WO2005097968A1

WO2005097968A1 - Microbe detector and method of microbe detection

Info

Publication number: WO2005097968A1
Application number: PCT/JP2005/005372
Authority: WO
Inventors: Tomonari Kogure; Kazuhiro Yamada; Hiraku Sasaki; Minoru Sakata; Akihiko Tokida
Original assignee: Bussan Nanotech Research Institute, Inc.
Priority date: 2004-04-06
Filing date: 2005-03-24
Publication date: 2005-10-20
Also published as: JP2007236202A

Abstract

A method of microbe detection, comprising, in microchannel chip (10) having channel (11) of ≤1 mm width (d), mixing a microbe-containing analyte solution with a luminescence solution capable of emitting light in the presence of microbial ATP in the channel (11); extracting microbial ATP from the microbe of the analyte solution by extraction means and causing the luminescence solution to emit light in the presence of the microbial ATP; and detecting the light emitted. Thus, there can be provided a method of microbe detection and microbe detector by which microbe detection can be performed easily within a satisfactorily short period of time.

Description

Specification

Bacteria detection device and bacteria detection method

Technical field

The present invention relates to a bacteria detection device and a bacteria detection method.

Background art

[0002] As a method for detecting bacteria, ATP in bacteria is extracted (Lysis step) using a surfactant or an electoporation method or the like, and the fluorescent light generated by removing enzymes such as luciferase therefrom is extracted. There is known a method of detecting the presence or absence of bacteria by receiving and measuring the light with a CCD or a photodiode (for example, see Patent Document 1 below).

[0003] Conventionally, this series of reactions has been performed in a test tube or the like. This method has an advantage that the detection result can be obtained easily and quickly without the necessity of preparing a medium, as compared with a culture method which is a general method for detecting bacteria.

Patent Document 1: JP-A-2000-189197

Disclosure of the invention

Problems to be solved by the invention

[0004] However, the method for detecting bacteria described in Patent Document 1 described above has the following problems.

[0005] In other words, the above-described method for detecting bacteria has a problem that several hours are required for detection where the reaction time is long. Such a problem is particularly serious in the food industry, which requires a bacteria detection test for many products, and for example, there has been a problem that fresh products cannot be shipped quickly.

[0006] Further, in the above bacterial detection method, a component such as avirase that removes free ATP, a component that inactivates avirase, a component such as luciferase that emits light by reacting with ATP, and bacterial flocs are dispersed. It is necessary to prepare components, components that break the cell wall of bacteria, and other necessary components separately in reagent bottles, etc.A prescribed amount is collected from each reagent bottle using a microsyringe, etc., and separated into sample tubes. The operation is extremely complicated. [0007] The present invention has been made in view of the above circumstances, and has as its object to provide a bacteria detection device and a bacteria detection method capable of easily detecting bacteria in a sufficiently short time.

Means for solving the problem

[0008] The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, it is not caused by the fact that the detection of bacteria is performed in a reaction vessel such as a force flask which requires a long time to detect bacteria as described above. I thought. In other words, the present inventors have found that in the above-described reaction vessel, the speed of the luminescence reaction is reduced due to insufficient contact probability between ATP in the bacterium and luciferase, and the detection of the bacterium takes a long time. I thought it might be. As a result of intensive studies, the present inventors have found that the above-mentioned problems can be solved by the following invention.

[0009] That is, the present invention relates to a first well for containing a sample solution containing bacteria, a second well for containing a luminescent solution that emits light in the presence of ATP in bacteria, and a method for containing ATP in bacteria and a luminescent solution. A microchannel chip having a light emitting section to be brought into contact, a first channel connecting the first well and the light emitting section and having a luminescent liquid introducing section, a microchannel chip having a second channel connecting the luminescent liquid introducing section and the second well, and a bacterial force Extraction means for extracting ATP in bacteria, light detection means for detecting light generated by contact of ATP in bacteria with luminescent liquid, and transfer of liquid contained in the first well to the light emitting part, and transfer to the second well Transfer means for transferring the contained liquid to the first channel, and wherein the channel width of the first channel is 1 mm or less (a first bacteria detection device).

[0010] The first bacteria detection device having such a structure does not require a sorting operation as in the related art, and has an effect of increasing convenience. That is, in the microchannel chip, since the first well containing the sample liquid and the light emitting section are connected by the first channel, the sample liquid can be sent to the light emitting section in a few seconds, and the second Since the channel is connected to the first channel, when the liquid contained in the first well is transferred to the light emitting unit by the transfer means, when the sample liquid is sent from the first well to the light emitting unit, the second liquid flows. The luminescent liquid contained in the well can be sequentially sent to the first channel, and each can be mixed in the first channel.

[0011] In addition, since the first bacteria detection device is provided with an extraction means for extracting bacterial force ATP in bacteria, it can extract ATP in bacteria contained in the sample solution and can extract ATP in the sample solution. The reaction between the bacterial ATP and the luminescent solution can be performed efficiently.

[0012] Further, the first bacteria detection device includes a transfer means for transferring the liquid in the well to the light emitting unit. In this case, the liquid can be reliably guided to the first channel by the transfer means.

[0013] Furthermore, since the channel width of the first channel is 1 mm or less, when the sample liquid and the luminescent liquid are mixed, diffusion takes place in a very short time, and bacterial power and ATP in the bacteria are extracted. Thereby, the reaction can be performed instantaneously. As a result, bacteria can be detected in a matter of seconds in the past, requiring at most one or two hours.

[0014] Further, the second bacteria detection device is the same as the first bacteria detection device, wherein the first channel has an extract introduction portion into which an extract for extracting ATP in the bacterial force and bacteria is introduced. The means includes a third well contained in the microchannel chip and containing the extract, and a third channel connecting the extract introducing section and the third well, and the transfer means has It is preferable that the liquid contained in the first well can be transferred to the light emitting unit, and the liquid contained in the third well can be transferred to the first channel.

[0015] That is, the second bacterium detection device comprises a first well for containing a sample liquid containing bacteria, a third well for containing an extract for extracting bacterial force and ATP in bacteria, and a second ATP for bacteria. A second well for containing a luminescent solution that emits light in the presence of a luminescent solution, a light emitting unit that makes ATP in bacteria contact with the luminescent solution, a first channel that connects the first well and the light emitting unit, and a first channel. It has a third channel connecting the extraction liquid introduction part and the third well, and a second channel connecting the luminescence liquid introduction part downstream of the extraction liquid introduction part of the first channel and the second well. A microchannel chip, light detecting means for detecting light generated by contact between ATP in bacteria and a luminescent liquid, and a liquid contained in a first well are transferred to a light emitting section, and contained in a second and third well. Transfer means for transferring the liquid to the first channel. Preferably, the channel width of the first channel is 1 mm or less.

[0016] The second bacteria detection device having such a structure does not require a sorting operation as in the conventional art, and has an effect of increasing convenience. That is, since the first well containing the sample liquid and the light emitting unit are connected by the first channel in the microchannel chip, the liquid contained in the first well is transferred to the light emitting unit by the transfer means. And sample fluid for several seconds Can be sent to the light emitting section. Also, at this time, since the second channel and the third channel are connected to the first channel, when the sample liquid is sent to the first well power emitting unit, the extract contained in the third well and the second well are connected. The luminescent liquid contained in the 2-well can be sequentially sent to the first channel, and each is mixed in the first channel. Thereby, the reaction between the test solution, the extract solution, and the luminescent solution can be efficiently performed. That is, the sample solution in the first well, the luminescent solution in the second well, and the extract in the third well can be mixed together by the transfer means.

[0017] Further, the second bacteria detection device includes a transfer means for transferring the liquid in the well to the light emitting unit. In this case, the transfer means can collectively and surely introduce the liquids of each pneumatic force into the first channel.

[0018] Furthermore, since the channel width of the first channel is 1 mm or less, when the sample solution, the extract solution, and the luminescent solution are mixed, diffusion takes place in a very short time, and as a result, the reaction is instantaneously performed. It can be carried out. As a result, bacteria can be detected in seconds, which used to take up to one and a half hours.

[0019] Further, in the third bacterium detection device, in the second bacterium testing device, the first channel is configured such that the first channel has an inactive portion for introducing a removing solution containing an ATP removing agent and an inactive ATP removing agent. A dehydrating liquid introducing portion into which an inactivating liquid containing an inactivating agent to be deactivated is introduced, and a fourth well for containing a microchannel chip force removing liquid; The apparatus further includes a fifth well for storing, a fourth channel connecting the removing solution introduction part to the fourth well, and a fifth channel connecting the deactivating liquid introduction part to the fifth well. In the first channel, the removing liquid introduction section is provided upstream of the extraction liquid introduction section, and the inactivating liquid introduction section is downstream of the removal liquid introduction section and upstream of the extraction liquid introduction section. And the transfer means transfers the liquid contained in the fourth and fifth wells to the first well. Preferably, it can be transferred to a channel. Here, the “upstream side” refers to the first well side on the first channel, and the “downstream side” refers to the light emitting unit side on the first channel.

[0020] In this case, the removing solution for removing free ATP is introduced from the fourth well into the first channel via the fourth channel and the removing solution introducing portion, whereby free ATP in the first channel is removed. Removed. For this reason, noise light due to free ATP can be removed and only light due to bacterial ATP can be detected, and the measurement accuracy of bacterial ATP can be improved.

[0021] Furthermore, if the removing solution is introduced into the first channel and remains forever, ATP in bacteria may be destroyed by the removing agent. According to the present invention, the deactivating agent is introduced into the first channel via the fifth channel and the deactivating liquid introduction part, and the removing liquid is deactivated by the deactivating liquid. The destruction of intracellular ATP by the liquid is sufficiently prevented. Therefore, intracellular ATP can be accurately determined.

[0022] Further, the removing liquid introducing section is provided upstream of the extracting liquid introducing section, and the inactivating liquid introducing section is upstream of the extracting liquid introducing section and downstream of the removing liquid introducing section. Therefore, even if the amount of bacteria in the detection solution is very small, the error can be sufficiently suppressed even when the intracellular ATP measurement accuracy is high as described above.

Further, in this case, the transfer means can also transfer the liquid contained in the fourth and fifth wells to the first channel. Therefore, the liquid contained in the first to fifth wells can be collectively and surely introduced into the first channel by the transfer means.

[0024] Furthermore, in the first channel of the second bacterium detection device, the temperature of at least one part downstream of the removal liquid introduction part is controlled to 50 ° C or more to inactivate the ATP removing agent. It is preferable to further include temperature control means for causing the temperature to be reduced.

[0025] That is, in the fourth bacterium detecting device, in the second bacterium detecting device, the first channel further includes a removing solution introducing portion into which a removing solution containing an ATP removing agent is introduced, and the microchannel chip Force 4th well for containing the removing solution, 4th channel connecting the 4th well with the removing solution inlet, and control the temperature of the removing solution to 50 ° C or higher to inactivate the ATP remover And a temperature control unit for controlling the temperature in the first channel, wherein the removal liquid introduction unit is provided upstream of the extraction liquid introduction unit in the first channel. It is possible to control the temperature of the downstream side of the removal liquid introduction section and the upstream side of the extraction liquid introduction section, and the transfer means transfers the liquid contained in the fourth well to the first channel. It should be one that can be transported .

[0026] In this case, the extra removal is performed only by controlling the temperature without introducing the inert inert liquid into the first channel. The removed liquid can be deactivated. Therefore, since the inactive dandelion liquid is not required, a well for storing the inactive danjiri liquid and a channel for sending the inactive danjiri liquid can be omitted, and the structure of the microchannel chip can be simplified. Can be

[0027] Further, it is preferable that the temperature control means controls the temperature of a portion on the downstream side of the removal liquid introduction portion and on the upstream side of the extraction liquid introduction portion (hereinafter, referred to as "temperature control portion"). By controlling the temperature in this manner, it is possible to obtain the same effect as in the case where the above-described inert irrigation solution is introduced into the first channel.

[0028] Furthermore, the liquid contained in the fourth well by the transfer means can be transferred to the first channel via the fourth channel and the removal liquid introducing section. Therefore, the liquid contained in the first to fourth wells can be introduced into the first channel collectively and surely by the transfer means.

Here, the sixth channel for accommodating the amplification solution for amplifying ATP in bacteria, the microchannel chip force of the third bacteria detection device, the amplification solution introduction section for the first channel, and the sixth well And a sixth channel connecting the amplifying liquid and the amplifying liquid introducing section is preferably provided downstream of the inactive irrigation liquid introducing section.

That is, in the fifth bacterium detection device, the first channel of the third bacterium detection device further has an amplifying solution introduction part into which an amplifying solution for amplifying ATP in bacteria is introduced, and The channel chip further includes a sixth well for accommodating the amplification solution, and a sixth channel for connecting the sixth solution with the amplification solution introduction unit, wherein the amplification solution introduction unit is inactive in the first channel. It is preferable that the transfer means is provided on the downstream side of the liquid inlet, and the transfer means can transfer the liquid contained in the sixth well to the first channel.

[0031] In this case, free ATP is sufficiently removed by the removing solution, and free ATP is not amplified, so that the measurement accuracy can be further improved. Further, since the sixth channel power amplification solution is introduced into the first channel via the sixth channel and the amplification solution introduction part, it is difficult for bacteria to emit light (further minute bacteria or bacteria with poor nutritional status). Can also be detected because it is amplified by the amplification solution. ATP amplification reagents include those disclosed in, for example, JP-A-2001-299390. [0032] Further, the liquid contained in the sixth well by the transfer means can also be transferred to the first channel via the sixth channel and the amplification liquid introduction unit. Therefore, the liquid contained in the first to sixth wells can be introduced into the first channel collectively and surely by the transfer means.

[0033] In the bacterium detection device according to the above No. 2-5, the seventh channel containing the decomposing solution for decomposing the microchannel chip force bacteria floc, the first channel decomposing solution introducing section and the seventh well are formed. Preferably, the apparatus further comprises a seventh channel for connection, and the decomposition liquid introduction section is provided upstream of the extract introduction section.

[0034] That is, in the sixth bacterium detection apparatus, in the bacterium detection apparatus according to the above-mentioned 2-5, the first channel further includes a decomposition solution introducing section into which a decomposition solution for decomposing bacterial flocs is introduced. The microchannel chip further includes a seventh well for containing the decomposed liquid, and a seventh channel for connecting the decomposed liquid introduction unit to the seventh well, wherein the first channel includes a decomposed liquid introduction unit. Is preferably provided upstream of the extraction liquid introduction section, and the transfer means is capable of transferring the liquid contained in the seventh well to the first channel. .

[0035] In this case, the decomposed liquid can be introduced into the first channel through the seventh channel through the seventh channel and the dispersion liquid introduction section, and the decomposed liquid can disperse the bacterial floc. As a result, the contact efficiency between the bacteria and the extract can be increased, and the extraction reaction can be promoted.

[0036] Further, the liquid contained in the seventh well by the transfer means can also be transferred to the first channel via the seventh channel and the dispersion liquid inlet. Therefore, at least the liquid contained in the first, third, and seventh wells can be collectively and surely introduced into the first channel by the transfer means.

[0037] In the above-mentioned bacteria detection device, the luminescent liquid introduction part may be provided on the downstream side of the first well with respect to the luminescent liquid introduction part, and the luminescence liquid introduction part may be located at a position closer to the extraction liquid introduction part than the extract liquid introduction part. It may be provided downstream of one well.

[0038] Further, in the microbial channel chip of the fifth bacterium detection device, the seventh bacterium detection device may be configured such that any one of the second, third, and sixth wells is replaced by another well. May also be used. In this case, the configuration of the bacteria detection device is extremely simple. It can be. Therefore, it is possible to provide the bacteria detection device at low cost.

[0039] Further, in the eighth bacteria detection device, the 2nd to 7th wells contain a luminescent solution, an extract solution, a removal solution, an inactivating solution, an amplification solution, and a decomposition solution corresponding to the well. It is preferable that they are accommodated.

[0040] That is, in the eighth bacterium detection device, in the third bacterium detection device, the first channel is a decomposition solution for decomposing the amplification solution introduction portion into which the amplification solution for amplifying ATP in the bacteria is introduced and the bacterial floc. The microchannel chip further includes a sixth well for containing the amplified solution, a seventh well for containing the decomposed solution, and a sixth well for the amplified solution. And a seventh channel connecting the decomposed liquid introduction section and the seventh well, wherein the decomposed liquid introduction section is located upstream of the extract introduction section in the first channel. In the first channel, the amplification liquid introduction section is provided downstream of the inactivation liquid introduction section, and the second, third, fourth, fifth, and fifth wells are provided. 6 El and 7 Elka At least one Ueru, extracts corresponding to the Ueru, luminous solution, removing liquid, inactivating solution, the amplification solution, and decomposition liquid is accommodated, preferred that a shall.

[0041] If these liquids are stored in advance, bacteria can be detected only by introducing a sample liquid, and measurement with high detection accuracy can be easily performed regardless of the environment. These wells can contain only the necessary liquids according to the characteristics of the bacteria to be detected and the accuracy of the detected values, and if the wells are unnecessary, the wells can of course be omitted. It is.

Here, it is preferable that the ninth bacterium detection device in the first to eighth bacterium detection devices is provided with control means for controlling the temperature of the liquid in the microchannel chip.

[0043] By controlling the temperature of the liquid in the microchannel chip, it is possible to provide an optimal environment for the reaction performed in the ninth bacterium detection device. For example, the reaction rate can be further increased by setting the temperature at an optimum value for the extract or the luminescent liquid, and further, the reaction can be stabilized by maintaining the temperature at that value. .

[0044] Further, in a tenth bacterium detection apparatus according to the present invention, in the first bacterium detection apparatus, the extraction means includes a heating means for heating the first channel, and an electric field sign for applying an electric field to the first channel. It is preferable that the apparatus further comprises at least one or more of the adding means and the ultrasonic wave applying means for applying ultrasonic waves to the first channel.

[0045] That is, the tenth bacterium detection device includes a first well for containing a sample solution containing bacteria, a second well for containing a luminescent solution that emits light in the presence of ATP in bacteria, and a bacterium. A light-emitting part for contacting the ATP with the light-emitting liquid, a first channel for connecting the first well to the light-emitting part, and a second channel for connecting the light-emitting liquid introduction part of the first channel to the second well. A microchannel chip, a light detecting means for detecting light generated by contact between the ATP in the bacteria and the luminescent liquid, and a liquid contained in the first well being transported to the light emitting section, and a liquid contained in the second well being removed. A transfer means for transferring the first channel; and a heating means for heating the first channel, wherein the channel width of the first channel is lmm or less, and an electric field for applying an electric field to the first channel. Applying the ultrasonic wave to the applying means and the first channel Preferably further comprises at least one or more means of sound wave applying means.

The tenth bacteria detection device having such a structure performs heating, electric field application, or ultrasonic application on at least a part of the first channel instead of having the third channel and the third well. Means. That is, since the first well for storing the sample liquid and the light emitting unit are connected by the first channel in the microchannel chip, the liquid contained in the first well is transferred to the light emitting unit by the transfer means. In addition, the sample liquid can be sent to the light emitting section in a few seconds. Since the first channel is provided with means for heating, applying an electric field, or applying an ultrasonic wave, when the sample liquid is sent from the first well to the light-emitting part, the bacterial force in the sample liquid also reduces the bacterial ATP. Can be extracted. Since the second channel is connected to the first channel, the luminescent liquid contained in the second well can be sequentially sent to the first channel and mixed with the sample liquid in the first channel. As a result, the reaction between bacterial ATP and the luminescent solution can be efficiently performed.

[0047] Therefore, the tenth bacterium detection device has the heating, electric field application or ultrasonic application means as the extraction means, so that the bacterial force and the bacterial ATP can be extracted without using the extract. be able to. Note that there may be a plurality of means for heating, applying an electric field, or applying an ultrasonic wave.

[0048] Further, the eleventh bacteria test apparatus is the same as the tenth bacteria test apparatus, wherein the first channel The removal liquid introduction part into which the removal liquid containing the ATP removal agent is introduced, and the inert deodorization liquid introduction part into which the deactivation liquid containing the deactivator for inactivating the ATP removal agent is introduced are further provided. A fourth channel for accommodating the microchannel chip force removing solution, a fifth channel for accommodating the inactivating liquid, and a fourth channel for connecting the removing solution introduction section to the fourth reservoir. The apparatus further comprises a fifth channel for connecting the inactive liquid introducing part and the fifth well, and in the first channel, the removing liquid introducing part is provided with a heating means, an electric field applying means, or an ultrasonic wave applying means. Power Provided upstream of the extraction unit for extracting ATP in bacteria, Inactivated liquid introduction unit is provided downstream of the removal liquid introduction unit and provided upstream of the extraction unit, and transported Means transfer liquid contained in 4th and 5th wells to 1st channel Making it possible to Rukoto, preferred to be a shall U ,.

[0049] In this case, when the removing solution for removing free ATP is introduced into the first channel through the fourth channel and the removing solution introducing portion, the free ATP is removed by the removing solution. For this reason, noise light due to free ATP can be removed and only light due to intracellular ATP can be detected, and the measurement accuracy of intracellular ATP can be improved.

[0050] Furthermore, if the removing solution is introduced into the first channel and remains forever, ATP in bacteria may be destroyed by the removing agent. According to the present invention, the deactivator is introduced into the first channel via the fifth channel and the fifth channel and the inert liquid introducing section, and the removing liquid is inerted by the inert liquid. As a result, destruction of intracellular ATP by the removing solution is sufficiently prevented. Therefore, intracellular ATP can be accurately determined.

[0051] Further, since the removing liquid introducing section is provided upstream of the extracting section, and the inactivating liquid introducing section is provided upstream of the extracting section and downstream of the removing liquid introducing section. Even if the amount of bacteria in the detection solution is very small, the error can be sufficiently suppressed even when the intracellular ATP measurement accuracy is high as described above.

[0052] Further, the liquid stored in the fourth and fifth wells by the transfer means can also be transferred to the first channel. Therefore, the liquids contained in the first, second, fourth, and fifth wells can be collectively and surely introduced into the first channel by the transfer means.

[0053] Further, in the tenth bacterium detection device, the first channel is located between the removing liquid introduction part and the first channel. It is preferable to further include temperature control means for controlling the temperature of at least one part of the downstream side to 50 ° C. or higher to inactivate the ATP removing agent.

That is, in the twelfth bacterium detection device, in the twelfth bacterium detection device described above, the first channel further includes a removal solution introduction part into which a removal solution containing an ATP removing agent is introduced, The chip has a fourth well for containing the removing solution, a fourth channel connecting the removing solution introduction section and the fourth well, and a control of the temperature of the removing solution to 50 ° C or higher to prevent the use of the ATP removing agent. Temperature control means for activating, in the first channel, the removal liquid introduction section is upstream of the extraction section for extracting bacterial force ATP in bacteria by heating means, electric field application means or ultrasonic application means. The temperature control unit, the temperature of which is controlled by the temperature control means, is capable of controlling the temperature of the portion downstream of the removal liquid introduction unit and upstream of the extraction unit, In addition, the transfer means removes the liquid stored in the fourth well. Preferably, it is possible to transfer to the first channel.

[0055] In this case, it is possible to inactivate the excess removing liquid only by controlling the temperature without introducing the inactivating liquid into the first channel. Therefore, since the inactive dandelion liquid is not required, a well for storing the inactive danjiri liquid and a channel for sending the inactive danjiri liquid can be omitted, and the structure of the microchannel chip can be simplified. Can be

When this temperature control unit is provided in the temperature control unit, the same effect as in the case where the above-described inert inert liquid is introduced into the first channel can be obtained by controlling the temperature.

Further, the liquid contained in the fourth well by the transfer means can also be transferred to the first channel via the fourth channel and the removal liquid introducing section. Therefore, the liquid contained in the first, second, and fourth wells can be collectively and surely introduced into the first channel by the transfer means.

Here, in the eleventh bacterium detection device, the sixth channel for accommodating the amplification solution for amplifying the microchannel chip force ATP in the bacterium, the first channel amplification solution introduction section and the sixth channel And a sixth channel connecting the amplifying liquid and the amplifying liquid introducing section is preferably provided downstream of the inactivating liquid introducing section.

[0059] That is, in the thirteenth bacterium detection device, in the twelfth bacterium detection device, the first channel further includes an amplification solution introduction section into which an amplification solution for amplifying ATP in bacteria is introduced. The microchannel chip further includes a sixth well for containing the amplification solution, and a sixth channel connecting the sixth solution with the amplification solution introduction unit. In the first channel, the amplification solution introduction unit is provided. It is preferable that the liquid is provided downstream of the inert liquid inlet and that the transfer means is capable of transferring the liquid contained in the sixth well to the first channel. .

[0060] In this case, free ATP is sufficiently removed by the removing solution, and free ATP is not amplified, so that the measurement accuracy can be further improved. Further, since the sixth channel power amplification solution is introduced into the first channel via the sixth channel and the amplification solution introduction part, it is difficult for bacteria to emit light (further minute bacteria or bacteria with poor nutritional status). Can also be detected because it is amplified by the amplification solution.

[0061] In addition, the liquid contained in the sixth well by the transfer means can be transferred to the first channel via the sixth channel and the amplification liquid introduction unit. Therefore, the liquid contained in the first, second, fourth, and sixth wells can be collectively and surely introduced into the first channel by the transfer means.

[0062] In the bacterium detection apparatus of Nos. 10-13, the seventh channel containing the digestion solution for decomposing the microbial chip force bacteria floc, the digestion solution introduction section of the first channel, and the seventh tube are provided. Preferably, the apparatus further comprises a seventh channel for connection, and the decomposition liquid introduction section is provided upstream of the luminescence liquid introduction section.

That is, a fourteenth bacterium detection device according to the tenth to thirteenth bacterium detection device, further includes a decomposing solution introducing portion into which a decomposing solution for decomposing the first channel force bacterial floc is introduced, The channel chip further includes a seventh well for containing the decomposed liquid, and a seventh channel connecting the decomposed liquid introduction unit and the seventh well. In the first channel, the decomposed liquid introduction unit includes a heating unit. The transfer means is provided upstream of the extraction unit that extracts ATP in bacteria from bacteria by electric field application means or ultrasonic application means, and the transfer means transfers the liquid contained in the seventh well to the first channel. It is preferable that this is possible.

In this case, the decomposition solution can be introduced into the first channel through the seventh channel and the seventh channel and the dispersion liquid introduction section, and the bacterial floc can be dispersed by the decomposition solution. Thereby, the contact efficiency between the bacteria and the extract can be increased, and the extraction reaction can be promoted.

[0065] Further, the liquid contained in the seventh well by the transfer means can be transferred to the first channel via the seventh channel and the dispersion liquid inlet. Therefore, at least the liquid contained in the first, second, and seventh wells can be collectively and reliably introduced into the first channel by the transfer means.

[0066] In the fifteenth bacteria detection device, the second, fourth, and seventh wells contain a luminescent solution, a removing solution, an inactivating solution, an amplifying solution, and a decomposing solution corresponding to the well. Is preferred.

That is, in the fifteenth bacteria detection device, the first channel is the same as the eleventh bacteria detection device, wherein the first channel is connected to the amplification solution introduction section into which the amplification solution for amplifying ATP in the bacteria is introduced and the bacterial block. The microchannel chip further includes a digestion solution inlet for introducing the digestion solution to be decomposed, and the microchannel chip has a sixth well for containing the amplification solution, a seventh well for containing the digestion solution, and an amplification solution introduction unit. A sixth channel for connecting the sixth well and a seventh channel for connecting the decomposition solution introduction portion to the seventh well are further provided. In the first channel, the decomposition solution introduction portion includes a heating means and an electric field application. Is provided upstream of the extractor for extracting ATP in bacterial force by means or ultrasonic application means, and in the first channel, the amplification liquid introduction section is provided downstream of the inactive irrigation liquid introduction section. And 2nd and 4th At least one well selected from the group consisting of a well, a fifth well, a sixth well, and a seventh well, and the luminescent, removing, inactivating, amplifying, and decomposing solutions corresponding to that well. Is contained! It's preferable to be! /.

[0068] If these liquids are stored in advance, bacteria can be detected only by introducing a sample liquid, and measurement with high detection accuracy can be easily performed regardless of the environment. It should be noted that these wells can be omitted if it is not necessary to contain the necessary liquid according to the characteristics of the bacteria to be detected and the accuracy of the detected value. It is.

[0069] Here, it is preferable that the sixteenth bacterium detection device is the same as the twelfth to fifteenth bacterium detection device, but further provided with a control means for controlling the temperature of the liquid in the microchannel chip.

[0070] By controlling the temperature of the liquid in the microchannel chip, the bacterial test of the present invention can be performed. It is possible to provide an optimum environment for the reaction performed in the discharge device. For example, the reaction rate can be further increased by setting the temperature at an optimum value for the light emitting liquid, and the reaction can be stabilized by maintaining the temperature at that value.

[0071] The present invention also provides a method for extracting bacterial ATP from bacteria from a sample solution containing bacteria in a channel of a microchannel chip having a channel having a channel width of 1 mm or less, in the presence of bacterial ATP. This is a bacteria detection method (first bacteria detection method) in which a luminescent liquid is caused to emit light and the light emission is detected.

[0072] This first bacteria detection method can be effectively implemented by the above-described bacteria detection device. Since the luminescent solution reacts with bacterial ATP, when bacteria and luminescent solution are mixed before extracting bacterial ATP, the bacterial ATP is subsequently extracted from the bacteria and emits light at the same time. Since the liquid reacts with ATP in bacteria, the object of the present invention can be achieved without any problem.

[0073] Further, in the second bacteria detection method, the sample solution of the first bacteria detection method, an extract solution for extracting intracellular ATP from bacteria, and a luminescent solution emitting light in the presence of intracellular ATP are provided. It is preferable to mix ATP and extract the ATP in the bacterium with the bacterial force using the extract.

That is, in the second bacteria detection method, in a channel of a microchannel chip having a channel having a channel width of 1 mm or less, a sample solution containing bacteria and an extract solution for extracting intracellular ATP from bacteria from bacteria. Mixed with a luminescent solution that emits light in the presence of bacterial ATP, extracts bacterial ATP from the bacteria with the extract, emits the luminescent solution in the presence of bacterial ATP, and detects the luminescence. Is preferred.

[0075] The second bacteria detection method can be effectively implemented by the above-described bacteria detection device. In the channel, a method of mixing the sample solution and the extract solution and then mixing with the luminescent solution, a method of mixing the sample solution and the luminescent solution in the microchannel chip and then mixing with the extract solution, and extraction Any method may be used in which the liquid and the luminescent liquid are mixed and then mixed with the sample liquid in the microchannel chip. Since the luminescent solution reacts with ATP in bacteria, if bacteria and luminescent solution are mixed before extracting ATP in bacteria, then if the extract is mixed with bacteria and luminescent solution, bacterial Since the luminescent solution reacts with the ATP in the bacterium at the same time that the ATP is extracted, the object of the present invention can be achieved without any problem.

[0076] In the third method for detecting bacteria, the extract of the second method for detecting bacteria is used for a specific cell. It is preferred that the extract be a specific extract that selectively extracts ATP.

[0077] There are many types of bacteria, and it may be desired to selectively detect only specific bacteria. Therefore, if the extract is the above-mentioned specific extract, for example, in a food test, only bacteria that may cause food poisoning (eg, Vibrio parahaemolyticus, Salmonella, Staphylococcus aureus, Bacillus cereus or Campylobacter) are selectively selected. Can be detected.

[0078] In the fourth bacteria detection method, the specific extract of the third bacteria detection method is preferably a phage or an antibiotic.

[0079] If the specific extract is a phage II antibiotic, only bacteria that are susceptible to the phage II antibiotic are destroyed. By selecting the phage II antibiotic, ATP in the bacterium can be obtained only from the desired bacteria. Can be extracted.

[0080] In the fifth bacteria detection method, the sample solution in the second to fourth bacteria detection methods is mixed with a removing solution containing an ATP removing agent before being mixed with the extract solution and the luminescent solution. It is preferable that free ATP is removed from the sample solution using a removing solution, and further mixed with an inactivating solution containing an inactivating agent to inactivate the removing solution.

[0081] As described above, by introducing the removing solution for removing free ATP,

The noise light due to ATP can be removed, and the introduction of the inactive solution can prevent the removal solution from destroying ATP in bacteria. Therefore, measurement accuracy of ATP in bacteria can be improved.

[0082] The removing liquid and the inactivating liquid are introduced before the sample liquid and the extract liquid are mixed. By doing so, only unnecessary free ATP can be removed, thereby preventing the ATP in bacteria from being adversely affected.

Here, it is also possible to inactivate the ATP removing agent by controlling the temperature to 50 ° C. or higher in place of the inactive liquid in the above-mentioned method for detecting bacteria 2-4.

That is, in the sixth bacteria detection method, before mixing the extract solution and the luminescent solution in the above-described second to fourth bacteria detection methods, the sample solution is mixed with a removal solution containing an ATP removing agent.

Alternatively, it is possible to remove the ATP removing agent by removing the sample fluid free ATP with the removing solution and controlling the temperature of the mixed solution with the removing solution to 50 ° C. or more. [0085] In this case, since it is not necessary to prepare an inactivating solution in advance, it is possible to inactivate the excess ATP removing agent only by controlling the temperature, so that the measurement accuracy of ATP in bacteria can be easily increased. And monkey.

[0086] In the fifth bacterial detection method, an amplification solution for amplifying intracellular ATP in the channel may be mixed.

[0087] That is, in the seventh bacterium detection method, the sample solution obtained in the fifth bacterium detection method is mixed with an amplification solution that inactivates the removal solution and then amplifies ATP in the bacterium. ATP in the bacteria of the bacteria contained in the solution may be amplified.

[0088] At this time, the amplification solution is introduced after inactivating the removal solution. Then, only the ATP in the bacterium can be amplified by the amplification solution, and the measurement accuracy can be further improved.

[0089] In the second to seventh bacteria detection methods, a decomposition solution for decomposing bacterial flocs in the channel may be further mixed.

That is, in the eighth bacterial detection method, the sample liquid in the second to seventh bacterial detection methods is mixed with a decomposition solution for decomposing bacterial flocs before mixing the extract solution and the luminescent solution. Therefore, the sample solution may also break down the bacterial flocs by the digestion solution.

[0091] At this time, the decomposing solution is performed before extracting intracellular ATP from the bacteria. Then, the floc of bacteria can be dispersed by the decomposed solution, and the contact efficiency between the bacteria and the extract can be increased, so that the extraction reaction can be promoted.

[0092] In the ninth bacterium detection method, the ATP in the bacterium is removed from the bacterium by applying at least one of heating, electric field application, and ultrasonic wave to the sample liquid in the first bacterium detection method. It is preferred to extract.

That is, in the ninth bacterium detection method, in a channel of a microchannel chip having a channel having a channel width of 1 mm or less, a sample liquid containing bacteria is subjected to heating, electric field application, and ultrasonic application. It is preferable to extract bacterial ATP in the bacterium by performing at least one of them, to cause the luminescent solution to emit luminescence in the presence of the ATP in the bacterium, and to detect the luminescence.

[0094] In the ninth bacterium detection method, the bacterium detection device can be effectively implemented. That is, in the above invention, the sample solution is heated instead of the extract solution, and the electric field is applied. ATP can be extracted from the bacterium by performing any one of the above and applying ultrasonic waves.

[0095] In the tenth bacteria detection method, the sample solution in the ninth bacteria detection method is mixed with a removal solution containing an ATP remover before heating, applying an electric field, or applying ultrasonic waves. It is preferable to remove free ATP from the sample solution using a removing solution, and further inactivate the removing solution by mixing with an inactivating solution containing an inactivating agent.

[0096] By introducing the removing solution for removing free ATP as described above, noise light due to free ATP can be removed as described above, and further, by introducing an inactive solution, the removing solution can be removed. Can destroy ATP in bacteria. Therefore, measurement accuracy of ATP in bacteria can be improved.

[0097] Here, it is also possible to inactivate the ATP-removing agent by controlling the temperature to 50 ° C or higher instead of the inactivator in the ninth bacterium detection method.

[0098] That is, in the eleventh bacteria detection method, the sample solution is subjected to a removing solution containing an ATP removing agent before heating, applying an electric field or applying an ultrasonic wave to the sample solution in the ninth bacteria detection method. The ATP remover can be removed by removing the ATP removal reagent by removing the sample fluid free ATP with the remover, and controlling the temperature of the mixture with the remover to 50 ° C or higher. .

[0099] In this case, since it is not necessary to prepare an inactivating solution in advance, the excess ATP remover can be inactivated by temperature control alone, so that the measurement accuracy of ATP in bacteria can be easily increased. And monkey.

[0100] Further, an amplification solution for amplifying ATP in bacteria in the channel in the tenth bacteria detection method may be mixed.

[0101] That is, in the twelfth bacterium detection method, after inactivating the removal solution in the sample solution in the tenth bacterium detection method, the sample solution is combined with an amplification solution that amplifies ATP in bacteria. By mixing, the ATP in the bacteria of the bacteria contained in the sample solution may be amplified.

[0102] At this time, the amplification solution is introduced after inactivating the removal solution. Then, only the ATP in the bacterium can be amplified by the amplification solution, and the measurement accuracy can be further improved.

[0103] Further, a decomposition solution for decomposing bacterial flocs in the channel in the ninth and twelfth bacterium detection methods may be further mixed. That is, in the thirteenth bacterium detection method, before heating, applying an electric field or applying an ultrasonic wave to the sample liquid in the ninth and twelfth bacterium detection methods, the sample liquid is subjected to bacterial flocculation. It may be mixed with a decomposition solution to be decomposed, and the decomposition solution may decompose bacterial flocs contained in the sample solution.

[0105] At this time, the digestion is performed before extracting intracellular ATP from the bacteria. Then, the floc of bacteria can be dispersed by the decomposed solution, and the contact efficiency between the bacteria and the extract can be increased, so that the extraction reaction can be promoted.

The invention's effect

According to the present invention, it is possible to provide a bacteria detection device and a bacteria detection method capable of easily detecting bacteria in a sufficiently short time.

Brief Description of Drawings

FIG. 1 is a perspective view showing a microchannel chip which is a main part of a first embodiment of a bacterium detection device of the present invention.

FIG. 2 is a partial cross-sectional view along a thickness direction of a main body constituting the microchannel chip of FIG. 1.

FIG. 3 is a cross-sectional view showing first and second modes of a first channel, a second channel, and a third channel.

FIG. 4 is a schematic view showing a first embodiment of the bacteria detection device of the present invention.

FIG. 5 is a schematic view showing a second embodiment of the bacteria detection device of the present invention.

FIG. 6 is a schematic view showing a third embodiment of the bacteria detection device of the present invention.

FIG. 7 is a schematic diagram showing a fourth embodiment of the bacteria detection device of the present invention.

FIG. 8 is a schematic diagram showing a fifth embodiment of the bacteria detection device of the present invention.

FIG. 9 is a schematic view showing a sixth embodiment of the bacteria detection device of the present invention.

FIG. 10 is a schematic view showing a seventh embodiment of the bacteria detection device of the present invention.

FIG. 11 is a schematic view showing an eighth embodiment according to the bacteria detection device of the present invention. Explanation of symbols

[0108] 1 ··· 1st Lell, 2, 2b… 3rd Lell, 3, 3b, 3c… 2nd Lell, 4 ··· 4th Lell, 5 ···

5 ゥ, 6… 6th, 7… 7 ゥ, 8… Reaction field (light emitting part), 8a… Light detection means, 1 0 · · · Microchannel chip, 10a ... body, 10b ... canopy, 11, lib, 11c, l lh---1st channel, 12, 12b ... 3rd channel, 13, 13b, 13c ... 2nd channel, 14, 14h---4th channel, 15, 15h… 5th channel, 16 ··· 6th channel, 17, 17h… 7th channel, 21a, 21b… channels, 23a, 23b, 24a— 24c ... substrate, 30 · · · filter, 31a ... extraction liquid introduction part, 3 lb ... luminescence liquid introduction part, 31c ... removal liquid introduction part, 3 Id ... inert deodorant liquid introduction part, 31e ... amplification liquid introduction part, 31f: Decomposed liquid introduction part, 35 · · · cavity, 36 · · · air vent hole, (1 · · · width of channel.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, preferred embodiments of the bacteria detection device according to the present invention will be described in detail with reference to the drawings.

FIG. 1 is a perspective view showing a microchannel chip, which is a main part of the first embodiment of the bacteria detection device of the present invention. As shown in FIG. 1, the bacteria detection device of the present embodiment has a microchannel chip 10, and the microchannel chip 10 has a rectangular flat main body 10a and a cover 10b that covers the surface of the main body 10a. And On one surface of the main body 10a, a first well 1 and a reaction field (light emitting part) 8 are formed at both ends, and the first well 1 accommodates a sample liquid containing bacteria. ing. The first well 1 and the reaction field 8 are connected by a first channel 11 formed on one surface of the main body 10a. By introducing the sample liquid into the first well 1, the first well 1ゥ It is now sent from the well 1 to the reaction field 8 via the first channel 11!

[0111] Further, a third well 2 and a second well 3 are formed along the first channel 11 in the main body 10a, and the third well 2 has an extract solution for extracting bacteria and ATP in bacteria. However, the second well 3 is designed to contain a luminescent solution that emits light in the presence of ATP in bacteria.

[0112] The third well 2 is connected to the extract introduction portion 31a of the first channel 11 by the third channel 12, and the second well 3 is connected to the reaction field 8 by the second channel. The third channel 12 and the second channel 13 are both formed on one surface of the main body 10a. Then, the reaction field 8 is arranged downstream of the extract introduction part 31a. Therefore, when the sample liquid is sent to the reaction field 8 through the first channel 11, the extract is sent from the third well 2 to the third channel 12, and the light is emitted from the second well 3 to the second channel 13. The liquid is sent. The sent extract solution and luminescent solution are mixed with the detection solution in the first channel 11 to perform a desired reaction. In the bacteria detection device of the present embodiment, the reaction field

Reference numeral 8 also serves as a luminescent liquid introduction part 31b.

[0113] FIG. 2 is a partial cross-sectional view of the main body 10a along the thickness direction. In FIG. 2, the cross-sectional shape of the first channel 11 is rectangular, and the channel width d of the first channel 11 is 1 mm or less. The “channel width” means a length in a direction perpendicular to the thickness direction of the main body. When the cross-sectional shape of the first channel 11 is quadrangular, it refers to the horizontal length, and when it is trapezoidal or triangular, it refers to the longest length in the direction. In the case of a circular or semicircular shape, the length of the diameter is used. Although not shown, the cross-sectional shapes of the third channel 12 and the second channel 13 are the same as those of the first channel 11, and the channel width is the same as that of the first channel 11.

[0114] The first level 1, the third level 2 and the second level 3 are holes formed along the direction perpendicular to the surface force so that the sample liquid, the extract liquid and the luminescent liquid can be accommodated.

[0115] Further, a cavity 35 is formed in the main body 10a on the opposite side to the first well 1 with respect to the reaction field 8. The cavity 35 has an air vent 36 extending to the edge of the main body 10a.

[0116] Further, in the microchannel chip 10, a light detecting means (not shown!) Is arranged adjacent to the reaction field 8.

[0117] Furthermore, the above-described cover 10b is provided so as to seal the third well 2, the second well 3, the first channel 11, the third channel 12, and the second channel 13. The cover 10b is preferably transparent. In this case, it is convenient to visually check whether or not the liquid in each of the wells 1, 2, and 3 has been introduced into the first channel 11.

[0118] In the bacterium detection apparatus thus configured, the main body 10a is covered with the cover 10b while the third well 2 contains the extract and the second well 3 contains the luminescent liquid. In the cover 10b, the portion of the hollow portion 35 is pressed with a finger to release air from the space of the hollow portion 35 through the air vent hole 36 and close the air vent hole 36 by some method. If you lift your finger after dropping the sample liquid in the first well 1, the space in the cavity 35 becomes negative pressure, so the sample liquid in the first well 1, the extracted liquid in the third well 2, and the second well 3 The luminescent liquid is drawn into the cavity 35. As a result, the sample The effluent is mixed at the extract inlet 31a, and bacterial ATP is extracted from the bacteria in the sample liquid.Then, the mixture and the luminescent solution are mixed at the luminescent solution inlet 3 lb, and the bacterial ATP is Reacts with the luminescent liquid to emit light. This light emission is detected by light detection means (not shown).

[0119] According to the bacterium detection device having such a structure, the sorting operation as in the related art is not required, and there is an effect that convenience is increased. That is, in the microchannel chip 10, the first well 1 containing the sample solution and the reaction field 8 are connected by the first channel 11, so that the sample solution is sent to the reaction field 8 in a few seconds. Since the third channel 12 and the second channel 13 are connected to the first channel 11, when the sample solution is sent from the first well 1 to the reaction chamber 8, it is stored in the third well 2. The extracted liquid and the luminescent liquid contained in the second well 3 can be sequentially sent to the first channel 11, and each is mixed in the first channel 11 to efficiently react with the sample liquid. Can do well.

Further, it is preferable that the channel width d of the first channel 11 is 1 mm or less, and the channel width d of the third and second channels 12 and 13 is 1 mm or less. In this case, when the sample liquid, the extract liquid, and the luminescent liquid are mixed, the diffusion is performed in an extremely short time, and as a result, the reaction can be performed instantaneously. As a result, bacteria can be detected in seconds, which used to take at most 12 hours at the maximum. Further, the channel width d is more preferably 0.1 to 500 m.

[0121] In the microchannel chip 10, the main body 10a is hermetically sealed by the cover 10b, so that the invasion of bacteria from the outside is sufficiently suppressed. Therefore, it is extremely convenient because it does not require inspection in a clean environment or strict disinfection as in the past.

[0122] As the light detecting means, known means can be used, and for example, a CCD, a photodiode, or a photomultiplier tube can be used. The light detecting means detects light generated by the reaction of ATP in bacteria with the luminescent solution in the reaction field 8. Therefore, the installation position of the light detection means is not particularly limited, but is preferably installed near the reaction field 8. In addition, it is also possible to install in the main body 10a of the microchannel chip 10.

[0123] Examples of the bacteria contained in the sample liquid include, but are not particularly limited to, bacteria that may cause food poisoning (eg, Vibrio parahaemolyticus, Salmonella, Staphylococcus aureus, Bacillus cereus or Bacillus pylobacter). [0124] The component contained in the extract may be any component capable of extracting ATP in bacteria, and may be lysozyme, a mixture of ethanol and ammonia, methanol, ethanol, a surfactant (benzetonium chloride, benzalco-chloride). Plum, Triton X-100, etc.), trichloroacetic acid, perchloric acid, phage, antibiotics and the like.

[0125] Further, the component contained in the luminescent solution may be any component that emits light in the presence of ATP, and examples thereof include luciferase and luciferin.

[0126] Note that these sample liquid, extract liquid and luminescent liquid may be a suspension or emulsion as long as they are liquid.

FIGS. 3A and 3B are cross-sectional views showing the first, third, and second aspects of the first channel 11, the third channel 12, and the second channel 13. FIG.

[0128] The channel 21a according to the second embodiment shown in Fig. 3A is a flow path formed by laminating two base materials. That is, a groove is provided in a lower portion of one base material 23a and an upper portion of the other base material 23b, and the channels 21a are formed by bonding. The channel 21b according to the third embodiment shown in FIG. 3B is formed by laminating three base materials. That is, a channel 21b is formed by providing a through groove in the base material 24b as an intermediate layer, and laminating the base materials 24a and 24c on the upper and lower portions so as to sandwich the base material 24b. The channel 21a and the channel 21b correspond to the first channel 11, the third channel 12, and the second channel 13, respectively.

[0129] These substrates 23a, 23b, 24a-24c can be arbitrarily determined, and may be glass such as quartz glass or Pyrex (registered trademark) glass, polymers such as PDMS, polycarbonate or polyimide, iron, stainless steel, and aluminum. Metal such as nickel, copper or the like, or silicon or the like can be used. When laminating, the respective substrates may be of the same type or different types.

It is preferable that the channel width of the first channel 11 is 1 mm or less, and the channel width of the third channel 12 and the second channel 13 is 1 mm or less.

[0131] When the channel width is within the above range, when the sample solution, the extract solution, and the luminescent solution are mixed in the channel, the contact surface area per unit volume increases, and thus the reaction may be promoted. it can. That is, diffusion takes place in a very short time, and as a result, the reaction Can be performed instantaneously. On the other hand, if the channel width exceeds lmm, the diffusion control cannot be sufficiently reduced, so that the object of the present invention cannot be achieved. More preferably, the channel width is 0.1-500 / zm.

[0132] Further, the microchannel chip 10 according to the first embodiment is configured such that the force of storing the extract in the third well 2 and the luminescent liquid in the second well 3 can be reversed. To use it.

That is, when the sample liquid in the first well 1 is sent to the reaction field 8 through the first channel 11, the luminescent liquid is sent from the third well 2 through the third channel 12, and further, the second well 3 Similarly, when the extract is sent through the second channel 13, the sample liquid of the first well 1 is sent to the reaction field 8 through the first channel 11, and the third well 2 A mixed liquid of the extract and the luminescent liquid may be sent through the flow passage 12.

[0134] Since the extract is a solution for extracting ATP in bacteria, it does not react when mixed with a luminescent solution containing no bacteria, but the luminescent solution emits light in the presence of ATP. This is because the reaction solution does not contain ATP and does not react even when mixed with the extract. In addition, when the sample liquid and the luminescent liquid are mixed and then mixed with the extract, the ATP in bacteria can be extracted from the sample liquid and emit light at the same time. When the extract solution and the luminescent solution are mixed and then mixed with the sample solution, the number of wells and the number of channels can be reduced, and the bacteria detection device can be simplified.

FIG. 4 to FIG. 10 are schematic diagrams showing a bacterium detection device according to the eleventh embodiment, and FIG. 11 is a schematic diagram showing an bacterium detection device according to an eighth embodiment of the present invention.

FIG. 5 is a schematic view showing a second embodiment of the bacteria detection device according to the present invention. FIG. 5 shows a mode in which the first channel 11 of the microchannel chip 10 according to the first embodiment is branched into the first channel 11 and the channel l ib, one of which is similar to the first embodiment, while the other is The fuel cell system includes a well 2b, a well 3b, and a reaction field 8, and the well 2b and the channel l ib are connected by a channel 12b, and the well 3b and a channel l ib are connected by a channel 13b. The bottle 2b contains a specific extract for extracting bacterial ATP from a specific bacterium, and the bottle 3b contains a luminescent solution for emitting bacterial ATP. Therefore, on the one hand, it is possible to detect bacteria contained in the sample solution, and on the other hand, it is possible to detect specific bacteria contained in the sample solution. it can.

FIG. 6 is a schematic diagram showing a third embodiment of the bacteria detection device according to the present invention. FIG. 6 shows a mode in which the first channel 11 of the microchannel chip according to the first embodiment is branched into the first channel 11 and the channel 11c, one of which is similar to that of the first embodiment, and the other of which is a well 3c. A reaction field 8 is provided, and the cell 3c and the channel 11c are connected by a channel 13c. Lulu 3c contains a luminescent solution that emits ATP in bacteria. Therefore, on the one hand, bacteria contained in the sample solution can be detected, and on the other hand, free ATP contained in the sample solution can be detected, and the measurement accuracy can be improved.

FIG. 7 is a schematic diagram showing a fourth embodiment of the bacteria detection device according to the present invention. FIG. 7 shows the microchannel chip according to the first embodiment, which is provided with a fourth well 4 and a fifth well 5, and the fourth well 4 has a removing liquid force for removing free ATP. Contains an inert liquid for inactivating the removing liquid. The fourth well 4 is the most upstream and is connected by the first channel 11 and the fourth channel 14, and the fifth well 5 is located downstream of the fourth well 4 and upstream of the third well 2. 11 and 5th channel 15 are connected. Therefore, when the sample solution is sent to the reaction field 8 through the first channel 11 and the removing solution is sent through the fourth channel 14 and the fourth channel 14, the sample solution is released, which reduces the measurement accuracy. ATP can be removed, and when the inactive solution is sent from the fifth well 5 through the fifth channel 15, the excess removing solution can be inactivated. That is, free ATP can be removed and ATP in bacteria can be accurately detected, so that measurement accuracy can be improved.

[0139] Examples of the components contained in the removing solution include adenosine phosphate deaminase, avirase, alkaline phosphatase, acid phosphatase, hexokinase, and adenosine triphosphatase.

[0140] Examples of the components contained in the inert solution include coformycin, EDTA, dithiothreitol, ammonium sulfate, HEPES, MES, and Tricine.

FIG. 8 is a schematic diagram showing a fifth embodiment of the bacteria detection device according to the present invention. FIG. 8 includes a sixth well 6 in addition to the microchannel chip according to the first embodiment, and the sixth well 6 contains an amplification solution for amplifying ATP in bacteria. The sixth well 6 is connected upstream of the third well 2 by the first channel 11 and the sixth channel 16. Therefore, When a body fluid is sent to the reaction field 8 through the first channel 11 and the amplification solution is sent from the sixth well 6 through the sixth channel 16, the amount of ATP in bacteria can be increased. In other words, even a minute amount of bacteria or bacteria having poor nutritional status can be made to remarkably emit light, and since these bacteria can be detected, measurement accuracy can be improved.

[0142] In the sixth well 6 according to the fifth embodiment, the order of sending the third well 2 extract and the second well 3 luminescent solution may be different.

[0143] The components contained in the amplification solution are not particularly limited !, but are adenylate kinase, polyphosphate kinase, polyphosphate, adenosine monophosphate, pyruvate orthophosphate dikinase, phosphoenolpyruvate, and pyrroline. Acids and magnesium ions are exemplified.

FIG. 9 is a schematic diagram showing a sixth embodiment of the bacteria detection device according to the present invention. FIG. 9 shows a microchannel chip provided with fourth and fourth wells 4 and 5 according to the fourth embodiment and sixth well 6 according to the fifth embodiment. Therefore, since ATP in bacteria can be amplified at the same time as free ATP can be removed, measurement accuracy can be improved. Care must be taken in the order of the flow. That is, in order not to amplify the free ATP, it is necessary to remove the free ATP, inactivate the removing solution, and then send the amplification solution, the extract solution, and the luminescent solution.

FIG. 10 is a schematic view showing a seventh embodiment of the bacteria detection device according to the present invention. FIG. 10 includes a seventh well 7 in addition to the microchannel chip according to the first embodiment, and the seventh well 7 contains a decomposition solution for decomposing bacterial flocs. Further, the seventh well 7 is connected to the first channel 11 and the seventh channel 17 on the upstream side of the third well 2. Therefore, when the sample solution is sent to the reaction field 8 through the first channel 11 and the digestion solution is sent through the seventh channel 7 to the seventh channel 17, the bacterial floc can be decomposed. Since the bacterial flocs can be dispersed, the contact efficiency between the bacteria and the extract can be increased, and the extraction reaction can be promoted.

[0146] The components contained in the decomposition solution are not particularly limited !, but include Tris-Hcl, HEPES, PBS, MES, Tricine and the like.

FIG. 11 is a schematic diagram showing an eighth embodiment of the bacteria detection device according to the present invention. Figure 11 shows When the sample liquid is introduced into the first well 1 containing the sample liquid, the first to fifth paths A to E are sent and the individual liquids can be individually detected. First, the introduced sample solution removes insoluble components through a filter 30 and then mixes with a removing solution to remove free ATP, and further mixes with an inactivating solution to inactivate excess removing solution. . In addition, the floc of the bacteria is dispersed with the decomposition solution. The mixture thus obtained is branched to the first to fifth paths A to E. Then, in the first route A, the ATP in the bacterium is extracted and amplified by the amplifying solution and the extract, the luminescence is emitted by the luminescent solution, and the bacterium can be detected by the light detecting means 8a. In the second pathway B, the bacterial ATP of the specific bacterium A is extracted and amplified by the amplifying solution and the specific extract A, and the luminescence is emitted by the luminescent solution. Can be. Further, in the third route C, it is possible to extract and amplify ATP in the bacterium of the specific bacterium B with the amplification solution and the specific extract B, emit the luminescence with the luminescent solution, and detect the bacterium B with the light detection means 8a. it can. In the fourth route D, free ATP can be detected by the amplification solution and the luminescence solution. Then, in the fifth route E, ATP is introduced, and the activity of the reagent can be confirmed by the amplification solution and the luminescence solution.

[0148] Therefore, all bacteria can be detected by pathway A, specific bacteria A and B can be detected by pathways B and C, and free ATP detection and reagents can be detected by pathways D and E. Can be confirmed. In addition, the diffusion in this route is performed in a very short time. As a result, the reaction can be performed instantaneously, and the bacteria can be detected quickly and accurately.

[0149] The bacteria detection device of the present invention is not limited to the above embodiment. For example, in the first embodiment, a negative pressure portion is formed in the hollow portion 35 by pressing the cover 10b with a finger, whereby the liquid from each well is introduced into the first channel 11. That is, the cover 10b, the cavity 35, and the air vent 36 constitute the transfer means, but the bacteria detection device of the present invention may use a valve, a pump, or the like as the transfer means. Even in such a case, it is possible to collectively and surely and easily introduce the liquids of each pneumatic force into the first channel. It is also useful to provide a check valve. These can be installed in a microchannel chip.

[0150] By providing the transfer means as described above, the liquid contained in each well is transferred to the light emitting unit. It becomes easy to send. In addition, these transfer means can collectively and surely introduce the liquids of each gell force into the first channel without affecting the number of wells and the number of channels.

[0151] Further, the bacteria detection device of the present invention includes a heating means for heating the first channel 11, an electric field applying means for applying an electric field to the first channel 11, and an ultrasonic wave application for applying an ultrasonic wave to the first channel 11. It is preferable to provide at least one or more of the means.

[0152] When the bacteria detection device is provided with these means, ATP in bacteria can be extracted from bacteria by these means instead of the extract. Therefore, since the third well is not required, the structure of the device can be further simplified. These can be applied not only to the extraction reaction, but also to the reaction for emitting light. Also, a plurality of these means can be installed in parallel, and can be installed in a microchannel chip.

[0153] Furthermore, the first channel may further include temperature control means for controlling the temperature of at least one portion downstream of the removing solution introduction portion to 50 ° C or more to inactivate the ATP removing agent. Is preferred. In this case, it is possible to inactivate the excess removing solution only by controlling the temperature. Therefore, since the inactive liquid is unnecessary, a channel for sending the inactivating liquid for containing the inactive liquid can be omitted, and the structure of the microchannel chip can be reduced. Can be simplified.

In the above embodiment, when bacteria are detected, the extraction liquid and the luminescent liquid are stored in the third and second wells, respectively. It is preferable that the liquid is stored. If the extract solution and the luminescent solution are stored in advance, bacteria can be detected simply by introducing the sample solution into the first well, and measurement with high detection accuracy can be easily performed regardless of the environment. Furthermore, a removing solution, an inactivating solution, an amplifying solution, and a decomposing solution can be stored in advance as necessary.

[0155] Further, in this case, it is preferable to provide a control means for controlling the temperatures of the extraction liquid and the luminescent liquid. The temperature control means can be provided so as to sandwich the microchannel chip from above and below with a heater block, and a temperature sensor is electrically connected to the heater block. Then, the temperature of the heater block is controlled based on the temperature obtained by the temperature sensor.

[0156] By controlling the temperature in this manner, it is possible to optimize the reaction performed in the apparatus of the present invention. Environment can be provided. For example, the reaction rate can be further increased by setting the temperature at an optimum value for the extract, the luminescent liquid, etc., and furthermore, the reaction can be stabilized by maintaining the temperature at that value. it can.

[0157] Further, the microchannel chip is preferably frozen and stored by a freezer (temperature control means) or the like. If stored frozen, bacteria can be stored for a long period of time without multiplying or inactivating bacteria. Also, frozen microchannel chips can be used by thawing.

Industrial applicability

[0158] Since the bacteria detection device and the bacteria detection method of the present invention can easily detect bacteria in a sufficiently short time, they can be particularly applied to the food industry and the like when a bacteria detection test is required for many commodities.

Claims

The scope of the claims

[1] 1st well for containing a sample solution containing bacteria,

A second well for containing a luminescent solution that emits light in the presence of the intracellular ATP; a light emitting unit that contacts the intracellular ATP with the luminescent solution;

A first channel connecting the first well to the light emitting unit and having a luminescent liquid introduction unit; a second channel connecting the luminescent liquid introduction unit to the second well;

A microchannel chip having

Extraction means for extracting ATP in bacteria,

Light detection means for detecting light generated by the contact between the ATP in the bacteria and the luminescent liquid; transferring the liquid stored in the first well to the light emitting unit; Means for transferring to the first channel;

, And

The bacteria detection device, wherein a channel width of the first channel is 1 mm or less.

[2] The first channel has an extract introducing portion into which an extract for extracting ATP in the bacterial capilla is introduced,

The extraction means includes a third well included in the microchannel chip and containing the extract, and a third channel connecting the extract introduction part and the third well. And

The transfer means is capable of transferring the liquid stored in the first well to the light emitting unit and transferring the liquid stored in the third well to the first channel. The bacterium detection device according to claim 1.

[3] The first channel is provided with a removing solution introducing portion into which a removing solution containing an ATP removing agent is introduced, and an inert solution containing an inactivating agent for inactivating the ATP removing agent. Further comprising an activation liquid introduction part,

The microchannel chip,

A fourth well for containing the removal solution;

A fifth well for containing the inert liquid;

A fourth channel connecting the removal solution introduction section and the fourth well, A fifth channel connecting the inactive liquid introduction part and the fifth well;

In the first channel, the removal liquid introduction section is provided upstream of the extract introduction section,

The inactivating liquid introduction section is provided downstream of the removal liquid introduction section and upstream of the extract introduction section, and

3. The bacteria detection device according to claim 2, wherein the transfer means is capable of transferring the liquid contained in the fourth and fifth wells to the first channel.

[4] The first channel further has a removing solution introducing portion into which a removing solution containing an ATP removing agent is introduced,

The microchannel chip,

A fourth well for containing the removal solution;

A fourth channel connecting the removal solution introduction section and the fourth well,

Temperature control means for controlling the temperature of the removing solution to 50 ° C. or higher to inactivate the ATP removing agent,

Is further provided,

A temperature control unit whose temperature is controlled by the temperature control unit can control a temperature of a portion downstream of the removal liquid introduction unit and upstream of the extraction liquid introduction unit; and

3. The bacteria detection device according to claim 2, wherein the transfer means is capable of transferring the liquid contained in the fourth well to the first channel.

[5] The first channel further has an amplification solution introduction part into which an amplification solution for amplifying ATP in bacteria is introduced,

The microchannel chip,

A sixth well for containing the amplification solution,

A sixth channel connecting the amplification solution introduction section and the sixth well, Is further provided,

In the first channel, the amplification liquid introduction section is provided downstream of the inactivation liquid introduction section, and

4. The bacteria detection device according to claim 3, wherein the transfer means is capable of transferring the liquid contained in the sixth well to the first channel.

[6] The first channel further has a decomposition solution introducing section into which a decomposition solution for decomposing bacterial flocs is introduced,

The microchannel chip,

A seventh well containing the decomposition solution;

A seventh channel connecting the decomposition solution introduction section and the seventh well,

Is further provided,

In the first channel, the decomposition solution introduction section is provided upstream of the extract solution introduction section, and

6. The bacteria detection device according to claim 2, wherein the transfer means is capable of transferring the liquid contained in the seventh well to the first channel.

[7] The microchannel chip according to claim 5, wherein any one of the second well, the third well, and the sixth well also serves as another well. Bacteria detection device.

[8] The first channel further includes an amplification solution introduction section into which an amplification solution for amplifying ATP in bacteria is introduced, and a decomposition solution introduction section into which a decomposition solution for decomposing bacterial flocs is introduced, Channel chip

A sixth well for containing the amplification solution,

A seventh well containing the decomposition solution;

A sixth channel connecting the amplification solution introduction section and the sixth well,

Is further provided,

In the first channel, the decomposition solution introduction section is provided upstream of the extract solution introduction section, In the first channel, the amplification liquid introduction section is provided downstream of the inactivation liquid introduction section, and

The extract corresponding to at least one well selected from the group consisting of the second well, the third well, the fourth well, the fifth well, the sixth well, and the seventh well, and the extract corresponding to the well. 4. The bacteria detection device according to claim 3, wherein the luminescence solution, the removal solution, the inactivation solution, the amplification solution, and the decomposition solution are accommodated.

[9] The bacteria detection device according to any one of claims 118, further comprising control means for controlling a temperature of a liquid in the microchannel chip.

[10] The extraction means may include a heating means for heating the first channel, an electric field applying means for applying an electric field to the first channel, and an ultrasonic applying means for applying ultrasonic waves to the first channel. The bacterium detection device according to claim 1, further comprising at least one or more means.

[11] The first channel is provided with a removing solution introducing portion into which a removing solution containing an ATP removing agent is introduced and an inert solution containing an inactivating agent for inactivating the ATP removing agent. Further comprising an activation liquid introduction part,

The microchannel chip,

A fourth well for containing the removal solution;

A fifth well for containing the inert liquid;

A fifth channel connecting the inactive liquid introduction part and the fifth well;

In the first channel, the removal liquid introduction unit is provided upstream of an extraction unit that extracts ATP in bacteria from bacteria by the heating unit, the electric field application unit, or the ultrasonic application unit,

The deactivating liquid introduction part is provided downstream of the removal liquid introduction part and upstream of the extraction part, and

11. The bacteria detection device according to claim 10, wherein the transfer means is capable of transferring the liquid contained in the fourth and fifth wells to the first channel.

[12] The first channel further includes a removing solution introducing section into which a removing solution containing an ATP removing agent is introduced. Have

The microchannel chip,

A fourth well for containing the removal solution;

Is further provided,

A temperature control unit whose temperature is controlled by the temperature control means is capable of controlling the temperature of a portion downstream of the removing liquid introduction unit and upstream of the extraction unit; and

11. The bacteria detection device according to claim 10, wherein the transfer means is capable of transferring the liquid contained in the fourth well to the first channel.

[13] The first channel further has an amplification solution introduction section into which an amplification solution for amplifying ATP in bacteria is introduced,

The microchannel chip,

A sixth well for containing the amplification solution,

Is further provided,

12. The bacteria detection device according to claim 11, wherein the transfer means is capable of transferring the liquid contained in the sixth well to the first channel.

[14] The first channel further has a digestion solution introducing section into which a digestion solution for decomposing bacterial flocs is introduced,

The microchannel chip, A seventh well containing the decomposition solution;

Is further provided,

In the first channel, the decomposition solution introduction unit is provided upstream of an extraction unit that extracts ATP in bacteria from bacteria by the heating unit, the electric field application unit, or the ultrasonic application unit, and

14. The bacteria detection device according to claim 10, wherein the transfer means is capable of transferring the liquid contained in the seventh well to the first channel.

[15] The first channel further includes an amplification solution introduction section into which an amplification solution for amplifying ATP in bacteria is introduced, and a decomposition solution introduction section into which a decomposition solution for decomposing bacterial flocs is introduced, The tip is

A sixth well for containing the amplification solution,

A seventh well containing the decomposition solution;

Is further provided,

In the first channel, the decomposition solution introduction unit is provided upstream of an extraction unit that extracts ATP in bacteria from bacteria by the heating unit, the electric field application unit, or the ultrasonic application unit,

At least one well selected from the group consisting of the second well, the fourth well, the fifth well, the sixth well, and the seventh well is also provided with the light emitting liquid corresponding to the well and the removal. 12. The bacteria detection device according to claim 11, wherein the bacteria detection device contains a solution, the inactivation solution, the amplification solution, and the decomposition solution.

[16] The bacteria detection device according to any one of claims 10 to 15, further comprising control means for controlling a temperature of a liquid in the microchannel chip. Extract the ATP in the bacteria from the sample solution containing the bacteria in the

A method for detecting bacteria, which comprises causing a luminescent solution to emit light in the presence of ATP and detecting the emitted light.

[18] The sample solution, the extract for extracting the bacterial force ATP in the bacterium, and the luminescent solution emitting light in the presence of the ATP in the bacterium are mixed, and the extract is used to separate the bacterium from the bacteria 18. The method for detecting bacteria according to claim 17, wherein ATP is extracted.

19. The bacteria detection method according to claim 18, wherein the extract is a specific extract that selectively extracts ATP only for specific bacteria.

[20] The method for detecting bacteria according to claim 19, wherein the specific extract is a phage or an antibiotic.

[21] Before mixing the extract solution and the luminescent solution, the sample solution is mixed with a removing solution containing an ATP removing agent, and the removing solution removes the sample solution free ATP, and further inactivates the sample solution. The method for detecting bacteria according to any one of claims 18 to 20, wherein the elimination solution is inactivated by mixing with an inactivation solution containing a activating agent.

[22] Before mixing the extract solution and the luminescent solution, the sample solution is mixed with a removing solution containing an ATP removing agent, and the removing solution removes the sample solution free ATP, and further removes the ATP. The method for detecting bacteria according to any one of claims 18 to 20, wherein the temperature of the mixed solution with the solution is controlled to 50 ° C or higher to inactivate the ATP removing agent.

23. The method according to claim 21, wherein the sample solution is mixed with an amplification solution for amplifying intracellular ATP after inactivating the removing solution to amplify intracellular ATP of bacteria contained in the sample solution. Bacteria detection method.

[24] Before mixing the sample liquid with the extract and the luminescent liquid, the sample liquid is mixed with a decomposing solution for decomposing bacterial flocs, and the decomposing solution decomposes the floc of the sample liquid capillar bacteria. The method for detecting bacteria according to any one of claims 18 to 23.

25. The bacteria detection method according to claim 17, wherein the bacterial force also extracts the intracellular ATP by performing at least one of heating, electric field application, and ultrasonic application to the sample solution.

[26] Before performing the heating, the electric field application, or the ultrasonic application to the sample solution, the sample solution is mixed with a removing solution containing an ATP removing agent, and the free ATP is removed using the removing solution. Then, the mixture is mixed with an inactivating liquid containing an inactivating agent to inactivate the removing liquid. The method for detecting bacteria according to claim 25.

[27] Before performing the heating, the electric field application, or the ultrasonic application to the sample solution, the test solution is mixed with a removing solution containing an ATP removing agent, and is separated from the sample solution by the removing solution. 26. The method for detecting bacteria according to claim 25, wherein ATP is removed, and the temperature of the mixed solution with the removing solution is controlled to 50 ° C. or higher to inactivate the ATP removing agent.

[28] After inactivating the removal solution in the sample solution, the sample solution is mixed with an amplification solution for amplifying ATP in bacteria to amplify ATP in bacteria of bacteria contained in the sample solution. 27. The method for detecting bacteria according to claim 26, wherein the bacteria are detected.

Before performing the heating, the application of the electric field, or the application of the ultrasonic wave to the sample solution, the sample solution is mixed with a decomposing solution for decomposing bacterial flocs, and is contained in the sample solution by the decomposing solution. The method for detecting bacteria according to any one of claims 25 to 28, wherein the method comprises decomposing bacterial flocs.