WO2005090592A1 - Method of high-sensitivity detection of microbe, and luminescence measuring reagent kit and system for microbe detection - Google Patents

Abstract

A method of measuring microbial proliferation and/or activity, characterized in that a microbe is incubated together with oxidized quinone in a culture solution, and that any product is reacted with a chemiluminescence reagent, followed by quantitative determination. This reaction is carried out in the presence of ions of at least one metal selected from the group consisting of molybdenum, manganese, nickel and cobalt. There is provided a luminescence measuring reagent kit for microbe detection, including oxidized quinone, a chemiluminescence reagent and at least one compound being a supply source of ions of a metal selected from the group consisting of molybdenum, manganese, nickel and cobalt. There is further provided a luminescence measuring system for microbe detection, including this kit and a device for concentrating a microbe solution. The provided method of microbe detection and luminescence measuring reagent kit and system realize a high detection limit for microbes, are suitable for speedy measuring of a multiplicity of test subjects and permit measuring over a wide range of pH.

Description

 Specification

 Highly sensitive detection method of microorganisms, reagent kit and system for luminescence measurement for microorganism detection

 Technical field

 The present invention relates to a method for detecting microorganisms with high sensitivity, a reagent kit and a system for measuring luminescence for detecting microorganisms.

 Background art

 [0002] A microbial test that is routinely performed is a method in which a culture solution or an extract containing microorganisms is mixed with an agar medium and cultured in an incubator until colonies are visually observed. The power of this method has been considered a highly accurate method. Recently, the presence of living but uncultivable microorganisms has become known, and a new method for measuring living microorganisms has become necessary.

 [0003] As a method for measuring a living microorganism without relying on a culture method, there is a method for measuring an enzyme activity or ATP of a cell. The ATP method is a method of measuring the concentration of microbial microorganisms by measuring the concentration of adenosine triphosphate (ATP) in cells by biochemiluminescence using D_noreciferin and luciferase. However, differences in the operation method may result in inability to determine whether the microorganism is alive or dead. It has been pointed out that dead microorganisms may also contain ATP as a cause, and that ATP other than microorganisms is mixed during experimental operations. In particular, removal of non-microbial ATP is the biggest problem, and treatment with ATPase is one of the methods for removing ATP existing outside the body of microorganisms. However, even after this operation, ATP equivalent to several hundred microorganisms remains. As a result, the detection sensitivity of microorganisms could not be further improved.

 [0004] Japanese Patent Application Laid-Open No. 1-169499 (Document 1) discloses a method for measuring the amount of hydrogen peroxide generated when quinone is added to living cells using chemiluminescence. However, since this method quantifies only hydrogen peroxide, accurate measurement was not possible when 0- was the main product.

[0005] Japanese Patent Publication No. 2883149 (Reference 2) discloses that living cells or tissues can be prepared simply by adding a drug. A method for measuring the number of viable cells using menadione and an iron chelator has been disclosed as a method for nondestructively and rapidly performing quantitative measurements on living cells with continuous light emission. . In this method, menadione, luminol, and an iron chelating agent are simultaneously added under alkaline conditions, and each sustained luminescence is measured for several minutes. For this reason, many samples could not be measured quickly, the activity could not be maintained in an alkaline solution, and they were unsuitable for microorganisms with low reactivity with microorganisms and menadione.

[0006] Japanese Patent Application Laid-Open No. 2001-120299 (Literature 3) discloses the growth of a microorganism characterized by incubating the microorganism with oxidized quinone in a culture solution and quantifying the product by a chemiluminescence method. And methods for measuring Z or activity are disclosed. This method allows the use of a variety of culture media to determine the growth and activity of a wide variety of microorganisms, and does not interfere with the measurement of catalase or other enzymes from microorganisms and does not affect the presence or absence of oxygen. This is the method.

 [0007] Today, a wide variety of foods are produced, and in order to manage these food hygiene, a method that enables quick and simple microbial testing at a manufacturing site has been required. In order to cope with this, the ATP method has been devised in various ways, but has not reached the point where it can be easily used by users in terms of cost, cost and technology. Furthermore, any of the methods described in Literatures 13 to 13 has a problem that the detection limit is still high as a quick and simple test of microorganisms performed at a manufacturing site to control the food hygiene.

 [0008] Rather, there is a kit on the market that checks the degree of cleaning at the manufacturing site by measuring ATP derived from foodstuffs, which is not related to the determination of dead bacteria. However, this kit only measures food-derived ATP and does not test for viable microorganisms.

[0009] Accordingly, an object of the present invention, the detection limit for microorganisms, than conventional methods are suitable for high ingredients and rapid measurement of the conventional method and the same or more than that number analytes, and Hirore, _P H region It is an object of the present invention to provide a method for detecting a microorganism which can be measured in a microorganism.

 Another object of the present invention is to provide a luminescence measurement reagent kit and a system for detecting microorganisms for use in such a method.

[0010] The inventor of the present invention has proposed a method in which a suitable quinone is allowed to act under the optimal pH conditions of microorganisms, and active oxygen generated there can be instantaneously and efficiently quantified with high sensitivity. The present inventors have found that the above objects can be achieved by using the present invention, and have completed the present invention.

Disclosure of the invention

 The present invention for solving the above problems is as follows.

 [I] A method for measuring the growth and / or activity of a microorganism, comprising incubating the microorganism with an oxidized quinone in a culture solution, and quantifying the product by reacting the product with a chemical luminescent reagent. The method in the presence of at least one metal ion selected from the group consisting of molybdenum, manganese, nickel and cobalt.

[2] The method according to [1], wherein a chelating agent is used in combination with the metal ion.

[3] The method according to [1] or [2], wherein the product is reduced quinone and / or superoxide ion (active oxygen).

 [4] The method according to [1], wherein the chemiluminescent reagent is luminol or a derivative thereof.

 [5] The method according to any one of [1] to [4], wherein the oxidized quinone is menadione or coenzyme Q1.

 [6] The method according to any one of [1] to [5], wherein the concentration power of the metal ion is in a range of 100 / iM.

 [7] Microbial power S incubated with the oxidized quinone, obtained by culturing microorganisms and collecting the precipitated microorganisms by centrifugation. [1] The method according to any of the above.

 [8] Microbial power S incubated with the oxidized quinone, obtained by culturing microorganisms, dehydrating them, and concentrating the bacterial solution [1]. The method described in.

 [9] The method according to [7] or [8], wherein the cultured microorganism is a living microorganism.

 [10] A luminescence measurement reagent kit for detecting microorganisms, comprising at least one compound serving as a metal ion supply source selected from the group consisting of oxidized quinone, chemiluminescent reagent, molybdenum, manganese, nickel and cobalt.

 [10] The kit according to [10], further comprising a chelating agent.

[12] A microorganism comprising the kit according to [10] or [11] and an apparatus for concentrating a microorganism solution. Luminescence measurement system for detection.

 [13] The system according to [12], further comprising a fluorescence emission measuring device.

[0012] According to the method, kit and system of the present invention, live microorganisms can be directly, rapidly and simply measured with sensitivity comparable to the ATP measurement method. In particular, according to the method, kit and system of the present invention, it is possible to easily and quickly perform a sterility test of a product that is not limited to detection of microbial contamination at a food production site.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0013] The method of the present invention is a method for measuring the growth and / or activity of a microorganism, which comprises incubating the microorganism with an oxidized quinone in a culture solution and reacting the product with a chemiluminescent reagent to quantify the product. is there.

[0014] The microorganism to be measured in the method of the present invention is not particularly limited, and examples thereof include intestinal bacteria, Pseudomonas aeruginosa, influenza, staphylococci, hemolytic streptococci, enterococci, bacillus, batateroides, Clostridium and the like.

[0015] A respiratory chain is present in the cell membrane of microorganisms, and oxidizes gnorecose and alcohol to secure energy for life support. In the respiratory chain, there are proteins with many electron transfer functions including cytochrome components. Also, quinone reductase is present in the periplasmic cytoplasm of gram-negative bacteria. Therefore, when quinone is added from outside the cell, oxidized quinone is reduced by microorganisms, and the reduced product further reacts with dissolved oxygen in the culture solution to generate unstable superoxide ion. Guessed. In addition, if there is no dissolved oxygen, it is assumed that the reduced form of quinone will continue to exist in the culture solution as it is. That is, the product obtained by incubating the microorganism with oxidized quinone is presumed to be reduced quinone and z or superoxide ion (active oxygen).

In the method of the present invention, when an oxidized quinone such as menadione is added to a bacterium such as a gram-negative bacterium or a gram-positive bacterium and incubated, under aerobic conditions, superoxydionion (〇-) is produced. Occurs in the culture. Then, by quantifying the superoxide ion by a chemiluminescence method, it is possible to measure the growth and activity of the bacteria. Specifically, bacterial growth and activity can be quantified from the luminescence intensity of a chemiluminescence reaction using a luminescent reagent such as luminol or a derivative thereof. Luminol derivatives include, for example, Sonoremino ¹ ~~ nore and 8— Amino— 5— chloro— 7— phenylpyriod [3,4—]

 pyridazine-1,4- (2H, 3H) dione sodium salt and the like.

Further, in the method of the present invention, the reaction between the product and a chemiluminescent reagent is carried out in the presence of at least one metal ion selected from the group consisting of molybdenum, manganese, nickel and cobalt. From the viewpoint of measuring with high sensitivity, the concentration of the metal ion is suitably in the range of 100 μΜ, and preferably in the range of 550 μΜ.

 Compounds serving as molybdenum ion sources include, for example, Na NaοΜ 2Η0, MoF, MoO

, MoS-2HO can be power S.

 The compound serving as a source of manganese ions is, for example, MnCl4Η0, (CHCOO) Μη

4H0, Mn (NO) 6Η0, MnSO5Η〇.

 Compounds serving as a source of nickel ions include, for example, NiCl · 6Η〇, NiCO, (HCOO) Ni

• 2H 0, Ni (NO) · 6Η〇, Ni (S〇-6H O)

 Compounds serving as sources of cobalt ions include, for example, CoCl.sub.6Η0, Co (NO) 6Η0,

CoSO · 7Η 0, Co (ΡΟ) · 8Η Ο.

 [0018] In this reaction, a chelating agent is preferably used in combination. As a chelating agent, for example, EDTA (ethylenediaminetetraacetic acid), EDDP, Bicine, DPTA, EDDP, DTPA, EGTA,

 Methyl-EDTA, NTA, NTP, NTPO can be mentioned.

 It is desirable that the selection of the metal ion and the chelating agent and their concentrations be appropriately performed according to the type of the specimen. The concentration of the chelating agent can be, for example, 10 times the metal salt concentration.

 [0020] In the method of the present invention, the reaction with a chemiluminescent reagent is made of molybdenum, manganese, nickel and cobalt so that active oxygen generated by the reaction between a living microorganism and quinone can be detected efficiently by gamma luminescence. Performed in the presence of at least one metal ion selected from the group. More specifically, it is possible to make the quinone-containing solution contain a compound that is a source of the metal ions. Further, this quinone-containing solution can also contain the above-mentioned chelating agent.

In the present invention, by using the specific metal ion as a catalyst, the background value is reduced, and it becomes possible to track a slight change in the light emission intensity. Previously, peroxidase was used as a catalyst. The concentration of microorganisms 10 times higher than the detection sensitivity was the detection limit. Also, Japanese Patent No. 2883149 discloses a method for measuring the number of viable cells using menadione and an iron chelating agent. However, the method of the present invention is more detectable than the method using an iron chelating agent. Limit values can be lowered. This point will be described in Examples.

[0022] As the oxidized quinone, for example, menadione or coenzyme Q1 can be used. Menadione reacts with most cells (animal cells, plants, yeasts, bacteria) and effectively produces reactive oxygen. However, for cells with high fat content, it is preferable to use Coenzyme Q1. For example, reacting a congener Q1 with a high fat content bacterium, such as Mycobacterium tuberculosis, promotes the production of active oxygen more efficiently than menadione and improves the measurement sensitivity.

 [0023] Specifically, the method of the present invention is carried out, for example, by adding a quinone-containing liquid (oxidized quinone and molybdenum, manganese, nickel and nickel) to a sample liquid (for example, a liquid in which microorganisms may exist). (Contains at least one metal ion selected from the group consisting of cobalt.) 50 / i 1 is mixed, incubated at a predetermined temperature for 10 minutes, and further mixed with a chemiluminescent reagent 100 / i 1 to emit light for several seconds. By measuring the strength, the growth and / or activity of the microorganism can be measured. Thus, the method of the present invention is a simple and quick method. The number of viable microorganisms that can be detected by the method of the present invention is several hundred CFU (colony forming units) / 50 μl in the case of bacteria, and several tens of CFU / 50 il in the case of yeast. For this operation, for example, a 96-well plate or the like can be used, so that many types of samples can be measured at a time.

 [0024] The emission intensity can be measured using a commercially available tube-type and microplate-type luminometer. When a 96-well plate or the like is used, a microplate-type luminometer is used to measure the emission intensity. A meter can be used.

 [0025] In the method of the present invention, the sample liquid (for example, a liquid containing microorganisms) is obtained by culturing live microorganisms, centrifuging the microorganisms, and collecting the precipitated microorganisms. Can be something. Alternatively, the sample liquid (for example, a liquid in which microorganisms are present) can be obtained by culturing living microorganisms, dehydrating them, and concentrating the bacterial liquid. The detection sensitivity can be increased by using the microorganism-containing specimen solution thus concentrated.

[0026] Specifically, as described above, a low concentration that cannot be detected by a reaction with quinone for 10 minutes is used. As described above, the detection sensitivity can be increased by further culturing the microorganism to increase the concentration, or by increasing the concentration of the microorganism in the medium by centrifugation or dehydration treatment (or a combination thereof). The former requires a centrifuge, but for example, centrifugation at 4000g for 5 minutes completes the operation and is convenient. The latter requires a water absorption time of about 30 minutes, but it is simple because a disposable water-absorbing agent can be used and a centrifuge is not required. Combining cultivation and these concentration operations can further improve sensitivity.

[0027] For example, Escherichia coli is designated as a food contamination indicator, and there are many foods that must be E. coli negative. Furthermore, if the sterility test of other bacteria including Escherichia coli can be performed, the safety of food can be confirmed. In the chemiluminescence method of the present invention, the sterility test can be completed in about 4 to 5 hours by combining the culturing and the concentration operations. A sterility test using a conventional agar medium takes two days, and the ATP assay determines ATP after culturing for several hours under different culture conditions.

 [0028] The present invention provides a luminescence measurement for detecting microorganisms, comprising at least one kind of a compound serving as a source of a metal ion selected from the group consisting of oxidized quinone, a chemiluminescent reagent, molybdenum, manganese, nickel and covanolate. Reagent kit. As the compound serving as a supply source of a metal ion selected from the group consisting of oxidized quinone, chemiluminescent reagent, molybdenum, manganese, nickel and cobalt, those described in the method of the present invention can be used. The kit of the present invention can further include a chelating agent in addition to the above. As the chelating agent, those described in the method of the present invention can be used. This kit of the present invention can be used in the above-described method of the present invention.

 [0029] The chemiluminescent reagent is a powder, which is dissolved in an alkaline buffer and used at the time of measurement.

 Long-term cold storage is possible, and the performance of the luminescent reagent is stable.

 Oxidized quinone is a powder, which is dissolved in ethanol and stored. When measuring, mix with a buffer containing a metal salt and a chelating agent.

 [0030] As an example of these uses, first, 50 µl of a bacterial solution is put into each well of a 96-well plate, and then 50 µl of a mixed buffer of oxidized quinone 'metal salt' chelating agent is added for 10 minutes. Incubate at the specified temperature. Then, add 100 µl of luminescent reagent and immediately measure the fermentation intensity.

[0031] The present invention further provides a microorganism comprising the above-described kit of the present invention, comprising a device for concentrating a microorganism solution. Includes a luminescence measurement system for detection. The apparatus for concentrating a microorganism solution can be a centrifuge or a water-absorbing agent (for example, preferably a disposable type).

. Specific examples of the water absorbing agent include “Mizubutori-kun” AB-1100 biological solution sample concentrating agent for research, which is sold by Atoichi Co., Ltd.

[0032] The luminescence measurement system of the present invention can further include a fluorescence luminescence measurement device.

[0033] As described above, the system in which the luminescence measurement reagent kit of the present invention is combined with a concentrating operation device enables a microbial assay to be carried out by a simple operation, and is a product that can be detected only by detecting microbial contamination at a food manufacturing site. Can be easily and quickly performed.

 Example

 Hereinafter, the present invention will be described in detail with reference to examples.

 Example 1

 (Comparison of catalytic effects of various metals)

 Escherichia coli (Escherichia coli ATCC25922) was pre-incubated overnight in a cation-adjusted Mueller-Hinton medium, and then diluted with the same medium, and the detection limit was determined by a chemiluminescence method. The number of viable bacteria in the diluent was cultured overnight in TSA medium, and CFU / ml was determined from the number of colonies. The operation of the chemical luminescence method was performed as follows.

 (1) Mix 50 µl of the diluted bacterial solution with 50 µl of menadione (10 mg / l) / metal (2-200 µM / EDTA (2 (T 2000 µΜ) mixed solution).

 (2) Leave at 37 ° C for 10 minutes.

 (3) Inject 100 μl of luminol reagent.

 (4) Measure the luminescence intensity for 2 to 5 seconds immediately after the injection.

 [0035] The results in Table 1 show only a part of the effect of the metal used as the catalyst. However, the detection sensitivity is improved as compared with the case where no metal is added when the addition of 20 is used. 30 times higher sensitivity. Thus, it can be seen that the metals in the table have a catalytic effect. Furthermore, it can be seen that molybdenum, manganese, Eckhenore and cobalt have higher sensitivity than iron.

[Table 1] Table 1 Detection limits of Escherichia coli using various metals

 Detection limit (CFU / ml)

 At 2 M

FeS0 ₄ 1.6 X 10 ⁴

NiCl ₂ ■ 6H ₂ 0 1.6 10 ⁴

Na ₂ Mo0 ₄ ■ 2H ₂ 0 4.1 X 10 ³

MnCl ₂ 4H ₂ 0 1.6 X 10 ⁴

CoCl ₂ ■ 6H ₂ 0 6.6 10 ⁴

 At 20 jU M

FeS0 ₄ 3.3 10 ⁴

NiCl ₂ ■ 6H ₂ O 8.2 X 10 ³

Na ₂ Mo0 ₄ ■ 2H ₂ 0 4.1 X 10 ³

MnCl ₂ ■ 4H ₂ 0 4.1 X 10 ³

CoCl ₂ ■ 6H ₂ 0 8.2 10 ³

1.3 10 ^{5 with} no metal added

Example 2

 (Creating a standard curve for E. coli)

 The Escherichia coli cultured overnight in a Myura Hinton medium was diluted and quantified by the chemiluminescence method of Example 1. The metal and chelating agent used this time were 20 μ μ Na MoO2 · and 200 μΜ

 2 4 2

EDTA. As shown in FIG. 1, quantification was possible from about 200 CFU / 50 / il (4,000 CFU / ml) to 10 ⁶ CFU / 50 / i1 (2 × 10 ⁷ CFU / ml) in 10 minutes of measurement.

Example 3

 (Reduction of E. coli detection time)

An attempt was made to shorten the detection time of Escherichia coli after overnight culture by the chemiluminescence method of Example 2. Figure 2 shows the relationship between the culture time and the number of initial bacteria when the luminescence intensity of the background reached 1.5 times or more. In the figure, XI indicates that the bacteria were not concentrated by centrifugation, X10 indicates that the bacterial solution was concentrated 10-fold by centrifugation (4000 g for 5 minutes), and X100 indicates that the bacteria were concentrated 100-fold. ing. Tens of thousands of CFU / ml without enrichment culture CFU / 50/1 was detected immediately and several hundred CFU / ml was detected at 100-fold concentration. In the enrichment culture, the initial number of bacteria was detected after 6 hours when the initial number of bacteria was several CFU / ml, and was detected after 4 hours in the 100-fold concentration operation.

Example 4

 (Creating a standard curve for M. bovis)

 Mycobacterium tuberculosis is rich in fat components and could not be quantified with menadione. Therefore, according to the method of Example 2, the medium was changed to Middlebrook 7H9, and 300 μl instead of menadione.

As shown in Fig. 3, it was found that the use of Coenzyme Q1 of Μ can detect bovine tuberculosis bacteria up to 100,000 zOO bacteria solution or 2000 CFU / ml bacteria solution. Mycobacterium bovis CFU was measured after 3 weeks of culture on Middlebrook 7H9 agar. Thus, it was found that tuberculosis bacterium can be quantified by chemiluminescence much faster than the measurement on a conventional agar medium.

 Industrial applicability

[0040] The method of the present invention can be used for microbial testing at a manufacturing site to control food hygiene.

 Brief Description of Drawings

FIG. 1 is a standard curve of Escherichia coli obtained in Example 2.

 FIG. 2 shows the relationship between the culture time and the number of initial bacteria when the luminescence intensity of the background obtained in Example 3 reaches 1.5 times or more of the luminescence intensity.

 FIG. 3 is a standard curve of Mycobacterium bovis obtained in Example 4.

Claims

The scope of the claims  [I] A method for measuring the growth and / or activity of a microorganism, comprising incubating the microorganism with an oxidized quinone in a culture solution, and quantifying the product by reacting the product with a chemical luminescent reagent. The method in the presence of at least one metal ion selected from the group consisting of molybdenum, manganese, nickel and cobalt. [2] The method according to claim 1, wherein a chelating agent is used in combination with the metal ion. 3. The method according to claim 1, wherein the product is reduced quinone and / or superoxide ion (active oxygen). [4] The method according to any one of [13] to [13], wherein the chemiluminescent reagent is luminol or a derivative thereof.  [5] The method according to any one of [1] to [14], wherein the oxidized quinone force is S menadione or Coenzam Q1.  [6] The method according to any one of claims 15 to 15, wherein the concentration power of the metal ion is in a range of 100 / iM.  7. The method according to claim 1, wherein the microorganism to be incubated with the oxidized quinone is obtained by culturing the microorganism, centrifuging the microorganism, and collecting the precipitated microorganism. The described method.  8. The method according to claim 4, wherein the microorganism incubated with the oxidized quinone is obtained by culturing the microorganism, dehydrating the microorganism, and concentrating the bacterial solution. the method of.  [9] The method according to claim 7 or 8, wherein the cultured microorganism is a living microorganism. [10] oxidized quinone,  Chemiluminescent reagent,  At least one compound that is a source of metal ions selected from the group consisting of molybdenum, manganese, nickel and cobalt;  A luminescence measurement reagent kit for detecting microorganisms, comprising:  [11] The kit according to claim 10, further comprising a chelating agent. [12] A microorganism test comprising the kit according to claim 10 or 11 and a device for concentrating a microorganism solution. Emission measurement system for exit.  13. The system according to claim 12, further comprising a fluorescence measurement device.