WO2000065022A1 - Instrument for testing specimen and instrument for wipe test - Google Patents

Abstract

An instrument for testing a specimen whereby vital cells are collected from a specimen and ATP originating in the cells is measured at a high accuracy. This instrument is provided with a test tube-form measurement container, a tubular container for an ATP eliminator fixed at the upper part in the top opening of the measurement container, and a tubular container for an extraction agent fixed below the ATP-eliminator container in the opening of the measurement container. A phosphorescent reagent is put into the measurement container. The container for the ATP eliminator is provided with a sealing material adhered to both of the top and bottom openings and a free ATP eliminator is enclosed therein. The container for the extracting agent is provided with a sealing material adhered to both of the top and bottom openings and an extracting agent for extracting the above-described ATP is enclosed therein.

Description

 Description Specimen test equipment and wipe test equipment Technical field

 The present invention relates to a device for collecting biological cells in a liquid or on a solid surface, removing free adelicin triphosphate (hereinafter abbreviated as free ATP), and measuring the amount thereof by a luminescence reaction. Background art

 Measurement of Escherichia coli, yeast, lactic acid bacteria, and other biological cells is very important in fields such as food hygiene, biotechnology, clinical testing, medicine, ultrapure water, and the environment.

 In general, biological cells are measured using a hemocytometer under a microscope (microscope method), turbidity measurement method, weighing method, packed 'volume (packed volume) ^] method, and colony counting method ( Hereinafter, the pour culture method) is known.

 However, the microscopic method, the turbidity measuring method, the gravimetric method, the packed volume method, and the like have a problem that the sensitivity is low and it is impossible to distinguish dead cells from viable cells. The mixed culture method requires the cultivation of biological cells, so measurement usually takes one day or more, and is not suitable for obtaining rapid results.

 Cell measurement in the above fields requires rapid and highly sensitive measurement. For example, in the field of food hygiene, testing for microbial contamination of products is essential for product shipment. Traditionally, this test is performed by the pour culture method, but since the test takes more than one day, the product must be kept in a warehouse until the results are obtained. This not only poses a problem in terms of distribution efficiency, but also increases the possibility of microbial contamination of milk and other products as storage time increases. In addition, microorganisms that pose a problem of contamination in foods are generally low in concentration, so highly sensitive inspection is required.

As a method for measuring the concentration of microorganisms that satisfies the above requirements, there is known a method for measuring ATP, which is always present in living microorganisms, using a bioluminescence method. This method A sample containing biological cells is contacted with an extractant containing tricloacetic acid (TCA), Tris buffer, ethanol, or a lytic enzyme to extract intracellular ATP outside the cell, and then extract The resulting ATP is contacted with a luminescent reagent containing luciferin, a substrate for firefly luminescence, and luciferase, an enzyme. Then, luciferin, luciferase, and ATP (bioluminescence is generated by an enzymatic reaction, and the amount of generated light is measured to measure intracellular ATP to measure biological cells.

 ATP is originally contained in the cells of all living organisms, albeit in varying amounts, and exists as so-called somatic cells not only in microorganisms, but also in single-celled organisms, and in cells of animal and plant tissues. In addition, free (free) ATP exists around living cells.

 Therefore, even if an attempt is made to detect ATP present only in a living cell from a sample containing a certain living cell, the ATP in the living cell and free ATP present in the vicinity thereof are detected together. In other words, even if we consider using ATP as an index for measuring biological cells, the free ATP other than the biological cells just described is simultaneously measured as background light emission (noise), leading to a decrease in detection accuracy. I had it.

 As a technique for removing the free ATP, the present applicant has previously proposed "ATP elimination agent, ATP elimination method, reagent for measuring biological cells and biometric method using the same," (Japanese Patent Publication No. 9-182600). ).

 The present invention relates to the use of adenosine phosphate deaminase alone or to the group consisting of avilase, allyl phosphatase, acid phosphatase, hexokinase, and adenosine triphosphatase together with the adenosine phosphate deaminase. An ATP elimination method and an ATP elimination agent using at least one selected from the group consisting of a reagent for measuring biological cells and a method for measuring biological cells using the same. Disclosure of the invention

Therefore, the present inventors have conducted further research, and by incorporating a free ATP eliminator, an ATP extractant, and a luminescent reagent into a test device, it is possible to easily measure a biological cell of a specimen with high accuracy. The inventors of the present invention have found that the present invention is present, and have completed the present invention. A first concept of the present invention (according to the above, a sample testing instrument for measuring the amount of biological cells by collecting biological cells from a sample and causing the cells to emit light, comprising: a free ATP scavenger; There is provided a sample testing instrument comprising: an extractant for extracting cell-derived ATP; and a luminescent reagent for causing the ATP to act on the extracted ATP to emit light.

 For example, according to the embodiment, the sample testing instrument has a test tube shape, a measurement container containing the luminescent reagent, a tube shape, and a sealing material at each of the upper and lower openings. An ATP erasing agent container with a free ATP erasing agent sealed inside, and a tube-shaped seal material attached to each of the upper and lower openings, and an extractant sealed inside. And an extractant container, wherein the ATP scavenger container is fitted above the upper opening of the measurement container with the extractant container positioned below.

 A breaker for breaking the sealing material is provided at a lower part inside the ATP erasing agent container and the extracting agent container. This breaker breaks through the sealing material by being bent downward from below the swab. In order to easily break the sealing material, for example, according to the embodiment, it is preferable to form a projection projecting downward on the lower surface of the tip of the breaker.

 A breaking force for breaking a seal material located above the ATP erasing agent container is further provided at an upper end portion of the measurement container.

 According to a second concept of the present invention, there is provided a wiping test instrument for measuring the amount of biological cells by collecting biological cells from a specimen and causing the cells to emit light. A specimen wiping tool having a detached main body, a holding member detachably inserted into an upper end opening of the main body, a cotton swab held by the holding member, a free ATP eliminating agent, and a biological cell-derived ATP. And a luminous reagent containing a luminescent reagent for causing the ATP to act on the extracted ATP to emit light. The present invention provides a wiping inspection instrument which is fitted in a lower end opening of a wiping inspection device.

The configuration of the sample test device is the same as the configuration of the test device described above. According to a third aspect of the present invention, a biological cell is collected from a specimen and luminescent. A wiping test instrument for measuring the amount of biological cells by applying a breaker breaking member having a 銳 -square shape at the lower end thereof, a tubular body having upper and lower openings, and an upper opening of the body. A sample wiping tool having a holding member detachably inserted into the part, a cotton swab held by the holding member, an ATP erasing agent container and an extractant container provided at a lower portion in the main body, and a test tube A measuring container having a shape and being vertically movably inserted into an opening at the lower end of the main body. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a sectional view of a wiping inspection instrument according to a first embodiment of the present invention.

 FIG. 2 is an enlarged cross-sectional view of the sample testing instrument shown in FIG.

 FIG. 3 is an enlarged cross-sectional view taken along line m-m in FIG.

 FIG. 4 is an enlarged sectional view taken along line IV-IV in FIG.

 FIG. 5 is a cross-sectional view showing another embodiment of the sample testing instrument. FIG. 6 is a sectional view showing another embodiment of the wiping inspection instrument.

 FIG. 7 is a cross-sectional view showing still another embodiment of the sample testing instrument. BEST MODE FOR CARRYING OUT THE INVENTION

 In the present invention, first, free ATP is decomposed with a free ATP scavenger from a sample collected by wiping with a cotton swab or the like, and then ATP derived from a biological cell is extracted with an extractant. Then, a luminescent reagent is allowed to act on the extracted ATP derived from biological cells, and ATP, that is, biological cells are detected based on the amount of luminescence.

 Examples of the free ATP scavenger include reagents obtained by using a single or a combination of avirase, alkaline phosphatase, acid phosphatase, hexokinase, adenosine triphosphatase, and adenosine phosphate deaminase.

As an ATP extraction reagent as an extractant, for example, a mixed solution of ethanol and ammonia, methanol, ethanol, a surfactant, trichloroacetic acid, perchloric acid and the like can be used. Detergents have high ATP extraction efficiency, so ATP extraction Suitable as a reagent. Surfactants include anionic surfactants (eg, sodium dodecyl sulfate (SDS), potassium lauryl sulfate, sodium monolauroyl phosphate, sodium alkyl benzene sulfonate), surfactants (For example, benzalkonium chloride (BAC), benzatonium chloride (BZC), cetylpyridinium chloride, cetyltrimethylammonium bromide, myristyl dimethyl benzyl benzylammonium), amphoteric 1) Surfactants (for example, Twittergent Detergent 3-08,3-10,3-12,3-14,3-16, Tego), nonionic surfactants (for example, Tween20, 60,80, Span 60,80, Triton X-45, X-100, poly (xylene ethylene ether), poly (xylene ethylene lauryl ether) can be used.

 Examples of the luminescent reagent include a reagent containing luciferin which is a substrate of firefly luminescence and luciferase which is an enzyme.

 In FIG. 1, a wiping inspection instrument 1 according to a first embodiment of the present invention includes a main body 2 mainly formed in a tubular shape with upper and lower openings, and an attaching / detaching itself through an upper opening 3 of the main body 2. The apparatus includes a sample wiping tool 4 to be inserted, and a biological cell sample test device 6 fitted into the lower opening 5 of the main body 2.

 The specimen wiping tool 4 includes a swab 9 and a holding member 10 for holding the swab 9. The cotton swab 9 has a rod-shaped cotton shaft 7 and a cotton portion 8 formed in an oval shape at the lower end of the cotton shaft 7. The specimen 4 is removed from the main body 2 and the surface of the specimen is wiped with a cotton swab 9 to collect biological cells.

 The holding member 10 includes a lower portion 11 having a lower diameter and a larger portion 12 having an upper portion. The diameter of the small portion 11 is such that the holding member 10 stops at an arbitrary position of the main body 2 when the holding member 10 is inserted into the main body 2. When the sample wiping tool 4 is moved downward, the stepped portion 13 formed by the small portion 11 and the large diameter portion 12 abuts on the upper end portion 14 of the main body 2, and the sample wiping is performed. Tool 4 acts as a stop to prevent further lowering. As shown in FIG. 4, a plurality of linear projection members 15 extending in the axial longitudinal direction are provided on the upper inner wall surface of the main body 2. The distal end 16 of these projection members 15 and the outer peripheral edge of the flange-shaped member 17 provided at the middle part of the holding member 10 are in sliding contact with each other, and the movement of the holding member 10 relative to the main body 2 is made smooth. I have.

FIG. 2 shows the sample test device 6. This sample test device 6 A measuring container 20 formed in the shape of a test tube, an ATP erasing agent container 22 fitted to the opening 21 of the measuring container 20 and positioned above, and a lower portion of the ATP erasing agent container 22 Extraction agent container 23. As shown in FIG. 1, the sample testing instrument 6 is pushed into the main body 2 until it comes into contact with a stepped portion 24 formed at the upper portion of the lower opening 5 of the main body 2.

 The ATP erasing agent container 22 has a cylindrical shape, and has a sealed chamber 27 formed by sticking sealing materials 25 and 26 to upper and lower openings. In the closed chamber 27, a free ATP removing agent 28 for removing free ATP attached to the cotton portion 8 is stored.

 The ATP erasing agent container 22 has a breaker 29 at the lower portion inside. The breaker 29 is composed of, for example, six breaker pieces 31 each having an arrow shape extending from the inner wall surface of the ATP eraser container 22 toward the center as shown in FIG. The tip of each breaker piece 31 has a projection 30 projecting downward. By pushing the cotton swab 9, each breaker piece 31 is pushed downward and bent, and the sealing material 26 is pierced by the projection 30. At this time, the sealing material 33 above the extractant container 23 described later is also broken at the same time.

 As shown in FIG. 1, a breaker 32 having a structure similar to that of the breaker 29 described above is provided near the step portion 24 of the main body 2. The breaker 32 penetrates the seal material 25 provided at the upper opening of the ATP eraser container 22 by the downward movement of the swab 9.

 The breaker 29 of the ATP removing agent container 22 removes the surface of the cotton portion 8 provided at the tip of the cotton swab 9 to remove free ATP attached to the cotton portion 8 to the free ATP removing agent. 2. Effectively migrate to 8. The swab 9 receives a slight downward movement resistance due to the breaker 29, but this resistance keeps the contact time between the free ATP scavenger 28 and the cotton section 8 longer, and causes the cotton section 8 to adhere to the cotton section 8. Transfer of free ATP to free ATP scavenger 28 is effective. Further, in order to prolong the contact time between the free ATP eliminating agent 28 and the cotton portion 8, the downward movement of the cotton swab 9 may be artificially stopped.

The structure of the extractant container 23 is the same as that of the ATP scavenger container 22 described above. You That is, the extractant container 23 has a cylindrical shape, and its upper and lower openings are sealed with sealants 33 and 34 to have a closed chamber 35. The closed chamber 35 contains an extractant for extracting ATP from biological cells collected with a cotton swab 9.

3 6 are stored. The extractant container 23 has a breaker 37 at its lower part. The breaker 37 has the same structure as the shake force 29 provided in the ATP eraser container 22. The lower movement of the swab 9 causes the breaker 37 to bend downward and break through the sealing material 34. When the breaker 37 is pressed downward and bent, the breaker 37 removes the surface of the cotton portion 8.

 As described above, since the ATP erasing agent container 22 and the extractant container 23 are fitted so as to be embedded in the upper part of the measurement container 20, the closed chamber 38 is provided in the measurement container 20. It is formed. A luminescent reagent 39 is stored in the closed chamber 38.

 When the ATP eliminator container 22 and the extractant container 23 are fitted into the measurement container 20, the upper end surface 41 of the ATP eliminator container 22 located above the extractant container 23 is measured. It is preferable that the upper end surface 42 of the container 20 be flush with the upper surface 42, because the size of the sample testing instrument 6 can be reduced. Furthermore, the top surface of the measuring container 20 is sealed with a sealing material 25.

By sealing up to 42, the opening of the sample testing instrument 6 is completely sealed, and when the sample testing instrument 6 is distributed independently as described later, it is necessary to prevent invasion of various bacteria from outside. It is useful and becomes a measurement container containing three types of reagents.

 The breakers 29, 32, and 37 are not necessarily provided if the sealing material 25, 26, 33, or 34 is easily broken by the swab 9, and should be provided as necessary. do it.

 Next, how to use the wiping greeting device 1 shown in FIG. 1 will be described. First, the sample wiping device 4 is provided in a state where it is inserted partway into the main body 2. Luminescent reagent 39 is enclosed in measurement container 20, free ATP quencher 28 is enclosed in ATP quencher container 22, and extractant 36 is enclosed in extractant container 23. It is.

 Next, the specimen wiping tool 4 is pulled out from the main body 2, and the specimen surface is wiped with the cotton part 8 provided at the tip of the swab 9, and the biological cells are collected.

The specimen wiping tool 4 from which the biological cells have been collected is inserted into the main body 2 again. Then, the sample wiping tool 4, that is, the cotton swab 9 is pushed down, and the breaker 3 2 (the seal material 25 shown in FIG. 2 is broken, and the cotton portion 8 is immersed in the free ATP scavenger 28. When pushing down, the cotton swab 9 receives the pushing-down resistance by the breaker 29, but detects this pushing-down resistance and removes the free ATP without breaking the sealing material 26 by the instruction of the indicator (not shown) provided in the main body 2. The cotton portion 8 is kept soaked in the free ATP scavenger 28 until the breaker 32 is opened by the cotton swab 9 into a petal shape. Since there is a step of removing with the tip of 32, the free ATP attached to the surface of the cotton portion 8 is efficiently transferred to the free ATP scavenger 28.

 When the swab 9 is further pushed down, the sealers 26 and 33 are broken by the breaker 29, and the cotton portion 8 is immersed in the extractant 36. In this process, when the cotton swab 9 is further pushed down, the cotton portion 8 comes into contact with the breaker 37, and the cotton swab 9 receives resistance to be pushed down by the breaker 37. Inevitably increases the contact time of ATP, and guarantees the termination of ATP extraction and elimination from biological cells

 Then, the sample wiping tool 14 is pushed down until the step 13 of the holding member 10 shown in FIG. 1 and the upper end 14 of the main body 2 come into contact with each other. By this pushing-down operation, the sealing material 34 is broken by the breaking force 37, and the extractant 36 containing biological cells falls into the measuring container 20.

 Finally, by gently shaking the entire device 1 for wiping inspection, the luminescent reagent 39 and the extracting agent 36 come into contact, and the action of the luminescent reagent 39 causes a luminescent reaction to detect biological cells. .

As described above, the wiping test device 1 according to the first embodiment has been described. However, in the present invention, the sample test device 6 alone can be used as a test device. That is, the swab may be a commercially available swab, and after wiping the sample with the swab, piercing from the upper end of the sample test device 6 and tearing off the sealing materials 25, 26, 33, and 34 sequentially, as described above. If the procedure is performed as described above, the living cells of the specimen can be inspected. When the sample testing instrument 6 is used alone in this way, the sample testing instrument 6 shown in FIG. 2 may be used, and as shown in FIG. The sample testing instrument 6 provided with 2 may be used. FIG. 6 shows a device for wiping inspection according to a second embodiment of the present invention. The wiping inspection instrument 1a according to the second embodiment is configured such that the measurement container 20a is vertically movable with respect to the main body 2a, and the ATP eraser container 22 and the extractant container 23 are connected to the main body 2a. This is an example of insertion on the step portion 24 of FIG.

 The opening of the measuring container 20a is sealed with a sealing material 43, and the luminescent reagent 39 is stored inside the measuring container 20a. The main body 2 integrally has a pipe-shaped seal breaking member 44 whose tip is formed in a rectangular shape at the lower part of the step portion 24. The seal breaking member 44 pushes up the measuring container 20a to break the sealing material 43 of the measuring container 20a and open the measuring container 20a.

 In the lower part of the main body 2, a breaker 32a, an ATP eliminator container 22 sealed with a free ATP eliminator 28, and an extractant container 23 sealed with an extractant 36 are sequentially provided from above. ing.

 In the above-mentioned wiping inspection instrument 1a, first, the free ATP is erased by moving down the swab 9, and then the ATP extraction and the elimination reaction are stopped by the living cells, and finally the sealing material 3 of the extractant container 23 4 is broken. The extractant 36 containing ATP derived from biological cells falls and flows into the measurement container 20a through the communication hole 45 of the seal breaking member 44. Then, the extractant 36 containing ATP comes into contact with the luminescent reagent 39, and the biological cells are detected by the luminescent reaction.

 FIG. 7 shows another embodiment of the sample testing instrument 6. This embodiment shows an example in which the opening 21 is sealed with a sealing material 25a larger than the outer diameter of the container 20 at the opening 21 of the measuring container 20 of the specimen inspection instrument 6. ing.

 According to this embodiment, the portion 25 b protruding from the outer diameter of the measuring container 20 is picked up with a finger, and the sealing material 25 a is peeled off. Can be omitted. Furthermore, a durable material can be used as the sealing material 25a, and the quality is improved as a single product. Industrial applicability

As described above, according to the wiping test device of the present invention, free ATP can be eliminated and only the target biological cells can be measured. Useful as a test tool.

Claims

The scope of the claims 1. A sample testing instrument that collects biological cells from a sample and emits light to measure the amount of biological cells.  A free A T P scavenger,  An extractant for extracting ATP derived from the biological cell;  And a luminescent reagent for causing the extracted ATP to act to emit light. 2. The sample testing instrument is  A measurement container having a test tube shape and containing the luminescent reagent,  An ATP erasing agent container having a tubular shape, a sealing material attached to each of the upper and lower openings, and a free ATP erasing agent sealed therein;  An extractant container having a tubular shape, a sealing material is attached to each of the upper and lower openings, and an extractant is enclosed therein; The method according to claim 1, wherein the ATP erasing agent container is fitted at an upper position of an upper opening of the measurement container, and the extractant container is located at a lower position. Specimen test equipment. 3. The specimen inspection instrument according to claim 2, wherein a breaker for breaking the sealing material is provided at a lower portion inside the ATP erasing agent container and the extractant container. 3. The specimen inspection instrument according to claim 2, wherein a breaker for breaking a seal member located above the ATP erasing agent container is provided at an upper end portion of the measurement container. 5. The sample testing instrument according to claim 2, wherein an opening of the measurement container is sealed with a sealing material having a diameter larger than an outer diameter of the measurement container. 6. A wiping test device for measuring the amount of biological cells by wiping the specimen to collect biological cells and causing the cells to emit light,  A free A T P scavenger,  An extractant for extracting ATP derived from the biological cell;  A luminescent reagent for causing the extracted ATP to act to emit light, and a wiping test device comprising: 7. The wiping inspection instrument further comprises:  A tube-shaped body with upper and lower openings  A holding member detachably inserted into the upper end opening of the main body, and a sample wiping tool having a cotton swab held by the holding member;  A sample test device for containing the free ATP eliminating agent, the extractant, and the luminescent reagent; 7. The wiping test device according to claim 6, wherein the sample test device is fitted into a lower end opening of the main body. 8. The sample testing instrument is  A test container in the shape of a test tube, in which the luminescent reagent is stored; and a seal material in each of the upper and lower openings, in the shape of a tube, and the inside thereof (ATP erasing agent container enclosing the free ATP erasing agent,  An extractant container having a tubular shape, a sealing material attached to each of the upper and lower openings, and an extractant enclosed therein; The method according to claim 7, wherein the measurement container is fitted with the ATP erasing agent container at an upper position of an upper opening of the measurement container and the extractant container at a lower position. Equipment for wiping inspection. 9. The wiping inspection instrument according to claim 8, wherein a breaker for breaking a seal material on an upper portion of the ATP erasing agent container is provided at a lower portion inside the main body. 10. The wipe according to claim 8 or 9, wherein a breaker for breaking the seal material is provided below the inside of the A A-eraser container and the extractant container. Inspection equipment. 1 1. A wiping test instrument that collects biological cells from a specimen and emits light to measure the amount of biological cells.  A tubular body having a breaker breaking member having a 先端 angled tip at the bottom, and an upper and lower opening;  A holding member detachably inserted into the upper opening of the main body, and a sample wiping tool having a cotton swab held by the holding member;  An ATP erasing agent container and an extracting agent container provided at a lower portion in the main body, a measurement container having a test tube shape, and being vertically movably inserted into an opening at a lower end portion of the main body; A device for wiping inspection, comprising: