CN1091804C - Microbiological test method and reagents - Google Patents

Abstract

The present invention relates to a method for detecting microbes and/or the existence of substances in microbe cells and/or the content of the substances in a sample. The present invention comprises that the content of adenylate kinase in the sample is detected by means of the capacity of the adenylate kinase that adenosine diphosphate (ADP) is converted into adenosine triphosphate (ATP) when added magnesium ions exist in the adenylate kinase; the detection is associated with the microbes and/or the existence of the substances in the microbe cells and/or the content of the substances. The method has higher sensitivity than the existing luciferase/luciferin detecting method. Reagents comprise purified ADP and luciferase without adenylate kinase, and the purified ADP, the luciferase, a reagent detecting box comprising the reagents and a device used for automatically operating the method are provided together.

Description

Microorganism detection method and reagent

The present invention relates to detect and measure method of microorganism, be used for the reagent and the detection kit of this method, comprise the main agents of implementing present method in this test kit.

All organisms of living all utilize Triphosaden (ATP) as chemical energy source, and have known to react with Luci/luciferin that ATP drives and measured ATP.The light that this enzymatic reaction produces can be used photometric determination, and it is associated with the ATP amount that exists.People just know that ATP (sees: the luminous editors such as fast method (ATP LuminescenceRapid Methods in Microbiology) (1989) Stanley of ATP in the microbiology as the purposes of microbe quantity figureofmerit since mid-term nineteen sixties; Blackwell Science Press, London, 1-10 page or leaf), its major advantage is quick and sensitive.Use the measuring method of this form, simple sample can approximately carry out assay determination in the several minutes, and for complex sample, also only use takes half hour usually, and the detection sensitivity that is provided can be low to moderate 10 ^-12The ATP of M.But, need still to provide more that the sensitive method is used to measure microorganism or their inclusion, and keep the quick, easy of working method.

The inventor has been found that by measuring target and turns to the enzyme, particularly Myokinase that produces ATP from ATP that speed and sensitivity based on the ATP method might be significantly increased.Myokinase is used for adenosine diphosphate (ADP) (ADP) is transformed into the enzyme of Triphosaden (ATP) by all organisms.With this kind of enzyme, rather than make target, and, can make the sensitivity of measuring mark Myokinase in the cell be reduced to 10 at least by with the preferred method of the present invention, reagent and test kit with ATP ^-20Mole.

Known that available Luci/luciferin system measures Myokinase and (sees: Brolin etc., biological chemistry and bio-physical method magazine, 1, (1979) 163-169, with Shutenko etc., Biotekhnologiya, No 4. (1988) 542-547), be used to detect the active of this kind of enzyme and some Mammals and plant tissue studied (for example see Rodionova etc., Fiziologiya Rastenii (1978), 25,4, P731-734).But also nobody proposes this mensuration system is used for detecting and measuring microorganism, and the people of research this kind of enzyme itself also do not understand the superiority of doing like this, and promptly this method can provide higher sensitivity.

Though the amount of Myokinase is littler than ADP or ATP, but with its biological markers as microorganism, measure its existence by the ATP of its generation, to obtain higher sensitivity, typically can obtain 400,000 times amplification, that is to say, in time, every mole of enzyme can make 400,000 moles of ADP be transformed into ATP at 10 minutes incubations.Like this, by measuring the substrate or the product of this kind of enzyme catalyzed reaction, estimation can provide and be low to moderate 10 ^-20The detection sensitivity of mole.

Applicant's common unsettled PCT application WO 94/17202 relates to the general method that the ability that per sample ADP is transformed into ATP is come microorganism in the working sample, and the existence of this ability with microorganism or its intracellular matter is associated.This patent application illustration certain methods, wherein magnesium ion is that the reaction of two molecule ADP and each adenylate kinase 3 enzyme active sites is necessary, but adds magnesium ion with the reagent form in the method, but by being present in the Mg in the bacterial cell ²⁺Provide magnesium ion with impurity component as other reagent.In the embodiment that uses this technology, it approximately is 10 that the cell quantity of being measured is proved to be ², more rational result is 10 on the statistics ³Or it is more; And can obtain linear relationship between luminous counting and the cell quantity.

The present invention relates to a kind of improved technology, in that transformation reaction provides under the reaction conditions of magnesium ion to ADP, mode with the best is measured the activity of Myokinase, and wherein used reagent is handled, and removes Myokinase, make reagent reach higher degree, whereby, can determined microbe population be about tens in per 200 μ l samples, rather than hundreds of be individual, and the reading of linear relationship might be reduced to 10 cells between the light that cell and ATP produce.

A first aspect of the present invention has provided microorganism and/or the existence of its intracellular matter and/or the method for content in a kind of test sample, this method is characterised in that, the amount of Myokinase is measured by following method in the sample: sample is mixed with adenosine diphosphate (ADP) (ADP), determine amount by the Triphosaden (ATP) of sample ADP generation, and the existence of the amount of the ATP that will produce like this and Myokinase/or content be associated, and then be associated with the existence or the content of microorganism and/or its intracellular matter, wherein ADP is transformed into ATP and carries out under the condition that magnesium ion exists, and the volumetric molar concentration of this magnesium ion is enough to allow that ADP is transformed into ATP to greatest extent.The preferred content of magnesium ion is enough to provide 1 mole of magnesium to 1 mole of ADP, thereby all ADP molecules can both be associated with at least one magnesium ion.

In this preferred embodiment on the one hand of the present invention, the sample that provides is aqueous suspensions or solution form, and come Myokinase in the working sample by sample being added ADP and magnesium ion, and then microorganism and/or its intracellular matter measured, with this understanding, all Myokinases that exist in the sample all will participate in ADP is transformed into ATP, to preset time of sample incubation to promote this transition process, add Luci and luciferin reagent, the luminous quantity of working sample, and make it to link with the existence and the content of Myokinase.

The amount of that added and ADP sample mix, preferably be enough in mixture, provide ADP concentration, more preferably surpass 0.01mM, and most preferably surpass 0.08mM above 0.005mM, in the mixture of conversion step, particularly preferred ADP amount is about 0.1mM.

Contain the magnesium ion remover in use, for example under the situation as reagent such as the EDTA of sequestrant/sequestering agent and phosphate buffered saline buffers, should be realized that, for ADP being provided enough magnesium ions, make it to reach the transformation of the best, excessive magnesium ion preferably is provided.Be transformed in the process of ATP at ADP, for above-mentioned preferred ADP concentration, in suspension or the solution preferred magnesium ion concentration be 1mM or more than, more preferably 5mM or more than, and most preferably 10mM or more than.Magnesium ion can provide with the form of any magnesium salts, preferably magnesium acetate.

A preferred scheme of the present invention is when incubation begins luciferin/Luci luminescence assays reagent to be joined in the sample, preferably adds as single agents with ADP and magnesium ion source.Luci preferably should with the extraction agent separate storage.

In the solution of the present invention, when the transition process that is transformed into ATP at ADP in this way begins, just comprised all reagent, and/or as an independent step, continue the photometer counting and measure after adding luciferin/Luci, magnesium ion can provide by luciferin/Luci reagent.But,, be necessary to determine the content of magnesium ion by pilot study or calculating because magnesium ion can combine with EDTA and phosphoric acid salt.Those skilled in the art can recognize, join the suitableeest magnesium salts amount in given ADP, sample and luciferin/luciferin enzyme mixture, can contain the known quantity bacterium with one, sample as intestinal bacteria (E.coli), experiment by routine easily determines, and can obtain maximum signal like this.Following Fig. 3 is given in the index of the optimal dose that joins the magnesium acetate in the mixture in following examples.

Because Mg ²⁺Ion can promote ADP consumption because the pollutent Myokinase exists, so preferably before use, they should not be kept among the solution jointly; Sequestrant such as EDTA can be included among the ADP, to stop this process.Magnesium ion and ADP can preferably mix before facing use, perhaps mixed in the ADP conversion step.When reagent combines preservation, preferably make them remain on the lyophilize state, be transformed into ATP prematurely to avoid ADP.

As mentioned above, though available other measuring method, ATP preferably measures with luciferin/luciferin enzyme system, but it can provide the optics measured signal of ATP content in the indication sample.Those skilled in the art knows luciferin/luciferin zymin and the method that is used to measure ATP, and they are (for example the seeing Brolin etc.) that can buy.Typical formulation comprises for example Luci of 0.1-10mg/l, 15-1000 μ mol/l D-luciferin, and following reagent MgCl for example ₂(2.5-25mmole), the damping fluid of EDTA, BSA and pH7 (seeing for example EP 054676).

For the single agents in the adenylate kinase 3 enzyme assay described herein, it all is the suitableeest that its pH preferably should be adjusted to two kinds of enzymes, and this is for when ADP is transformed into ATP, can continue to carry out a kind of compromise proposal that luminous counting is measured.This pH can determine by the experiment of routine with the sample of a known bacterium number.

Sample, ADP and magnesium ion source can be mixed in any damping fluid that is suitable for the Myokinase reaction pH value can be provided; Do not need other reagent.Therefore, can provide the damping fluid of pH5.5-8.5 to use, and best pH scope is between pH6-7, preferably pH6.5.The example of the damping fluid that is suitable for comprises Tris damping fluid and phosphoric acid buffer.Optimal way is sample is collected in sort buffer liquid and/or to be diluted, and gets ready for implementing mensuration of the present invention.

The same with other amplification assay methods, the sensitivity of adenylate kinase 3 enzyme assay of the present invention also is subjected to the restriction of reagent purity.In this mensuration, the pollutent that material impact is arranged is ATP in the ADP substrate and the Myokinase in the luciferin zymin.In order to be used for the sensitive microbiological method, particularly when these microorganisms may be deleterious, and need measure under micro-situation the time, for the material of reaction with it in this assay method, the purity of every kind of reagent all must be high as much as possible.

For first problem, preferably use highly purified commodity ADP (purity＞99.5%), be further purified processing with column chromatography before using.It is necessary doing like this, even all may cause high background reading because a spot of ATP pollutes.For example, available diethylaminoethyl cellulose chromatography column is also made elutriant with 0.02mM hydrochloric acid.Compare with ADP, ATP is gone out by wash-out from chromatography column slowlyer, reaches the degree that the two is separated basically.Use the combination of other chromatography media and elutriant also can reach similar effects, for example available Nucleosil (registered trademark, RTM) column filling (can be from Technicol, Stockport Cheshire UK obtains), as Nucleosil3 (RTM) and Nucleosil 5 (RTM), with pH6 contain 5mM sulfuric acid tetrabutylammonium with 77: the 0.06M KH of the ratio of 23v/v preparation ₂PO ₄: methyl alcohol is made elutriant, and (HPLC) separates by high performance liquid chromatography.It is standby that collection has the fraction of high ADP/ATP ratio, and its purity can be measured by the noclilucence of luciferin/Luci reagent mediation, measures ADP content after the Myokinase effect, measures the ATP pollutant load there not being Myokinase to do the time spent.

Can also be with preferred Econopaq Q (RTM) reinforcing yin essence ion-exchange gel post (Biorad-RTM), with 20mM potassiumphosphate balance under pH4.6, make the KPi gradient elution, concentration is to 400mM, find that ADP is firmly kept (strongly retained), can be used as a successive peak and gone out by wash-out, ATP is gone out by wash-out subsequently.By this method, can obtain containing on the ATP molecular fraction and be limited to 2 * 10 ^-8ADP.The purest ADP that the applicant knows from document is 0.001% (seeing above-mentioned Shutenko etc.), and the ADP that provides in the inventive method, contains molecular fraction less than 0.001 ATP, and more preferably molecular fraction is less than 2 * 10 ^-8Or still less.

Another method of removing ATP from substrate A DP is to use the enzyme of energy specificity degraded ATP, as Luci or apyrase.These enzymes can be used to be further purified the ADP of chromatography purification, perhaps by another kind of method, the ADP that crosses through enzyme purification can be handled with column chromatography purification again.Should be noted that apyrase also is a kind of ADP enzyme, but because it has higher activity to ATP, and the concentration of ADP is very high, does not therefore have big problem.

For second problem, as the Myokinase of a kind of " house keeper (housekeeping) " enzyme, in fact be present in all organisms basically, therefore also be present in the luciferin zymin usually.Though may be the pollution of trace, because the target of measuring is exactly the low-down Myokinase of concentration in the sample, so its existence in Luci may be a limiting factor.In fact the applicant measure (definition this kind of enzyme 1 activity unit (U) be: at 20 ℃, pH7.8 exists 0.5mM ADP and 4.5mM Mg ²⁺The time, per minute is transformed into 1 μ molADP the enzyme amount of 1 μ mol ATP), commercially available Luci may contain 10 ^-7U/ml or higher adenylate kinase 3 enzymic activity, and in its substrate luciferin if any, content is also very low.Therefore, need usually to make the Luci stable reagentization with stablizer, normally a kind of protein of stablizer, as bovine serum albumin (BSA), this commercial preparation that the applicant measured all has significant adenylate kinase 3 enzymic activity.

The molecular weight of Luci and Myokinase is significantly different, is respectively 61kD and 21kD.And Luci is a kind of membrane bound protein, be hydrophobic relatively therefore, and Myokinase is a lyoenzyme.So just can be by for example molecular exclusion chromatography method, the reversed phase chromatography method is perhaps used this two kinds of methods simultaneously, removes Myokinase from the luciferin zymin.On the other hand, perhaps in addition, Myokinase is polluted the problem of Luci, can also avoid the Myokinase of any like this pollution generation remarkable influence that all have no time by adding bioluminescent reagents (Luci and luciferin) before visiting is surveyed or when just having begun to detect.

The proper method of purifying Luci is to carry out fractional separation by column chromatography, for example the gel of available low porosity such as dextran G-25 (RTM) (Sephadex G-25) (see Nielsen and Rasmussen, Acta Chemica Scandinavica, 22 (1968) P1757-1762); Can also use Sephadex (RTM) post and agarose (Sepharose successively, RTM) post (as Blue Sepharose) chromatography, and/or come purifying (to see Devine etc. by the SDS electrophoresis, biological chemistry and Acta Biophysica Sinica 1172 (1993) 121-132), perhaps enzyme is worn out (aging) for some time therein by improving envrionment temperature.

In order from reagent such as bovine serum albumin, to remove the adenylate kinase 3 enzymic activity, can use column chromatography equally.Proved that in this respect effective another kind of treatment process is that BSA is done chemical treatment, the ability of handling its stable Luci of back still keeps, but the adenylate kinase 3 enzymic activity reduces or eliminated fully.Any conventional chemical facture that is used for eliminating the protein enzymic activity can similarly be used for this purpose.On the other hand, the nonprotein luciferase stabiliser, as glycerine, can replenishing or substitute as BSA.

For example, the applicant determines after measured, only by make heat treated under acidity or alkaline pH, just can make adenylate kinase 3 enzymic activity among the commercially available BSA be reduced to that it is former activated below 2% or lower.Appropriately and effectively handle is that under pH5.6 or pH10,50 ℃ to BSA heating 24 hours.Another source of no Myokinase BSA is chemically treated acetylize BSA reagent, can obtain from Sigma and BDH.Those skilled in the art will recognize, chemically treated BSA also is suitable for other method.

Act on mutually in order to make all Myokinases relevant measure reagent with ADP of the present invention, magnesium ion and Luci/luciferin with the target microorganism, be necessary microbial lytic, make it the effect that discharges intracellular matter or make it to be subject to reagent.This cracking is handled and can be realized with mechanical means such as ultrasonic generator, also can use the osmotic pressure ballistic method, randomly in conjunction with the cold punching striking, perhaps uses N,O-Diacetylmuramidase reagent, or is to use stain remover to realize more easily.This stain remover can be bought and obtain, and is commonly called " extraction agent (extractant) ".Typical extraction agent comprises general cationic detergent such as CTAB (cetyl trimethylammonium bromide), and patented product such as Biotrace (RTM) XM extraction agent (can be from Biotrace, Bridgend, UK obtains), CelcisUK positively charged ion extraction agent and Lumac NRM (RTM) (Nucleotide releasing agent, can be from LumacBV, Holland obtains).When using CTAB, suitable preparation comprises the CTAB aqueous solution of 0.01-1%, and is for example available 0.2%, but those skilled in the art also can use other concentration.

Therefore, ADP and Luci/luciferin reagent are joined suspect in the testing sample that contains microorganism before, preferably, its cell content can be contacted with luminescence assays reagent by using cracking agent with microbial lytic.If require differentiation target cell and other as the fungal spore cell, can make two groups of mensuration simultaneously, handle with nonionic detergent for one group, it can only those spores of cracking and multicellular animals " body " cell (for example Triton TX-100 (RTM)), another group is handled with above-mentioned cationic detergent " extraction agent ", can make all cells cracking.If between working cycle, add the ATP enzyme with detergent-treatment/add Luci/measure, as apyrase, these two groups mensuration can be carried out on same sample, a circulation is handled with nonionic detergent in the round-robin first step, another is handled with cationic detergent, and filtration step is arranged between the two.

Know, extraction agent is importantly (to see for example Simpson etc. (1991) to the effect of Luci/luciferin system, J.Biolumin Chemilumin 6 (2) PP 97-106), known cationic detergent can make reaction strengthen, but can cause Luci inactivation gradually, anionic detergent meeting inhibited reaction, and non-ionic detergent and zwitterionic detergent can promote reaction widely.Discovery with 0.15% cationic detergent and 0.25% tertiary diamine tensio-active agent (from Celcis, Cambridge, the UK acquisition) mixture, it is gratifying being used for this purpose, but, those skilled in the art can find other " extraction agent " undoubtedly, when making it to coexist as same solution, forms Myokinase and Luci the suitableeest active mixing.

After all basic steps are finished, be that ADP is transformed into ATP, after Luci acts on luciferin, the light that sends from mixture can be measured by whole samples being retained in the photometer pipe in the photodetector for example, adding Luci and luciferin, or other can promote the reagent of this basic step to measure immediately or simultaneously afterwards.

Second aspect present invention provides a detection kit, and it comprises the needed main agents of the inventive method, i.e. adenosine diphosphate (ADP), a kind of magnesium ion source and preferred Luci and luciferin.This test kit preferably should comprise all these reagent, and Luci and luciferin be to provide as independent reagent solution, also has the stain remover that is suitable for cracking target cell to be measured in the test kit.Only need cationic detergent when being generally used for detecting microorganism, if and fungal spore and somatocyte may be preponderated, in order to measure their quantity, can also comprise nonionic detergent, this test kit is an independent packaging form preferably, have about how operating the specification sheets of the inventive method, reagent is provided in the container, and its concentration is suitable for directly using or the use of dilution back.

If before ADP begins to be transformed into ATP, add magnesium ion, so, it may be suitable adding magnesium ion with Luci/luciferin reagent, but, in this case, magnesium ion should be excessive, surpasses to be incorporated into EDTA or phosphatic magnesium ion quantity in reagent, and should be not only suitable Myokinase but also the optimum quantity that is fit to the luciferin enzyme require.When being used to measure microorganism, magnesium ion preferably adds with sample collection or dilution buffer liquid, but for special application, preferably adopts other mode.Most convenient be that magnesium ion adds with sample collection or dilution buffer liquid, ADP adds with stain remover and surfactivity extraction agent, also can randomly add stablizer such as EDTA, Luci and luciferin add simultaneously, and a kind of detection kit of three reagent so just is provided.Perhaps by another kind of method, can adopt cryodesiccated single agents form that these reagent is provided, they can not interact before using and cause degraded as ADP like this.

The preferred detection kit of the present invention comprises that purity is higher than 99.999% ADP, and contain BSA's, and there is not the active Luci of Myokinase/luciferin reagent basically, on the other hand, the ratio of the amount of the Luci/luciferin that is adopted, should make the luciferin endonuclease capable act on the luciferin substrate fully apace, make after initial luminous end, all Lucis can both combine with adenosine aldehyde kinases product A TP, this ratio and/or their relative concentration have dated in the working instructions of test kit, thus, the microorganism that produces Myokinase will be by kinetic reaction fast, and pollutent ATP is instructed to out by luminous.

Preferred purified reagent can obtain by above-mentioned method.Be noted that the adenylate kinase 3 enzymic activity in the Luci can also eliminate by the time of Luci being placed some months or several years.

Now will be only according to following non-limiting example and accompanying drawing, by example method of the present invention, instrument, reagent and test kit are described.Can further visualize other embodiments of the present invention according to these for those skilled in the art.

Accompanying drawing:

Fig. 1: with the improved assay method of the present invention, intestinal bacteria (E.coli) in the 200 μ l samples are measured, incubation adds luciferin/Luci after 1 minute and 5 minutes respectively, with the logarithm of photometer detection signal the logarithm of intestinal bacteria bacterium number is made curve.

Fig. 2: the logarithm with the photometer detection signal when not having magnesium ion is made curve to the logarithm of Bacillus coli cells number.

Fig. 3: show the influence of magnesium ion concentration to the photometer detection signal, signal is when pH7.5 and pH8.0, measures from the Pseudomonas aeruginosa (P.aeruginosa) of fixed qty, shows that signal has strengthened 10 times when not adding magnesium ion.

Embodiment 1: the preparation of the Adenosine diphosphate glycoside reagent of purifying

Be further purified high purity (＞99.95%) commodity ADP (Sigma) with liquid phase chromatography, use 5ml Econopac Q extraction column (cartridge) (RTM) (Biorad-RTM), with 20mMpH4.6 potassiumphosphate balance, the ADP (2.1mg) of the 5ml 1mM that packs into.Carry out the KPi gradient elution, concentration is to 400mM, and ADP firmly is retained on the post, and can be gone out by wash-out as a peak at about 340mMKPi.A pump (5ml/ minute) and a gradient mixer are installed in this system, form 50 to 1M KPi gradient in the 200ml total amount, are one with 5ml and collect fraction.ADP is gone out by wash-out as a sharp peak between fraction 12 and 17, begins to occur ATP at the terminal point of this gradient, goes out residual ATP at [KPi] concentration to wash-out between the 1M.The purest ADP fraction that obtains from post only contains molecular fraction and is less than 2 * 10 ^-8ATP.

Embodiment 2: the preparation of the Luci reagent of no Myokinase

(Biotrace HM RTM), eliminates wherein adenylate kinase 3 enzymic activity by burin-in process, comprises with drying regime and placing 12 months, wherein places several months down at high ambient temperature (about 30 ℃) to luciferin/Luci reagent of buying.

Embodiment 3: the another kind of preparation method of no kinases Luci reagent

Press the method for Devine etc. (1993), the Luci of buying with the column chromatography purification that uses above-mentioned Blue Sepharose (RTM) post.

Embodiment 4: the preparation of the BSA of no Myokinase

(RIA Grad, Cat.No.A-7888) BSA are mixed with the solution of 1% (weight/volume) in the 200ml sterilized water, making its initial pH is 5.6 with Sigma Fraction V.Get the BSA sample of two 50ml, place respectively in the Duran bottle of 100ml, remaining BSA solution is adjusted to pH10 with 5M NaOH, gets 50ml respectively and places in other two Duran bottles.Add thiomersal(ate), making its final concentration is 0.02%, to stop microorganism growth as sanitas, again with this bottle 37 ℃ or 50 ℃ of following incubations 24 hours, properly use then 5M HCI or 5MNaOH transfer to 7.6 with the pH of each bottle.The active measuring method of Myokinase is as follows: the BSA sample that 100 μ l are as above prepared mixes with 100 μ l 30mM magnesium acetate solution, this mixture is placed in the photometric 35ml photometer pipe, add ADP solution and 100 μ l luciferin/Luci reagent (Celcis that 100 μ l prepare in embodiment 1, Cambridge UK), this reagent is by having carried out going the adenylate kinase 3 enzymic activity to handle with column chromatography and the chemically treated BSA of adding, after the delay in 5 seconds, can detect accumulative total luminous more than 10 seconds and be stored in the computer, carry out 10 successive readings in 10 second altogether measuring the speed that ATP produces, and carry out replication.To 4 replications of luminous work from the ATP aqueous solution 5 μ l (91 fly mole) of 10ng/ml, calculate: the average signal that whenever flies mole is 2950.

The result: the BSA sample is still limpid behind 37 ℃ of incubations, then generates precipitation behind 50 ℃ of incubations, and is slight in the pH10 precipitation, very serious in the pH5.6 precipitation.PH10 and 50 ℃ slight variable color is arranged.The adenylate kinase 3 enzymic activity that remains in these samples is as shown in table 1, with the photometer readings signify of per minute.

Gentle more deactivation form is used in suggestion, uses the long timed interval, if perhaps plan long storage time, BSA should be frozen drying immediately, because at pH10, only will can not use owing to the increase of discoloration after 2 weeks under 50 ℃.This variation can not take place at 37 ℃ of following samples, therefore, this just provides a chance preferably, can reduce stable no Myokinase BSA by increasing the incubation time.Biotrace HM (RTM) reagent has been lost activity with drying regime after 40 ℃ are deposited, this fact table is understood this possibility.

Table 1:d[ATP]/dt processing counting t5-15 t95-105 difference (fm.sec -1)
	9,350 41,727 23,207 7.1 9,845 26041 37/5.6 (mean value) 11,896 32,945 37,/10 7,192 30,602 17,943 5.5 5,047 20,557 4,469 19,377 50/5.6 606 1,191 595 0.18 343 948 50,/10 460 1,014 500 0.15 342 847 314 754

Embodiment 5: have the preparation of luciferin/Luci reagent of BSA

The commodity preparation of luciferin/Luci contains B5A usually; with chemically treated BSA among the top embodiment 4 or acetylize-BSA (as buying) of buying from BDH or Sigma; press normal mixed with the Luci of no Myokinase; and mix some other standard C elcis reagent, measure in volume (i.e. 300 μ l) adenylate kinase 3 enzymic activity less than 10 thereby provide a kind of at it ^-9The Celcis LDR luciferin/Luci luminescence reagent of unit (U).

Embodiment 6: detection kit of the present invention

The invention provides the detection kit of forming by following part:

(i) container that the 15mM magnesium acetate solution that is used for sample collection or dilution is housed;

(ii) the container that purified ADP solution is housed is (for ATP, the purity of ADP＞99.99999998% in the solution), solution prepares as described in example 1 above, at potassiumphosphate (7.5mM, pH6.5) in the damping fluid, concentration is 0.3mM, and the mixed extraction agent that contains 0.2mM EDTA and be made up of 0.15% cationic detergent and 0.25% tertiary diamine tensio-active agent.

(iii) one be equipped with luciferin/Luci LDR (Celcis, Cambridge, the UK) container of bioluminescent reagents, adenylate kinase 3 in reagent alcohol is active in 10 ^-8U/100 μ l.

In kit package, can comprise randomly that also is equipped with a nonionic detergent solution (TritonX-100 (TRM), 0.2% or Equivalent) container, and/or container that ATP enzyme such as apyrase are housed, this kind of enzyme be used to decompose owing to nonionic detergent to the ATP that sample effect discharges, make it to be applicable to by adding cationic detergent and measure again.

Embodiment 7: measure known quantity E.coli with method of the present invention

Make original bacteria liquid (stock) with E.coli (intestinal bacteria) broth culture of cultivating for 1 week, contain about 2.2 * 10 in the solution of the phosphate buffered of its per 200 μ l pH7.4 ⁷Individual microorganism, ((i) among the embodiment 6 does 10 serial dilutions, obtains containing 10 in per 200 μ l samples with the sample collection/dilution reagent that contains magnesium ion ⁷Series of samples to 0.1 microorganism.

200 μ l samples are added in the photometer pipe of 3.5ml, add 100 μ l ADP/ extraction agents (among the embodiment 6 (ii)), amount of the mixture is 300 μ l, and at room temperature incubation is 1 minute or 5 minutes.After finishing, incubation adds the improved Celcis LDR of 100 μ l bioluminescent reagents (among the embodiment 6 (iii)) immediately, with Biotrace M3 (RTM) photometer, measure earlier at interval luminous in first 10 seconds, per then 10 seconds measuring spaces once, up to 1 minute, measure luminous increase in the mode of accumulative total.Deduct the start signal value from last reading, the signal measuring result who obtains is with the readings signify of per minute.

With reference to figure 1 usefulness of the inventive method as can be seen, as seen from the figure, after the sample that contains 10 and 10 above bacteriums for every duplicate samples and the ADP mixing incubation 5 minutes, and after every duplicate samples sample of containing 100 and 100 above bacteriums and the ADP mixing incubation 1 minute, all can obtain the linear response of coincidence statistics between the quantity of E.coli and sample are luminous, the determination limit of both of these case all is about 10 bacteriums.Present method is compared with the method for WO 94/17202, seem very superior, the latter only had 26cpm (count per minute) difference in 1 minute afterwards to the sample incubation that contains 100 bacteriums, the sample that contains 1000 bacteriums only there is the 67cpm difference, only under the situation of 1000 or 1000 above cells, just can obtains linear response.By contrast, by method of the present invention, every part of sample that contains 1000 cells can obtain the signal increased value of several thousand cpm in 1 minute behind the incubation.

Be to be appreciated that, for the sample of a unknown microbe population is measured with present method, can be shown in Fig. 1 and Fig. 2 (for example with logarithmic value), dose known amounts with microorganism is mapped to luminous counting, make a calibration curve,, obtain counting from microorganism (the comprising the zero microorganism) sample that contains unknown number with same mensuration scheme, then can estimate microbial numbers in the sample, reading value identical on this quantity and the curve is corresponding.

The technology people of this area should be realized that, is present in a certain peculiar microorganism, as the amount of the Myokinase in the bacterium, may be with other microorganism different.For example, according to its size, yeast contains more Myokinase than bacterium, and in fact, available this method is measured single yeast.Therefore, for a certain given microorganism, may need to make a specific calibration curve, and for the same microorganism of different states, as the microorganism that is weakened, the microorganism that is in pH or oxygen Stress, also may need to make specific calibration curve.But another advantage that the inventive method is better than existing method based on ATP is, the coexist ATP content of the alterable height that is consumed in the cellular metabolism of the content of Myokinase is compared, and with the quantity of cell relation more closely arranged.

Claims (38)

1. microorganism and/or the existence of its intracellular matter and/or the method for content in the working sample, this method is characterised in that, the content of Myokinase is measured by following method in the sample: make sample and adenosine diphosphate (ADP) (ADP) and the magnesium ion source that adds as reagent mixes, the amount of the Triphosaden (ATP) that test sample is generated by ADP, and the amount of the ATP that produces is associated with the existence and/or the content of microorganism and/or its intracellular matter.

2. the process of claim 1 wherein that magnesium ion adds with the volumetric molar concentration that is enough to make ADP change ATP to greatest extent into.

3. the process of claim 1 wherein that the content of the magnesium ion that adds is enough to provide 1 mole of magnesium to 1 mole of ADP, all may be associated all ADP molecules with at least one magnesium ion.

4. the method for claim 1, wherein sample is that form with aqueous suspensions or solution provides, the mensuration of microorganism and/or its intracellular matter is carried out as follows: add ADP and magnesium ion to sample, used condition makes the Myokinase of any existence all will make ATP be transformed into ATP, to preset time of sample incubation, to promote this transition process, add luciferin enzyme and luciferin reagent, mensuration is from the luminous quantity of sample, and makes it to be associated with the existence and the content of microorganism and/or its intracellular matter.

5. the amount with the ADP of sample mix of the process of claim 1 wherein is enough to provide the ADP concentration above 0.005mM in mixture.

6. the method for claim 5, wherein ADP surpasses 0.08mM.

7. the method for claim 5, wherein the concentration of ADP is about 0.1mM.

8. the process of claim 1 wherein to be transformed in the process of ATP that the concentration of magnesium ion in suspension or solution is 1mM or higher at ADP.

9. the method for claim 8, wherein the concentration of magnesium ion in suspension or solution is 10mM or higher.

10. the method for arbitrary claim in the claim 1, wherein magnesium ion is that form with magnesium acetate provides.

11. the method for arbitrary claim in the claim 4, wherein luciferin/luciferin enzyme luminescence reagent is when incubation begins, and adds in the sample as the single agents that has ADP and magnesium ion source.

12. the method for arbitrary claim in the claim 1, wherein magnesium ion source and ADP preserve with the exsiccant form or in independent solution, before facing use, or in the conversion step of ADP, their are mixed or make the aqueous solution.

13. the process of claim 1 wherein that magnesium ion source and sample are mixed before adding ADP.

14. the method for claim 13, wherein sample is to be collected in containing the solution of magnesium ion source or to dilute.

15. the process of claim 1 wherein that the process that ADP is transformed into ATP is to carry out under the condition of pH5.5-8.5.

16. the process of claim 1 wherein that the molecular fraction of the ATP that ADP contains is lower than 0.001%.

17. the method for claim 16, wherein ADP contains 2 * 10 ^-8Mole %ATP or still less.

18. the method for claim 16, wherein ADP was stored under the condition of sequestrant before using, and prematurely it was transformed into ATP to prevent the pollutent Myokinase.

19. the method for claim 4, wherein luciferin enzyme/luciferin reagent contains and is less than 10 ^-7The Myokinase of U/ml.

20. the method for claim 19, wherein luciferin enzyme/luciferin reagent contains through chemical treatment and has eliminated the active bovine serum albumin of its Myokinase.

21. the process of claim 1 wherein and their Myokinase is contacted the sample extraction agent processing of cracking microorganism cells with ADP with magnesium ion.

22. the method for claim 21, wherein cell is fungal spore or eukaryotic cell, and extraction agent contains nonionic detergent.

23. the method for claim 21 wherein will detect and/or quantitatively, extraction agent contains cationic detergent whole cells.

24. the method for claim 23, wherein extraction agent also comprises surfactant.

25. the method for claim 21, wherein cell is a bacterial cell, and wherein from being deducted the ATP that is discharged by nonionic detergent the ATP of cationic detergent and tensio-active agent release, residual content is associated with bacterial cell quantity.

26. one kind is detected with the method for arbitrary claim among the claim 1-25 and/or the detection kit of quantitative assay microorganism and/or its intracellular matter, it comprises ADP and magnesium ion source, and a kind of extraction agent, the microorganism Myokinase contacts with them and makes ADP change ATP into.

27. the detection kit of claim 26, it also comprises luciferin enzyme and the luciferin that exists with the bioluminescent reagents form, and they can be luminous when ATP exists.

28. the detection kit of claim 27, it comprises a kind of magnesium ion source as sample collection or diluting soln.

29. the detection kit of claim 28 is wherein collected or dilution buffer liquid comprises magnesium acetate.

30. the detection kit of arbitrary claim among the claim 27-29, wherein ADP and stain remover and/or tensio-active agent extraction agent exist jointly.

31. the detection kit of arbitrary claim among the claim 27-30, ADP wherein, magnesium ion source and bioluminescent reagents with three independently container provide.

32. the detection kit of arbitrary claim among the claim 27-30, whole reagent wherein provide with single freeze dried form.

33. the detection kit of arbitrary claim among the claim 26-32, wherein for ATP, the purity of ADP is higher than 99.999%.

34. the detection kit of claim 26, this test kit comprise a kind of adenylate kinase 3 enzymic activity less than 10 ^-7The bioluminescent reagents of U/ml.

35. the detection kit of claim 33, wherein bioluminescent reagents comprises through chemical treatment and has eliminated the active bovine serum albumin of Myokinase.

36. used reagent in the detection kit of arbitrary claim among the claim 26-35, wherein for ATP, the purity of the ADP that it is contained is higher than 99.999%.

37. the reagent of claim 36, it also comprises is enough to the sequestrant that antipollution Myokinase changes ADP into ATP.

38. the reagent of claim 37, wherein sequestrant comprises EDTA.

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