WO2006095697A1

WO2006095697A1 - Method of detecting microorganism and bacteriolytic reagent

Info

Publication number: WO2006095697A1
Application number: PCT/JP2006/304296
Authority: WO
Inventors: Tadahiro Matsuno; Akio Kuroda
Original assignee: Bussan Nanotech Research Institute, Inc.; National University Of Corporation Hiroshima University
Priority date: 2005-03-10
Filing date: 2006-03-06
Publication date: 2006-09-14
Also published as: JP2006246792A

Abstract

It is intended to provide a method of detecting a microorganism which comprises the extraction step of contacting microbial cells with a bacteriolytic reagent and thus extracting intracellular ATP from the microbial cells, the amplification step of amplifying the intracellular ATP thus extracted, and the light emission step of contacting the intracellular ATP with luciferin and luciferase for light emission, wherein the bacteriolytic reagent contains a lytic enzyme, a nonionic surfactant and a chelating agent. Thus, a method of detecting a microorganism, whereby a microorganism even in an extremely small amount can be sufficiently detected, and a bacteriolytic reagent are provided.

Description

Specification

Microbial detection method and lysis reagent

Technical field

The present invention relates to a microorganism detection method and a lysis reagent.

Background art

[0002] In the food industry and the like that handle raw meat and dairy products, there is a demand for a method for detecting microorganisms quickly and with high sensitivity from the viewpoint of food poisoning prevention. In recent years, as a method for detecting microorganisms, a method for detecting microorganisms by measuring biochemiluminescence generated by the reaction of ATP with luciferin and luciferase using ATP (adenosine triphosphate) present in living organisms as an index. Is used.

[0003] In order to measure intracellular ATP using this method, it is necessary to extract ATP contained in the cell. Methods for extracting ATP in cells include adding microorganisms to an aqueous solution of triclonal acetate (TCA method), extracting cells with an aqueous surfactant solution (surfactant method), and 90 ° C. Known methods include adding cells to Tris buffer heated to a suitable temperature (Tris buffer method), methods using ethanol (ethanol method), methods using lytic enzymes such as lysozyme (enzyme method), etc. . However, in the above TCA method and Tris buffer solution method, it is necessary to carry out a neutralization treatment or to increase the temperature of the sample. That is, it is necessary to prepare the _P H and temperature, it is difficult to quickly extract the stable amount of ATP. On the other hand, in the surfactant method and the ethanol method, the enzyme for biochemiluminescence may be inhibited by the surfactant or ethanol, and the sensitivity of biochemiluminescence tends to decrease. On the other hand, the enzymatic method has a problem that it takes time to extract intracellular ATP.

[0004] As described above, any of the above ATP extraction methods has merits and demerits, and the present situation is that they are properly used according to circumstances. Of these, a surfactant method using a cationic surfactant that can effectively extract ATP from microorganisms that may inhibit enzymes as a lysis reagent is often used. For example, in Patent Document 1 below, in a method for measuring intracellular ATP of a sample containing cells by a biochemiluminescence method, the sample is referred to as a cationic field. A method for measuring intracellular ATP in contact with an ATP extraction reagent containing a surfactant and an enzyme reaction inhibition inhibitor containing a protein or glycoside to inhibit enzyme reaction inhibition by a cationic surfactant Is disclosed. According to this method, the effect that the light emission inhibition by the cationic surfactant is suppressed can be obtained.

Patent Document 1: Japanese Patent No. 3175018

Disclosure of the invention

Problems to be solved by the invention

[0005] By the way, in a method for detecting a microorganism by measuring biochemiluminescence generated by the reaction of intracellular ATP with luciferin and luciferase, there is a method of amplifying intracellular ATP when the microorganism is extremely small. Effectively, this amplification is performed using an amplification reagent, and an amplification enzyme and an amplification substrate are generally used as the amplification reagent.

[0006] However, in the detection method described in Patent Document 1 described above, when an amplification substrate composed of an anionic compound such as a polyphosphate compound is present, a cationic surfactant and An anionic compound forms an insoluble salt, and as a result, the lysis effect tends to be remarkably reduced, and microorganisms cannot be sufficiently detected. In addition, even when an enzymatic reaction is performed using an anionic compound and bacterial ATP as a substrate after the start of the lysis reaction, it tends to be difficult to maintain the lysis reaction. In this case, since the amount of luminescence decreases with time, the amount of luminescence may vary greatly depending on the time of measurement, and microorganisms cannot be detected with good reproducibility.

[0007] When an anionic surfactant is used as a surfactant instead of a cationic surfactant, the enzyme itself tends to be denatured, the lysis reaction itself does not proceed, and microbiology occurs. Objects can hardly be detected. On the other hand, when a nonionic surfactant is used as a surfactant, there is a problem that it cannot be detected when the amount of luminescence with a slow lysis reaction is very small.

[0008] The present invention has been made in view of the above circumstances, and an object thereof is to provide a microorganism detection method and a lysis reagent that can sufficiently detect even a very small amount of microorganism.

Means for solving the problem

[0009] In order to solve the above-described problems, the microorganism detection method of the present invention uses a lysis cell for microbial cells. An extraction step of extracting intracellular ATP from a microorganism cell, contacting with a drug, an amplification step of amplifying the extracted intracellular ATP, and contacting the amplified intracellular ATP, luciferin and luciferase And a luminescent reagent, wherein the lysis reagent contains a lytic enzyme, a nonionic surfactant and a chelating agent.

In the microorganism detection method of the present invention, intracellular ATP is extracted when a microorganism cell is brought into contact with a lysis reagent. At this time, since the nonionic surfactant is used as the surfactant contained in the lysis reagent, it is sufficiently suppressed that the surfactant inhibits or denatures the lytic enzyme. Moreover, since the said lysis reagent contains a chelating agent, lysis reaction is accelerated | stimulated.

[0011] Furthermore, since the lysis reagent contains a lytic enzyme in addition to the nonionic surfactant and the chelating agent, the cells are sufficiently lysed and the intracellular ATP is sufficiently extracted. Since the extracted intracellular ATP is amplified, a sufficient amount of luminescence can be obtained in a short time by a luminescence reaction with luciferin and luciferase even if the amount of microorganisms is very small. Moreover, in the microorganism detection method of the present invention, as described above, inhibition and denaturation of the lytic enzyme are sufficiently suppressed in the lysis reaction. As described above, nonionic surfactants are used as surfactants as described above, so that insoluble salts are sufficiently formed even in the presence of anionic compounds such as polyphosphoric acid used in amplification reagents. Is prevented. Accordingly, the temporal decrease in the amount of luminescence can be sufficiently suppressed, and the reproducibility of the detection result of the microorganism can be improved.

[0012] Although the reason why the chelating agent can promote the lysis reaction has not been clarified, the chelating agent accelerates the process of extracting intracellular ATP from the cell surface. This is presumed to function as a so-called catalyst.

[0013] In the microorganism detection method, the lytic enzyme is preferably an enzyme that hydrolyzes a cell wall. In this case, in biochemiluminescence, the decrease in the amount of luminescence is more sufficiently suppressed.

[0014] In the microorganism detection method, the lysis reagent preferably further contains a saccharide.

. In this case, the decrease in the activity of the lytic enzyme is more sufficiently suppressed, and the amount of luminescence is further increased.

[0015] In the microorganism detection method, the concentration of the nonionic surfactant is such that 0.1 mass% to 1.0 mass% with respect to the amount, and the concentration of the lytic enzyme is 1. OX 10 ⁵ units / mL to OX 10 ⁶ units / mL with respect to the total amount of the lysis reagent, The concentration of the chelating agent is preferably 5 mM to 10 mM with respect to the total amount of the lysis reagent. Here, lunit refers to the amount of enzyme that decreases the absorbance (640 nm) by 0.001 per minute when lysozyme is produced using a phosphate buffer suspension of dried micrococcus lysodeikticus as a substrate.

[0016] When the concentration of the nonionic surfactant, lytic enzyme, and chelating agent contained in the lysis reagent is within the above range, the extraction of intracellular ATP is further promoted compared to the case where the concentration is outside the above range. Thus, a sufficient amount of light emission can be obtained in a short time, and the temporal decrease in the amount of light emission can be more sufficiently suppressed.

[0017] The lysis reagent of the present invention is characterized by containing a lytic enzyme, a nonionic surfactant and a chelating agent. When this lysis reagent is brought into contact with a microorganism, intracellular ATP can be sufficiently extracted from the cells of the microorganism in a short time. Note that the microorganisms to be lysed by the lysis reagent are not particularly limited.

The invention's effect

[0018] According to the microorganism detection method of the present invention, even a very small amount of microorganism can be sufficiently detected in biochemiluminescence.

Brief Description of Drawings

FIG. 1 is a graph showing a temporal change in light emission amount in Example 1.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, preferred embodiments of the present invention will be described in more detail.

[0021] The microorganism detection method of the present invention comprises an extraction step of extracting intracellular ATP of microorganisms using a lysis reagent containing a lytic enzyme, a nonionic surfactant and a chelating agent, and the extracted intracellular An amplification step for amplifying ATP; and a luminescence step for reacting the amplified intracellular ATP with luciferin and luciferase.

[0022] [Extraction process]

The extraction step is a step of extracting intracellular ATP from the cell force of the microorganism.

[0023] In the extraction step, a lysis reagent containing a lytic enzyme, a nonionic surfactant and a chelating agent is used. By using such a lysis reagent, intracellular AT P can be sufficiently extracted in a short time.

[0024] The lytic enzyme is not particularly limited, and examples thereof include lysozyme, chitinase, chitosanase, achromopeptidase, β-1,3-glucanase, and hexosaminidase. Among these, lytic enzymes such as lysozyme that hydrolyze cell walls are preferable. These lytic enzymes can hydrolyze the cell wall. In this case, the decrease in the amount of luminescence is more sufficiently suppressed in biochemiluminescence. Lysozyme is more useful because it is relatively inexpensive and easy to obtain.

[0025] The lysis reagent includes a nonionic surfactant. By using a nonionic surfactant as the surfactant, it is suppressed that the surfactant inhibits the lytic enzyme contained in the lysis reagent. Further, as will be described later, when amplification of the extracted intracellular cells is performed, it is prevented that the surfactant and the amplification reagent form an insoluble salt. The nonionic surfactant is not particularly limited, and examples thereof include polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene sorbitan ester, and sorbitan fatty acid ester.

[0026] The lysis reagent includes a chelating agent. Chelating agents play a role in promoting the lysis reaction. Although it does not specifically limit as a chelating agent, For example, EDTA, EGTA, CyDTA, DTPA, NTA, TTHA, IDA, NTPO, BAPTA etc. are mentioned. Among these, EDTA, EGTA, CyDTA, 0? I prefer 8 or 1 ^ ¾8. If the chelating agent is EDTA, EGTA, CyDTA, DTP A or TTH A, the lysis reaction is further accelerated.

[0027] The lysis reagent preferably further contains a saccharide. In this case, the lysis rate becomes faster and the decrease in the activity of the lytic enzyme is further suppressed. Accordingly, the temporal decrease in the amount of luminescence is sufficiently suppressed, and the reproducibility of the detection result of the microorganism is further increased. The saccharide is not particularly limited, and examples thereof include sucrose, glucose, fructose, maltose, galactose, ratatose, trehalose, mannose and the like. Among these, sucrose or trehalose is preferable. If the saccharide is sucrose or trehalose, the decrease in enzyme activity is further suppressed.

[0028] It should be noted that if the lysis reagent does not inhibit the activity of the lytic enzyme described above, It may further contain a reagent such as tylene glycol, glycerol, 2-mercaptoethanol, dithiothreitol and the like.

[0029] The microorganism to be lysed by the lysis reagent is not particularly limited. In the microorganism detection method of the present invention, the target microorganism is not particularly limited, and examples thereof include Escherichia coli, coliforms, Salmonella, Staphylococcus aureus and the like.

[0030] Next, a preferred concentration range of the lytic enzyme, nonionic surfactant and chelating agent contained in the lysis reagent described above will be described.

[0031] The concentration of the lytic enzyme is preferably from 1. OX 10 ⁴ units / mL to OX 10 ^? Units / mL with respect to the total amount of the lysis reagent. 1. 0 X 10 ⁵ units / mL to l. OX 10 ⁶ units / mL is more preferable. If the concentration of the lytic enzyme is less than 1. OX 10 ⁵ unitsZmL, the lytic enzyme function is insufficient compared to the case where the concentration is in the above range, and it tends to take time to extract intracellular ATP. When the concentration of the lytic enzyme exceeds 1. OX 10 ⁶ unitsZmL, there is no difference in the function of the lytic enzyme compared to the case where the concentration is in the above range, and the lytic enzyme tends to be used wastefully.

[0032] Surfactants generally tend to inhibit enzymes, but the degree of inhibition increases as the surfactant concentration increases. In addition, the extraction ability increases as the surfactant concentration increases, and the lower the concentration, the lower the inhibition, but the extraction ability becomes insufficient. Therefore, the concentration of the nonionic surfactant used in the present invention is important. That is, it is preferable that the concentration of the nonionic surfactant is 0.01% by mass to 5.0% by mass with respect to the total amount of the lysis reagent. 0.1% by mass to 1.0% by mass More preferably. When the concentration of the nonionic surfactant is less than 0.1% by mass, the lysis rate tends to be slower than when the concentration is within the above range, and the concentration of the nonionic surfactant is low. If it exceeds 1.0 mass%, the nonionic surfactant tends to inhibit the enzyme reaction of the enzyme (such as a lytic enzyme) as compared with the case where the concentration is in the above range.

[0033] The concentration of the chelating agent is preferably 5 mM to 10 mM, more preferably ImM or more with respect to the total amount of the lysis reagent. When the chelating agent concentration is less than 5 mM, the lysis reaction tends to be slower than when the concentration is in the above range, and when the chelating agent concentration exceeds 10 mM, the concentration is in the above range. Compared to enzyme reactions (especially luminescence) Reaction) tends to be inhibited.

[0034] Further, when the lysis reagent contains saccharides, the concentration of the saccharides is preferably 0.1% by mass or more based on the total amount of the lysis reagent. More preferably, it is ˜10% by mass. When the saccharide concentration is less than 0.5% by mass, it is difficult to obtain an effect of increasing the rate of lysis compared to the case where the concentration is within the above range. When the saccharide concentration exceeds 10% by mass, Compared with the case where is in the above range, the degree of inhibition of the lytic enzyme activity tends to increase.

[0035] For the lysis reagent, the concentration of the nonionic surfactant is 0.1% by mass to 1.0% by mass with respect to the total amount of the lysis reagent, and the concentration of the lysis enzyme is It is preferable that it is 1. OX 10 ⁵ units / mL to OX 10 ⁶ units / mL with respect to the total amount of the reagent, and the concentration of the chelating agent is ⁵ mM to 10 mM with respect to the total amount of the lysis reagent.

[0036] When the concentration range of the nonionic surfactant, lytic enzyme, and chelating agent contained in the lysis reagent is also within the above range, compared to the case where either one is outside the above range, the intracellular ATP Extraction is further promoted, a sufficient amount of light emission can be obtained in a short time, and a temporal decrease in the amount of light emission can be sufficiently suppressed.

[0037] [Amplification process]

The amplification step is a step of amplifying the intracellular ATP extracted in the extraction step.

[0038] The amplification reagent includes an amplification substrate and an amplification enzyme. When polyphosphate is used as the amplification substrate and adenylate kinase and polyphosphate kinase are used as the amplification enzymes, the amplification mechanism of ATP is as follows. It is. That is, AMP (adenosine monophosphate) is reacted with adenylate kinase in the presence of ATP to form two molecules of ADP (adenosine nitric acid), and in the presence of polyphosphate compound, ADP reacts with polyphosphate kinase to form two molecules of ATP and a polyphosphate compound. Thus, one molecule of ATP can be converted to two molecules of ATP, and ATP can be amplified by repeating this reaction.

[0039] An amplification reagent for amplifying ATP is composed of an amplification substrate and an amplification enzyme, and the amplification reagent is composed of polyphosphate as an amplification substrate, adenylate kinase and polyphosphate kinase as amplification enzymes. Consists of. In addition, instead of polyphosphate kinase, An acid kinase can also be used. Of these, those using polyphosphate as an amplification substrate and adenylate kinase and polyphosphate kinase as amplification enzymes are preferred. In this case, since polyphosphoric acid acts as a phosphate donating substrate, there is an advantage that reaction efficiency can be improved by using polyphosphoric acid having a long chain length.

[0040] [Light emission process]

The luminescence process is a process in which amplified ATP is brought into contact with luciferin and luciferase to generate biochemiluminescence. This luminescence step can be performed by mixing ATP with luciferin and luciferase.

[0041] By detecting this luminescence with a light detection device or the like, microorganisms can be detected. As the photodetection device, a known device can be used. For example, a CCD, a photodiode, or a photomultiplier tube can be used.

[0042] The microorganism detection method of the present invention may further include other steps as long as it includes an extraction step, an amplification step, and a luminescence step. The other processes include a decomposition process for decomposing bacterial flocs before extracting intracellular ATP from microorganisms, a removal process for removing unnecessary ATP that does not depend on microorganisms using a removal liquid, and this removal liquid. At least one of the inactivation steps for inactivating the material is included. Example

[0043] Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited to these examples.

[0044] (Preparation of lysis reagent A)

Lysis reagent A was prepared by the following method.

Lysozyme (derived from chicken egg white) as a lytic enzyme, Triton X-100 (registered trademark) as a nonionic surfactant, sucrose as a saccharide, and ethylenediaminetetraacetic acid (as a chelating agent) EDTA) was used. Lysozyme, Triton X-100, sucrose, and EDTA are mixed in 10 mM Tris buffer (pH 8.0) to a concentration of 8 mgZmL, 0.1 mass%, 0.8 mass%, and 50 mM, respectively. Reagent A.

[0045] (Preparation of lysis reagent B) A commercially available ATP extraction reagent (Lucifer 250+, manufactured by Kikkoman Co., Ltd.) was used as lysis reagent B.

[0046] (Preparation of amplified substrate solution and amplified luminescent enzyme solution)

An amplification substrate solution and an amplified luminescent enzyme solution were prepared by the following method.

The AMP and polyphosphate, so concentration of each 4 X 10 ^_5 M and ImM, mixed with 50 m M Tris buffer (pH 7. 4), was amplified substrate solution. In addition, PPK-ADK (amplification enzyme) was mixed with Lucifer 250+ (luminescence reagent, manufactured by Kikkoman Co., Ltd.) so as to have a concentration of 40 gZmL to obtain an amplification luminescence enzyme solution.

[0047] [Example 1]

About 25 μL of PBS solution containing approximately lOOcellsZmL of E.coli JM109 is added to 25 μL of lysis reagent 試, 50 μL of amplification substrate solution, and 100 μL of amplified luminescent enzyme solution. The change in the amount of luminescence obtained over time was measured using the Perkin-Elmer Wallac 1420 ARVO

MX

Measurement was performed using a bell microplate counter. Figure 1 shows the results obtained.

[0048] [Comparative Example 1]

The amount of luminescence was measured according to Example 1 except that lysis reagent B was used instead of lysis reagent A.

[0049] When the lysis reagent B was used, the reaction solution immediately became cloudy when mixed with the amplification substrate solution, and it was impossible to perform amplified luminescence even using the amplified luminescent enzyme solution. That is, it was found that the lysis reagent B cannot be applied to the amplified luminescence reaction. In addition, FIG. 1 shows that, according to Example 1, the presence of a trace amount (several Z measurement) of microorganisms can be detected by combining ATP extraction using lysis reagent A and ATP amplified luminescence reaction. It was done.

[0050] From the above, according to the present invention, it was confirmed that a microorganism detection method and a lysis reagent capable of sufficiently detecting even a very small amount of microorganisms can be provided.

Industrial applicability

[0051] The microorganism detection method and lysis reagent of the present invention can be used for detection of microorganisms such as bacteria using ATP as an index.

Claims

The scope of the claims

[1] An extraction step of contacting a microorganism cell with a lysis reagent to extract intracellular ATP from the microorganism cell;

An amplification step for amplifying the extracted intracellular ATP;

A light emitting step of contacting the amplified intracellular ATP, luciferin and luciferase to emit light, and

A microorganism detection method, wherein the lysis reagent contains a lysis enzyme, a nonionic surfactant and a chelating agent.

[2] The method for detecting a microorganism according to claim 1, wherein the lytic enzyme is an enzyme that hydrolyzes a cell wall.

[3] The microorganism detection method according to claim 1 or 2, wherein the lysis reagent further contains a saccharide.

[4] The concentration of the nonionic surfactant is 0.1% by mass to 1.0% by mass with respect to the total amount of the lysis reagent, and the concentration of the lytic enzyme is with respect to the total amount of the lysis reagent. 1.0 X 10 ⁵ units / mL to OX 10 ⁶ units / mL, and the concentration of the chelating agent is ⁵ mM to 10 mM with respect to the total amount of the lysis reagent. The microorganism detection method according to any one of the above.

[5] a lysis reagent comprising a lysis enzyme, a nonionic surfactant and a chelating agent