
2. kit according to claim 1, is characterized in that: described every box kit comprises one group of 1ml dummy, one group of 1ml test agent and two groups of each 2ml developers, and each group reagent is all encapsulated in 5ml ampoule.
3. the preparation method of a kit claimed in claim 1: it is characterized in that comprising:
(1) prepare distilled water aseptic packaging and testing agent;
(2) dummy preparation: take mercuric chloride or mercuric sulfate by concentration 0.02mol-0.05mol/l requirement, be dissolved in sterile packaged after distilled water;
(3) developer preparation:
A, the preparation of phosphotungstic acid reagent, take sodium tungstate 40g-60g, is dissolved in distilled water 300ml-500ml, add 85% phosphoric acid 30ml-50ml, reflux and boil 2 hours, be cooled to room temperature, adding distil water is settled to 1000ml, adds lithium sulfate 30g-50g to dissolve, and is placed in 2 ℃ of-8 ℃ of stored refrigerated standby;
B, buffer solution preparation: buffer solution is PH5.4-PH5.8, by pH value requirement preparation sodium acetate/acetic acid buffer solution or sodium dihydrogen phosphate/disodium hydrogen phosphate buffer solution, be placed in 2 ℃ of-8 ℃ of stored refrigerated standby;
C, phosphotungstic acid reagent solution prepared by step a and step b and buffer solution by volume 1:1 mix, and are placed in 2 ℃ of-8 ℃ of stored refrigerated sterile packaged standby.
4. the application detection method of a kit claimed in claim 1: it is characterized in that comprising: get respectively in the dummy and test agent that urina sanguinis sample 0.5ml splashes into a box kit, shake up, in standing a moment, add respectively one group of developer; Observe the change color of dummy solution and test agent solution, if two solution colours are unanimously judged to be feminine gender; If two solution colours are different, and the blueness that test agent color shows is more deeply judged to be the positive than dummy color.
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| CN201410390094.8A CN104181155A (en) | 2014-08-10 | 2014-08-10 | Kit and preparation method thereof |
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| CN201410390094.8A CN104181155A (en) | 2014-08-10 | 2014-08-10 | Kit and preparation method thereof |
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Cited By (11)
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| CN104714030A (en) * | 2015-03-05 | 2015-06-17 | 杭州欣叶生物科技有限公司 | Urine detection kit for cervical cancer and using method of urine detection kit |
| CN105115966A (en) * | 2015-08-18 | 2015-12-02 | 江苏东博生物医药有限公司 | Detection reagent box for sulfydryl compounds in urine and preparation method and application |
| CN105158246A (en) * | 2015-10-14 | 2015-12-16 | 智优生物科技（上海）有限公司 | Preparation method for urine monohydroxyphenyl derivative detection reagent |
| CN106226295A (en) * | 2016-07-05 | 2016-12-14 | 苏州锐霖生物科技有限公司 | A kind of urine sulfhydryl compound detectable and preparation method |
| CN106468715A (en) * | 2016-06-21 | 2017-03-01 | 安徽康乐美生物科技有限公司 | A kind of cervical cancer urine detection reagent and its preparation method and application |
| CN107024472A (en) * | 2017-02-20 | 2017-08-08 | 北京众生合生物科技有限公司 | The single hydroxyl amphyl of urine determines the method for preparation and use of reagent |
| CN107576652A (en) * | 2017-02-06 | 2018-01-12 | 刘�东 | Detection reagent of sulfhydryl compound and preparation method thereof and purposes in a kind of urine |
| CN110927156A (en) * | 2019-12-26 | 2020-03-27 | 欧迪玛(湖北)生物科技有限公司 | Urine detection kit for cervical cancer and preparation method thereof |
| CN111830023A (en) * | 2019-08-19 | 2020-10-27 | 杭州爱光医疗器械有限公司 | Sulfhydryl compound detection reagent, detection test paper, kit, test paper box and preparation method thereof |
| CN114441523A (en) * | 2022-02-10 | 2022-05-06 | 梅宗泉 | Preparation method of kit for detecting sulfhydryl metabolites in urine |
| CN115290904A (en) * | 2022-10-09 | 2022-11-04 | 山东三医生物技术有限公司 | In-situ hybridization HPV detection test strip, kit and preparation method thereof |
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104714030A (en) * | 2015-03-05 | 2015-06-17 | 杭州欣叶生物科技有限公司 | Urine detection kit for cervical cancer and using method of urine detection kit |
| CN105115966A (en) * | 2015-08-18 | 2015-12-02 | 江苏东博生物医药有限公司 | Detection reagent box for sulfydryl compounds in urine and preparation method and application |
| CN105115966B (en) * | 2015-08-18 | 2016-11-09 | 江苏东博生物医药有限公司 | Thiol compound detection kit and preparation method and application in urine |
| CN105158246A (en) * | 2015-10-14 | 2015-12-16 | 智优生物科技（上海）有限公司 | Preparation method for urine monohydroxyphenyl derivative detection reagent |
| CN106468715A (en) * | 2016-06-21 | 2017-03-01 | 安徽康乐美生物科技有限公司 | A kind of cervical cancer urine detection reagent and its preparation method and application |
| CN106226295A (en) * | 2016-07-05 | 2016-12-14 | 苏州锐霖生物科技有限公司 | A kind of urine sulfhydryl compound detectable and preparation method |
| CN107576652B (en) * | 2017-02-06 | 2021-05-18 | 深圳华创生物医药科技有限公司 | Detection reagent for sulfhydryl compounds in urine, preparation method and application thereof |
| CN107576652A (en) * | 2017-02-06 | 2018-01-12 | 刘�东 | Detection reagent of sulfhydryl compound and preparation method thereof and purposes in a kind of urine |
| CN107024472A (en) * | 2017-02-20 | 2017-08-08 | 北京众生合生物科技有限公司 | The single hydroxyl amphyl of urine determines the method for preparation and use of reagent |
| CN111830023A (en) * | 2019-08-19 | 2020-10-27 | 杭州爱光医疗器械有限公司 | Sulfhydryl compound detection reagent, detection test paper, kit, test paper box and preparation method thereof |
| WO2021032103A1 (en) * | 2019-08-19 | 2021-02-25 | 杭州爱光医疗器械有限公司 | Sulfydryl compound detection reagent, test paper, reagent kit, test paper box and preparation thereof |
| CN110927156A (en) * | 2019-12-26 | 2020-03-27 | 欧迪玛(湖北)生物科技有限公司 | Urine detection kit for cervical cancer and preparation method thereof |
| CN114441523A (en) * | 2022-02-10 | 2022-05-06 | 梅宗泉 | Preparation method of kit for detecting sulfhydryl metabolites in urine |
| CN115290904A (en) * | 2022-10-09 | 2022-11-04 | 山东三医生物技术有限公司 | In-situ hybridization HPV detection test strip, kit and preparation method thereof |
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