CN101126718A - Surface cleaning detection reagent for sanitation monitoring - Google Patents
Surface cleaning detection reagent for sanitation monitoring Download PDFInfo
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- CN101126718A CN101126718A CNA2006101124115A CN200610112411A CN101126718A CN 101126718 A CN101126718 A CN 101126718A CN A2006101124115 A CNA2006101124115 A CN A2006101124115A CN 200610112411 A CN200610112411 A CN 200610112411A CN 101126718 A CN101126718 A CN 101126718A
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Abstract
The utility model discloses a surface cleanliness detection reagent used in health supervision, which is based on the combination of ATP biological fluorescence method and enzymatic cycling amplification technology to realize rapid detection and field assessment of health surface cleanliness. The utility model comprises ATP releaser and a fluorescent inducer with ATP regeneration enzyme; wherein, the ATP releaser can release microorganisms, somatic cells and ATP in product residues; the fluorescent inducer can react with the ATP to produce quantitative fluorescence, and then combined with the ATP regeneration enzyme to amplify the produced fluorescent signals further. The utility model, which has the advantages of convenient use of reagent, simple operation, fast response, high sensitivity and low detection cost, is widely used in health monitoring and health field supervision in food industry, cosmetic industry, medical industry and other industries.
Description
Technical field
The invention belongs to the health and epidemic prevention technical field, relate to health and detect and monitoring, relate in particular to a kind of combining and realize the reagent of sanitary surface cleanliness factor fast detecting based on ATP bioluminescence method and enzyme circulation amplifying technique.
Background technology
In the normal operation process of industries such as food, cosmetics, electronics, medical treatment, to sanitary controls such as operating personnel's hand, using appliance and work tops with detect most important.At present, most of enterprises and Health Inspection Authorities mainly come the Evaluation Environment sanitary condition with the micro organism quantity of body surface, and depend on tradition or improved agar plate method when detecting, its detection speed is slow, efficient is low, at least need 24～48 hours from collected specimens to obtaining the result, can't reach the requirement that enterprise and hygienic quality supervisory organ obtain environmental microorganism information fast.On the other hand, often not only there is microorganism in body surface, and a large amount of body cells on it or product residue thing are also incited somebody to action the greatly clean-up performance of ectocrine surface.Therefore, need to develop a kind of quick, accurate, easy sanitary surface cleaning method for quick, come the environmental health situation is carried out detecting in real time and effectively and monitoring.
Atriphos (ATP) bioluminescence method is the fast method that is expected to realize detecting immediately the sanitary surface clean-up performance at present.This method is fluorescence and the linear within the specific limits principle of ATP concentration of utilizing ATP and fluorescein in the biosome, luciferase reaction to produce, the existence that comes the detection of biological body.This method need not incubation, easy and simple to handle, highly sensitive, can obtain testing result in several minutes, therefore in detecting, microbial contamination and biomass obtained developing rapidly, many countries are with its on-the-spot supervision detects as environmental health effective means, and domesticly still are in the starting stage.Yet ATP bioluminescence method is because the fluorescence signal that reaction generates is more weak and decay is very fast, and its sensitivity does not often reach the detection requirement, need improve detection sensitivity further combined with other means.
Enzyme circulation amplifying technique is a kind of the most direct, easy technology of improving the sensitivity of ATP bioluminescence method.It mainly is, to utilize the catalytic action of ATP regeneration enzyme to impel the product A MP (adenosine monophosphate) of ATP bioluminescence reaction to be converted into ATP again, thereby amplify the fluorescence signal that the ATP biological respinse produces when compound exists at phosphate (or phosphate).In recent years, domestic existing patent CN1338003 relates to the detection that the ATP regeneration enzyme is applied to corpse or other object for laboratory examination and chemical testing pollutant, but used phosphate compound poor heat stability and consumption are big, may have low, the uneconomic problem of ATP regeneration efficiency.
Summary of the invention
Purpose of the present invention is exactly at above deficiency, for environmental sanitary quality control provide a kind of easy and simple to handle, detection speed is fast, highly sensitive and detect the low detectable of cost, can be used for field quick detection, be used for the body surface cleanliness factor fast detecting of industries such as food, cosmetics, medical treatment and the on-site sanitation supervision of hygienic quality supervisory organ.
For reaching above purpose, solution of the present invention provides a kind of atriphos (ATP) bioluminescence method and enzyme circulation amplifying technique are combined and realizes the reagent of clean fast detecting of sanitary surface and site assessment, and this detectable mainly comprises the ATP releasing agent and contains the fluorescence induction agent of ATP regeneration enzyme.
The surface cleaning detection reagent that is used for sanitation monitoring that provides of the present invention is made up of atriphos releasing agent and the fluorescence induction agent that contains the atriphos regeneration enzyme; Wherein:
By every milliliter, contain in the atriphos releasing agent: trishydroxymethylaminomethane (Tris) damping fluid of the ionic surfactant of 0.005～2mg, the non-ionics of 0.01～12mg and 20～200mM;
Contain in the fluorescence induction agent of atriphos regeneration enzyme by every milliliter, contain: the neutralizing agent of the enzyme stabilizers of the fluorescein of 0.005～3mg, the luciferase of 0.005～1.2mg, 1～15mM solubility inorganic magnesium salt, 0.5～80mM, the fluorescence-enhancing agent of 0.1～8mM, 0.1～15mM, the compound polyphosphate of 0.001～1.3mM, the atriphos regeneration enzyme of 0.002～100U and the TRIS buffer of 20～500mM, the pH=7.2-7.8 of this buffering agent.
Described detectable, wherein, ionic surfactant is from DTAB (DTAB), dodecyl dimethyl benzyl ammonium bromide (BDDAB), benzethonium chloride (BZC), hexadecylpyridinium chloride quaternaries such as (CPC), and chooses a kind of composition at least in the sulfonate surfactant such as sodium dodecylsulphonate (SDS), dodecyl sodium sulfate, neopelex.
Described detectable, wherein, non-ionic surfactant is to choose a kind of composition at least from aliphatic alcohol polyethenoxy (15) ether (AEO-15), alkylphenol-polyethenoxy (15) ether (OP-15), octyl phenyl polyoxyethylene ether (TritonX-100), Nonyl pheno (10) ether polyethylene glycol type surfactants such as (NP-10).
Described detectable, wherein, enzyme stabilizers is to choose a kind of composition at least from bovine serum albumin(BSA), gel, glycerine, vinyl ethylene glycol, ammonium sulfate.
Described detectable, wherein, fluorescence-enhancing agent is to choose a kind of composition from coacetylase, pyrophosphate, dithiothreitol (DTT), mercaptoethanol, cysteine, polyglycol, diethylaminoethyl dextran at least.
Described detectable, wherein, neutralizing agent is to choose a kind of composition at least from cyclodextrin, tween, ethylenediamine tetraacetic acid, cyclohexene diamino tetraacethyl.
Described detectable, wherein, the atriphos regeneration enzyme is to choose a kind of composition at least from pyruvate orthophosphate dikinase, pyruvate kinase, adenylate kinase, phosphate transferase, polyphosphoric acid synzyme etc.
Embodiment
Provided by the invention ATP bioluminescence method and enzyme circulation amplifying technique are combined realized the reagent of sanitary surface cleanliness factor fast detecting and site assessment, mainly comprises the ATP releasing agent and contains the fluorescence induction agent of ATP regeneration enzyme.
The ATP releasing agent, mainly contain DTAB (DTAB), dodecyl benzyl dimethyl ammonium bromide (BDDAB), benzethonium chloride (BZC), hexadecylpyridinium chloride quaternaries such as (CPC), and sodium dodecylsulphonate (SDS), dodecyl sodium sulfate, at least a ionic surfactant in the sulfonate surfactants such as neopelex, and aliphatic alcohol polyethenoxy (15) ether (AEO-15), alkylphenol-polyethenoxy (15) ether (OP-15), octyl phenyl polyoxyethylene ether (Triton X-100), at least a non-ionics in Nonyl pheno (10) the ether polyethylene glycol type surfactants such as (NP-10).
Its preparation process is: get the ionic surfactant of 0.005～2g and the non-ionics of 0.01～12g and be dissolved in the Tris damping fluid (regulating pH=7.2-7.8 with hydrochloric acid or 10% NaOH) of 20～200mM, making its final volume is 1L.Here, all reagent all adopt to be analyzed purely, and mM is meant a kind of concentration unit (be mM/liter).This releasing agent all discharges the ATP in microorganism, body cell and the product residue in the sample in the 2min at ambient temperature, but the reactive material ATP of detection by quantitative is provided for the reaction of ATP bioluminescence.
Contain the fluorescence induction agent of ATP regeneration enzyme, form by fluorescein, luciferase and solubility inorganic magnesium salt, stabilizing agent, fluorescence-enhancing agent, neutralizing agent, compound polyphosphate, ATP regeneration enzyme and Tris damping fluid (pH=7.8).Wherein, fluorescein, luciferase and solubility inorganic magnesium salt are the essential reagent of ATP bioluminescence reaction; Enzyme stabilizers and fluorescence-enhancing agent are the auxiliary reagent that is used for stopping reaction reagent, improves reactivity, choose a kind of composition at least respectively from bovine serum albumin(BSA), gel, glycerine, vinyl ethylene glycol, ammonium sulfate and in the coacetylase, pyrophosphate, dithiothreitol (DTT), mercaptoethanol, cysteine, polyglycol, diethylaminoethyl dextran.Neutralizing agent is to choose a kind of composition at least from cyclodextrin, tween, ethylenediamine tetraacetic acid, cyclohexene diamino tetraacethyl, is mainly used in the inhibiting effect that residual A TP releasing agent reacts bioluminescence in the alleviation system; The ATP regeneration enzyme, then be from pyruvate orthophosphate dikinase, pyruvate kinase, adenylate kinase, phosphate transferase, polyphosphoric acid synzyme, to choose a kind of composition at least, be mainly used to the product A MP (adenosine monophosphate) of catalysis ATP bioluminescence reaction and combine with phosphate radical in the compound polyphosphate and form ATP, thereby make ATP obtain the recycling fluorescence signal that reacts generation that further amplifies.
Its preparation process is: get the solubility inorganic magnesium salt of 1～15mM, the enzyme stabilizers of 0.5～80mM, the fluorescence-enhancing agent of 0.1～8mM and the neutralizing agent of 0.1～15mM earlier and be dissolved in the Tris damping fluid (regulating PH with hydrochloric acid or acetic acid is 7.8) of 20～500mM, making the solution final volume is 10mL, and then add the fluorescein of 0.05～30mg and the luciferase of 0.05～12mg makes its dissolving, and solution is carried out packing, keeps in Dark Place with standby.All reagent all adopt analysis pure, so just, obtained to contain the ATP bioluminescence reaction system of neutralizing agent, this system can be directly used in detection by quantitative ATP, also can further add the compound polyphosphate of 0.001～1.3mM and the atriphos regeneration enzyme of 0.002～100U/mL on this basis and form more highly sensitive fluorescence induction agent.
Present invention is described below in conjunction with specific embodiment:
Embodiment 1:
A preferred cover detectable, its composition and concentration are as follows.
The ATP releasing agent consists of:
DTAB 0.2mg/ml
Aliphatic alcohol polyethenoxy (15) ether 1.2mg/ml
Tris-HCl(20mM) PH7.4
The consisting of of fluorescence induction agent that does not contain the ATP regeneration enzyme:
Fluorescein 0.3mg/mL
Luciferase 0.05mg/mL
Magnesium chloride 10mM
Bovine serum albumin(BSA) 0.11mM
Ethylenediamine tetraacetic acid 2mM
Tetra sodium pyrophosphate 0.02mM
Dithiothreitol (DTT) 1mM
Cyclodextrin 6mM
Tris-HCl(50mM) PH7.8
With above ATP releasing agent with do not contain the fluorescence induction agent of ATP regeneration enzyme, by shown in concentration make a cover detectable, come the surface cleanliness of the breadboard weighing instrument of fast detecting.During detection, earlier to object under test with cotton swab smear back and forth on its whole surface take a sample for 2～3 times after, with sample dispersion in 5mL solution, the acquisition sample solution; The sample solution that takes out 0.05mL then places test tube, vibration a little behind the ATP releasing agent of adding 0.05mL; Add the fluorescence induction agent of 0.3mL again, the combined with fluorescent detector detects, and RLU represents measured value with relative light unit.The fluoroscopic examination value that records balance on goods and services, weighing scoop and measuring cup correspondence is respectively 79RLU, 6RLU and 37RLU.
Embodiment 2:
A preferred cover detectable, its composition and concentration are as follows.
The ATP releasing agent consists of:
Sodium dodecylsulphonate 0.1mg/ml
Triton x-100 1mg/ml
Tris-HCl(20mM) PH7.8
The consisting of of fluorescence induction agent of containing the ATP regeneration enzyme:
Fluorescein 0.25mg/mL
Luciferase 0.04mg/mL
Magnesium chloride 10mM
Bovine serum albumin(BSA) 0.11mM
Ethylenediamine tetraacetic acid 2mM
Coacetylase 0.05mM
Dithiothreitol (DTT) 1mM
Compound polyphosphate 0.05mM
Polyphosphoric acid synzyme 0.1U/mL
Adenylate kinase 0.5U/mL
Tris-HCl(50mM) PH7.8
With above ATP releasing agent with contain the fluorescence induction agent of ATP regeneration enzyme, by shown in concentration prepare respectively and obtain a cover detectable, come the surface clearness of laboratory work environment is carried out fast detecting; Preparation does not simultaneously contain the fluorescence induction agent of ATP regeneration enzyme, does contrast and detects.During detection, earlier 100cm is smeared with cotton swab in surface to be measured
2Area take a sample, to the not enough 100cm of surface area
2Door handle then on whole surface, smear back and forth and take a sample for 2～3 times with cotton swab, obtain sample solution; The sample solution that takes out 0.05mL then places test tube, adds the fluorescence induction agent of ATP releasing agent and the 0.3mL of 0.05mL successively, and the combined with fluorescent detector detects, and RLU represents measured value with relative light unit, records to the results are shown in Table shown in 1.
As can be seen, when the luminous quantity detected value when having the ATP regeneration enzyme to exist will be starkly lower than no ATP regeneration enzyme, this was that this fluorescence decay takes place easily and instability because the fluorescence volume that obtains during no ATP regeneration enzyme is to be caused by the ATP in the sample fully; And in case add ATP regeneration enzyme and corresponding compound polyphosphate, just can impel the AMP of ATP bioluminescence reaction to be converted into ATP and the participation fluorescence reaction that circulates once more, stablize the ATP in the sample to a certain extent and participated in the fluorescence volume that reaction obtains, thereby improved the sensitivity that detects effectively.
Comparison and detection result when table 1 has or not the ATP regeneration enzyme
| Detected object | Sampling area (cm 2) | Luminous quantity detected value (RLU) | |
| The ATP regeneration enzyme is arranged | No ATP regeneration enzyme | ||
| 1 pond, door handle 1 door handle, 2 ponds, 2 work tops, 1 work top, 2 work tops 3 | ＜100 ＜100 100 100 100 100 100 | 245 167 630 480 298 220 52 | 56 23 270 190 161 63 7 |
Claims (7)
1. a surface cleaning detection reagent that is used for sanitation monitoring is made up of atriphos releasing agent and the fluorescence induction agent that contains the atriphos regeneration enzyme; Wherein:
By every milliliter, contain in the atriphos releasing agent: the TRIS buffer of the ionic surfactant of 0.005～2mg, the non-ionics of 0.01～12mg and 20～200mM;
Contain in the fluorescence induction agent of atriphos regeneration enzyme by every milliliter, contain: the neutralizing agent of the enzyme stabilizers of the fluorescein of 0.005～3mg, the luciferase of 0.005～1.2mg, 1～15mM solubility inorganic magnesium salt, 0.5～80mM, the fluorescence-enhancing agent of 0.1～8mM, 0.1～15mM, the compound polyphosphate of 0.001～1.3mM, the atriphos regeneration enzyme of 0.002～100U and the TRIS buffer of 20～500mM;
The pH=7.2-7.8 of trishydroxymethylaminomethane buffering agent.
2. detectable as claimed in claim 1, wherein, ionic surfactant is to choose a kind of composition from DTAB, dodecyl dimethyl benzyl ammonium bromide, benzethonium chloride, hexadecylpyridinium chloride, sodium dodecylsulphonate, dodecyl sodium sulfate, neopelex at least.
3. detectable as claimed in claim 1, wherein, non-ionic surfactant is to choose a kind of composition at least from aliphatic alcohol polyethenoxy (15) ether, alkylphenol-polyethenoxy (15) ether, octyl phenyl polyoxyethylene ether, Nonyl pheno (10) ether.
4. detectable as claimed in claim 1, wherein, enzyme stabilizers is to choose a kind of composition at least from bovine serum albumin(BSA), gel, glycerine, vinyl ethylene glycol, ammonium sulfate.
5. detectable as claimed in claim 1, wherein, fluorescence-enhancing agent is to choose a kind of composition from coacetylase, pyrophosphate, dithiothreitol (DTT), mercaptoethanol, cysteine, polyglycol, diethylaminoethyl dextran at least.
6. detectable as claimed in claim 1, wherein, neutralizing agent is to choose a kind of composition at least from cyclodextrin, tween, ethylenediamine tetraacetic acid, cyclohexene diamino tetraacethyl.
7. the described detectable of claim 1, wherein, the atriphos regeneration enzyme is to choose a kind of composition at least from pyruvate orthophosphate dikinase, pyruvate kinase, adenylate kinase, phosphate transferase, polyphosphoric acid synzyme.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2006101124115A CN101126718B (en) | 2006-08-16 | 2006-08-16 | Surface cleaning detection reagent for sanitation monitoring |
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| CN2006101124115A CN101126718B (en) | 2006-08-16 | 2006-08-16 | Surface cleaning detection reagent for sanitation monitoring |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107505311A (en) * | 2017-09-25 | 2017-12-22 | 江苏中新医药有限公司 | The quick method and biological indicator for determining sterilization effect |
| CN111089859A (en) * | 2018-10-23 | 2020-05-01 | 北京中医药大学 | Novel adenosine triphosphate bioluminescence determination method and application thereof |
| CN114350626A (en) * | 2021-12-21 | 2022-04-15 | 合肥巅峰生物科技有限公司 | Luciferase freeze-dried powder dilution reaction liquid |
| CN116555390A (en) * | 2023-05-18 | 2023-08-08 | 新疆紫晶川梭高新农业股份有限公司 | Microorganism detection reagent and detection method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100462714C (en) * | 2005-05-23 | 2009-02-18 | 西安交通大学 | Multi-sample microbial pollution fast screening method |
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2006
- 2006-08-16 CN CN2006101124115A patent/CN101126718B/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107505311A (en) * | 2017-09-25 | 2017-12-22 | 江苏中新医药有限公司 | The quick method and biological indicator for determining sterilization effect |
| CN111089859A (en) * | 2018-10-23 | 2020-05-01 | 北京中医药大学 | Novel adenosine triphosphate bioluminescence determination method and application thereof |
| CN114350626A (en) * | 2021-12-21 | 2022-04-15 | 合肥巅峰生物科技有限公司 | Luciferase freeze-dried powder dilution reaction liquid |
| CN116555390A (en) * | 2023-05-18 | 2023-08-08 | 新疆紫晶川梭高新农业股份有限公司 | Microorganism detection reagent and detection method |
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| CN101126718B (en) | 2010-09-15 |
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