JPH10165200A - Microbial atp assay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for assaying microbial ATP, capable of efficiently and accurately conducting a microbial assay for a sample rich in free ATP in a short time. SOLUTION: This method for microbial ATP assay is characterized by that an ATP-decomposing reagent is added to a sample rich in free ATP not derived from microorganism to decompose the free ATP in the sample followed by adding an ATP-extractive reagent to the resultant sample to extract ATP from the microorganisms. At this time, simultaneously, the extractive reagent deactivates the ATP-decomposing reagent added to the sample previously, and subsequently, a luminescent reagent containing a reagent suppressing the inhibition of an enzymatic reaction is brought into contact with the resultant sample, and the light generated by biological chemiluminescent reaction is assayed with a relevant device.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a microbial ATP for measuring ATP in microbial cells of a sample containing a microbe using a biochemiluminescence method using luciferase and luciferin.
The present invention relates to a method for measuring Escherichia coli, yeast, lactic acid bacteria and other microorganisms in fields such as food hygiene, biotechnology, clinical testing, medicine, ultrapure water, and the environment.

[0002]

2. Description of the Related Art Measurement of Escherichia coli, yeast, lactic acid bacteria and other microorganisms is very important in fields such as food hygiene, biotechnology, clinical testing, medicine, ultrapure water, and environment. Generally, the measurement of microorganisms is performed by counting colonies in a growth medium, measuring under a microscope using a hemocytometer, measuring turbidity, and the like.

[0003] The method of measuring colonies in a growth medium is excellent in that it is highly sensitive, can measure only living cells, and can fix microbial cells by using a selective medium, but requires a medium. The time required for the measurement is usually one day or more, which is not suitable for quickly obtaining results. Since the measurement method using a hemocytometer uses a microscope, it has disadvantages such as complicated operations and difficulty in automation.
The turbidity measurement method is excellent in that it is quick and easy to automate, but it has low sensitivity, cannot distinguish between live cells and dead cells, and cannot be applied to samples with high turbidity such as fermented milk. Has problems.

[0004] The measurement of microorganisms in the above fields requires quick and highly sensitive measurement. For example, in the field of food hygiene, microbial contamination inspection of products is indispensable for shipping products. Conventionally, this test is performed by the colony counting method, but since the test requires one day or more, the product must be stored in a warehouse until a result is obtained, which not only has a problem in distribution efficiency, but also has a problem. For products such as milk, the longer the storage time, the higher the potential for microbial contamination. In addition, since the concentration of microorganisms that cause contamination in food is generally low, a highly sensitive test is required.

As a method for measuring microorganisms satisfying such requirements, there is known a method for measuring adenosine triphosphate (ATP), which is always present in living microorganisms, using a biochemiluminescence assay. This method utilizes the fact that the concentration of ATP contained in microbial cells is proportional to the microbial concentration, and is a very effective method because the measurement time is short and the sensitivity is high.

However, most foods and the like contain ATP (free ATP) not derived from microorganisms, and it is difficult to measure only ATP of microorganisms. In addition, free ATP is often orders of magnitude higher than microbial ATP, making measurement more difficult. For this reason, the biochemiluminescence method has not been widely applied to food measurement.

[0007]

Therefore, as a means for solving these problems, apyrase, which is an ATPase, is added to a sample as an ATPase reagent, and free ATP not derived from microorganisms is decomposed beforehand before measurement. A way to do that has been developed. This measurement method includes a step of decomposing free ATP by applying apyrase to a sample, a step of extracting ATP from a microorganism, and a step of measuring ATP by causing luciferase and luciferin to act.

In this measuring method, it is necessary to sufficiently increase the concentration of apyrase in order to efficiently decompose free ATP in a short time. However, if the apyrase concentration is too high, ATP extracted from the microorganism in the next step will be decomposed at the same time, resulting in a problem that the ATP concentration becomes zero in all samples. In order to solve this problem, it is conceivable to reduce the rate of ATP decomposition by lowering the concentration of apyrase. However, this method requires a longer time for decomposition, and has a problem in terms of speed.
Apyrase is used to decompose ATP,
A method for extracting ATP after adding an apyrase inhibitor has also been developed. However, this method not only increases the number of steps for adding the inhibitor but also decreases the efficiency, but also decreases the measurement sensitivity due to dilution. There was also a problem.

Accordingly, an object of the present invention is to provide a method for measuring microbial ATP that can efficiently, accurately, and in a short time measure a microorganism containing a large amount of free ATP.

Another object of the present invention is to make it possible to sufficiently increase the concentration of an ATP-decomposing reagent for decomposing free ATP, and therefore to improve sensitivity without requiring extra dilution. At the same time, the operation of the microorganism AT is simpler in terms of operation without lowering the extraction ability of the microorganism ATP.
It is to provide a method for measuring P.

[0011]

The above object is achieved by the method for measuring ATP of a microorganism according to the present invention. In summary, the present invention relates to a method for measuring ATP in a microbial cell of a sample containing a microorganism using a biochemiluminescence method with luciferase and luciferin, comprising:
The sample is contacted with an ATP decomposing reagent to decompose free ATP not derived from microorganisms, and then (b) the sample is contacted with an ATP extraction reagent to extract ATP from the microorganism and deactivate the ATP decomposing reagent. And then (c)
Microbial ATP, wherein an ATP is measured by bringing an enzyme reaction inhibition inhibitor and a luminescent reagent into contact with the sample.
Measurement method.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Next, the method for measuring microbial ATP of the present invention will be described in more detail. According to the present invention,
First, free AT that is not derived from microorganisms, for example, food.
An ATP decomposition reagent is added to a sample (sample) containing a large amount of P to decompose free ATP in the sample. Next, an ATP extraction reagent is added to the sample in which free ATP has been decomposed, and ATP is extracted from the microorganism. At this time, ATP
The extraction reagent inactivates the ATP degradation reagent previously added to the sample. Thereafter, the sample is brought into contact with a luminescent reagent containing an enzyme reaction inhibition inhibitor, and the light generated by the biochemiluminescence reaction is measured by a biochemiluminescence measuring device.

As an ATP decomposing reagent, ATP is converted to AMP.
Apyrase known as an enzyme that decomposes to (adenosine monophosphate) can be used.

As the ATP extraction reagent, a cationic surfactant is used. Examples of the cationic surfactant include alkyldimethylbenzylammonium chloride, benzalkonium chloride, benzethonium chloride and cetyltrimethylammonium chloride. obtain.

As the enzyme reaction inhibition inhibitor, silicone can be used, and any silicone can be used as long as it is used as an antifoaming agent. oil,
Alternatively, a modified product of silicone resin and silicone oil may be used. Specifically, as the silicone resin,
For example, dimethylpolyxan or the like can be used. As the silicone oil, for example, linear dimethylpolyxane or the like can be used. As the modified silicone resin or silicone oil, for example, a modified dimethylpolysiloxane can be used. One or more of these silicones can be selected and used. Furthermore, as the enzyme reaction inhibition suppressing reagent, cyclodextrin described in JP-A-6-504200 can also be used.
As the cyclodextrin, any one or a plurality of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin or a modified form thereof can be selected and used.

As described above, the method for measuring the ATP of a microorganism according to the present invention uses the free ATP of a sample as an ATP containing apyrase.
After decomposing with a TP-decomposing reagent, it is brought into contact with an ATP extraction reagent containing a cationic surfactant to extract ATP from microorganisms, deactivate apyrase, and add silicone to suppress the enzyme reaction inhibition of the surfactant. According to the method of the present invention, not only can the concentration of apyrase be sufficiently high to decompose free ATP, but also the step of inactivating apyrase and Since the extraction step is performed at the same time, it is possible to efficiently measure the microorganisms of a sample containing a large amount of free ATP such as food. In the method of the present invention, a cationic surfactant is used to inactivate apyrase. However, the higher the concentration of the surfactant, the higher the ability to inactivate apyrase. However, cationic surfactants inhibit the luciferase reaction. Therefore, in the present invention, in order to solve this problem, silicone is used as an enzyme reaction inhibition suppressing reagent.

Next, the microorganism AT of the present invention will be described according to Examples.
The method for measuring P will be described in more detail.

Example 1 In this example, the method for measuring microbial ATP of the present invention was carried out using a batch-type biochemiluminescence measuring apparatus. or,
In this example, an ATP decomposing reagent containing apyrase, an ATP extraction reagent containing dodecyldimethylbenzylammonium chloride (benzalkonium chloride) as a cationic surfactant, and KS-502 (trade name, Shin-Etsu Chemical Co., Ltd.) as silicone Modified silicone oil 100
% Containing) was used. The measurement operation was as follows.

First, 100 μl of a 100 nmol / l ATP aqueous solution is placed in a measuring cuvette, and 0.5 U
100 ml of a reagent (decomposition reagent A) prepared by dissolving 1 / ml apyrase and 1 mM EDTA (ethylenediaminetetraacetic acid) in a HEPES buffer solution at pH 7.75 was added and reacted for 2 minutes.

Then, 0.1% benzalkonium chloride and 1 mM EDTA were added to the cuvette at pH 7.75 HE.
Extraction reagent (extraction reagent A) dissolved in PES buffer was added to 10
0 μl was added and stirred.

Furthermore, 10% KS-5 was used as an inhibitory reagent in a luminescent reagent obtained by dissolving 1 mM EDTA, firefly luciferase and luciferin in a HEPES buffer at pH 7.75.
100 μl of the reagent (Luminescent reagent A) containing 02 was added to the cuvette and stirred.

Next, the cuvette was set in a biochemiluminescence measuring device, and the light generated by the biochemiluminescence reaction was measured. After setting the cuvette, the luminescence amount was 10
Light emission from the second was measured every 0.1 second for 10 seconds, and integrated and found.

EDTA added to extraction reagent A and luminescence reagent A
Is used for the purpose of suppressing the action of an enzyme such as alkaline phosphatase that degrades ATP when cells are contacted with an extraction reagent. pH 7.75 HEPES buffer
It is necessary to keep the enzyme that measures ATP using the biochemiluminescence method in an optimal state.

Table 1 shows the luminescence amount and the relative luminescence amount for ATP measurement. In order to demonstrate the effects of the present invention, a reagent obtained by removing apyrase from the decomposition reagent A (decomposition reagent B), a reagent obtained by removing benzalkonium chloride from the extraction reagent A (extraction reagent B), and a KS- Combinations with reagents other than 502 (luminescent reagent B) are shown as comparative samples.

[0025]

[Table 1]

The “combination 1” of the decomposing reagent B, the extracting reagent B, and the luminescent reagent B used as a blank did not contain apyrase, benzalkonium chloride, and KS-502, and thus did not inhibit the luminescent reaction and decomposed ATP. Therefore, the relative light emission amount is considered to be 100%. In the “combination 2” of the decomposition reagent A, the extraction reagent A, and the luminescence reagent A according to the present invention, since ATP is decomposed by apyrase, the relative luminescence amount as compared with the “combination 1” is 0.00.
ATP is about 100% degraded. However, in the case of the “combination 3” of the decomposition reagent B, the extraction reagent A, and the luminescence reagent A containing no apyrase, the relative luminescence amount was 75.7%, indicating that ATP was not decomposed and there was no significant interference.

Next, the measurement was performed by changing the measurement procedure.
That is, 0.5 U / ml apyrase and 1 mM EDTA were added to p.
100 μl of a reagent (decomposition reagent A) dissolved in H7.75 HEPES buffer, 0.1% benzalkonium chloride and 1
After 100 μl of an extraction reagent (extraction reagent A) in which mM EDTA was dissolved in a pH 7.75 HEPES buffer was added and stirred, 100 μl of a 100 nmol / 1 aqueous ATP solution was placed in a measurement cuvette and reacted for 2 minutes. Next, 100 μl of a reagent (luminescent reagent A) obtained by adding 1 mM EDTA, firefly luciferase, and luciferin to a luminescent reagent dissolved in a pH 7.75 HEPES buffer and 10% KS-502 as an inhibitory reagent was added to the cuvette and stirred. The cuvette thus obtained was set in a biochemiluminescence measuring device, and the light generated by the biochemiluminescence reaction was measured. The amount of luminescence was determined by measuring the luminescence from 10 seconds after the cuvette was set, measuring every 10 seconds for 10 seconds, and integrating.

Table 2 shows the luminescence amount and the relative luminescence amount for the ATP measurement. In order to demonstrate the effects of the present invention, a reagent obtained by removing apyrase from the decomposition reagent A (decomposition reagent B), a reagent obtained by removing benzalkonium chloride from the extraction reagent A (extraction reagent B), and a KS- Combinations with reagents other than 502 (luminescent reagent B) are shown as comparative samples.

[0029]

[Table 2]

The “combination 1” of the decomposing reagent B, the extracting reagent B, and the luminescent reagent B used as a blank does not contain apyrase, benzalkonium chloride, and KS-502, and thus does not inhibit the luminescent reaction and decomposes ATP. Therefore, the relative light emission amount is considered to be 100%. In “combination 2” of decomposition reagent A, extraction reagent A, and luminescence reagent A,
Since apyrase was inhibited by the extraction reagent, ATP was not decomposed and the relative luminescence was 70.2%. In "Combination 3" using the extraction reagent B containing no benzalkonium chloride, the relative luminescence was 2.5%, indicating that apyrase still functions even after the addition of the extraction reagent and ATP is decomposed. . Further, in the case of “combination 4” of the luminescent reagent B containing no KS-502, the relative luminescence amount is 19.8%, and the luminescence amount is about ４ as compared with “combination 2”. This is a phenomenon that occurs because the luminescent reagent is inhibited by benzalkonium chloride.

From these facts, 0.5 U / ml apyrase has the ability to decompose 100 nmol / 1 ATP in about 2 minutes, but it is necessary to add a substance that inhibits the action of apyrase after the apyrase has been acted on. , Microbial A
Decomposition of ATP also occurs after extraction of TP, and A
It turns out that TP cannot be measured accurately. In addition, it was found that benzalkonium chloride used as an extraction reagent has the ability to inhibit apyrase, but benzalkonium chloride inhibits the biochemiluminescence reaction of luciferase and luciferin used in the next step. It is necessary to suppress the inhibition. It is understood that silicone has the effect of suppressing this inhibition.

In this example, the higher the concentration of apyrase, the higher the rate of decomposing ATP, but if the concentration is too high, the effect of apyrase cannot be suppressed by benzalkonium chloride. Here, if the concentration of benzalkonium chloride is increased, apyrase can be inactivated even if the concentration of apyrase is high, but if the concentration of benzalkonium chloride is too high, the biochemiluminescence reaction of KS-502 is caused by KS-502. It is important to select an appropriate concentration of apyrase, benzalkonium chloride, and KS-502, because the inhibition cannot be suppressed. In this combination, apyrase was 0.1 to 1 U / ml, benzalkonium chloride was 0.04 to 0.1 wt%, and KS-502 was 5 to 5 U / ml.
20 wt% is appropriate, but preferably apyrase is 1%.
U / ml, 0.1 wt% benzalkonium chloride, KS
-502 is preferably 20 wt%.

The EDTA and the pH buffer to be added to the decomposition reagent, the extraction reagent and the luminescence reagent need not necessarily be added to all the reagents, but may be added to any of them. If the extraction time is short, the addition of EDTA is not necessary,
It is not necessary to use a pH buffer if the pH of the sample to be measured is near neutrality.

Example 2 In this example, a sample in which free ATP was added to a culture of lactic acid bacteria was measured according to the method of the present invention. Also in this example, a batch-type biochemiluminescence measuring apparatus was used. In this example, an ATP decomposing reagent containing apyrase, an ATP extraction reagent containing cetyltrimethylammonium chloride as a cationic surfactant, and KM-72 as silicone (trade name, silicone resin manufactured by Shin-Etsu Chemical Co., Ltd.) (Containing 30%). The measurement operation was as follows.

First, 1 × 10 ⁴ , 1 × 10 ⁵ , 1 × 1 to which 100 nmol / 1 of ATP was added as free ATP.
0 ⁶ , 1 × 10 ⁷ cells / ml of a lactic acid bacterium culture solution was put into each measurement cuvette in an amount of 100 μl, and 0.1 U / m
1 apyrase and 1 mM EDTA in HE at pH 7.75
100 μl of reagent (decomposition reagent C) dissolved in PES buffer
Was added and reacted for 2 minutes. Thereafter, 100 μl of an extraction reagent (extraction reagent C) in which 0.1% cetyltrimethylammonium chloride and 1 mM EDTA were dissolved in a pH 7.75 HEPES buffer solution was added, and the mixture was stirred.
Was extracted. Further, 20% of KM-72 was added as a suppressing reagent to a luminescent reagent obtained by dissolving 1 mM EDTA, firefly luciferase and luciferin in a pH 7.75 HEPES buffer.
Was added to the cuvette and stirred, and the cuvette was set in a biochemiluminescence measuring apparatus, and light generated by the biochemiluminescence reaction was measured. The amount of luminescence was determined by measuring the luminescence from 10 seconds after the cuvette was set, measuring every 10 seconds for 10 seconds, and integrating. In addition, the amount of luminescence was determined in advance using a 100 nmol / 1 ATP standard reagent, and the ATP concentration was calculated. FIG.
Shows the measurement results.

"Combination 1" in FIG. 1 is a plot of the number of lactic acid bacteria and ATP concentration measured using the decomposition reagent C, extraction reagent C, and luminescence reagent C according to the present invention. It is the thing which plotted the result measured using the reagent (decomposition reagent B) which removed apyrase from C.

In "Combination 1", added free ATP
Is completely degraded, so that the ATP concentration is directly proportional to the number of bacteria. However, in the "combination 2" using the degradation reagent B to which apyrase is not added, free ATP is not decomposed, and thus regardless of the number of bacteria. It can be seen that the ATP concentration becomes a substantially constant value, and a direct proportional relationship cannot be obtained.

Thus, according to the present invention, it is possible to accurately measure the number of bacteria in a bacterial suspension containing a large amount of free ATP.

The time required for the extraction of ATP varies depending on the type of microorganism, but it is 10 seconds or more for E. coli and general bacteria.
In the case of yeast, 30 seconds or more are preferable. If the sample is left after the extraction, the ATP concentration gradually decreases. Therefore, it is preferable to measure the luminescence amount within 5 minutes after the extraction, especially when using an extraction reagent containing no EDTA. Is preferably measured immediately after the extraction is completed.

In the above embodiment, the addition of the reagent is performed manually. However, if the reagent is automatically added using a pump or an automatic shilling, the intracellular ATP can be measured more easily.

Also in this example, the higher the concentration of apyrase, the higher the rate of decomposing ATP, but if the concentration is too high, the effect of apyrase cannot be suppressed by cetyltrimethylammonium chloride.
Here, if cetyltrimethylammonium chloride is increased, apyrase can be inactivated even when the concentration of apyrase is high, but if the concentration of cetyltrimethylammonium chloride is too high, inhibition of the biochemiluminescence reaction by KM-72 is prevented. It cannot be suppressed. Therefore, it is important to select appropriate concentrations of apyrase, cetyltrimethylammonium chloride and KM-72. In this combination, apyrase is 0.1-
0.2 U / ml, cetyltrimethylammonium chloride 0.05-0.1 wt%, KM-72 20-30
0 wt% is appropriate. More preferably, apyrase is 0.1 U / ml, cetyltrimethylammonium chloride is 0.1 wt%, and KS-72 is 20 wt%.

[0042]

As described in detail above, the method for measuring ATP of a microorganism according to the present invention is free from AT which is not derived from a microorganism.
An ATP degradation reagent is added to a sample (sample) containing a large amount of P to decompose free ATP in the sample, and then an ATP extraction reagent is added to the sample in which free ATP has been decomposed to extract ATP from the microorganism. At this time, AT
The P extraction reagent deactivates the ATP degradation reagent previously added to the sample, and then contacts the sample with a luminescent reagent containing an enzyme reaction inhibition inhibitor, and emits light generated by the biochemiluminescence reaction using a biochemiluminescence measurement apparatus. In the present invention, the number of microorganisms in a sample containing a large amount of free ATP such as food can be efficiently and accurately measured, and the present invention does not require extra dilution. Therefore, not only can the sensitivity be improved, but also the AT is simpler in terms of operation without lowering the extraction ability.
A P measurement method can be constructed. Therefore, the present invention can greatly contribute to microbial testing in fields such as food hygiene, biotechnology, clinical testing, medicine, ultrapure water, and environment.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the number of lactic acid bacteria and ATP concentration.

Claims (13)

[Claims] An AT in a microorganism cell of a sample containing a microorganism.
A method for measuring microbial ATP, wherein P is measured using a biochemiluminescence method using luciferase and luciferin,
(A) contacting the sample with an ATP degrading reagent to decompose free ATP not derived from microorganisms; and (b) contacting the sample with an ATP extraction reagent to remove ATP from the microorganisms.
And inactivate the ATP degradation reagent,
(C) A method for measuring microbial ATP, comprising contacting an enzyme reaction inhibition inhibitor and a luminescent reagent with the sample to measure ATP. 2. The method according to claim 1, wherein the ATP-decomposing reagent is a reagent containing apyrase. 3. The method according to claim 1, wherein the ATP extraction reagent is a reagent containing a cationic surfactant. 4. The method for measuring microbial ATP according to claim 1, wherein the enzyme reaction inhibition suppressing reagent is a reagent containing silicone or cyclodextrin. 5. The method according to claim 1, wherein the luminescent reagent is a reagent containing luciferase and luciferin. 6. The method according to claim 3, wherein the cationic surfactant is alkyldimethylbenzylammonium chloride, benzalkonium chloride, benzethonium chloride or cetyltrimethylammonium chloride. 7. The method for measuring microbial ATP according to claim 4, wherein the silicone is a silicone resin, a silicone oil, or a modified product of a silicone resin and a silicone oil, and any one or a plurality of the silicones are selected and used. . 8. The microorganism according to claim 8, wherein the silicone resin is dimethylpolyxane, the silicone oil is linear dimethylpolyxane, and the modified silicone resin or silicone oil is a modified dimethylpolysiloxane. ATP measurement method. 9. The microorganism according to claim 4, wherein the cyclodextrin is α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or a modified product thereof, and any one or a plurality thereof is selected and used. ATP measurement method. 10. The microorganism ATP according to claim 1, wherein the ATP decomposing reagent is a reagent containing apyrase, the ATP extraction reagent is a reagent containing benzalkonium chloride, and the enzyme reaction inhibition suppressing reagent is denatured silicone oil. Measurement method. 11. The apyrase has a concentration of 0.1 to 1 U / m.
1, the benzalkonium chloride is 0.04 to 0.1 wt
%, The modified silicone oil (containing 100%) is 5 to 5%.
The method according to claim 10, wherein the amount is 20 wt%. 12. The microorganism ATP according to claim 1, wherein the ATP decomposition reagent is a reagent containing apyrase, the ATP extraction reagent is a reagent containing cetyltrimethylammonium chloride, and the enzyme reaction inhibition inhibitor is a silicone resin. Measuring method. 13. The apyrase is 0.1 to 0.2 U /
ml, 0.05 to 0.1 wt% of the cetyltrimethylammonium chloride, the silicone resin (30%
Content of the microorganism A is 20 to 30% by weight.
Method for measuring TP.