CN1876829A - Kit for anti-interference quick detection of microbe quantity by bioluminescence method - Google Patents
Kit for anti-interference quick detection of microbe quantity by bioluminescence method Download PDFInfo
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- CN1876829A CN1876829A CNA2006100343859A CN200610034385A CN1876829A CN 1876829 A CN1876829 A CN 1876829A CN A2006100343859 A CNA2006100343859 A CN A2006100343859A CN 200610034385 A CN200610034385 A CN 200610034385A CN 1876829 A CN1876829 A CN 1876829A
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Abstract
The invention relates to a rapid test method for microbial biomass using ATP bioluminescence and detection reagent cell, in which the said detection reagent cell consists of luciferase and its protecting agents, D-fluorescein, standard ATP, light-emitting detection buffer solution, non-cellular ATP remover, microbial cell ATP extractant and other agents. The rapid test standard method for microbial biomass using the detection reagent cell includes doing ATP normal curve in selected biological light-emitting device and selected system, obtaining linear regression equation after taking the logarithm, performing ATP biological light-emitting detection to specimen liquid by non-purpose ATP removal and micro-organism ATP extraction with the same enzyme in the same device and system, simultaneously doing blank control, then obtaining the specimen and blank CPM or RLU value, after taking the logarithm of the de-margined net relative luminescence value, and obtaining the mother ATP concentration with built regression equation. The ATP concentrations of bacteria, yeast, fungus and unicellular microalgae can be converted into the cell numbers of the said bacteria, yeast, fungus and unicellular microalgae, or homologous to the bacteria quantity.
Description
[technical field]
The present invention relates to method and detection kit that a kind of ATP of utilization biloluminescence method carries out the microbe population rapid detection, belong to biological technical field.
[background technology]
Biloluminescence method detects microorganism cells quantity and has fast simple advantage, whole process only is tens minutes, and conventional bacterium, yeast and total number of molds are measured the dull and stereotyped counting process of cultivating of employing, need several days from beginning to cultivate macroscopic bacterium colony, therefore have very strong advantage aspect the microbe population rapid detection.The The determination technology of being undertaken by the ATP biloluminescence method has higher sensitivity owing to do not need culturing process, can be used for factory and carries out the online detection of microbial count in production process, for the quality control of product provides the rapid detection means.Existing sensitive noclilucence instrument of at present external minority developed country and very highly purified detection kit, no matter yet highly sensitive luminous detection instrument or highly purified luminous detection reagent, its price is all very high, simple instrument is generally 5～100,000 yuan/platform, its good slightly price reaches 10～400,000 yuan/platform, and the test kit price supporting with instrument is 30～50 yuan/time, and this is for the situation of China, it is too high to detect cost, therefore is not applied fully at home.Realize the large-scale application of ATP biloluminescence method in China, must solve instrument and luminescence reagent two big key problems, the research and development of bioluminescent detection technology and bioluminescent detection instrument are devoted in this research in recent years always, the bioluminescent detection instrument has been developed model machine at present, therefore makes up the detection technique of bioluminescent detection test kit and creation facilities programization for applying the particularly important that seems.
[summary of the invention]
The objective of the invention is to make up highly sensitive ATP bioluminescent detection test kit, set up the detection technique of microorganism and alga cells quantity in standardized bacterium, yeast, mould and the tap water, realize the rapid detection of microbial count, for sanitary inspection and foodstuff production quality control provide gordian technique.
The invention provides a kind of quick detection kit of utilizing biloluminescence method rapid detection microbe population, this detection kit comprises reagent such as luciferase and protective material thereof, D-fluorescein, standard A TP, luminous detection damping fluid, acellular ATP remover, microorganism cells ATP extractant.(1) luciferase must be purified, and the vigor background ratio reaches 50～500, and vigor requires to add 0.1mL 1 * 10 in the 0.5mL luminescent detection system
-7The led pulse value CP6S of mol/L ATP is not less than 7000, and vigor detects and add 0.1mL luciferase, 0.1mL 1 * 10 in the 0.5mL luminescent detection system
-7The luminous detection damping fluid of mol/L ATP and 0.3mL 25mmol/L pH7.8 carries out, and background replaces standard A TP to carry out in the 0.5mL luminescent detection system with aseptic ultrapure water.(2) enzymatic protective reagent: adopt the mixing system of trehalose, PEG6000 and BSA, contain 10～100mg/L trehalose, 5～100mg/L PEG, 1～20mg/L BSA.Enzyme liquid and enzymatic protective reagent were by 10: 1 mixed.(3) luminous detection damping fluid (be called for short GB): can adopt a kind of in glycylglycine, Tricine, Tris, Glycine amide, Phosphate, the buffering salts such as MOPS, TES to be mixed with the damping fluid that concentration is 25mmol/L, pH7.2, preferred glycylglycine, it is formulated as contains the 25mmol/L glycylglycine, 0.5mg/mL BSA, 0.5mmol/L EDTA, 0.5mmol/L DTT, the damping fluid of pH7.2.(4) the luminous vigor of D-fluorescein must reach the level that is equivalent to Sigma L9504.(5) non-purpose cell ATP remover AE: with containing 50mmol/L Tris-HCl, 10mmol/L MgSO
4, 1mmol/LEDTA, 1mg/mL BSA, damping fluid preparation Pyrophosphate phosphohydrolase to the concentration of pH6.8 is 0.1～10mg/mL.(6) microorganism cells ATP releasing agent Ec: contain 1～30g/L TritonX-100,0.1～5.0g/L hexadecane trimethyl ammonium bromide (CTAB), 0.1～3.0g/L methyl-sulphoxide (DMSO), 0.01～0.1g/L ethylenediamine tetraacetic acid (EDTA) (EDTA), 0.01～0.1g/L sal epsom (MgSO
4).
Detection kit of the present invention can be got luciferase 100mL in advance in proportion; the luminous detection damping fluid that add enzymatic protective reagent 10mL, contains 50～150mgD-fluorescein (contains the 25mmol/L glycylglycine; 0.5mg/mL BSA; 0.5mmol/L EDTA; 0.5mmol/L DTT; pH7.2) 10mL obtains the ATP bioluminescent reagents of 120mL, with brown bottled.
For improving the immunity from interference of this bioluminescent reagents box, can also increase anti-interference substratum.Anti-interference substratum comprises that anti-anticorrosion formulation, anti-sterilization formulation and anti-ozone type liquid full-page proof detect substratum, they can eliminate sanitas Sorbic Acid and potassium sorbate, phenylformic acid and Sodium Benzoate respectively, the interference of sterilizing agent dioxide peroxide, Peracetic Acid, clorox and hydrogen peroxide and ozone and chlorine residue, when detection contains noisy sample, compare with the common liq substratum, can improve the sensitivity of detection greatly, detailed directions sees Table 1.
Interfering substance and concentration thereof that the anti-interference microbial liquid substratum of table 1 can be eliminated
| The substratum title | The interfering substance that can eliminate | The concentration of the interfering substance that can eliminate |
| Anti-anticorrosion form liquid full-page proof detects substratum | Phenylformic acid, Sodium Benzoate Sorbic Acid, potassium sorbate | 2.0g/L(kg) |
| Anti-sterilization form liquid full-page proof detects substratum | Dioxide peroxide Peracetic Acid clorox hydrogen peroxide | 150mg/L 400mg/L 700mg/L 180mg/L |
| Antiozonidate type liquid full-page proof detects substratum | The ozone chlorine residue | 10.0mg/L |
Using this detection kit, to carry out the standard method of microbe population rapid determination be by do the ATP typical curve in selected noclilucence instrument and selected system, obtain equation of linear regression after taking the logarithm, to remove and the extractive sample liquid of microbial atp through non-purpose ATP, in same instrument and system, use and carry out the ATP bioluminescent detection with a collection of enzyme, do blank simultaneously, obtain sample and barren CPM or RLU value, after net phase after the removal blank is taken the logarithm to luminous value, obtain the ATP concentration of sample by the regression equation of setting up, according to bacterium, yeast, the ATP content of microorganism such as mould and unicellular little algae can be converted into bacterium, yeast, the cell count of mould spores or unicellular little algae, or only meter is equivalent to bacterial number.As existing bacterium, yeast, mould or little algae in the sample and must measure respectively the time, then must there be one according to the filtering process of cell size fractionation.
Using this detection kit sets up the concrete grammar of microbe population rapid detection programization and is:
1. the making of standard A TP curve
In noclilucence instrument 0.5mL detection system, carry out 10 with ATP bioluminescent reagents box
-12～10
-6Mol/L series standard ATP concentration luminous detection, and make logarithmic curve and equation of linear regression.Detection system is 0.1mL luciferase (FL)+0.1mL ATP+0.3mL luminous detection damping fluid, carry out series standard ATP and replace not adding the barren led pulse value (CP6S or CPM value are measured) of ATP with sterilized water on light-emitting appearance, the net phase of removal blank value is done broken line graph, Trendline and regression equation to luminous value (Δ CP6S or Δ CPM) on log-log coordinates.
2. The pretreatment
The pretreatment comprises the removal of non-target ATP, the classified filtering of microorganism cells, the removal of interfering components in the sample, diluted sample or be concentrated to the sensing range that is fit to the ATP biloluminescence method etc. is according to form, character and the inclusion of sample and detect target and carry out respective handling.The removal of non-target ATP: unite the way that adopts millipore filtration and ATP remover, can save reagent.The classified filtering of microorganism cells: according to sample impurities situation, adopt the big aperture millipore filtration of 20 μ m to remove impurity earlier, under vacuum condition, pass through the aseptic filtering with microporous membrane of 8 μ m, 3 μ m, 0.45 μ m and 0.22 μ m then.The removal of interfering components in the sample: can add aseptic ultrapure water washing methods or carry out by centrifugal with the way of filtering with microporous membrane.For samples such as the food that contains sanitas, sterilizing agent or ozone, beverages, can be when needing higher detection sensitivity with carrying out preincubate to improve immunity from interference and detection sensitivity with anti-interference substratum.Diluted sample or concentrated: available aseptic ultrapure water dilution concentrates and can carry out the way of also available millipore filtration classified filtering by 8000～12000r/min centrifugal method.
3. the extracting of microorganism cells ATP
To carry out the extraction of ATP with microorganism cells ATP extractant Ec through pretreated sample, detailed process is after Ec and liquid sample add in 1: 1 ratio, room temperature effect 1.5～2.0min; Microorganism cells ATP extracts and then adds 1～2ml Ec on the filter membrane, and room temperature effect 1.5～2.0min gets final product.Wherein the preparation method of ATP extractant Ec is: add 1～30g TritonX-100,0.1～5.0g CTAB, 0.1～3.0g DMSO, 0.01～0.1g EDTA, 0.01～0.1g MgSO in every liter of aseptic ultrapure water
4, heating makes its dissolving, and it is even to shake.
4.ATP noclilucence test
Get 0.1mL ATP extracting solution, adding contains in the luminotron of 0.3mL luminous detection damping fluid, shakes up, add ATP bioluminescent reagents 0.1ml, put immediately after shaking up and carry out luminous detection in the luminous detection instrument, read led pulse count value CP6S or CPM, carry out the contrast test of blank sample simultaneously.Wherein luminous detection damping fluid (GB) contains: 25mmol/L glycylglycine, 0.5mg/mL BSA, 0.5mmol/L EDTA, 0.5mmol/L DTT, pH7.2.ATP bioluminescent reagents: get luciferase 100mL in proportion, add enzymatic protective reagent 10mL, contain the luminous detection damping fluid 10mL of 100mg D-fluorescein, obtain the ATP bioluminescent reagents of 120mL.
5. the calculating of corresponding microorganism cell ATP content in the sample
After net phase after the sample luminous value removal blank after the ATP noclilucence test is taken the logarithm to luminous value, by regression equation (lg[ATP]=A+Blg (Δ CP6S) or lg[ATP]=A+Blg (Δ CPM), dependency R greater than 0.98 for well, wherein A and B are the coefficient of regression equation), corresponding ATP concentration in the calculation sample.
6. bacterium, yeast, mould and unicellular algae quantity or in the calculating of the microbial count of bacterium in the sample
The early-stage Study result according to the present invention, the ATP content of bacterial cell is 3 * 10
-16G/cell; Saccharomycetic ATP content is 3 * 10
-14G/cel; The average A TP content of mould spores is 3 * 10
-15G/cell; The ATP content of single-cell algae is 3 * 10
-14G/cell is converted into the cell count of bacterium, yeast, mould and unicellular algae, or only counts the total quantity that is equivalent to bacterium.
Annotate: 505 is the molecular weight of ATP in the formula.
[description of drawings]
Fig. 1: in the 0.5mL system, detection system is 0.1mL bioluminescent reagents (FL)+0.1mL standard A TP+0.3mL luminous detection damping fluid (GB) on light-emitting appearance HKM-NRD (1) machine of last marine upright noclilucence instrument SHG-C and Huankai Microbiological Science ﹠ Technolgy Co., Guangdong's development.X-coordinate is by being added standard ATP concentration (10
-12～10
-6The mol/L scope) Lg[ATP], ordinate zou is the Lg Δ CP6S of the relative light unit of correspondence
1
[embodiment]
Embodiment 1: the structure of biloluminescence method quick detection kit (luminescence reagent is FL04113)
(1) preparation in advance of reagent
The luciferase protective material: get 500mg trehalose, 500mg PEG6000 and 15mg BSA, be dissolved in the 10mL sterile distilled water, the aseptic filtering with microporous membrane degerming by 0.22 μ m after shaking up gets final product.
Standard A TP solution: adopt the disodium salt A-2383 55.1mg of Sigma company, be dissolved in that 10mL is aseptic to be heated up in a steamer in the water, being made into concentration is 1 * 10
-2Mol/L is with the aseptic centrifuge tube dress of 1.5ml, every pipe 0.1ml.
Acellular ATP remover AP: its concrete composition and making processes are with containing 50mmol/L Tris-HCl, 10mmol/LMgS0
4, 1mmol/L EDTA, 1mg/mL BSA, damping fluid preparation Pyrophosphate phosphohydrolase to the concentration of pH6.8 is 5mg/ml, and passes through the aseptic filtering with microporous membrane of 0.22 μ m.
Microorganism cells ATP extracting solution E
c: add 20g TritonX-100,2.0g CTAB, 2.0gDMSO, 0.05g EDTA, 0.05g MgSO in every liter of aseptic ultrapure water
4, heating makes its dissolving, and it is even to shake.
Luminous detection damping fluid (being called for short GB): contain the 25mmol/L glycylglycine, 0.5mmol/L EDTA, 0.5mmol/LDTT, pH7.2.
(2) preparation of bioluminescent reagents (FL04113)
Get the luciferase 100mL of purifying, add enzymatic protective reagent 10mL, contain the luminous detection damping fluid 10mL of 100mg D-fluorescein, obtain 120mL solution, divide to install in 60 little brown bottles every bottle of 2mL, freeze-drying.
(3) structure of biloluminescence method quick detection kit
This detection kit comprises 4 bottles of bioluminescent reagents (FL04113), every bottle of 2mL; 0.1mL concentration is 1 * 10
-2Standard A TP solution 10 pipes of mol/L; 4 bottles of sterile distilled waters, every bottle of 25mL; 4 bottles of luminous detection damping fluids, every bottle of 20mL; 1 bottle of 20mL microorganism cells ATP extracting solution E
c1 brown bottled acellular ATP remover AP 5mL.
This test kit is used for the detection of embodiment 2～6.
Embodiment 2:FL04113 ATP noclilucence standard A TP curve
In the 0.5mL system, the examination criteria concentration range is 10 on light-emitting appearance HKM-NRD (1) machine of last marine upright noclilucence instrument SHG-C and the development of the triumphant microorganism in Guangdong scientific ﹠ technical corporation
-12～10
-6The ATP luminous value of mol/L, detection system are 0.1mL FL+0.1mL standard A TP+0.3mL GB, and make logarithmic curve and equation of linear regression, the results are shown in Table 1 and accompanying drawing 1.
As seen from Table 1: on the SHG-C light-emitting appearance, carry out luminous detection, when ATP concentration 10
-10～10
-6Better linear during mol/L, the regression curve in the 0.5mL luminescent system is Lg[ATP]=-12.4792+1.0374Lg Δ CPM, R=0.9977, n=5; On HKM-NRD (I) light-emitting appearance, when ATP concentration 10
-10～10
-6Better linear during mol/L, regression curve is Lg[ATP]=-10.8382+0.9547Lg Δ CP6S
1, R=0.9951, n=5.Detection sensitivity on two instruments is 10
-10Mol/L.
The luminous value of the ATP that table 1 FL041130 detects on SHG-C and HKM-NRD (I) machine
| Add [ATP] mol/L | SHG-C | HKM-NRD(1) | |||
| CP6S 1 | CPM | ΔCP6S 1 | CP6S 1 | ΔCP6S 1 | |
| H 2O 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 | 370 343 366 403 619 2950 25164 132482 | 3472 3312 3600 3674 5954 28545 241452 1294765 | - 128 202 2482 25073 237980 1291293 | 409 318 409 414 543 1677 8737 97535 | 0 5 134 1268 8328 97126 |
Embodiment 3: the luminous detection of the artificial sample of yeast (SHG-C)
(1) luminous detection on SHG-C
Carry out the yeast saccharomyces cerevisiae sample detection in the 0.5mL system on SHG-C, the dull and stereotyped cultivation results of artificial yeast's sample is 1.44 * 10
7Cfu/mL (the stoste letter is Y0), detection system: 0.1mL FL+0.1mL standard A TP or H
2O sample+0.3mL GB, luminous detection the results are shown in Table 2.
The artificial sample of table 2 yeast luminous detection result on SHG-C
| Sample | Luminescence method detects | |||||
| CPM | ΔCPM | LgΔ CPM | Lg[ATP] | [ATP]mol/ ml | Conversion cell count cells/ml | |
| H
2O standard | 3472 241452 1096208 144266 19182 5020 3753 | 1092736 140794 15710 1548 281 | 6.0385 5.1486 4.196 3.190 2.4487 | -6.2328 -7.1380 -8.1263 -9.170 -9.9389 | 5.8×10 -10 7.3×10 -11 7.5×10 -12 6.8×10 -13 1.2×10 -13 | 1.1×10 7 1.3×10 6 1.4×10 5 1.2×10 4 2.2×10 3 |
Annotate: yeast contains the ATP amount by 3 * 10
-14The g/cell meter.Standard A TP adopts 1 * 10
-7Mol/L.
The result shows: when the concentration of yeast sample 10
3Cells/m～10
7During cells/mL, detect than reflecting real level with the ATP biloluminescence method.
(2) luminous detection on HKM-NRD (1) machine
Carry out the artificial sample detection of yeast saccharomyces cerevisiae on encircling on triumphant HKM-NRD (1) machine in Guangdong in the 0.5ml system, the dull and stereotyped cultivation results of artificial yeast's sample is 1.44 * 10
7Cfu/mL (the stoste letter is Y0), detection system: 0.1mL FL+0.1mLATP or H
2O sample+0.3mL GB, luminous detection the results are shown in Table 3.
The artificial sample of table 3 yeast is the luminous detection result on HKM-NRD (1) machine
| Sample | Luminescence method detects | |||||
| CP6S 1 | Δ CP6S 1 | LgΔ CP6S 1 | Lg[ATP] | [ATP]moL /mL | Conversion cell count cells/mL | |
| H
2O standard | 180 14000 93000 8500 1100 280 210 | 92820 8320 920 100 30 | 4.9676 3.9201 2.9638 2.0 1.4771 | -6.1056 -7.2095 -8.2144 -9.2287 -9.7790 | 7.8×10 -10 6.2×10 -11 6.1×10 -12 5.9×10 -13 1.7×10 -13 | 1.43×10 7 1.14×10 6 1.12×10 5 1.08×10 4 3.12×10 3 |
Annotate: yeast contains the ATP amount by 3 * 10
-14The g/cell meter.
The result shows: the detected result of two instruments is more approaching, when the concentration of yeast sample 10
3Cells/m～10
7During cells/mL, detect than reflecting real level with the ATP biloluminescence method.
Embodiment 4: the luminous detection of bacteria artificial sample (SHG-C)
Carry out the yeast saccharomyces cerevisiae sample detection in the 0.5ml system on SHG-C, the dull and stereotyped cultivation results of artificial bacteria samples ATCC25922 is 6.4 * 10
8Cfu/mL (the stoste letter is Y0), detection system: 0.1mL FL+0.1mLATP or H
2O sample+0.3mLGB, luminous detection the results are shown in Table 4.
Table 4 bacteria artificial sample luminous detection result on SHG-C
| Sample | CPM | ΔCPM | LogΔ CPM | Luminescence method detects | ||
| Log[ATP] | [ATP]mol/ml | Conversion cell count cells/ml | ||||
| H 2O standard A TP E0 10 -1E0 10 -2E0 10 -3E0 10 -4E0 10 -5E0 | 3472 241452 691081 84802 11816 4326 3564 3457 | 687609 81330 8344 854 92 -15 | 5.8373 4.9102 3.9214 2.9314 1.9638 | -6.4236 -7.3853 -8.4111 -9.4382 -10.4420 | 3.77×10 -10 4.12×10 -11 3.88×10 -12 3.65×10 -13 3.61×10 -14 | 6.35×10 8 6.94×10 7 6.53×10 6 6.14×10 5 6.08×10 4 |
Annotate: the average A TP content of bacterium is 3 * 10
-16G/cell, standard A TP adopts 1 * 10
-7Mol/L.
The result shows: when detecting microbe population with the ATP biloluminescence method, speed is very fast, and concrete testing process only needs tens minutes, and the ATP content conversion relation by standard regression equation and bacterium can draw cell count, is 10 at the bacterial cell number
4Cells/mL ~ 10
8Cells/mL can reflect real level.
Embodiment 5: the ATP luminous detection of the artificial sample of mould
Carry out the artificial sample detection of mould MIG3.95 spore in the 0.5mL system on SHG-C, MIG3.95 spore count microscopic count value is 9.45 * 10
7Cfu/mL (stoste is called for short M0), detection system: 0.1mL FL+0.1mLATP or H
2O sample+0.3mL GB, the ATP luminous detection the results are shown in Table 4.
The luminous test result of ATP of the artificial sample of table 5 mould
| Sample | Luminescence method detects | |||||
| Average CPM | Δ CPM | LgΔ CPM | Lg[ATP] | [ATP]mol/ml | Conversion cell count cells/ml | |
| H
2O standard | 3472 241452 556031 54241 8292 3940 3483 | 552559 50769 4820 468 11 | 5.7424 4.7056 3.6830 2.6702 1.0414 | -6.5220 -7.5976 -8.6584 -9.7091 -11.3993 | 3.006×10 -10 2.526×10 -11 2.196×10 -12 1.95×10 -13 3.99×10 -14 | 5.06×10 7 4.25×10 6 3.70×10 5 3.28×10 4 6.72×10 3 |
Annotate: the average A TP content of mould spores is by 3 * 10
-16G/cell calculates, and standard A TP adopts 1 * 10
-7Mol/L.
The result shows: when detecting mould spores and count with the ATP biloluminescence method, when spore count 10
3～10
8Result that the ATP biloluminescence method converts between the cells/mL and microscopic counting result difference are not very big.
1, water sample: 1
#Source water factory tank water, 2
#5 gallons of natural spring, 3 of drinking
#5 gallons of direct drinking waters, 4
#The preceding water (1 of manganese sand is crossed in the mineral water water treatment
#～4
#Sample is by suspecting that product has the bottled drinking water producer of algae pollution to provide), 5
#Reservoir water, 6
#The balcony tank water.
2, water sample pre-treatment
Drink with water sample 100～1000mL (clean level per sample and decide) with reference to the routine sampling method, jolting makes sample dispersion even, vacuumize then and pass through 8 μ m, 3 μ m and 0.45 μ m millipore filtration respectively, and use 50mL aseptic deionized water washing and filtering funnel and millipore filtration respectively through sterilising treatment.
3. the extraction of sample ATP
The extraction of membrane retention cell ATP: the various filter membranes behind the suction filtration sample are taken off, place the aseptic small beaker of 50mL respectively, add the aseptic ultrapure water of 1mL, add 1mL cell ATP extract Ec again, inhale gently with the rifle head and beat, effect 1.5min.The ATP standard substance of getting the 1mL different concns respectively replace the 1mL aseptic deionized water and do same processing as standard; Blank millipore filtration is done same processing as blank sample.
4. luminous test
In the 0.5ml system, on SHG-C, carry out ATP noclilucence test.Draw 0.1mL sample ATP extracting solution in luminotron, add in the 0.3mL luminous detection damping fluid, add the bioluminescent reagents (FL04113) that 0.1mL prepares again, shake up at once, place 25 ℃ of bioluminescent detection instrument to carry out the led pulse counting.Do 1 * 10 simultaneously
-7The luminous detection of mol/L ATP standard substance and blank sample.
5. ATP concentration is calculated in the sample
According to the net phase of specimen to luminous value Δ CP6S, according to the ATP concentration of regression equation calculation sample.
6. the corresponding microorganism cell count is calculated in the sample
In the different microorganisms cell of on the filter membrane of classified filtering, holding back behind the ATP content, be converted into corresponding cell count according to the ATP content of different cells.
7. experimental result
Adopt the filter membrane of 8 μ m, 3 μ m and 0.45 μ m that the tap water sample is carried out classified filtering, detect membrane retention ATP and measure microalgae cell number and the bacterial count (table 5) that is converted in the sample.The result shows, the little algae in the sample is mainly by the membrane retention of 8 μ m, the membrane retention of 3 μ m and 0.45 μ m mainly be bacterium.From the detected result of table 5, sample has also polluted bacterium and fungi in pollution algae, the cleaning and sterilizing imperfection of prompting water technology or wrapping material.Because what exist in the tap water sample causes the interferential material less to luminous detection, comparatively close with membrane retention microorganism cells luminous detection result and colony counting method, therefore the purpose of the microorganism that pollutes in the rapid detection tap water be can reach, production scene monitoring and product analysis helped.
Table 6 filter membrane method is held back little algae and bacterium hierarchical detection result in the tap water
| Sample number into spectrum | Processing mode | Be converted into cell ATP concentration (mol/L) in the stoste | Be converted into cell count (cells/mL) | Plate count (cells/mL) | ||
| Bacterium | Fungi | Algae (counting) | ||||
| 1# | Stoste 8 μ m 3 μ m 0.22 μ m | 4.69×10 -11 1.67×10 -12 8.66×10 -13 | A7.88×10 2 B2.81×10 3 B1.45×10 3 | 3.11×10 3 | 6 | 5.25×10 2 |
| 2# | Stoste 8 μ m 3 μ m 0.22 μ m | 4.24×10 -11 2.52×10 -13 2.02×10 -13 | A7.12×10 2 B4.23×10 2 B3.39×10 2 | 4.87×10 2 | 15 | 7.00×10 2 |
| 3# | Stoste 8 μ m 3 μ m 0.22 μ m | 1.26×10 -12 4.72×10 -13 3.02×10 -13 | A21 B7.93×10 2 B5.07×10 2 | 8.23×10 2 | 0 | 15 |
| 4# | Stoste 8 μ m 3 μ m 0.22 μ m | 5.17×10 -12 3.09×10 -13 7.32×10 -13 | A87 B5.19×10 2 B1.23×10 3 | 1.65×10 3 | 0 | 105 |
| 5# | Former water | 1.77×10 -9 | 3.0×10 2 | 15 | 4 | |
| 8μm | 1.35×10 -9 | A2.27×10 4 | 5.25×10 3 | |||
| 3μm | 8.72×10 -12 | B1.46×10 4 | 0 | |||
| 0.45μm | 9.14×10 -12 | B1.54×10 4 | 3.1×10 2 | 0 | ||
| 6# | Former water | Can not survey | 1.64×10 8 | 0 | 0 | |
| 8μm | 3.98×10 -11 | A6.69×10 2 | ||||
| 3μm | 2.02×10 -11 | B3.39×10 4 | 0 | |||
| 0.45μm | 7.75×10 -11 | B1.30×10 5 | 0 |
Annotate: A represents to be converted into the quantity of microalgae cell in the table, and B represents to be converted into number of bacteria.
Claims (11)
1, a kind of biloluminescence method kit for anti-interference quick detection of microbe population is characterized in that comprising luciferase and protective material thereof, D-fluorescein, standard A TP, luminous detection damping fluid, acellular ATP remover, microorganism cells ATP extractant.
2, a kind of microbe population noclilucence quick detection kit; it is characterized in that getting in proportion luciferase 100mL; add enzymatic protective reagent 10mL, contain the luminous detection damping fluid 10mL of 50～150mg D-fluorescein, be pre-mixed and be mixed with the ATP bioluminescent reagents.
3, claim 1 or 2 described a kind of microbe population noclilucence quick detection kit, wherein luciferase must be through purifying, and the vigor background ratio reaches 50～500, adds 0.1mL 1 * 10 in the 1mL luminescent detection system
-7The led pulse value CP6S of mol/L ATP is not less than 7000.
4, claim 1 or 2 described microbe population noclilucence quick detection kit, wherein the luminous vigor of D-fluorescein must reach the level that is equivalent to Sigma L9504.
5, claim 1 or 2 described biloluminescence method quick detection kit, wherein the luciferase protective material contains the BSA of 50～75% trehaloses, 10～15% PEG6000 and 10～15%.
6, claim 1 or 2 described biloluminescence method quick detection kit, used luminous detection damping fluid adopt a kind of in glycylglycine, Tricine, Tris, Glycine amide, Phosphate, the buffering salts such as MOPS, TES to be mixed with the damping fluid that concentration is 25mmol/L, pH7.2.
7, the described biloluminescence method quick detection kit of claim 6, used luminous detection damping fluid contains 25mmol/L glycylglycine, 0.5mg/mL BSA, 0.5mmol/L EDTA, 0.5mmol/L DTT, pH7.2.
8, the described biloluminescence method quick detection kit of claim 1, the compound method of acellular ATP remover is: will contain 50mmol/L Tris-HCl, 10mmol/L MgSO
4, 1mmol/L EDTA, 1mg/ml BSA, pH6.8 damping fluid preparation Pyrophosphate phosphohydrolase to concentration be 0.1～10mg/mL.
9, the described biloluminescence method quick detection kit of claim 1, microorganism cells ATP remover contains 1～30g/LTritonX-100,0.1～5.0g/L hexadecane trimethyl ammonium bromide, 0.1～3.0g/L methyl-sulphoxide, 0.01～0.1g/L ethylenediamine tetraacetic acid (EDTA), 0.01～0.1g/L sal epsom.
10, claim 1 or 2 described a kind of microbe population noclilucence quick detection kit is characterized in that also containing one or more the anti-interference substratum in anti-anticorrosion formulation, anti-sterilization formulation or the anti-ozone type liquid full-page proof detection substratum.
11, utilize claim 1 or 2 described biloluminescence method quick detection kit to carry out the method for rapid detection, undertaken by following step:
(1) making of standard A TP curve
In noclilucence instrument 0.5mL system, carry out 10
-12～10
-6Mol/L series standard ATP concentration luminous detection, and make logarithmic curve and equation of linear regression: Lg[ATP]=A+BLg Δ CP6S or lg[ATP]=A+Blg (Δ CPM), wherein A and B are the coefficient of regression equation;
(2) The pretreatment
According to form, character and the inclusion of sample and detect target and carry out the respective sample pre-treatment: comprise the removal of interfering components in the classified filtering, sample of removal, the microorganism cells of non-target ATP, at last with diluted sample or be concentrated to the sensing range that is fit to the ATP biloluminescence method etc.;
(3) extracting of microorganism cells ATP
To carry out the extraction of ATP with microorganism cells ATP extractant Ec through pretreated sample, detailed process is after Ec and liquid sample add in 1: 1 ratio, room temperature effect 1.5～2.0min; Microorganism cells ATP extraction then is that the various filter membranes behind the suction filtration sample are taken off on the filter membrane, places the aseptic small beaker of 50mL respectively, adds 1～2mL cell ATP extract Ec, room temperature effect 1.5～2.0min;
(4) ATP noclilucence test
Get 0.1mL ATP extracting solution, add in the luminotron that contains 0.3mL luminous detection damping fluid and shake up, add bioluminescent reagents 0.1mL again, put immediately after shaking up and carry out luminous detection in the luminous detection instrument, read led pulse count value CP6S or CPM, carry out the contrast test of 1mL aseptic deionized water blank sample simultaneously.
(5) calculating of corresponding microorganism cell ATP content in the sample
After net phase after the sample luminous value removal blank after the ATP noclilucence test is taken the logarithm to luminous value, by the typical curve regression equation, corresponding ATP concentration in the calculation sample.
(6) bacterium, yeast, mould and unicellular algae quantity or in the calculating of the microbial count of bacterium in the sample
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