CN103336000A - Metering method for rapid measurement of mould in stored grain through ATP bioluminescence method and applications - Google Patents
Metering method for rapid measurement of mould in stored grain through ATP bioluminescence method and applications Download PDFInfo
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- CN103336000A CN103336000A CN2013102194539A CN201310219453A CN103336000A CN 103336000 A CN103336000 A CN 103336000A CN 2013102194539 A CN2013102194539 A CN 2013102194539A CN 201310219453 A CN201310219453 A CN 201310219453A CN 103336000 A CN103336000 A CN 103336000A
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Abstract
The invention relates to a metering method for rapid measurement of mould in stored grain through the ATP bioluminescence method. The metering method comprises the following steps. (1) 25 g grain and 10-20 glass beads are put into 225 mL sterile water and oscillated to obtain a bacterial suspension, standby. (2) 50-200 mL of the bacterial suspension is subjected to suction filtration through filter membranes with a pore size of 0.22 mum, the filter membranes are then transferred to a BAC solution which is 0.1 times the bacterial suspension in volume, the BAC solution is oscillated to extract ATP, and a reaction solution to be measured is obtained. (3) 100 muL of the reaction solution from step (2) is added into 100 muL of the rL/L reagent and mixed evenly, and luminescence values are measured. The metering method does not need cultivation processes to measure mould quantity through the ATP bioluminescence method and has simple operations and high sensitivity. Measurement results can be obtained in short time, and the metering method has obvious advantages compared with other measurement methods for microorganisms.
Description
Technical field
The present invention relates to mould detection technique field, relate to metering method and the application thereof of a kind of ATP biloluminescence method fast detecting grain storage mould.
Background technology
ATP is the abbreviation of chemical substance atriphos, is present in all biosomes (from microorganism to high animals and plants).The main effect of ATP is to provide energy in the biological cell body.Test the correlates between ATP concentration and the microorganism concn that are unequivocally established at present in a large number, and can be by measuring the actual conditions of ATP concentration reflection microorganism concn.
The grain storage mould detects the mensuration that refers to mould quantity the grain storage from quantitative angle, sees it is various properties and characteristicses according to showing the mould active procedure from qualitative angle, the active state of monitoring mould.Tradition mould detection technique mainly is to be basic means with the separation and purification cultivation, differentiates mould on the cellular level aspect from aspects such as morphological feature, biochemical reactions; Novel mould detection technique mainly comprises immunological technique, Protocols in Molecular Biology, biologically active zymotechnic, bioluminescence technique, CO
2Gas detection technology etc.
ATP bioluminescent detection method is to utilize ATP content constant in the microorganism particular growth process, and ATP and fluorescein-luciferase effect send bioluminescence, and the principle of luminous value and the linear correlationship of content of microorganisms is used for the quick mensuration of grain storage fungi count.At present, there is not the ATP biloluminescence method to be applied to the research report that the grain storage mould detects as yet.
Summary of the invention
At the problems referred to above, the purpose of this invention is to provide the metering method that a kind of grain storage mould Fast Detection Technique-ATP biloluminescence method detects the grain storage mould.
ATP biloluminescence method of the present invention detects the metering method of grain storage mould, comprises the steps:
(1) in the 225mL sterilized water, adds 25
gGrain is put into 10-20 grain beaded glass, and vibration obtains bacteria suspension, and is standby;
(2) get the 50-200mL bacteria suspension and carry out suction filtration, the used filter membrane of suction filtration is that pore size is the filter membrane of 0.22um, then filter membrane is transferred to the BAC(benzalkonium chloride of 0.1 times of bacteria suspension volume) in the solution, ATP is extracted in vibration, obtains reactant liquor, and is to be measured;
(3) reactant liquor with 100 μ L steps (2) joins 100 μ LrL/L(fluorescein-luciferases) in the reagent, measure luminous value behind the mixing.
Preferably, step (1) is for getting 25g grain, join in the conical flask that 225mL sterilized water and 10-20 grain beaded glass are housed, and the shaking table vibration obtains bacteria suspension, and is standby.
In the step (1), the mould quantity in the mensuration 225mL sterilized water in the 25g grain is with reference to GB4789.15-2010.
Described duration of oscillation is 20-30min, and rotating speed is 120-160 commentaries on classics/min.Preferably, described duration of oscillation is 30min, and rotating speed is 140 commentaries on classics/min.
Preferably, in the step (2), get the 100mL bacteria suspension and carry out suction filtration, the used filter membrane of suction filtration is that pore size is the filter membrane of 0.22um, then filter membrane is transferred in the 10mL BAC solution, and ATP is extracted in vibration, obtains reactant liquor, and is to be measured.Step (2) is carried out 10 times of enrichments for the bacteria suspension that step (1) is obtained.
In the step (2), the concentration of described BAC solution is 0.05-0.15%, and preferably, the concentration of described BAC solution is 0.1%.
In the step (2), the extraction time that ATP is extracted in described vibration is 2-3min, and preferably, the extraction time that ATP is extracted in described vibration is 3min.
In the step (2), the extraction temperature that ATP is extracted in described vibration is 25-30 ° of C, and preferably, the extraction temperature that ATP is extracted in described vibration is 30 ° of C.
In the step (2), the extraction rotating speed that ATP is extracted in described vibration is 120-160 commentaries on classics/min, and preferably, the extraction rotating speed that ATP is extracted in described vibration is 140 commentaries on classics/min.
In the step (2), described rL/L reagent is fluorescein-luciferase reagent, is commercially available.
In the step (3), the mensuration of described luminous value adopts the bioluminescence module of multi-functional microplate reader TECANINFINITE200, measures temperature and is set at 30 ° of C.
Described grain comprises corn, wheat, paddy rice etc.
Described detection mould comprises aspergillus niger, fusarium moniliforme, penicillium oxalicum etc.
The present invention also provides the application of described method in the grain storage mould detects.
The metering method of ATP biloluminescence method fast detecting grain storage mould of the present invention has following beneficial effect:
(1) the present invention has verified by experiment that first the ATP biloluminescence method can be used for detecting grain storage mould quantity, need not incubation when using the ATP biloluminescence method to measure mould quantity, simple to operate, and it is highly sensitive, but obtain measurement result within a short period of time, have remarkable advantages than other microorganism detection method.
(2) of the present invention experiment showed, in the method that at room temperature discharges ATP, it is best that surfactant B AC extracts mould ATP effect.BAC extracts the optium concentration of ATP and Best Times and is respectively 0.1% and 3min.Mould ATP luminous intensity and total number of molds are carried out correlation analysis find that both have significant correlation, and only need 10min detection time,
Illustrate that it is feasible that the ATP biloluminescence method is measured the grain storage total number of molds, can be used as a kind of effective grain storage mould method for quick, thereby ensure grain security.
Description of drawings
Fig. 1 is that BAC concentration is to the influence of mould ATP extraction effect;
Fig. 2 is that extraction time is to the influence of mould ATP extraction effect;
Fig. 3 is the relation of aspergillus niger quantity and ATP luminous intensity;
Fig. 4 is the relation of fusarium moniliforme quantity and ATP luminous intensity;
Fig. 5 is the relation of penicillium oxalicum quantity and ATP luminous intensity;
Fig. 6 is the corn mould ATP luminous intensity variations of storage period water cut 14% and the variation of total number of molds;
Fig. 7 is the corn mould ATP luminous intensity of storage period water cut 14% and the correlation analysis of total number of molds.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, experiment material, reagent and instrument etc. used in the embodiment of the invention all are commercially available, if specifically do not indicate, used technological means is conventional means well-known to those skilled in the art among the embodiment.
As do not specialize, concentration of the present invention is mass concentration.
Surfactant B AB, BAC completely newly open up available from Beijing and reach Science and Technology Ltd.;
Reagent such as TCA, Triton-100, CTAB, SDS, acetone are purchased in Peking blue and are shooted a retrievable arrow chemical products Ltd;
Standard A TP is available from Pu Luomaige (Beijing) Bioisystech Co., Ltd;
RL/L reagent is fluorescein-luciferase reagent, purchases the Bioisystech Co., Ltd in Pu Luomaige (Beijing).
Embodiment 1 different ATP releasing agents discharge method and the effect of ATP to mould
Below different ATP releasing agents are described method and the corresponding action effect thereof that mould discharges ATP.
Get the 25g corn, join in the conical flask that 225mL sterilized water and 10 beaded glasses are housed, shaking table vibration 30min, rotating speed is 140 commentaries on classics/min, obtains bacteria suspension, and is standby.
1, BAC extraction method
Get the 100mL bacteria suspension and carry out suction filtration, the used filter membrane of suction filtration is that pore size is the 0.22um filter membrane, then filter membrane is transferred in the test tube that 10mL0.15%BAC solution is housed, and 3min is extracted in 140 commentaries on classics/min vibration in 30 ° of C water-baths, obtain reactant liquor, preserve to be measured under 4 ° of C.2, TCA method
Get the 100mL bacteria suspension and carry out suction filtration, then filter membrane is transferred to the TCA(trichloroacetic acid that 10mL10g/L is housed) in the test tube of solution, 5min is extracted in 140 commentaries on classics/min vibration in 30 ° of C water-baths, obtains reactant liquor, preserves to be measured under 4 ° of C.
3, boil the damping fluid method
8mLTricine damping fluid (25mmol/LTricine, 0.5mmol/LEDTA, 5mmol/LMgSO will be housed
4, 0.5mmol/LDTT, 500 μ g/LBSA, accent pH to 7.8) test tube is placed in the boiling water bath, get the 100mL bacteria suspension then and carry out suction filtration, rapidly filter membrane is transferred to behind the suction filtration in the damping fluid in the boiling water bath, boil extraction 5min(and need not vibration, boil extraction) after put into ice-water bath rapidly and cool off, transfer to the 10mL volumetric flask then, residual extract is transferred in the lump with a small amount of Tricine damping fluid flushing and is settled to 10mL in the volumetric flask in the test tube, mixed obtain reactant liquor after evenly, place preserve under 4 ° of C to be measured.
4, boil the KOH method
The test tube that 8mL KOH solution (100mmol/LKOH, 0.5mmol/LEDTA, 10mmol/LTris) will be housed is placed in the boiling water bath, get the 100mL bacteria suspension then and carry out suction filtration, rapidly filter membrane is transferred in the KOH solution of boiling water bath behind the suction filtration, boil extraction 5min(and need not vibration, boil extraction) after put into ice-water bath rapidly and cool off, transfer to 10mL volumetric flask (being neutralized to pH is 7.8) then, residual extract is with a small amount of Tricine damping fluid (25mmol/LTricine, 0.5mmol/LEDTA, 5mmol/L MgSO in the test tube
4, 0.5mmol/LDTT, 500 μ g/LBSA, transfer pH to 7.8) flushing transfers in the lump and is settled to 10mL in the volumetric flask, mixedly obtains reactant liquor after evenly, place preserve under 4 ° of C to be measured.
5, BAB extraction method
Get the 100mL bacteria suspension and carry out suction filtration, then filter membrane is transferred to the 10mL0.15%BAB(benzalkonium bromide is housed) in the test tube of solution, after 3min is extracted in 140 commentaries on classics/min vibration in 30 ° of C water-baths, obtain reactant liquor, preserve to be measured under 4 ° of C.
6, Surfactant CTAB facture
Get the 100mL bacteria suspension and carry out suction filtration, then filter membrane is transferred in the test tube that 10mL CTAB solution (10g/L) is housed, after 2min is extracted in 140 commentaries on classics/min vibration in 30 ° of C water-baths, obtain reactant liquor, preserve to be measured under 4 ° of C.
7, surfactant SDS facture
Get the 100mL bacteria suspension and carry out suction filtration, then filter membrane is transferred in the test tube that 10mL SDS solution (10g/L) is housed, after 2min is extracted in 140 commentaries on classics/min vibration in 30 ° of C water-baths, obtain reactant liquor, preserve to be measured under 4 ° of C.
8, surfactant Triton X-100 facture
Get the 100mL bacteria suspension and carry out suction filtration, then filter membrane is transferred in the test tube that 10mL TritonX-100 solution (10g/L) is housed, after 2min is extracted in 140 commentaries on classics/min vibration in 30 ° of C water-baths, obtain reactant liquor, preserve to be measured under 4 ° of C.
9, acetone treatment method
Get the 100mL bacteria suspension and remove supernatant after centrifugal, precipitation adds 10mL acetone, little 5min that boils on boiling water bath, treat the acetone volatilization after, add 10mL sterilization ultrapure water, obtain reactant liquor, preserve to be measured under 4 ° of C.
100 μ L reactant liquor to be measured in the above-mentioned 1-9 method is joined in the 100 μ LrL/L reagent, adopt the bioluminescence module of multi-functional microplate reader TECANINFINITE200, measure temperature and be set at 30 ° of C, measure luminous value.
Different releasing agents are handled ATP that the back the discharges intensity of giving out light to bacteria suspension, the results are shown in Table 1.
The various ATP method for releasing of table 1 compare the release action of mould ATP
Present embodiment has compared various ATP method for releasing to the releasing effect of mould ATP, result such as table 1.
Boil the damping fluid method and to boil KOH method method similar, boiling damping fluid method ratio as seen from the experiment, to boil KOH method extraction effect better.Boil the damping fluid method and be and discharge a kind of comparatively feasible method in the middle of the cell ATP technology, but need optional equipment and complex operation, very inconvenient when being applied to online detection.Simultaneously from this experimental result as can be seen, compare with other ATP method for releasing, its releasing effect for mould is not very good.
TCA has very strong inhibiting effect for fluorescein-luciferase, must suitably dilute reactant liquor after therefore extracting and could further carry out luminous detection, and this sensitivity that just will inevitably cause detecting reduces.Therefore, TCA method effect in actual applications has been subjected to very big restriction, now replaced gradually by other method for releasing, and also not very good for the extraction effect of mould ATP from the result TCA method of this test yet.
The releasing effect of Surfactant CTAB, SDS and the mycotic spore ATP of Triton X-100 is inequality, the better effects if of the more other two kinds of surfactants of CTAB releasing effect wherein, and the release of the mould ATP of SDS is not effect almost.
The effect that quaternary ammonium salt surface active agent BAB and BAC extract ATP is best in all extracting method, and it is even better and toxicity is lower that both compare the BAC effect.Therefore, as the method that discharges ATP under the room temperature, it is a kind of good mould ATP method for releasing that surfactant B AC handles mould, and it is more convenient to boil the damping fluid method, sensitiveer than the TCA method.
Acetone extracts comparatively favourable for the release of fatty and the material that protein is higher, but it has certain inhibiting effect for luciferase, thereby also influences the ATP extraction effect.
In sum, use the effect of the mould release of surfactant B AC ATP best.The top condition that the influence of the fluorescein-luciferase reaction system of embodiment 2BAC and mould ATP extract
1, the influence of the fluorescein-luciferase reaction system of BAC
With sterilized water with standard A TP (10
-7Mol/L) be diluted to 10 respectively
-8, 10
-9, 10
-10, 10
-11The ATP working fluid of mol/L.Prepare two kinds of reagent: reagent A (75 μ L sterilized waters,
In contrast); Reagent B(25 μ L0.1%BAC+50 μ L sterilized water).Respectively with 25
μThe ATP working fluid of the variable concentrations of L joins in two kinds of reagent of 75 μ L, gets 100 μ L mixed liquors, joins 100
xIn the LrL/L reagent, measure luminous value behind the mixing, the result sees table 2 for details.
Table 20.1%BAC reagent is to the influence of mould ATP bioluminescence reaction
As can be seen from Table 2, the ATP working fluid luminous value that adds 0.1%BAC is compared with control group, and difference is less, and the luminous recovery of luminous detection liquid can think that the fluorescein-luciferase reaction system of 0.1%BAC do not have obvious influence between 96.0%-104.4%.
2, the top condition of mould ATP extraction
(1) measures the optium concentration that BAC extracts mould ATP
Get the 100mL bacteria suspension and carry out suction filtration, then filter membrane is transferred in the test tube that 10mL BAC solution is housed, concentration gradient is 0.01%, 0.05%, 0.1%, 0.15%, 0.2%,
0.25%, after 1min is extracted in 140 commentaries on classics/min vibration in 0.3%, 30 ° of C water-bath, gets 100 μ L reactant liquors, join in the 100 μ L rL/L reagent, survey luminous value behind the mixing.
Result such as Fig. 1, Fig. 1 show that concentration is that 0.1% BAC solution is to ATP extraction effect the best of mould.
(2) measure the best duration that BAC extracts mould ATP
Getting the 100mL bacteria suspension carries out behind the suction filtration filter membrane being transferred in the test tube that 10mL0.1%BAC solution is housed, then respectively in 30 ° of C water-baths 140 commentaries on classics/ min vibration extract 1,2,3,4,6,8,10,15min, get 100 μ L reactant liquors, join in the 100 μ L rL/L reagent, survey luminous value behind the mixing.
Result such as Fig. 2, Fig. 2 show when BAC solution extraction time is 3min, to ATP extraction effect the best of mould.
3, experimental result
Above-mentioned experimental result shows, concentration is when 0.1% BAC solution extraction time being 3min, to ATP extraction effect the best of mould, and the fluorescein-luciferase reaction system do not had obvious influence.
The correlation analysis of embodiment 3ATP luminescence method and classic flat-plate counting method and the application in the experiment of corn storage
1, the ATP luminescence method detects the metering method of fungi count
(1) get the 25g corn, join in the conical flask that 225mL sterilized water and 10 beaded glasses are housed, shaking table vibration 30min, rotating speed is 140 commentaries on classics/min, obtains bacteria suspension, and is standby.
(2) get the 100mL bacteria suspension and carry out suction filtration, the used filter membrane of suction filtration is that pore size is the filter membrane of 0.22um, filter membrane is transferred to is equipped with in the 10mL0.1%BAC solution then, and 3min is extracted in 140 commentaries on classics/min vibration in 30 ° of C water-baths, obtains reactant liquor, and is to be measured;
(3) reactant liquor with 100 μ L steps (2) joins in the 100 μ LrL/L reagent, adopts the bioluminescence module of multi-functional microplate reader TECANINFINITE200, measures temperature and is set at 30 ° of C, measures luminous value.
2, colony counting method and ATP luminescence method detect the correlation analysis of mould bacterium amount
From corn, separate gain the upper hand mould bacterial classification aspergillus niger (Aspergillus niger), fusarium moniliforme (Fusarium moniliforme) and penicillium oxalicum (Penicillium oxalicum).
Three kinds of moulds are inserted the PDA inclined-plane respectively, under 28 ° of C, cultivate 4d, after treating that spore fully forms, the percent by volume that adds the 5mL sterilization in 3 test tubes respectively is 0.5%Tween80 solution, wash the spore of three kinds of moulds respectively with Tween80 solution, obtain the spore liquid of three kinds of moulds, be adjusted to 10 with the blood counting chamber counting and with sterilized water after manually vortex fully vibrates
7Individual/mL, preserve under 4 ° of C.
With stroke-physiological saline solution three kinds of mycotic spore liquid are diluted to 10
3, 10
4, 10
5, 10
6, 10
7The different gradients of individual/mL are carried out the correlation analysis between ATP luminescence method and the classic flat-plate counting method, and the result sees Fig. 3 (aspergillus niger) for details, Fig. 4 (fusarium moniliforme), Fig. 5 (penicillium oxalicum).
By Fig. 3-5 as can be known, the facies relationship number average is more than 0.98, and therefore, the metering method that ATP biloluminescence method of the present invention detects the grain storage mould has feasibility, and only needs 10min detection time.
3, the microorganism of corn simulation storage changes detection
With sterilized water corn moisture is modulated to the 14%(mass concentration), store under 28 ° of C,
Every 5d sampling once, make bacteria suspension with sterilized water vibration washing corn, adopt the method for plate culture count and ATP luminescence method that it is detected respectively, draw under water cut 14% condition the situation of change of corn mould ATP luminous intensity and total number of molds and the correlation analysis that is applied in ATP luminescence method in the storage experiment.
As shown in Figure 6, when mould ATP luminous value less than 3 * 10
3The time, can determine that tentatively total number of molds is lower than 8 * 10 in the corn
4Individual/g.
As shown in Figure 7, the related coefficient of total number of molds logarithm value and mould ATP luminous intensity logarithm value is 0.9553 (P＜0.05), has significant correlation, illustrate that ATP biloluminescence method of the present invention can be used for the mensuration of corn total number of molds, also show method and the condition of extraction mould ATP of the present invention on the other hand, be conducive to improve ATP biloluminescence method quantitative measurement total number of molds accuracy.
The metering method that ATP biloluminescence method of the present invention detects mould is equally applicable to the mensuration of total number of molds in other grains except corn.
Though, above used general explanation, embodiment and test, the present invention is described in detail, on basis of the present invention, can make some modifications or improvements it, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the metering method of an ATP biloluminescence method detection grain storage mould is characterized in that described method comprises the steps:
(1) add 25g grain in the 225mL sterilized water, put into 10-20 grain beaded glass, vibration obtains bacteria suspension, and is standby;
(2) get the 50-200mL bacteria suspension and carry out suction filtration, the used filter membrane of suction filtration is that pore size is the filter membrane of 0.22um, then filter membrane is transferred in the BAC solution of 0.1 times of bacteria suspension volume, and ATP is extracted in vibration, obtains reactant liquor, and is to be measured;
(3) reactant liquor with 100 μ L steps (2) joins in the 100 μ L rL/L reagent, measures luminous value behind the mixing.
2. method according to claim 1 is characterized in that, in the step (1), described duration of oscillation is 20-30min, and rotating speed is 120-160 commentaries on classics/min; Preferably, described duration of oscillation is 30min, and rotating speed is 140 commentaries on classics/min.
3. method according to claim 1 is characterized in that, in the step (2), the concentration of described BAC solution is 0.05-0.15%; Preferably, the concentration of described BAC solution is 0.1%.
4. method according to claim 1 is characterized in that, in the step (2), the extraction time that ATP is extracted in described vibration is 2-3min; Preferably, the extraction time of described vibration extraction ATP is 3min.
5. method according to claim 1 is characterized in that, in the step (2), the extraction temperature that ATP is extracted in described vibration is 25-30 ° of C; Preferably, the extraction temperature of described vibration extraction ATP is 30 ° of C.
6. method according to claim 1 is characterized in that, in the step (2), the extraction rotating speed that ATP is extracted in described vibration is 120-160 commentaries on classics/min; Preferably, the extraction rotating speed of described vibration extraction ATP is 140 commentaries on classics/min.
7. method according to claim 1 is characterized in that, in the step (3), the mensuration of described luminous value adopts the bioluminescence module of multi-functional microplate reader TECAN INFINITE200, measures temperature and is set at 30 ° of C.
8. according to each described method of claim 1-7, it is characterized in that described grain comprises corn, wheat, paddy rice.
9. according to each described method of claim 1-7, it is characterized in that described detection mould comprises aspergillus niger, fusarium moniliforme, penicillium oxalicum.
10. the application of each described method of claim 1-9 in the grain storage mould detects.
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| CN107384756A (en) * | 2017-08-01 | 2017-11-24 | 云南中烟工业有限责任公司 | A kind of collection device and method of essence spice for cigarette contaminating microorganisms |
| 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 |
| CN109632734A (en) * | 2018-11-30 | 2019-04-16 | 张丽英 | A method of utilizing Aspergillus flavus in ATP bioluminescence reaction detection food |
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| CN107384756A (en) * | 2017-08-01 | 2017-11-24 | 云南中烟工业有限责任公司 | A kind of collection device and method of essence spice for cigarette contaminating microorganisms |
| CN107384756B (en) * | 2017-08-01 | 2023-09-15 | 云南中烟工业有限责任公司 | Device and method for collecting tobacco essence and spice polluted microorganisms |
| 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 |
| CN109632734A (en) * | 2018-11-30 | 2019-04-16 | 张丽英 | A method of utilizing Aspergillus flavus in ATP bioluminescence reaction detection food |
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