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

Microbiological Assays and Reagents

The present invention relates to a method for detecting and measuring microorganisms, a reagent for the method and a detection kit, which contains the main reagents for implementing the method.

All living organisms utilize adenosine triphosphate (ATP) as a chemical energy source, and it is known that ATP can be measured using the ATP-driven luciferase/luciferin reaction. The light produced by this enzymatic reaction can be measured photometrically and can be correlated to the amount of ATP present. The use of ATP as an indicator of microbial populations has been known since the mid-1960s (see: ATP Luminescence Rapid Methods in Microbiology (1989) Stanley et al. eds.; Blackwell Scientific Publishers, London, pp. 1-10), whose main advantages are speed and sensitivity. Using this form of assay, simple samples can be assayed in about a few minutes, and usually only half an hour for complex samples, providing detection sensitivities as low as 10 ^-12 M ATP. However, there is still a need to provide more sensitive methods for the determination of microorganisms or their contents, while maintaining a fast and simple method of operation.

The present inventors have found that by shifting the assay target from ATP to ATP-producing enzymes, in particular adenylate kinase, the speed and sensitivity of ATP-based methods can be significantly improved. Adenylate kinase is an enzyme used by all organisms to convert adenosine diphosphate (ADP) to adenosine triphosphate (ATP). By targeting this enzyme, rather than ATP, and by using the preferred methods, reagents and kits of the invention, the sensitivity of measuring the intracellular marker adenylate kinase can be reduced to at least ^10-20 molar.

It is known that the luciferase/luciferin system can be used to measure adenylate kinase (see: Brolin et al., Journal of Biochemical and Biophysical Methods, 1, (1979) 163-169, and Shutenko et al., Biotekhnologiya, No 4. (1988) 542-547), for assaying the activity of this enzyme and for studies in certain mammalian and plant tissues (see for example Rodionova et al., Fiziologiya Rastenii (1978), 25, 4, p. 731-734). However, no one had proposed the use of this assay system for the detection and assay of microorganisms, and those who had studied the enzyme itself had not yet understood the advantage of doing so, which would provide greater sensitivity.

Although adenylate kinase is present in smaller amounts than ADP or ATP, using it as a microbial biomarker to determine its presence through the ATP it produces will result in higher sensitivity, typically 400,000-fold Amplification, that is, conversion of 400,000 moles of ADP to ATP per mole of enzyme within a 10 minute incubation time. Thus, detection sensitivities as low as ^10-20 molar are estimated to be provided by assaying the substrate or product of this enzyme-catalyzed reaction.

Applicant's co-pending PCT application WO 94/17202 relates to a general method for assaying microorganisms in a sample based on their ability to convert ADP to ATP and correlating this ability with the presence of the microorganism or its intracellular material. This patent application exemplifies methods in which magnesium ions are necessary for the reaction of two molecules of ADP with each adenylate kinase active site, but in which the magnesium ions are not added as a reagent, but via Mg ²⁺ and as impurity components of other reagents to provide magnesium ions. In an example using this technique, the number of cells measured proved to be about 10 ² , with a statistically more reasonable result of 10 ³ or more; and a linear relationship between luminescence counts and cell number was obtained .

The present invention relates to an improved technique for assaying the activity of adenylate kinase in an optimal manner under reaction conditions that provide magnesium ions for the ADP conversion reaction, and for treating the reagents used therein to remove adenylate Kinase, to achieve a higher purity of the reagent, whereby the number of microorganisms that can be measured per 200 μl sample is on the order of tens rather than hundreds, and the readout of the linear relationship between cells and the light produced by ATP is possible down to 10 cells.

The first aspect of the present invention is to provide a method for detecting the presence and/or content of microorganisms and/or their intracellular substances in a sample, which is characterized in that the amount of adenylate kinase in the sample is determined by the following method Assayed: mixing a sample with adenosine diphosphate (ADP), determining the amount of adenosine triphosphate (ATP) produced by the sample ADP, and correlating the amount of ATP thus produced to the presence/or amount of adenylate kinase, thereby Associated with the presence or content of microorganisms and/or their intracellular material, wherein the conversion of ADP to ATP is carried out in the presence of magnesium ions in a molar concentration sufficient to allow maximum conversion of ADP to ATP. The preferred amount of magnesium ions is sufficient to provide 1 mole of magnesium for 1 mole of ADP, thereby enabling all ADP molecules to associate with at least one magnesium ion.

In a preferred embodiment of this aspect of the invention, the sample provided is in the form of an aqueous suspension or solution, and the adenylate kinase in the sample is determined by adding ADP and magnesium ions to the sample, and then to microorganisms and/or their In this condition, all adenylate kinases present in the sample will participate in the conversion of ADP to ATP. The sample is incubated for a predetermined time to promote this conversion process. Add luciferase and Fluorescein reagent, which measures the amount of luminescence in a sample and correlates it to the presence and amount of adenylate kinase.

The amount of ADP added mixed with the sample is preferably sufficient to provide an ADP concentration in the mixture in excess of 0.005 mM, more preferably in excess of 0.01 mM, and most preferably in excess of 0.08 mM, in the mixture of the transformation step, especially A preferred amount of ADP is about 0.1 mM.

In cases where reagents containing magnesium ion scavengers such as EDTA and phosphate buffers are used as chelating/sequestering agents, it should be recognized that in order to provide sufficient magnesium ions to ADP for optimal transformation, preferably by providing an excess of magnesium ions. During the conversion of ADP to ATP, the preferred concentration of magnesium ions in the suspension or solution is 1 mM or above, more preferably 5 mM or above, and most preferably 10 mM or above for the above-mentioned preferred ADP concentrations. Magnesium ions may be provided in the form of any magnesium salt, preferably magnesium acetate.

A preferred solution of the present invention is to add the luciferin/luciferase luminescence assay reagent to the sample at the beginning of incubation, preferably as a single reagent together with ADP and magnesium ion source. Luciferase should preferably be stored separately from the extractant.

In the protocol of the present invention, all reagents are included when the conversion of ADP to ATP in this way begins, and/or are continued as a separate step after the addition of luciferin/luciferase For photometric counting determination, magnesium ions can be provided by luciferin/luciferase reagents. However, since magnesium ions can combine with EDTA and phosphate, it is necessary to determine the content of magnesium ions through preliminary experiments or calculations. Those skilled in the art will recognize that the optimum amount of magnesium salt to be added to a given ADP, sample and luciferin/luciferase mixture can be determined using a known amount of bacteria, such as Escherichia coli (E. coli) samples, which are easily determined by routine experimentation, will give the largest signal. Figure 3 below gives an indication of the optimum amount of magnesium acetate added to the mixture in the following examples.

Since Mg ²⁺ ions can promote ADP consumption due to the presence of contaminant adenylate kinase, preferably they should not be co-kept in solution prior to use; chelating agents such as EDTA can be included in ADP to prevent this process. Magnesium ions and ADP may preferably be mixed just before use, or in the ADP conversion step. When the reagents are stored together, they are preferably kept in a lyophilized state to avoid premature conversion of ADP to ATP.

As noted above, ATP is preferably measured using the luciferin/luciferase system, which provides an optically detectable signal indicative of the amount of ATP in the sample, although other assay methods may be used. Luciferin/luciferase preparations and methods for assaying ATP are well known to those skilled in the art and commercially available (see, eg, Brolin et al.). A typical formulation contains e.g. 0.1-10 mg/l luciferase, 15-1000 μmol/l D-luciferin, and reagents such as _MgCl (2.5-25 mmole), EDTA, BSA and a buffer at pH 7 (see e.g. EP 054676).

For a single reagent in the adenylate kinase assay described here, the pH should preferably be adjusted to be optimal for both enzymes in order to allow for continuous luminescence counting while ADP is being converted to ATP A measure of compromise. The pH can be determined by routine experimentation with a sample of known bacterial count.

The sample, ADP, and source of magnesium ions can be mixed in any buffer that provides a pH suitable for the adenylate kinase reaction; no other reagents are required. Therefore, any buffer that can provide a pH of 5.5-8.5 can be used, and the optimum pH range is between pH 6-7, preferably pH 6.5. Examples of suitable buffers include Tris buffer and phosphate buffer. Most suitably, samples are collected and/or diluted in this buffer in preparation for carrying out the assays of the invention.

As with other scale-up assays, the sensitivity of the adenylate kinase assay of the present invention is limited by the purity of the reagents. Contaminants of importance in this assay are ATP in the ADP substrate and adenylate kinase in the luciferase preparation. For use in sensitive microbiological assays, especially when these microorganisms may be deleterious and need to be assayed in trace amounts, each reagent must be as pure as possible for the substances it reacts with in this assay high.

For the first problem, it is preferable to use high-purity commercial ADP (purity>99.5%), and use column chromatography for further purification before use. This is necessary because even small amounts of ATP contamination can cause high background readings. For example, a diethylaminoethylcellulose chromatography column using 0.02 mM hydrochloric acid as the eluent can be used. ATP is eluted from the column more slowly than ADP to such an extent that the two are essentially separated. Similar effects can also be achieved using other chromatographic media and eluent combinations, for example available Nucleosil (registered trademark, RTM) column packing (available from Technicol, Stockport Cheshire UK), such as Nucleosil 3 (RTM) and Nucleosil 5 (RTM ), using 0.06M KH ₂ PO ₄ :methanol prepared at a ratio of 77:23 v/v containing 5 mM tetrabutylammonium sulfate at pH 6 as the eluent, and separated by high performance liquid chromatography (HPLC). Fractions with a high ADP/ATP ratio are pooled for later use, and their purity can be determined by luciferin/luciferase reagent-mediated bioluminescence, ADP content is measured after adenylate kinase action, and in the absence of adenylate kinase The ATP pollutant content is determined during the action.

Can also use preferred Econopaq Q (RTM) strong anion exchange gel column (Biorad-RTM), balance at pH4.6 with 20mM potassium phosphate, do KPi gradient elution, concentration reaches 400mM, find that ADP is retained firmly ( strongly retained), can be eluted as a continuous peak, followed by ATP. In this way, ADP containing an upper limit of 2 x 10 ^-8 mole percent of ATP can be obtained. The applicant knows from the literature that the purest ADP is 0.001% (see Shutenko et al. above), and the ADP provided in the method of the present invention contains ATP in a mole percent of less than 0.001, more preferably in a mole percent of less than 2 x 10 ^-8 or less.

Another method for removing ATP from the substrate ADP is to use an enzyme that specifically degrades ATP, such as luciferase or apyrase. These enzymes can be used to further purify the chromatographically purified ADP, or alternatively, the enzyme-purified ADP can be further purified by column chromatography. It should be noted that apyrase is also an ADPase, but since it is more active towards ATP, and the concentration of ADP is very high, it is not a big problem.

With regard to the second question, adenylate kinase, essentially a "housekeeping" enzyme, is present in virtually all organisms, and therefore usually also in luciferase preparations. Although trace contamination is likely, its presence in luciferase may be a limiting factor since the assay targets adenylate kinase at very low concentrations in the sample. In fact, the applicant has measured (1 activity unit (U) of definition this enzyme is: at 20 ℃, pH7.8, when there is 0.5mM ADP and 4.5mM Mg ²⁺ , change 1 μ mol ADP into 1 μ mol per minute Enzyme amount of ATP), commercially available luciferase may contain 10 ^-7 U/ml or higher adenylate kinase activity, and the content, if any, in its substrate luciferin is also very low. Therefore, it is usually necessary to use a stabilizer to stabilize the luciferase reagent. The stabilizer is usually a protein, such as bovine serum albumin (BSA). nucleotide kinase activity.

The molecular weights of luciferase and adenylate kinase are significantly different, 61 kD and 21 kD, respectively. Furthermore, luciferase is a membrane-bound protein and thus relatively hydrophobic, whereas adenylate kinase is a soluble enzyme. Thus, adenylate kinase can be removed from the luciferase preparation by, for example, size exclusion chromatography, reversed phase chromatography, or both. On the other hand, or in addition to this, the problem of adenylate kinase contamination of luciferase can also be solved by adding bioluminescent reagents (luciferase and luciferin) just before the detection or when the detection is just started. Avoid so that any contaminating adenylate kinase does not have time to have a noticeable effect.

A suitable method of purifying luciferase is fractionation by column chromatography, e.g. using a low-porosity gel such as Sephadex G-25 (RTM) (Sephadex G-25) (see Nielsen and Rasmussen, Acta Chemica Scandinavica , 22(1968) P1757-1762); can also use Sephadex (RTM) column and Sepharose (Sepharose, RTM) column (such as Blue Sepharose) chromatography sequentially, and/or purify by SDS electrophoresis (see Devine et al., Bio Acta Chem. Biophys. 1172 (1993) 121-132), or aging the enzyme in it for a period of time by increasing the ambient temperature.

To remove adenylate kinase activity from reagents such as bovine serum albumin, column chromatography can likewise be used. Another treatment that has proven effective in this regard is the chemical treatment of BSA in which the ability to stabilize luciferase is retained but adenylate kinase activity is reduced or completely eliminated. Any conventional chemical treatment used to eliminate enzymatic activity in proteins can equally be used for this purpose. On the other hand, non-protein luciferase stabilizers, such as glycerol, can be used as a supplement or substitute for BSA.

For example, the applicant has determined through measurement that the adenylate kinase activity in commercially available BSA can be reduced to 2% or less of its original activity only by heat treatment at acidic or alkaline pH. . A suitable and effective treatment is heating BSA at 50°C for 24 hours at pH 5.6 or pH 10. Another source of adenylate kinase-free BSA is chemically processed acetylated BSA reagent, available from Sigma and BDH. Those skilled in the art will appreciate that BSA chemically treated by other means is also suitable.

In order to make all adenylate kinases related to target microorganisms interact with ADP, magnesium ions and luciferase/luciferin assay reagents of the present invention, it is necessary to lyse the microorganisms to release intracellular substances or make it susceptible to the action of the reagent. This lysis treatment can be achieved by mechanical means such as a sonicator, also by osmotic shock, optionally in combination with cold shock, or by using a lysozyme reagent, or more conveniently by using a detergent. Such detergents are commercially available and are commonly referred to as "extractants". Typical extractants include common cationic detergents such as CTAB (cetyltrimethylammonium bromide), as well as proprietary products such as Biotrace (RTM) XM extractant (available from Biotrace, Bridgend, UK), CelcisUK Cationic Extractant and Lumac NRM (RTM) (Nucleotide Release Agent, available from Lumac BV, Holland). When CTAB is used, suitable formulations include 0.01-1% CTAB in water, for example 0.2% may be used, although other concentrations may be used by those skilled in the art.

Therefore, prior to adding ADP and luciferase/luciferin reagents to a sample suspected of containing microorganisms, it is preferable to lyse the microorganisms by using a lysing agent to allow their cellular contents to come into contact with the luminescent assay reagents . If it is desired to differentiate target cells from other, e.g. fungal spore cells, two sets of assays can be performed simultaneously, one treated with a non-ionic detergent that lyses only those spores and the "body" cells of multicellular animals (e.g. Triton TX-100( RTM)), the other group was treated with the above-mentioned cationic detergent "extractant", which lysed all cells. If an ATPase, such as apyrase, is added between the detergent treatment/luciferase addition/assay cycle, both sets of assays will be performed on the same sample, at the end of the cycle. One cycle in one step is treated with a nonionic detergent and the other with a cationic detergent, with a filtration step in between.

Extractants are known to be important for the action of the luciferase/luciferin system (see e.g. Simpson et al. (1991), J. Biolumin Chemilumin 6(2) PP 97-106), and cationic detergents are known to be able to Enhance the reaction, but cause the gradual inactivation of luciferase, anionic detergents will inhibit the reaction, non-ionic detergents and zwitterionic detergents can broadly promote the reaction. A mixture of 0.15% cationic detergent and 0.25% tertiary diamine surfactant (obtained from Celcis, Cambridge, UK) was found satisfactory for this purpose, however, those skilled in the art will no doubt find other The "extractant", when allowed to coexist in the same solution, forms an optimal mix of adenylate kinase and luciferase activities.

After all the basic steps are complete, i.e. the conversion of ADP to ATP and the action of luciferase on luciferin, the light emitted from the mixture can be measured by keeping the whole sample in a luminometer tube e.g. inside a light detector , immediately or simultaneously with the addition of luciferase and luciferin, or other reagents that facilitate this basic step.

The second aspect of the present invention provides a detection kit, which comprises the main reagents required by the method of the present invention, namely adenosine diphosphate, a source of magnesium ions, and preferably luciferase and luciferin. The kit should preferably include all of these reagents, and the luciferase and luciferin are provided as separate reagent solutions with a detergent suitable for lysing the target cells to be tested. Usually only cationic detergents are required for the detection of microorganisms, and if fungal spores and somatic cells may predominate, non-ionic detergents may also be included in order to determine their numbers. The kit is preferably in a separate packaged form, Accompanied by instructions on how to perform the method of the invention, the reagents are provided in containers at concentrations suitable for use directly or after dilution.

If magnesium ions are to be added before the conversion of ADP to ATP begins, then it may be appropriate to add magnesium ions together with the luciferase/luciferin reagent, however, in this case the magnesium ions should be in excess The amount of magnesium ions bound to EDTA or phosphate should be the optimal amount for both adenylate kinase and luciferase. For the determination of microorganisms, magnesium ions are preferably added together with the sample collection or dilution buffer, but for special applications, other methods are preferred. Most conveniently, magnesium ions are added together with the sample collection or dilution buffer, ADP is added together with the detergent and surfactant extractant, and optionally a stabilizer such as EDTA, luciferase and luciferin are added together , thus providing a three-reagent detection kit. Alternatively, the reagents can be provided as lyophilized single reagents so that they do not interact to cause degradation, eg, of ADP, prior to use.

The preferred detection kit of the present invention comprises ADP with a purity higher than 99.999%, and a luciferase/luciferin reagent containing BSA and substantially free of adenylate kinase activity. On the other hand, the adopted luciferase The ratio of the amount of luciferase/luciferin should be such that the luciferase can act on the luciferin substrate quickly enough that after the initial luminescence is over, all the luciferase can bind to the adenosyl aldokinase product ATP, the ratio and/or their relative concentrations are indicated in the instruction manual of the kit, thus, the microorganism producing adenylate kinase will undergo a rapid kinetic reaction, while the pollutant ATP will be eliminated by luminescence. Indicate it.

Preferred purification reagents are obtainable by the methods described above. It should be noted that adenylate kinase activity in luciferase can also be abolished by leaving the luciferase over a period of months or years.

The methods, apparatus, reagents and kits of the invention will now be described by way of example only, with reference to the following non-limiting examples and figures. From these further embodiments it will be possible for those skilled in the art to conceive of other embodiments of the invention.

Attached picture:

Figure 1: Using the improved assay method of the present invention, Escherichia coli (E.coli) in a 200 μl sample is assayed, and luciferin/luciferase are added after incubation for 1 minute and 5 minutes respectively, and the signal is detected by a photometer Curve the logarithm of the logarithm of the number of E. coli bacteria.

Figure 2: The logarithm of the photometric detection signal is plotted against the logarithm of the E. coli cell number in the absence of magnesium ions.

Figure 3: Shows the effect of magnesium ion concentration on the photometric detection signal measured from a fixed number of Pseudomonas aeruginosa (P.aeruginosa) at pH 7.5 and pH 8.0, indicating that The signal was enhanced 10-fold with magnesium ions.

Embodiment 1: Preparation of purified adenosine diphosphate reagent

High-purity (>99.95%) commercial ADP (Sigma) was further purified by liquid chromatography with 5 ml Econopac Q extraction column (cartridge) (RTM) (Biorad-RTM), equilibrated with 20 mM pH 4.6 potassium phosphate, loaded into 5 ml 1 mM ADP (2.1mg). With KPi gradient elution up to 400 mM, ADP is firmly retained on the column and can be eluted as a peak at approximately 340 mM KPi. A pump (5ml/min) and a gradient mixer were installed on the system to form a gradient of 50 to 1M KPi in a total of 200ml, with 5ml as a collection fraction. ADP eluted as a sharp peak between fractions 12 and 17, ATP started to appear at the end of the gradient, and residual ATP eluted between [KPi] concentrations to 1M. The purest ADP fraction obtained from the column contained only less than 2 x 10 ^-8 mole percent ATP.

Example 2: Preparation of luciferase reagent without adenylate kinase

For the purchased luciferin/luciferase reagent (Biotrace HM, RTM), eliminate the adenylate kinase activity in it through aging treatment, including placing it in a dry state for 12 months, and keeping it in a high ambient temperature (about 30°C ) for several months.

Embodiment 3: Another preparation method of kinase-free luciferase reagent

According to the method of Devine et al. (1993), the purchased luciferase was purified by column chromatography using the above-mentioned Blue Sepharose (RTM) column.

Example 4: Preparation of BSA without adenylate kinase

Use Sigma Fraction V (RIA Grad, Cat.No.A-7888) BSA to prepare a 1% (weight/volume) solution in 200ml sterile water to make its initial pH 5.6. Take two 50ml BSA samples and place them in 100ml Duran bottles respectively. The remaining BSA solution is adjusted to pH 10 with 5M NaOH, and take 50ml and place them in the other two Duran bottles. Sodium ethylmercury thiosalicylate was added to a final concentration of 0.02% to act as a preservative to prevent microbial growth, and the bottle was incubated at 37°C or 50°C for 24 hours, and then appropriately washed with 5M HCI or 5M NaOH adjusted the pH of each bottle to 7.6. The assay method of adenylate kinase activity is as follows: 100 μ l of the BSA sample prepared above is mixed with 100 μ l of 30 mM magnesium acetate solution, this mixture is placed in the 35 ml photometer tube of luminometer, adds 100 μ l of the ADP solution prepared in Example 1 and 100 μl of luciferin/luciferase reagent (Celcis, Cambridge UK), which had been treated with deadenylate kinase activity by column chromatography and addition of chemically treated BSA, was available after a 5 second delay. Luminescence was detected and accumulated over 10 seconds and stored in a computer, a total of 10 consecutive 10 second readings were taken to determine the rate of ATP production, and replicates were performed. The luminescence from 5 μl (91 femtomoles) of 10 ng/ml ATP aqueous solution was measured repeatedly, and the average signal per femtomoles was calculated to be 2950.

Results: The BSA sample was still clear after incubation at 37°C, but precipitated after incubation at 50°C. The precipitation was slight at pH 10 and very serious at pH 5.6. Slight discoloration at pH 10 and 50°C. The adenylate kinase activity remaining in these samples is shown in Table 1, expressed as photometric readings per minute.

It is recommended to use the more mildly inactivated form, using a longer time interval, or if it is intended to be stored for a long time, the BSA should be freeze-dried immediately, as after only 2 weeks at pH 10, 50°C it will fail due to increased discoloration used. This change did not occur in the samples at 37°C, so this provided a good opportunity to reduce the stable adenylate kinase-free BSA by increasing the incubation time. The fact that the Biotrace HM (RTM) reagent lost activity after storage at 40°C in a dry state suggests this possibility. Table 1: d[ATP]/dt Treatment Count t5-15 t95-105 Difference (fm.sec ^-1 ) 37/5.6 9350 4177 23207 7.19845 26041 (average) 11896 3294537/10 7192 306060 17943 5.55047 20574469 1937750/5.6 606 1195 0.18343 94850 1014 500.15342 847314 847314 847314

Example 5: Preparation of luciferin/luciferase reagent with BSA

Commercial preparations of luciferin/luciferase typically contain B5A by combining chemically treated BSA in Example 4 above, or commercially available acetylated-BSA (such as from BDH or Sigma), with adenylate kinase-free Luciferase is mixed in normal proportions and spiked with some other standard Celcis reagents to provide a Celcis LDR luciferin with less than 10 ^-9 units (U) of adenylate kinase activity in its assay volume (i.e. 300 μl) / luciferase luminescent reagent.

Embodiment 6: detection kit of the present invention

The invention provides a detection kit consisting of the following parts:

(i) a container containing a 15 mM magnesium acetate solution for sample collection or dilution;

(ii) A container containing a purified ADP solution (>99.99999998% purity of ADP in solution for ATP), prepared as described in Example 1, buffered in potassium phosphate (7.5 mM, pH 6.5) In the solution, the concentration is 0.3mM, and contains 0.2mM EDTA and a mixed extractant consisting of 0.15% cationic detergent and 0.25% tertiary diamine surfactant.

(iii) A container filled with luciferin/luciferase LDR (Celcis, Cambridge, UK) bioluminescence reagent, the adenylate kinase activity in the reagent is less than 10 ^-8 U/100 μl.

Within the kit package, optionally, a container containing a non-ionic detergent solution (TritonX-100 (TRM), 0.2% or equivalent), and/or a container containing an ATPase such as adenosine triphosphate Container for bisphosphatase, an enzyme used to decompose ATP released by the action of nonionic detergents on the sample, making it suitable for reassay by addition of cationic detergents.

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

Use the E.coli (Escherichia coli) broth culture cultured for 1 week as the original bacterial solution (stock), which contains approximately 2.2×10 ⁷ microorganisms in every 200 μl of pH7.4 phosphate buffered solution, and use magnesium ions The sample collection/dilution reagent ((i) in Example 6 was serially diluted 10 times to obtain serial samples containing 10 ⁷ to 0.1 microorganisms per 200 μl sample.

200 μl of the sample was added to a 3.5 ml luminometer tube, 100 μl of ADP/extractant ((ii) in Example 6) was added, the total amount of the mixture was 300 μl, and incubated at room temperature for 1 minute or 5 minutes. Immediately after the incubation was completed, 100 μl of the improved Celcis LDR bioluminescent reagent ((iii) in Example 6) was added, and with a Biotrace M3 (RTM) luminometer, the luminescence of the first 10-second interval was first measured, and then every 10-second interval Measured once, up to 1 min, the increase in luminescence was measured cumulatively. The initial signal value was subtracted from the final reading to obtain a signal measurement expressed in readings per minute.

Can find out the performance of the inventive method with reference to Fig. 1, as can be seen from the figure, after each sample contains 10 and more than 10 bacteria samples and ADP mixed incubation 5 minutes, and each sample contains 100 and more than 100 A statistically linear response was obtained between the number of E. coli and sample luminescence after a 1 min incubation of the bacterial sample with ADP, with a limit of determination of approximately 10 bacteria in both cases. Compared with the method of WO 94/17202, the method is very superior, the latter has only 26 cpm (counts per minute) difference after 1 minute incubation for samples containing 100 bacteria, and only 26 cpm (counts per minute) for samples containing 1000 bacteria. 67cpm difference, linear response only at 1000 or more cells. In contrast, according to the method of the present invention, a signal increase of several thousand cpm can be obtained after 1 minute incubation for each sample containing 1000 cells.

It should be appreciated that in order to use this method for the determination of a sample with an unknown number of microorganisms, a calibration curve can be constructed by plotting the known number of microorganisms against the luminescence counts as shown in Figures 1 and 2 (e.g. in logarithmic values) , using the same assay protocol to obtain counts from a sample containing an unknown number of microorganisms (including zero microorganisms), the number of microorganisms in the sample can be estimated, which corresponds to the same reading value on the curve.

Those skilled in the art will recognize that the amount of adenylate kinase present in a particular microorganism, such as bacteria, may differ from other microorganisms. For example, yeasts contain more adenylate kinase than bacteria, depending on their size, and in fact individual yeasts can be measured in this way. Therefore, for a given microorganism, a specific calibration curve may need to be prepared, and for the same microorganism in a different state, such as a weakened microorganism, a microorganism in a pH or oxygen stress state, a specific calibration curve may also need to be prepared . However, another advantage of the method of the present invention over existing ATP-based methods is that the amount of adenylate kinase is proportional to the number of cells compared to the highly variable amount of ATP that is consumed in cellular metabolism. closer relationship.

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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