WO1999011816A1

WO1999011816A1 - Reagents for testing cleanness and methods for testing cleanness

Info

Publication number: WO1999011816A1
Application number: PCT/JP1998/003761
Authority: WO
Inventors: Tatsuya Sakakibara; Seiji Murakami
Original assignee: Kikkoman Corporation
Priority date: 1997-08-28
Filing date: 1998-08-25
Publication date: 1999-03-11
Also published as: AU8750398A; JPH1169997A

Abstract

Reagents for testing cleanness, which permit the measurement of not only ATP serving as an indication of stain but also ADP, AMP and RNA difficultly measurable by the luciferin-luciferase luminescence reagents or the prior art, and which enable the detection even of a slight stain at high sensitivity and the evaluation of cleanness at high accuracy; and methods of testing cleanness with the same. The methods comprise bringing a stain-collecting support into contact with the place to be tested to recover the support bearing a stain adhering thereto, bringing aseptic water or an extractant for microbial intracellular components into contact with the resulting support, reacting the solution thus obtained with (1) a reagent for testing cleanness comprising pyruvate orthophosphate dikinase, phosphoenolpyruvic acid, pyrophosphoric acid, luciferin, luciferase and a metal salt or (2) a reagent for testing cleanness obtained by adding to the reagent (1) at least one member selected from the group consisting of enzymes catalyzing the formation of ATP from ADP and ribonucleases, and measuring the quantity of luminescence.

Description

Description Cleanliness test reagent and cleanliness test method Technical field

The present invention relates to an improvement of a cleanliness inspection method (hereinafter, referred to as a cleanliness inspection method by AT II method) using ATP as a contamination mark using a luciferin-luciferase luminescent reagent. More specifically, in addition to ATP, an indicator component of dirt, ADP, AMP and RNA, which were difficult to measure with conventional luciferin 'luciferase luminescent reagents, were measured at the same time. The present invention relates to a cleanliness test reagent capable of detecting and accurately evaluating cleanliness, and a cleanliness test method using the reagent.

Conventional technology

Conventionally, a cleanliness inspection method using the ATP method is known.

This method is generally performed according to the following procedure.

(1) Wipe the inspection area (10 cm ² ) with a cotton swab moistened with sterile water.

(2) Rinse swab in test tube with sterile water

(3) Take a predetermined amount of the rinse solution into a test tube for measurement

(4) Add predetermined amount of ATP extraction reagent

(5) Add a predetermined amount of luminescent reagent (luciferin / luciferase luminescent reagent). (6) Measure the amount of luminescence with a luminometer (luminometer).

On the other hand, ATP is degraded by ATPase immediately after the death of the organism to become ADP or AMP. Foods and beverages, their semi-finished products, and their raw materials contain significant amounts of ADP and AMP, but they do not react with the luciferin-norrecyclase luminescent reagent.

Therefore, the conventional cleanliness inspection method using the ATP method cannot detect AMP, ADP, and RNA other than ATP, and therefore cannot perform a highly accurate cleanliness inspection.

Technical issues

The present invention is based on the conventional Luciferin 'Lucif An object of the present invention is to provide a reagent for testing cleanliness, and a method for testing cleanliness using this reagent, with the accuracy of simultaneously measuring ADP, AMP and RNA, which was difficult to measure with a perase luminescent reagent.

Solution

In order to solve such problems, the present inventors include pyruvate orthophosphate dikinase (hereinafter, sometimes referred to as PPDK), phosphoenolpyruvate, pyrophosphate, luciferin, luciferase and a metal salt. Luminescent reagent

(Hereinafter sometimes referred to as “PPDK-containing luminescent reagent”), it is possible to measure AMP in addition to ATP, which is an indicator of contamination, and to detect small contamination with high sensitivity. As a result, it was found that high-precision cleanliness inspection can be performed. In addition, when the above-mentioned luminescent reagent containing PPDK is used in combination with “enzyme that catalyzes the reaction that generates 80 to 8” and Z or “RNA degrading enzyme”, in addition to ATP and AMP, AD P and RNA Was also measured at the same time, and it was found that even less dirt could be detected and a more accurate cleanliness inspection could be performed. That is, it has been found that a more accurate cleanliness test can be performed by measuring at least one selected from the group consisting of ADP, AMP and RNA and ATP as an index.

The present invention has been completed based on these findings.

That is, (1) the present invention is a cleanliness test reagent characterized by comprising a pyruvate orthophosphodiquinidase, a phosphoenolpenolepinoleic acid, a pyrophosphate, a luciferin, a luciferase and a metal salt.

(2) Further, the present invention provides at least one selected from the group consisting of an enzyme that catalyzes a reaction for producing ATP from ADP, a reagent for extracting a microbial intracellular component, and an RNA degrading enzyme.

A cleanliness inspection reagent characterized by comprising one or more of pyruvate orthophosphate dikinase, phosphoenolpyruvate, pivalate, luciferin, lumpulase and a metal salt,

(3) The present invention also provides a cleanliness inspection method characterized by measuring at least one kind selected from the group consisting of ADP, AMP and RNA and ATP as an index, (4) Further, the present invention provides a method for producing a dirt-collecting carrier by contacting the carrier with the dirt with the inspection site, and then contacting the carrier with the dirt with sterile water. A method for testing cleanliness, comprising reacting the reagent with a cleanliness test reagent containing lysine, phosphoenorubyric acid, pyrophosphate, luciferin, luciferase, and a metal salt, and measuring the amount of luminescence generated.

(5) In addition, the present invention provides a method for producing a carrier to which dirt is collected by bringing a carrier for collecting dirt into contact with an inspection site, and then contacting a reagent for extracting a component in a microbial cell with the carrier. At least one selected from the group consisting of an enzyme that catalyzes the reaction of producing ATP from ribonuclease and RNA degrading enzyme; pyruvate orthophosphoditokinase, phosphoenolpyruvate, pyrophosphate, noresiferin, and lumferase. And a cleanliness test reagent containing a metal salt and measuring the amount of luminescence generated.

(6) In addition, the present invention provides a method for testing cleanliness containing a pyruvate orthophosphodikinase, phosphoenorubyric acid, pyrophosphate, luciferin, luciferase, and a metal salt to a test object or a processed product thereof. A cleanness inspection method characterized by measuring the amount of light generated.

(7) Further, the present invention relates to a method for producing a test object or a processed product thereof, comprising: A cleanliness test reagent containing pyruvate orthophosphate dikinase, phosphoenolpyruvate, pyrophosphate, noresiferin, noresiferase and metal salts, and measuring the amount of luminescence generated. Cleanliness inspection method.

Embodiment of the Invention

Hereinafter, the cleanliness test reagent of the present invention will be described in detail, and then a cleanliness test method using the cleanliness test reagent will be described in detail.

First, pyruvate orthophosphate dikinase used in the present invention acts on AMP, phosphoenorubyric acid and pyrophosphate in the presence of magnesium ion to catalyze a reaction to produce ATP, pyruvate and phosphate. These enzymes are already known for their physicochemical properties and manufacturing methods and are readily available. Such enzymes are known from microorganisms and plants.

Examples of microorganism-derived microorganisms include microorganisms belonging to the following genera or species.

(1) Propionibacterium shelmani

(P r op i on i b a c t e r i um s he rman i i)

[B i o c h em i st r y 10, 72 1-729 (1 97 1)],

(2) Acetobakt-xylinum (Ac e t ob a c t e r xy 1 i n urn) [Jou r a n a l o f Ba c t e r i o l o gy (1 970)].

(3) Bacteroides sympiosas (Bacteriosides)

s ymb i o s u s) LMe t o o d s i n En z ymo 1 o gy 42, 1 99-2 1 2 (1 975)] and

(4) Microbispora (for example, Microbispora

Micr obi s p o r a t h e r mo s e a I FO 1 4047.

Examples of plant-derived plants include the following plants.

(1) Maize leaf origin [B i o c em i s s t r y 12, 2862-2867 (1973)] o

(2) Sugarcane leaf origin [Th e B i o c hemi c a l J ou rn a l 1 1 4, 1 1 7-1 25 (1 9 69)] o

An example of the production of pyruvate orthophosphoditokinase produced by a microorganism is shown below.

Yeast extract 0.2%, casamino acid 0.2%, ferrous sulfate 0.001%, potassium chloride 0.05%, dipotassium phosphate 0.1%, magnesium sulfate 0.05%, lactic acid 0 50 ml of a 3% medium (pH 7.0) was placed in a Sakaguchi flask (500 ml volume), and sterilized at 121 ° C for 15 minutes.

This medium was inoculated with Microbispora sammorosa (Microbispsp0rathrermorosea) IFO14047, and this was cultured at 45 ° C with shaking to obtain a culture.

50 ml of this culture was inoculated into a 5 liter Tohsaka flask containing 1 liter of a medium having the same composition as described above, and cultured overnight to obtain a culture. This culture was inoculated at 50 Om1 into 30 liters of a mentor containing 20 liters of medium having the same composition as described above, and the aeration rate was 20 liters / minute, and the mixture was stirred. Aeration and agitation culture was performed at 45 ° C. for 24 hours under the conditions of a speed of 300 r.p.m. After completion of the culture, cells were collected from 40 liters of the culture using a Microza (manufactured by Asahi Kasei Corporation). To a portion (200 g) of the cells, 20 mM HEPES buffer (pH 7.5) containing 5 mM EDTA, 1 ml MgS04 and 1 mM DTT (hereinafter referred to as buffer A) was added, and the cells were added. The suspension was sufficiently suspended to obtain 700 ml.

Purification of pyruvate orthophosphodic kinase

The above cell suspension (700 ml) was purified by the following procedure.

step 1

(Preparation of crude enzyme solution)

8.75 g of lysozyme (manufactured by Nagase Seikagaku Co., Ltd.) was added to the above-mentioned cell suspension (700 ml), and the mixture was allowed to stand at room temperature for 2 hours while stirring slowly, followed by diammonium phosphate 2 3.1 g was added, and the mixture was further stirred at room temperature for 2 hours to disrupt the cells.

This crushed liquid was centrifuged at 700 rpm for 15 minutes, and the supernatant was collected to obtain 620 ml of the liquid.

Step 2

(First Q A E—Sephadex / Chromatography I)

Ammonium sulfate was dissolved at a rate of 2 g / 100 ml in the above-mentioned solution (620 ml), and this was previously dissolved in the buffer A (pH 7.5) containing 0.15 M ammonium sulfate. The enzyme was adsorbed on about 700 ml of QAE-Sephadex resin equilibrated in the above). This was washed with the buffer A (pH 7.5) containing 0.15 M ammonium sulfate to remove unnecessary proteins, and then the buffer A (pH 7.5) containing 0.6 M ammonium sulfate was removed. Eluted.

Step three

(Second QAE-Sephadex · Chromatography I)

The eluate was concentrated using a hollow fiber ultrafiltration device (PAN 13-DX, manufactured by Asahi Medical Co., Ltd.), and then charged with QAE-Sephadex previously equilibrated with the buffer solution A (pH 7.5). Column (diameter 6 cm x 15 cm) The enzyme was adsorbed. Then, after washing with the buffer A containing 0.15 M ammonium sulfate (pH 7.5) to remove unnecessary proteins, the buffer A containing 0.15 M ammonium sulfate (pH 7.5 ) And a buffer containing 0.8 M ammonium sulfate (pH 7.5), and eluted with 3 liters of a concentration gradient buffer.

Step 4

(Putiltoyopearl. Chromatography)

After collecting the active fraction of pyruvate orthophosphoprotein kinase from the above eluate, adding ammonium sulfate to adjust the concentration of ammonium sulfate to 1 M, and then preparing the buffer A (pH 7.5) containing 1 M ammonium sulfate in advance. The enzyme was adsorbed on a column (diameter 4.5 cm x 15 cm) packed with butyl toyopearl (manufactured by Toso Ichisha) equilibrated in step 1.

This was eluted with 1.2 liters of a buffer (pH 7.5) having an ammonium sulfate concentration gradient (1.0 M to 0 M) in the buffer A (pH 7.5).

Step 5

(Gel filtration chromatography 1)

The active part was concentrated to 2 ml using an ultrafiltration membrane device (fraction: 100,000) manufactured by Namicon Co., Ltd. Gel filtration was performed on a TSK gel G300 SWXL column (0.76 cm x 30 cm x 2) equilibrated with 0 mM HE PES buffer (pH 7.5).

All the enzymes were subjected to gel filtration, and the active part was concentrated. The active part was again subjected to the same ram as described above to perform gel filtration. All the enzymes were subjected to gel filtration, and 5.4 ml of the eluted active fraction was collected.

This fraction is a sample of this enzyme determined to be homogeneous by SDS-polyacrylamide gel electrophoresis and has a total protein content of 6.65 mg, a total activity of 66.0 U, and a specific activity of 9 .92 UZmg.

A method for determining the valency of pyruvate orthophosphoditokinase.

(Method of quantifying generated ATP by luminescence method)

50 mM BIS-TR IS PRO PANE buffer containing 3 mM magnesium sulfate, 25 mM ammonium sulfate, 2 mM 2 mercaptoethanol, 2 mM pyrophosphate, 2 mM phosphoenorubyruvic acid and 0.1 mM AMP Liquid (pH 6.8) 1 Take 180〃1 into a microtube, bring the temperature equilibrium to 37 ° C, add the enzyme solution 201 with appropriate activity, react for 15 minutes, and add Boil for 3 minutes to stop the reaction.

An appropriate dilution of this reaction solution 501 is placed in a test tube, and 501 of “Lucifer® LU” (Kikkoman) solution is dropped therein, and the luminescence is measured. Separately, prepare a graph in which the relationship between the ATP standard solution of known concentration and the amount of luminescence was examined in advance.

Using this graph, the amount of enzyme that produces 1 量 mo 1 of ATP per minute at 37 ° C is defined as one unit.

Next, examples of the luciferin used in the present invention include luciferin, a norrecifulin derivative, a luciferin precursor, and the like.

Examples of the luciferin derivative include the following.

(1) D—luciferin—0—sulfate (D—luci ferrin—0—sulph ate),

(2) D-Luciferin methyl ester (D_ 1 uci ierin

me t hy l e s t e r,

(3) D—Luciferin monophosphate—D—luciferine—0—phosphate,

(4) D—Luciferin-L—Fenilalanine (D—Lu c i F e r i n—L—H e ny l a l an i n e)

(5) D—luciferin—L——α—arginine ₀

Examples of the luciferin precursor include 2-cyano-6-methoxybenzothiazole (2-cyano-6-methoxyxybenzothiazol, D-cysteine, and the like).

Next, as luciferase, for example, the following beetles (Coleoptera)

Luciferases derived from Coloptera), mutated luciferases thereof, and luciferases derived from non-beetles.

[Coleoptera-derived Lumbera Ize] (1) Noresiola * Lu ciolacruciata,

(2) Noresiola * Lateralis (Luciol a l a t e r a l i s),

(3) Noresiola 'Minglerica' (Luciolamngreelica),

(4) Rampiris * Naktinoré power (Lampy r i s n a c t i 1 u c a), (5) Photinus' piralis (Phot innus py r a li s),

(6) Photilis-Pencilvani force

p e nn s y l v an i c a),

(7) Hoyaria Pallabira (Hot a r i a p a r vu l a),

(8) Pyroco area “Pyrocoe 1 ia miyako”, (9) Pyrophorus ”Pyrophotarums (Pyrophorus)

1 agiophtha 1 amus) ₀

(10) A noresiferase produced by the above-described luciferase and mutant luciferase by a gene recombination method.

Next, examples of metal salts include divalent metal salts, for example, magnesium salts such as magnesium sulfate and magnesium chloride, and manganese salts such as manganese sulfate and manganese chloride.

The following are examples of enzymes that catalyze the reaction of producing ATP from ADP.

(1) Pyruvate kinase (Pyruv atekine sase)

(E.C. 2. 7. 1. 40)

Substrate: Phosphoenolpinolevic acid (Phospho eno 1pyr uv ate)

(2) Kinase acetate (Ac e t a t e k i n a se)

(E.C. 2.7.2.2.1)

Substrate: acetyl'phosphoric acid (Acety1 phosphate)

(3) Creatine kinase (Creatinekinease)

(E.C. 2.7.3.3.2)

Substrate: Creatine phosphate (4) Ketohe xo kinase (EC 2.7.1.3)

Substrate: D—fructoses 1-phosphate (D—fructosose1-phosphate)

(5) Phospholipin kinase (Phospho ribu 1okinase) Substrate: D—ribulose · 1,5-diphosphate (D—ribu10se1,

5—d i pho s ph a t e)

(6) Carb ama t e k i n a se

Substrate: Carbamoinolenic acid (Carb amoyle ep pho s p a te) (7) Phosphoglycerate kinase (Pho spho gly c e r a te)

k i n a s e

Substrate: 3-Phospho-D-glyceric acid (3-Phospho-D-glycererte)

(8) Aspartate kinase (Aspartatekinekase)

Substrate: L-aspartic acid 4-monophosphate (L-Aspartate4—phosphate) and the like.

In addition, examples of the reagents for extracting intracellular components of microorganisms include surfactants (benzetonium chloride, benzalkonium chloride, triton XI 00, etc.), methanol, ethanol, a mixed solution of ethanol and ammonium, an aqueous solution of trichloroacetic acid, and perchloric acid. Aqueous solution and the like can be mentioned.

The RNase refers to an enzyme that catalyzes a reaction for producing 5′-mononucleotide (AMP, GMP, CMP, UMP) from RNA, and examples thereof include those described below.

(1) Endonuclease S 'One (EC 3.1.30.1),

(2) Venom's exonuclease (Venomexonu c 1ease) (EC 3.1.1.15.1),

(3) Phosphodiesterase 'One (Phos pho

d e s t e r a s e l) (E C 3.1.4. 1).

In addition, the above nuclease 'S-Pin' has a nuclease 'Py' (Nuclease P,), Mung bean nuclease (Mu ngbeansnuc 1 ease), Neuroshola * classa 'nuclease (eurosporacrassa nu clease), etc. are strongly included. In addition, in the present invention, in addition to the above-described components, ATP which is a component inherently contained in an enzyme activator, an enzyme stabilizer, a buffer, and a cleanliness test reagent and which causes background luminescence is also included. It is preferable to add one or more of erasing agents. Examples of the activator for the enzyme include ammonium sulfate (an activator for pyruvate orthophospho-todikinase).

Enzyme stabilizers include dithiothreitol and EDTA (a luciferase stabilizer).

Other examples include monosaccharides (eg, glucose), disaccharides (eg, sucrose), polysaccharides and sugar alcohols (eg, glycerol). As the buffer, those which maintain the luminescence reaction solution within the optimal pH range of luciferase are preferable. Glycine-NaOH buffer, HE PES buffer, and tris (hydroxymethyl) aminomethane And a Tris buffer. Examples of the ATP elimination agent of the cleanliness test reagent include ATP-degrading enzymes, such as adenosine phosphate deaminase (a method for measuring enzyme activity: see Japanese Patent Application Laid-Open No. Hei 9-118600).

Next, the preferred concentration ranges of the components of the reagent for testing cleanliness of the present invention are shown below. (1) Pyruvate orthophosphate dikinase (P PDK):

0.001 U / m1 or more (final concentration), especially 0.02 to 100 U / m1 (final concentration).

(2) Phosphoenolpyruvate:

Concentration of 0.1 mM (final concentration) or more, especially 0.5-8.0 mM (final concentration) _c (3) Pyrophosphate:

Concentration above 1.0 uM (final concentration), especially between 50 and 100 M (final concentration) c

(4) Noresifulin: concentration _c above 5.0 U (final concentration), especially 50.0 to 10000 M (final concentration)

(5) Luciferase: 'A concentration of 0.1 mg / m 1 (final concentration) or more, especially 0.5 to 20 mg / ml (final concentration).

(6) Magnesium ion:

1. OmM (final concentration) A concentration that is higher than, especially, 5.0 to 10 OmM (final concentration). (7) Ammonium sulfate:

0.1 mM (final concentration) A concentration that is higher than or equal to 0.5 mM OmM (final concentration). (8) Dithiothreitol:

0.1 mM (final concentration) A concentration that is not less than 0.5 mM, especially 0.5 to 1 OmM (final concentration).

(9) EDTA:

0. I mM (final concentration) Above, especially 0.5-1 OmM (final concentration).

(10) HE PES buffer (pH 7.0):

1 OmM (final concentration) Above, especially 20 to 20 OmM.

(11) Adenosine phosphate kinase: a concentration of 0.001 UZm 1 or more, especially 0.01 U / m 1 to 10 U / m 1.

Next, the cleanliness inspection method of the present invention will be described.

In the cleanliness inspection method of the present invention, the carrier to which dirt is collected is brought into contact with the inspection site to obtain a carrier to which the dirt is adhered, and then sterile water is brought into contact with the carrier. The measurement is performed by adding a cleanliness test reagent containing phosphate dikinase, phosphoenorubyric acid, pyrophosphate, luciferin, lumferase and a metal salt, and measuring the amount of luminescence generated.

Alternatively, a carrier for collecting dirt is brought into contact with the inspection site to obtain a carrier to which the dirt has adhered, and then a reagent for extracting an intracellular component of the microorganism is brought into contact therewith. A cleanliness inspection reagent comprising at least one member selected from the group consisting of enzymes that catalyze the reaction of ribonucleic acid and RNase; The reaction is performed by measuring the amount of luminescence generated.

Alternatively, the test object or its processed product is combined with pyruvate orthophosphate dikinase, phosphoenolpyruvate, pyrophosphate, noresifurin, lucifer The reaction is performed by reacting with a cleanliness test reagent containing a razor and a metal salt, and measuring the amount of luminescence generated.

Inspection points include cooking utensils such as cutting boards, kitchen knives and shamogi, machinery for pharmaceutical production, and food production machinery.

A soil-collecting carrier such as a cloth such as a cotton swab or a gauze, a non-woven fabric, or a sponge is brought into contact with these inspection points to obtain a soiled carrier.

At this time, if the object to be inspected is in a solid or paste form and cannot be collected by contact with the carrier, remove it with a tool (spatula, spoon, knife, etc.) and dissolve in sterile water. To obtain an aqueous solution (processed product), or to mix with sterile water to grind or filter to form a suspension or extract (processed product), and then to test samples

Use as (sample).

In the case of accumulated water or dirty water in the form of droplets, collect it with a spot and use it as it is or after diluting it with sterile water and use it as a test sample.

Next, a contact liquid obtained by bringing the extract into contact with an extraction reagent for a component in a microbial cell is prepared.

The concentration of these microbial intracellular component extraction reagents is preferably 0.001% to 1% .c. Thus, a contact solution from which microbial intracellular components, such as ATP, ADP, AMP, and RNA, are extracted is prepared. .

Next, when this contact liquid is reacted with the above-mentioned reagent for testing cleanliness, the reagent acts on ATP, ADP, AMP, and RNA to cause a luminescence reaction, and the amount of luminescence generated is measured.

The luminescence reaction between the above-mentioned microbial cell component and the cleanliness test reagent will be described below.

First, the luminescence reaction between AMP and the cleanliness test reagent will be described.

This reaction has the general formula

As shown in the dashed box (a), the AMP, pyrophosphate, phosphoeno-rubyruvic acid and magnesium ions are reacted with pyruvate orthophosphite dikinase to produce ATP, pyruvate and phosphate. As shown by the dashed line (mouth), ATP, luciferin, dissolved oxygen and magnesium ions are reacted with luciferase to produce AMP, pyrophosphate, oxylluciferin, carbon dioxide and light. According to this reaction formula, which is characterized by a combination of reactions, first, ATP is consumed by the (mouth) reaction, light is emitted, and AMP and pyrophosphate are generated. ) Regenerated into ATP by the reaction of. This ATP is again subjected to the reaction of (mouth), and the ATP is consumed to emit light.

Thereafter, these two reactions are performed simultaneously, and the luminescence is maintained at a constant level at a high level without decay for at least 10 minutes.

Next, the luminescence reaction between ADP and the cleanliness test reagent will be described. The luminescence reaction has the general formula

As shown in (2), the presence of phosphoenorubyruvic acid and magnesium ion in the reaction (a) (as described above) and the reaction (mouth) (as described above) are newly indicated by the two-dot chain frame (c). It is characterized by a combination of a reaction that converts ATP and pyruvate by pyruvate kinase, an enzyme that catalyzes the reaction that generates ATP from ADP.

In addition, another enzyme such as acetate kinase or creatine kinase may be used instead of pyruvate kinase as the enzyme that catalyzes the reaction of generating ATP from ADP used here.

The reaction formulas for acetate kinase and creatine kinase are shown below. Reaction formula of acetate kinase

Λ

(Reaction 3) Acetate kinase

Reaction formula of creatine kinase

In the reaction 2 described above, first, as shown by the two-dot chain line, a reaction in which ADP is reacted with pyruvate kinase in the presence of magnesium ion and phosphoenolpyruvate to convert it to ATP and pyruvate ( C), whereby ADP is converted to ATP, and then the ATP is consumed by the reaction (mouth) to emit light, AMP is generated, and then the AMP is converted to ATP by the reaction (ii). Is converted.

This ATP is again supplied to the reaction (mouth), and the ATP is consumed to emit light and is converted to AMP. Hereinafter, these two reactions are simultaneously and continuously repeated.

In this series of ATP conversion reaction systems, luminescence generated by the ATP conversion reaction system is stable for at least 10 minutes at a high level without decay.

The reactions 3 and 4 show the same embodiment as described above, and are not described here because they overlap.

Next, the luminescence reaction between RNA and the cleanliness test reagent will be described.

This reaction has the general formula

RNA degradation

R NA * AMP + GMP + CMP + UMP

As shown in (a) and (b), the RNA shown in the three-dot chain frame was reacted with RNase, and AMP, GM It is characterized by a combination of reactions (2) that convert it to P, CMP and UMP.

In the above reaction, first, as shown by the three-dot chain frame, the RNA is reacted with RNase, and the reaction (2) is performed to convert it to AMP, GMP, CMP and UMP. The AMP is converted to ATP by the reaction (a), and then the ATP is consumed by the reaction (mouth) to emit light and to generate AMP.

This AMP is again subjected to the reaction (a) and is converted into ATP. This ATP is then consumed by the reaction (mouth), emits light, and is converted into AMP.

Hereinafter, these reactions are simultaneously and continuously repeated.

The luminescence generated in this series of ATP conversion reaction systems is stable for at least 10 minutes at a high level without decay.

Next, a cleanliness inspection method using the dirt collection carrier storage container will be described.

In FIG. 1, the inspection location (for example, 10 cm ² ) is wiped off with a cotton swab moistened with sterile water to obtain a swab 5 with dirt attached to the dirt collection section 5a. (1) Press downward into 1 and temporarily stop the dirt collecting section 5a of the cotton swab 5 by penetrating one wall 7a of the microbial cell component extraction reagent storage tank 7 and store the extraction reagent 6 Rinse in the soil to obtain an extract of dirt (microorganisms and other dirt components), especially microbial intracellular components.

On the other hand, the luminous reagent storage tank 4 is pushed up from below the cylinder 1, and the thin film member 4 a of the storage tank is pressed against the sharp portion of the projection 8 to be cleaved.

Next, the piston 2 is further depressed from the stop position, the wiping part 5a penetrates (cleaves) the other wall surface 7b of the extraction reagent storage tank 7, and the extract of dirt is dripped to store the cleanliness inspection reagent. The mixture is mixed with the luminescent reagent in tank 4 to perform a luminescent reaction, and the amount of generated luminescence is measured with a luminometer, and the cleanliness is measured from the result.

Hereinafter, the present invention will be described more specifically with reference to examples.

Example

Example 1

Preparation of cleanliness test reagent capable of measuring ATP and AMP The following components were contained. (1) A luminescence reagent was uniformly mixed to prepare a reagent for testing cleanliness.

1) Luminescent reagent

Pyruvate orthophosphate dikinase (PPDK) 1.8 U / m 1 Phosphoenolpyruvate 4.2 mM Sodium pyrophosphate 200 / iM Luciferin 1.5 mM Luciferase (manufactured by Kikkoman) 4.5 mg / m 1 Magnesium sulfate (metal salt) 15 mM

* HEPES (buffer) 50 mM (pH 7.0)

* Sucrose (enzyme stabilizer) 0.37% by weight * EDTA2Na (inhibitor of enzyme by metal) 0 mM * Dithiothreitol (enzyme stabilizer) 0 mM * Ammonium sulfate (enzyme activation) 5 mM * Adenosine phosphate kinase 1 U / m 1 Of the above components, the HE PES buffer with the symbol “purine *” is used to stabilize the pH of the reaction system, and sucrose is luciferase. For stabilization, EDTA is used to prevent enzyme inhibition by metal, dithiothreitol is used for enzyme stabilization, ammonium sulfate is used to enhance the activation of pyruvate orthophosphoditokinase, and adenosine phosphate is also used. Deaminase is used as an ATP scavenger for cleanliness test reagents, and is not an essential component.

Example 2

Preparation of cleanliness test reagents capable of measuring ATP and AMP, which are non-microbial contaminants and microbial intracellular components

A cleanliness test reagent consisting of the following combination of (1) a luminescent reagent and (2) a reagent for extracting components from microbial cells was prepared.

1) Luminescent reagent

Same as Example 1 above.

2) Microbial cell component extraction reagent Benzalkonium chloride 0.02% by weight

HEPE S buffer (pH 7.0) 10 mM

Example 3

Preparation of cleanliness test reagents capable of measuring ATP, AMP and RNA

A cleanliness test reagent consisting of the following combination of (1) a luminescent reagent and (3) an RNA-degrading enzyme reagent was prepared.

1) Luminescent reagent

Same as Example 1 above.

3) RNA degrading enzyme reagent.

Nuclease P 'One (RNA degrading enzyme manufactured by Sigma) 1.2 U / ml HEPES buffer (pH 7.0) 10 mM Example 4

Preparation of a cleanliness test reagent that can measure ATP, AMP and RNA, which are non-microbial dirt components and microbial intracellular components

The following cleanliness test reagents were prepared by combining (1) a luminescent reagent, (2) a reagent for extracting components from microorganism cells, and (3) an RNase reagent.

1) Luminescent reagent.

Same as Example 1.

2) Microbial cell component extraction reagent

Benzalkonium chloride 0.02% by weight

HEPES buffer (pH 7.0) 10 mM

3) RNase reagent.

Nuclease 'Pie One (RNA degrading enzyme manufactured by Sigma) 1.2 U / ml HEPES buffer (pH 7.0) 10 mM Example 5

Preparation of cleanliness test reagents that can measure non-microbial dirt components and microbial intracellular components ATP, ADP and AMP

A cleanliness test reagent comprising the following combination of (1) a luminescent reagent and (2) a reagent for extracting components from microorganism cells was prepared. 1) Luminescent reagent.

Same as Example 1 except that pyruvate kinase (manufactured by Sigma) 500 U / m 1 was added to the luminescent reagent of Example 1.

2) Microbial cell component extraction reagent

Benzalkonium chloride 0.0 2% by weight

HEPES buffer (pH 7.0) 10 mM

Example 6

Preparation of a cleanliness test reagent that can measure ATP, ADP, AMP and RNA, which are non-microbial contaminants and microbial intracellular components

1) Luminescent reagent.

Same as Example 1 except that pyruvate kinase (manufactured by Sigma) 500 U / ml was added to the luminescent reagent of Example 1.

2) Microbial cell component extraction reagent

Benzalkonium chloride 0.0 2% by weight

HEPES buffer (pH 7.0) 10 mM

3) RNase reagent.

Nuclease P'Wan (Sigma) 1.2 U / ml HEPES buffer (pH 7.0) 10 mM

Example 7

(Cleanliness inspection method)

The samples described in Table 1 were dissolved or suspended in sterilized ultrapure water and diluted to the concentrations (%) shown in Table 1 before use.

If the sample was too thin to be measured, a sample with a higher concentration was measured and converted to the luminescence at each concentration by proportional calculation.

(1) Transfer the diluted water (rinse liquid) 1001 to a test tube for measurement.

(2) To this, 1001 of ultrapure water and 100 zl of the reagent for testing cleanliness (luminous reagent containing PPDK) prepared in Example 1 above are added. (3) The amount of luminescence generated was measured after 10 seconds with a luminometer LB9501 (Luminometer) manufactured by Berthold.

The cleanliness test reagent prepared in Example 1 can measure ATP and AMP.

Comparative Example 1

(Preparation of cleanliness test reagent for comparison)

A cleanliness test reagent for comparison was prepared in exactly the same manner as in the cleanliness test reagent of Example 1 except that pyruvate orthophosphoditokinase was not contained. The cleanliness test reagent prepared in Comparative Example 1 can measure ATP, but cannot measure AMP.

Comparative Example 2

(A cleanliness inspection method using the cleanliness inspection reagent of Comparative Example 1)

In the cleanliness test method of Example 7 described above, the procedure was the same except that the “cleanness test reagent” prepared in Comparative Example 1 was used instead of the “cleanliness test reagent” prepared in Example 1. The luminescence was measured.

Table 1 summarizes the respective light emission amounts obtained in Example 7 and Comparative Example 2 and the ratio of the former light emission amount to the latter light emission amount.

table 1

Comparison of the luminescence of the cleanliness test reagent of the present invention and that of the comparative example

From the results in Table 1, the cleanliness test method using the conventional luciferin-luciferase luminescence reagent (the cleanliness test reagent of the comparative example) could not detect AMP, and the cleanliness test reagent of the present invention ( It shows a lower value of luminescence compared to (luminescence reagent), making it impossible to perform highly sensitive and accurate cleanliness tests.

On the other hand, when the cleanliness test reagent of the present invention was used, a luminescence amount of 14 to 60 million times higher than that of the comparative example was obtained. It can be seen that a cleanliness inspection can be performed.

In addition, particularly when using beef extract as a test object, when using the cleanliness test reagent of the comparative example, almost no dirt can be detected, but when using that of the present invention, very high sensitivity is obtained. It turns out that dirt can be detected.

Example 8 '' (Cleanliness inspection method)

The samples described in Table 2 were dissolved or suspended in sterilized ultrapure water, and diluted to 0.01% by weight.

If the ATP concentration of the sample is too low (1 X ^10-13 M or less) and cannot be measured (samples marked with * in Table 2), measure the sample at a concentration of 1% by weight. Then, it was converted to 0.001% by weight concentration by proportional calculation.

(1) Transfer the diluted water (sample) 100 Z1 into a test tube for measurement.

(2) Add ultrapure water \ 00 and the reagent for testing cleanliness (luminous reagent containing PPDK) 100 / zl prepared in Example 1 above.

(3) The luminescence amount after 10 seconds was measured with a luminescence amount measuring device LB9501 manufactured by Berthold.

Then, the total concentration of ATP and AMP in the sample was measured.

The total concentration was determined using a calibration curve showing the linearity obtained from the relationship between the ATP standard solution having a known concentration and the luminescence amount for the following reason.

Reason: The calibration curve obtained from the ATP concentration and its luminescence amount is almost the same as the calibration curve obtained from the AMP concentration and its luminescence amount, so the total concentration of ATP and AMP is the ATP concentration and its luminescence. It can be measured using a calibration curve obtained from the amount.

The results are shown in Table 2 in the "Example (a) ATP + AMP" column.

Comparative Example 3

For comparison, in the cleanliness test method of this example, except that the `` cleanness test reagent prepared in Comparative Example 1 '' was used instead of `` the cleanliness test reagent prepared in Example 1 '', The cleanliness test was performed in exactly the same way, and the ATP concentration in the sample was determined. The results are shown in the column of “Comparative example (mouth)”.

The value of this example (the ratio of ATP concentration) to the ATP concentration of Comparative Example 3 was determined, and the results are shown in the column of “Example (a) / Comparative example (mouth)”. Table 2

Comparison of ATP concentration between the cleanliness test reagent of the present invention and that of the comparative example. Example (a) Comparative example (mouth) ATP concentration ratio ATP + AMP ATP Example (a)

/ Comparative Example (Mouth) Yeast extract 4.7 1 X 10 2.14 X 10 0 2 2 0 * Beef extract 3.6 9 X 10 1.6 9 X 10 2 1 8 0 0 0 0 0 * Malt extract 1.3 7 X 10 6.97 7 X 10 19 7 * Beer 1.8 6 X 10 4.19 X 1044 4 Milk 4.76 X 10 9.29 X 10 * Rice 1.59 X 10 2.36 X 10 6 7 4 Pork 4.0 2 X 10 6.61 X10 7 From the results in Table 2, beef extract, malt extract, When samples of beer and rice are used, their ATP concentration is too low (1 X 1 (T ¹³ M or less)), and the conventional luciferin-luciferase luminescent reagent (the cleanliness test reagent of Comparative Example 1) Then, it turns out that measurement is not possible.

In addition, the cleanliness inspection method using the conventional ATP method cannot detect AMP as a pollution index, and shows a lower luminescence amount than the PPDK-containing luminescent reagent of the present invention, and provides highly accurate cleanliness. No inspection can be performed.

On the other hand, when the luminescent reagent of the present invention was used, a luminescence amount of 5 times (measurement object: milk) to about 20 million times (the same: beef extract) was obtained as compared with the comparative example. It is clear that dirt can be inspected with high sensitivity. That is, the same luminometer By measuring with-, it is possible to detect a very small amount of dirt of 1/5 to 1/20 million.

In particular, when beef extract is to be inspected, ATP is almost completely decomposed, and it can be seen that it is difficult to inspect for dirt by the conventional method of performing cleanliness inspection using ATP as an index. On the other hand, according to the present invention, since ATP and AMP are used as indices, it can be seen that a high-sensitivity test can be performed 20 million times higher than when ATP is used as an index.

Example 9

The samples described in Table 3 were dissolved or suspended in sterilized ultrapure water and diluted to 0.01% by weight.

Incidentally, the sample is too thin if (in Table 2, ※ marked with samples corresponds) can not be measured (1 X 1 0 ¹³ M or less) measures a sample of darker density, 0.1 in proportional calculation 0 0 Converted to 1% by weight concentration.

(1) Transfer the diluted water (sample) 1001 to a test tube for measurement.

(2) Add 100 u1 of the RNA degrading enzyme reagent prepared in Example 3 to this, and leave at room temperature for 15 minutes. Degrades RNA contained in the sample and converts it to AMP. (3) Add the cleanliness test reagent (PPDK-containing luminescent reagent) 1001 prepared in Example 3 above.

(4) The luminescence after 10 seconds was measured with a luminescence meter LB9501 (Noreminometer I) manufactured by Berthold.

Then, the total concentration of the components of ATP, AMP and AMP derived from RNA in the sample was measured.

The total concentration was measured using a calibration curve showing ATP concentration and luminescence for the following reasons.

Reason The calibration curve showing ATP concentration and luminescence is almost the same as the calibration curve showing AMP concentration and luminescence, so the total concentration of ATP and AMP is the same as the calibration curve showing ATP concentration and luminescence. For L, it can be measured.

The results are shown in Table 3 in the column of ATP + AMP + RNA.

In addition, this test was performed on the ATP concentration obtained using the cleanliness test reagent of Comparative Example 1. Comparative values of those of the examples were obtained, and the results are shown in the column of “Example (a) Z comparative example (mouth)”.

Table 3

Comparison of ATP concentration between the cleanliness test reagent of the present invention and that of the comparative example Sample Example (a) Comparative example (mouth) ATP concentration ratio

ATP + AMP ATP Example (a)

+ RNA / Comparative Example (mouth) yeast extract 9 5 8 X 1 0 -. . 9 2 1 4 X 1 0 1 '4 4 8

* Beef extract 5.45 X 10 0-"1.6 9 X 1 0 — ' ⁵ 3 2 2 0 0 0 0 0

* Malt extract 1.9 3 X 10 "" 6.9 7 X 10 ¹⁴ 2 7 7

※ beer ^{2 5 8 X 1 0 12 4} 1 9 X 1 0 -.... '5 6 1 6 milk 1 1 5 X 1 0 "9 2 9 X 1 0- 1 3 1 2

※ rice 3 7 0 X 1 0 -. '' 2 3 6 X 1 0 -.. 14 1 5 6 8 pork 1 7 1 X 1 0 - ' . 5.6 1 X 10 ¹² 30 From the results in Table 2 and Table 3, the cleanliness test reagents (ATP, AMP and RNA) prepared in Example 3 consisting of a combination of a luminescent reagent and an RNase reagent were used. Of yeast extract: about 2 times (4 48 Z22) compared to the cleanliness test reagent prepared in Example 1 (ATP and AMP can be measured) without using RNase reagent. 0), beef extract: about 1.5 times (about 30 million / about 20 million), malt extract: 1.4 times (27/7/197), beer: 1.3 times (6 16) / 4 4 4), Milk: 2.4 times (1 2 Z 5), Rice: 2.3 times (1 5 6 6/6 7 4), Pork: 4.5 times (30 Z 7) It was found that the sensitivity was good.

Example 10

(1) Wipe the surface of the rice scoop with a cotton swab moistened with sterile water (10 cm). This rice scoop is the rice scoop used to separate cooked rice, which is lightly rinsed with water.

(2) The swab was rinsed in a test tube filled with sterile water (ultrapure water).

Then, 5 ml of the rinsing liquid is obtained. (3) Fill the test tubes A and B with 100 µl of the rinse solution.

(4) Add 100p.1 of ultrapure water to one test tube A. To the other test tube B, add the RNA degrading enzyme reagent 1001 prepared in Example 3 and leave it at room temperature for 15 minutes (decompose the RNA contained in the sample and convert it to AMP).

(5) To test tube A and test tube B, add the cleanliness test reagent (luminescent reagent) 1001 prepared in Example 1 and Example 3, respectively.

(6) The light emission amount after 10 seconds was measured by a light emission amount measuring device LB9501 (Norenminometer-1) manufactured by Berthold.

Then, the total amount of the concentrations of ATP, AMP and RNA-derived AMP in the rinse was measured.

The AMP concentration was determined by converting it to ATP.

Table 4 summarizes the results.

Table 4 (Results of inspection of cleanliness of rice rinsing waste liquid)

Example 1 1

(1) The rice scoop (inspection area) (10 cm ² ) was wiped off with a cotton swab moistened with ultrapure water. This rice scoop is made by rinsing the rice scoop used to separate cooked rice gently with water and spraying a suspension of various bacteria separated from old cooked rice. is there.

(2) The swab was rinsed in a test tube containing 5 ml of the intracellular component extraction reagent solution prepared in Example 2.

5 ml of a rinsing liquid from which dirt other than the germs and ATP which is an intracellular component of the germs were respectively extracted was obtained.

(3) Rinse solution (sample) 10 Q fil was taken in each of four test tubes A, B, C and D for measurement.

(4) Then, ultrapure water 1001 was added to test tubes A and B, respectively, and the RNase reagents prepared in Examples 4 and 6 were added to test tubes C and D, respectively. Add 1001 and leave at room temperature for 15 minutes (decompose the RNA contained in the sample and convert it to AMP).

(5) Then, the luminescent reagents 1001 prepared in Examples 2, 5, 5, and 6 were added to Examiners A, B, C, and D, respectively, and the amount of luminescence measured by Berthold was measured. The amount of luminescence after 10 seconds was measured with a vessel LB9501 (Noreminometer).

Then, the total concentration of the components of ATP, ADP, AMP and AMP derived from RNA in the sample was measured.

The AMP concentration was determined by converting it to ATP.

Table 5 summarizes the results.

Table 5 (Test results of cleanliness of rice rinsing wastewater contaminated with various bacteria)

Example 1 2

(1) Using a cotton swab moistened with ultrapure water, the area near the injection hole of the minced meat making machine immediately after use was lightly rinsed with water and the surface was wiped off (10 cm ² ).

(2) The swab was rinsed in a test tube containing ultrapure water.

Then, 5 ml of a rinsing liquid was obtained.

(3) Rinse solution 100/1 was taken in each of test tubes A and B for measurement.

(4) Then, add 100 μl of ultrapure water to test tube A and 1001 of the RNA degrading enzyme reagent prepared in Example 3 to test tube B, and leave at room temperature for 15 minutes. (San Decompose the RNA contained in the pull and convert it to AMP).

(5) Then, the luminescent reagent 1001 prepared in Example 1 and Example 3 were added to test tubes A and B, respectively, and a luminescence amount meter L B9501 manufactured by Berthold was used.

(Luminometer 1), the luminescence amount after 10 seconds was measured.

The AMP concentration was determined by converting it to ATP.

Table 6 summarizes the results.

Table 6 (Results of inspection of cleanliness of rinse liquid of minced meat making machine)

Example 13

(1) Using a cotton swab moistened with ultrapure water, lightly rinse the area around the injection hole of the minced meat making machine immediately after use with water, and spray this surface with a suspension of various bacteria separated from old pork.

(Inspection location) (10 cm ² ) was wiped off.

5 ml of a rinsing liquid from which dirt other than the germs and ATP, AMP and RNA, intracellular components of the germs were respectively extracted, were obtained. (3) Rinse solution (sample) 100 C1 was taken in each of four measurement test tubes A, B, C and D.

(4) Then, test tube A and test tube B contain 100/1 ultrapure water, respectively, and test tube C and test tube D contain RNase prepared in Example 4 and Example 6, respectively. Add reagent 1001 and leave at room temperature for 15 minutes (decompose the RNA contained in the sample and convert it to AMP).

(5) Then, add the luminescent reagent 1001, prepared in Examples 2, 5, 5, and 6, respectively, to test tubes A, B, C, and D, and measure the luminescence from Berthold. The luminescence after 10 seconds was measured using a LB9501 (Nolemminometer-1).

The AMP concentration was determined by converting it to ATP.

Table 7 summarizes the results.

Table 7 (Results of the cleanliness inspection of the rinsing waste liquid of the minced meat manufacturing machine contaminated with various bacteria)

The invention's effect

Since the present invention uses a PDKK-containing luminescent reagent, ADP, AMP and RNA, which were difficult to measure with a conventional luciferin-lumpulase luminescent reagent, in addition to ATP, which is an indicator of contamination, were used. By measuring at the same time, even a small amount of dirt can be detected with high sensitivity and the cleanliness can be evaluated accurately. In addition, the enzyme that catalyzes the reaction to generate ATP from ADP and Z or ribonuclease are added to the PPDK-containing luminescent reagent. In addition, ADP and RNA are also measured at the same time, Unclean dirt can be detected with high sensitivity and more accurate cleanliness inspection can be performed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic view showing one specific example of an instrument for performing the cleanliness inspection method of the present invention.

1 cylinder

2 piston

3 Cleanliness test reagent

4 Storage tank for cleanliness test reagent

4a Thin film member

5 Dirt collection carrier (cotton swab)

5 a Dirt collection section

6 Microbial cell component extraction reagent

7 Microbial cell component extraction reagent storage tank

Ding a tffl

7 b ia

8 protrusion

Claims

The scope of the claims

1. A cleanliness test reagent characterized by containing pyruvate orthophosphate kinase, phosphoenorubyruvic acid, pyrophosphate, luciferin, luciferase and metal salts.

2. At least one selected from the group consisting of an enzyme that catalyzes the reaction of producing ATP from ADP, a microbial intracellular component extraction reagent, and an RNase, pyruvate orthophosphate dikinase, phosphoenorubyric acid, A reagent for detecting cleanliness, comprising pyrophosphate, noreciferin, luciferase and a metal salt.

3. A cleanliness inspection method characterized in that at least one selected from the group consisting of ADP, AMP and RNA and ATP are measured as indices.

4. Contacting the inspection site with a carrier for collecting dirt to obtain a carrier to which the dirt is attached, and then contacting it with sterile water, and contacting the resulting contact solution with pyruvate orthophosphate dikinase, phosphoenolpyruvate, A cleanliness test method comprising reacting a cleanliness test reagent containing pyrophosphoric acid, luciferin, norecipherase and a metal salt, and measuring the amount of luminescence generated.

5. Contact the dirt-collecting carrier with the inspection site to obtain the dirt-retained carrier, and then contact the microbial cell component extraction reagent with this. The resulting contact solution is reacted with ADP-forming ATP from ADP. Cleanliness inspection including at least one selected from the group consisting of catalyzing enzymes and ribonucleases, and pyruvate orthophosphate dikinase, phosphoenolpyruvate, pivalic acid, luciferin, norecyclase and metal salts A cleanliness inspection method characterized by reacting with a reagent and measuring the amount of luminescence generated.

6. Luminescence generated by adding a cleanliness test reagent containing a pyruvate orthophosphoditokinase, phosphoenorubyruvic acid, pyrophosphate, luciferin, luciferase and a metal salt to the test object or its processed product A cleanliness inspection method characterized by measuring the amount.

7. The reaction of ATP generation from ADP to the test object or its processed At least one selected from the group consisting of a catalyzing enzyme, a microbial cell component extraction reagent, and an RNase; A cleanliness inspection method characterized by adding a cleanliness inspection reagent containing, and measuring the amount of luminescence generated.