CN101368157A - Quick bacteria cell cracking agent prescription - Google Patents
Quick bacteria cell cracking agent prescription Download PDFInfo
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- CN101368157A CN101368157A CNA2007101202833A CN200710120283A CN101368157A CN 101368157 A CN101368157 A CN 101368157A CN A2007101202833 A CNA2007101202833 A CN A2007101202833A CN 200710120283 A CN200710120283 A CN 200710120283A CN 101368157 A CN101368157 A CN 101368157A
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Abstract
The invention discloses a reagent formula for fast cracking of bacterial cells, which relates to detection technical field. The reagent formula includes at least one acidic bacterial cell cracking reagent and at least one basic neutralization reagent, and at least one organic nutrient and at least one inorganic salt solvent which are added. The reagent has advantages of fast bacterial cell cracking speed, high adenosine triphosphate (ATP) output capacity with inhibition of adenosine triphosphate (ATP) digestive enzyme in the cell, convenient operation, easy preparation and low cost. The reagent formula is just suitable for fast cracking of bacterial cells in the fields of fast microbial detection.
Description
Technical field
The invention belongs to the microbial rapid detection technical field, a kind of quick bacteria cell cracking agent prescription is provided.
Background technology
Putrid and deteriorated, the contaminated degree of food of total plate count index and food have substantial connection, are a kind of signals that may work the mischief to HUMAN HEALTH, both at home and abroad this index as weighing food by the important indicator of microbial contamination degree.The total plate count index as the essential items for inspection of food sanitation safe, then must be carried out random sampling and deliver to the technique center and detect.Current, total plate count detects, and what generally adopt both at home and abroad is colony counting method, this method need be cultivated 24～48 hours microorganism, and round of visits is long, complex operation, waste time and energy, and need professional person's operation, be difficult for the on-the-spot working efficiency that detects, greatly influences.So develop a kind of quick, accurate, easy instrument that is used for the food sanitation field quick detection, in food sanitation safe control, will have great demand and realistic meaning.
Triphosaden (ATP) bioluminescence method is the present stage foreign study a kind of total plate count detection method more with application, because of this method need not to carry out microorganism culturing, and simple to operate, test speed is fast, so this method has been subjected to extensive concern at present.The theoretical basis of Triphosaden (ATP) bioluminescence method is based on food to be measured or the long-pending contained bacterium number of utensil certain surface is certain, and every kind of individual contained Triphosaden (ATP) amount of bacterium all is on the same order of magnitude under normal conditions.It detects ultimate principle is at first to utilize the bacteria cell cracking agent with the bacterial cell fragmentation, discharge Triphosaden (ATP) in the born of the same parents, under the katalysis of luciferase, Triphosaden (ATP) discharges fluorescence with luciferin reaction, fluorescence intensity and Triphosaden (ATP) amount is proportional, and total plate count is proportional in Triphosaden (ATP) amount and the sample, therefore according to detected bioluminescence amount, according to the corresponding relation of fluorescence intensity and total plate count, can learn total plate count in the sample.
In Triphosaden (ATP) bioluminescence method detected, its committed step was the cracking to bacterial cell, and the efficient of bacteria cell cracking agent cracking release Triphosaden (ATP) directly determines the accuracy of detection and the detectability of bioluminescence detection method.When carrying out bacteria cell cracking, there are three factors to need to consider, as the bacteria cell cracking agent, except discharging Triphosaden (ATP) by the cracking bacterial cell membrane; Should be able to restrain simultaneously Triphosaden (ATP) degrading enzyme in the born of the same parents, Triphosaden (ATP) degrading enzyme degradable Triphosaden (ATP) thus reduce fluorescence intensity; The last bacteria cell cracking agent of being introduced can not exert an influence to the reaction of final Triphosaden (ATP) bioluminescence.The bacteria cell cracking method of using has Ethanol Method, butanols method, surfactant method, supersonic method and electric field method etc. at present.In Ethanol Method and butanols method, ethanol and butanols reagent are comparatively gentle, and lysis efficiency is lower, cracking time 30min, and the introducing of ethanol and butanols can exert an influence to the fluorescent reagent of follow-up introducing, causes this law range of application to be restricted; Utilize tensio-active agent (as sodium laurylsulfonate, Triton etc.) that bacterial cell is carried out cracking, Triphosaden (ATP) output capacity is higher, but tensio-active agent can not be restrained Triphosaden (ATP) degrading enzyme, introducing with the duration carbon chain compound can produce comparatively significantly influence to fluorescent reagent, lowest detectable limit is higher when utilizing this method to carry out the bioluminescence detection, needs 10 usually
4～10
5The total plate count of CFU/ml concentration does not satisfy the actual detected requirement.Supersonic method and electric field method are that the physical method that utilizes that occurs in the recent period carries out the method for bacteria cell cracking, but utilize these methods need be equipped with instrumentation usually, make it detect application at the scene and are restricted.Therefore develop that a kind of rate of cleavage is fast, Triphosaden (ATP) output capacity height, restrain Triphosaden (ATP) degrading enzyme simultaneously, easy to operate bacteria cell cracking agent has important practical significance.
Summary of the invention
Purpose of the present invention is exactly the deficiency at above-mentioned cleavage method, give that Triphosaden (ATP) bioluminescence detection method provides that a kind of rate of cleavage is fast, Triphosaden (ATP) output capacity height and can restrain Triphosaden (ATP) degrading enzyme, easy to operate simultaneously, be easy to preparation and lower-cost bacteria cell cracking agent, thereby improve Triphosaden (ATP) bioluminescence method detection speed greatly, set up a kind of conveniently total plate count in-situ check and test method, can solve the difficult problem of the on-the-spot testing of a large amount of food sanitations.
Another purpose of the present invention provides a kind of reagent, and this reagent can be used in Triphosaden (ATP) the bioluminescence method bacterial cell being carried out quick cracking; Can restrain simultaneously Triphosaden (ATP) degrading enzyme in the bacterial cell; Triphosaden (ATP) output capacity height; Can carry out field quick detection, satisfy the needs of food hygiene detection.
For reaching above purpose, technical solution of the present invention is:
A kind of quick bacteria cell cracking agent prescription is used for Triphosaden (ATP) bioluminescence method bacterial cell is carried out quick cracking; Its agent prescription comprises at least a acid bacteria cell cracking agent and at least a alkaline neutralization reagent, and adds at least a organic nutrient aganic nutrient agent and at least a inorganic salt solvent;
The acid bacteria cell cracking agent is to choose a kind of composition from chloric acid, perchloric acid, sulfuric acid, nitric acid, trichoroacetic acid(TCA), tetra-sodium, hydrofluoric acid, pyrosulfuric acid, hydrochloric acid, acetate at least;
The alkalescence neutralization reagent is to choose a kind of composition from yellow soda ash, sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammoniacal liquor, calcium hydroxide, magnesium hydroxide at least;
Organic nutrient aganic nutrient agent, be from protein powder, L-2,6-hydrochloride diaminocaproate, N-methylol Methionine calcium salt, Gluconolactone, pantonine-hydroxybutyric acid, maltonic acid sodium, o-benzoyl sulphonamide sodium, chlorination-beta-hydroxyethyl-N, N chooses a kind of composition in N-trimethyl ammonium, glucose, acetate TMA (TriMethylAmine) inner salt, calglucon, the fructose at least;
The inorganic salt solvent is to choose a kind of composition at least from barium carbonate, sodium iodide, zirconyl chloride, Potassium monofluoride, six hydration magnesium bromides, langbeinite, sodium-chlor, Repone K, calcium chloride, salt of wormwood, saltpetre, SODIUMNITRATE.
Described agent prescription, its described acid bacteria cell cracking agent concentration is chosen between 0.01～1.0mol/L.
Described agent prescription, its described acid bacteria cell cracking agent, its bacteria cell cracking time is chosen between 1～10min.
Described agent prescription, its described alkaline neutralization reagent concentration is chosen between 0.01～1.0mol/L; Its pH value is between 6.5～8.5.
Described agent prescription, its described alkaline neutralization reagent, in it and the time between 1～60s, choose.
Described agent prescription, its described organotrophy agent concentration is chosen between 0.01～0.50mol/L.
Described agent prescription, its described inorganic salt solvent strength is chosen between 0.01～0.50mol/L.
Agent prescription of the present invention is used for Triphosaden (ATP) bioluminescence method bacterial cell is carried out quick cracking, can restrain Triphosaden (ATP) degrading enzyme in the bacterial cell simultaneously; Triphosaden (ATP) output capacity height; Can carry out field quick detection, satisfy the needs of food hygiene detection.
Description of drawings
Fig. 1 utilizes the colibacillary test comparison synoptic diagram of the quick cracking of this bacteria cell cracking agent prescription;
Fig. 2 utilizes the agent of this bacteria cell cracking to carry out the cracking detected result figure of bacterium gradient dilution sample.
Embodiment
The present invention proposes a kind of quick bacteria cell cracking agent prescription, this reagent comprises following four kinds of reagent at least:
At least a acid bacteria cell cracking agent, described acid bacteria cell cracking agent is chosen from chloric acid, perchloric acid, sulfuric acid, nitric acid, trichoroacetic acid(TCA), tetra-sodium, hydrofluoric acid, pyrosulfuric acid, hydrochloric acid, acetate, concentration is chosen between 0.01～1.0mol/L, preferred concentration 0.03mol/L, the cracking time is chosen between 1～10min, preferred cracking time 5min.
This reagent and bacterial cell effect, the voidage of expansion bacteria cell wall increases the permeability of bacterial cell membrane, thereby discharges Triphosaden (ATP) in the born of the same parents.
At least a alkaline neutraliser, described alkaline neutraliser is chosen from yellow soda ash, sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammoniacal liquor, calcium hydroxide, magnesium hydroxide, concentration is got fixed between 0.01～1.0mol/L, preferred concentration 0.03mol/L, in and the time between 1～60s, choose, preferred in and time 10s.
The introducing of this reagent can neutralize to bacteria cell cracking liquid, regulates reagent pH value between 6.5～8.5, preferred pH value 7.8.
At least a organic nutrient aganic nutrient agent; described organic nutrient aganic nutrient agent is from protein powder, L-2; 6-hydrochloride diaminocaproate, N-methylol Methionine calcium salt, Gluconolactone, pantonine-hydroxybutyric acid, maltonic acid sodium, o-benzoyl sulphonamide sodium, chlorination-beta-hydroxyethyl-N; N; choose in N-trimethyl ammonium, glucose, acetate TMA (TriMethylAmine) inner salt, calglucon, the fructose; concentration is chosen between 0.01～0.50mol/L, preferred concentration 0.20mol/L.
Selected organic nutrient aganic nutrient agent can make that bacterium can keep normal metabolic in the test sample, thereby makes in the bacterial cell Triphosaden (ATP) level keep a higher level, increases Triphosaden (ATP) output capacity.
At least a inorganic salt solvent, described inorganic salt solvent is chosen from barium carbonate, sodium iodide, zirconyl chloride, Potassium monofluoride, six hydration magnesium bromides, langbeinite, sodium-chlor, Repone K, calcium chloride, salt of wormwood, saltpetre, SODIUMNITRATE, used concentration is chosen between 0.01～0.50mol/L, preferred concentration 0.20mol/L.
Selected inorganic salt solvent has the function of regulating osmotic pressure, can make that bacterial cell is in the normal osmotic pressure scope in the sample, increases the stability of bacterial cell.
Present invention is described below in conjunction with specific embodiment:
Embodiment 1
From a kind of reagent of prepared at concentrations shown in the following compounds:
Compound concentration
Perchloric acid 0.10mol/L
Potassium hydroxide 0.10mol/L
Calglucon 0.15mol/L
Repone K 0.15mol/L
Embodiment 2
From a kind of reagent of prepared at concentrations shown in the following compounds:
Compound concentration
Sulfuric acid 0.10mol/L
Potassium hydroxide 0.20mol/L
Fructose 0.25mol/L
Sodium iodide 0.25mol/L
Embodiment 3
From a kind of reagent of prepared at concentrations shown in the following compounds:
Compound concentration
Trichoroacetic acid(TCA) 0.03mol/L
Sodium hydroxide 0.03mol/L
Glucose 0.20mol/L
Sodium-chlor 0.20mol/L
With above reagent by shown in concentration mix, and regulate pH between 6.5～8.5, thereby prepare the bacteria cell cracking agent.Utilize a kind of of mentioned reagent combination, to containing a certain amount of intestinal bacteria sample (1 * 10
7CFU/mL) have or not bacterium cracking agent contrast experiment.At first getting bacterial suspension 30 μ L in the experiment adds in the detection cell, introducing the bacteria cell cracking agent that 30 μ L prepare mixes, cracking time 5min, detection cell is moved in the fluorescence detector (as Japanese F4500, independent research total plate count detector TX2007-1), start detection utilizes injection device to inject reacted fluorogenic agent 100 μ L behind the 20s, the record fluorescent signal, the contrast test result as shown in Figure 1.As seen from Figure 1, the sample with respect to no bacteria cell cracking agent is introduced utilizes this bacteria cell cracking agent to carry out quick cracking to bacterial cell, can obtain higher Triphosaden (ATP) output capacity in the 5min.Fig. 2 utilizes the agent of this bacteria cell cracking to carry out the cracking detected result figure of bacterium gradient dilution sample, as seen from Figure 2, and in total plate count 10
1～10
7In the scope of CFU/mL, the agent of this bacteria cell cracking is by higher bacterium cracking ability, Triphosaden (ATP) output capacity height, luminous intensity and total plate count are linear under the logarithm situation, can detect the bacteria samples of 10CFU/mL concentration, can satisfy in microorganism Triphosaden (ATP) the bioluminescence detection method the quick cracked needs of bacterial cell.
Above embodiment is just in order to illustrate the present invention, rather than limitation of the present invention.On the basis of the above description, the present invention can be many modifications and changes.Within the scope of the appended claims, the present invention can have and is different from other above-mentioned implementation, selects other related reagent for use, changes methods such as reagent concentration all within patent claimed range of the present invention.
Claims (7)
1. a quick bacteria cell cracking agent prescription is used for Triphosaden (ATP) bioluminescence method bacterial cell is carried out quick cracking; It is characterized in that this agent prescription comprises at least a acid bacteria cell cracking agent and at least a alkaline neutralization reagent, and add at least a organic nutrient aganic nutrient agent and at least a inorganic salt solvent;
The acid bacteria cell cracking agent is to choose a kind of composition from chloric acid, perchloric acid, sulfuric acid, nitric acid, trichoroacetic acid(TCA), tetra-sodium, hydrofluoric acid, pyrosulfuric acid, hydrochloric acid, acetate at least;
The alkalescence neutralization reagent is to choose a kind of composition from yellow soda ash, sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammoniacal liquor, calcium hydroxide, magnesium hydroxide at least;
Organic nutrient aganic nutrient agent, be from protein powder, L-2,6-hydrochloride diaminocaproate, N-methylol Methionine calcium salt, Gluconolactone, pantonine-hydroxybutyric acid, maltonic acid sodium, o-benzoyl sulphonamide sodium, chlorination-beta-hydroxyethyl-N, N chooses a kind of composition in N-trimethyl ammonium, glucose, acetate TMA (TriMethylAmine) inner salt, calglucon, the fructose at least;
The inorganic salt solvent is to choose a kind of composition at least from barium carbonate, sodium iodide, zirconyl chloride, Potassium monofluoride, six hydration magnesium bromides, langbeinite, sodium-chlor, Repone K, calcium chloride, salt of wormwood, saltpetre, SODIUMNITRATE.
2. agent prescription as claimed in claim 1 is characterized in that, described acid bacteria cell cracking agent concentration is chosen between 0.01～1.0mol/L.
3. agent prescription as claimed in claim 1 is characterized in that, described acid bacteria cell cracking agent, and its bacteria cell cracking time is chosen between 1～10min.
4. agent prescription as claimed in claim 1 is characterized in that, described alkaline neutralization reagent concentration is chosen between 0.01～1.0mol/L; Its pH value is between 6.5～8.5.
5. agent prescription as claimed in claim 1 is characterized in that, described alkaline neutralization reagent, in it and the time between 1～60s, choose.
6. agent prescription as claimed in claim 1 is characterized in that, described organotrophy agent concentration is chosen between 0.01～0.50mol/L.
7. agent prescription as claimed in claim 1 is characterized in that, described inorganic salt solvent strength is chosen between 0.01～0.50mol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101202833A CN101368157A (en) | 2007-08-15 | 2007-08-15 | Quick bacteria cell cracking agent prescription |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101202833A CN101368157A (en) | 2007-08-15 | 2007-08-15 | Quick bacteria cell cracking agent prescription |
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| Publication Number | Publication Date |
|---|---|
| CN101368157A true CN101368157A (en) | 2009-02-18 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007101202833A Pending CN101368157A (en) | 2007-08-15 | 2007-08-15 | Quick bacteria cell cracking agent prescription |
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|---|---|
| CN (1) | CN101368157A (en) |
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2007
- 2007-08-15 CN CNA2007101202833A patent/CN101368157A/en active Pending
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Open date: 20090218 |