JP4116579B2 - Bacterial spore processing chip and bacterial spore processing device - Google Patents

Description

  The present invention relates to a bacterial spore processing chip for processing bacterial spores in a supplied sample.

  Conventionally, in order to analyze bacteria, a culture method in which the bacteria are transferred to a culture medium and cultured to observe whether the bacteria proliferate has been the mainstream. However, since it takes 2 to 3 days for the culture, a genetic test method for amplifying a bacterial gene and detecting the presence or absence of the target bacteria by detecting the gene has recently been introduced. Here, bacteria such as Bacillus anthracis and Bacillus cereus form spores within a few hours when the surroundings are low in water and depleted in nutrients. This spore is a very hard shell-like substance, and has a strong resistance to heat, chemicals, ultraviolet rays, etc., and therefore appropriate spore treatment is required. A method for sterilizing bacterial spores is disclosed in Patent Document 1 below. Patent Document 1 shows an example in which a germination inducer is added to bacterial spores to germinate the spores, and then a lactic acid fermentation broth containing bacteriocin is added to perform sterilization.

JP 2002-330740 A

  However, the bacterial spore sterilization method described in the above-mentioned known examples suppresses bacterial spore growth in the presence of a lactic acid fermentation broth, and does not focus on bacterial genetic testing. Furthermore, since germinated anthrax and Bacillus cereus have a great influence on the human body, it is desirable that genetic testing of bacterial spores be performed in a closed and automated system. In addition, since the cost of reagents is very high in genetic testing, downsizing of the analysis system is also a big issue.

  Accordingly, the present invention provides a bacterial spore processing chip that solves at least one of the above-mentioned problems.

  In order to solve the above problems, the present invention has the following aspects. Thereby, the treatment of bacterial spores can be easily performed on the chip in a short time with a small amount of reagent.

  According to the present invention, it is possible to provide a bacterial spore processing chip or a bacterial spore processing apparatus capable of performing a gene extraction process from bacterial spore safely and easily on a chip in a short time.

  Examples of the present invention will be described below. In addition, this invention is not limited to the content disclosed by this specification, and does not restrict | limit the change based on a well-known technique.

[Example 1]
As an embodiment of the present invention, an example will be described in which whether a target bacterium is present is detected by extracting a gene from a spore-forming bacterium and amplifying the gene by polymerase chain reaction. Here, the bacteria that form spores can be bacteria such as Bacillus and Clostridium.

(Principle of analysis)
The analysis step is roughly divided into a step of adding germination promoters to bacterial spores to germinate the bacterial spores and a step of extracting genes from the germinated bacteria. Here, gene extraction is performed by a generally known solid-phase extraction method. The solid-phase extraction method is a method in which a gene is first bound specifically to a solid surface, and then extracted by eluting only the gene into an aqueous solution in distinction from other substances. Hereinafter, the solid phase extraction method will be described step by step.

Step 1: Sprouting Germination A germination promoter is added to the bacterial spores, and the bacterial spores begin to germinate after a certain period of time. At the stage of germination, the bacteria break the spores themselves, and germination leaves the bacterial cell wall open.

Step 2 ... Lysis of cell membrane A sample is mixed with a solution containing chaotropic ions (a monovalent anion having a large molecular diameter), and the bacterial cell membrane is broken by the action of chaotropic ions. Further, chaotropic ions simultaneously denature many proteins contained in the sample and inhibit the action of nucleases (enzymes that degrade nucleic acids).

Step 3 ... Binding When silica is added to the mixture after dissolution, the gene and silica are specifically bound by the action of chaotropic ions. Generally, a method of passing the mixture through a glass filter is used.

Step 4 ... Washing If the protein or chaotropic ions contained in the sample are mixed in the extract, the detection of the gene by gene amplification is inhibited, so an operation for washing the gene-silica is required. Here, it is washed with high-concentration ethanol. Since genes are difficult to dissolve in high concentrations of ethanol, genes adsorbed on silica do not elute during this process.

Step 5 ... Elution After washing, water or a low salt solution is added to the gene-silica to elute the gene from the silica.

Step 6 ... Gene detection Primer (single-stranded DNA having the same base sequence as about 20 bases at both ends of the target DNA region), DNA synthase (polymerase) and four kinds of substrates (dNTP) ) And the like, and a temperature cycle “thermal denaturation—annealing—synthesis of complementary strand” is applied to amplify the gene (PCR: polymerase chain reaction). Here, in addition to the reagent, a fluorescent dye is injected in advance, and a temperature cycle is applied while irradiating excitation light, whereby gene amplification can be detected in real time.

(Configuration of bacterial spore processing chip)
The configuration of a bacterial spore processing chip for processing bacterial spores will be described with reference to FIG. FIG. 1 is a detailed view of the bacterial spore processing chip 101. In this example, a mode including a lysate storage tank and a gene amplification reagent will be described.

  The bacterial spore processing chip 101 includes a germination promoting liquid storage tank 111 for storing a germination promoting liquid, a solution storage tank 112 for storing a cell membrane lysate, and a sample inlet 102 (may be used as a waste liquid tank). A gene elution tank 103 (which may be used as a waste liquid tank) in which germinated bacterial spores are dissolved by a lysing solution to elute bacterial genes, a gene extraction area 113 in which a flow path is filled with a gene-binding carrier, and a washing solution. The washing liquid storage tank 114 to be stored, the eluent storage tank 115 to store the gene eluent, the reaction tank 104 to detect the gene, and the liquid stored in the flow path constituting each tank flow more than the position. The chip port (121 to 128) is located on the end side of the path and serves as a contact point with an external flow path. Here, the fluid inside and outside the chip is communicated. The fluid is a gas such as air. In some cases, it may be a liquid such as water. In the present embodiment, an example in which a gene amplification reagent storage tank 116 that stores gene amplification reagents is further provided is shown. These chip ports allow fluid to communicate between the inside and outside of the chip. Most of these chip components are fine flow paths that have been pattern-transferred by microfabrication technology.

Here, a method for producing the bacterial spore processing chip 101 will be described. As a material for the bacterial spore processing chip 101, a resin excellent in disposal property is preferable to glass that is expensive to process and easily broken. Although the kind of resin is not particularly limited, polydimethylsiloxane (PDMS: manufactured by Dow Corning Asia Ltd., Sylpot 184) having the following excellent properties was used in this example. The chip preferably has the following characteristics.
-Good biocompatibility (normal silicone rubber is physiologically inert)
・ Transfer of mold with submicron accuracy (Because of low viscosity and high fluidity before curing, it can penetrate fine details of complex shapes.)
-Low cost (can be reduced to 1/100 or less compared to Pyrex glass, which is a conventional general-purpose micro device material)
・ Can be easily disposed of by incineration

  FIG. 2 shows a flow of creating a bacterial spore processing chip 101 using a resin substrate (hereinafter, a case where PDMS is used as an example) is shown. The bacterial spore processing chip can be formed by producing a pattern that describes the constituent elements of the bacterial spore processing chip by a photolithography technique, and transferring and molding this pattern onto a resin. The process is roughly divided into [1] pattern formation to be transferred to PDMS, [2] pattern transfer to PDMS, and [3] bonding of PDMS to each other. Hereinafter, each process will be described step by step.

[1] Molding of pattern to be transferred to PDMS Photosensitive thick film resist 207 (Micro, Chem, NANO SU-8) is applied to silicon wafer 208 as a pattern material (step 201), and photomask 209 is photosensitive thick. A micropattern is formed through exposure (step 202) and development process (step 203) on the film resist 207. Unlike conventional photoetching by wet etching, it has the advantage that a curved shape can be formed while maintaining a short cross section.

[2] Pattern Transfer to PDMS PDMS 210 and a curing agent are mixed at a weight ratio of 10: 1, applied onto the pattern, and heated at 100 ° C. for 1 hour to cure PDMS 210 (step 204). A concave PDMS 210 is obtained from the convex micropattern (step 205).

[3] Bonding of PDMSs The surface of the PDMS 210 to which the pattern has been transferred is subjected to oxygen plasma treatment, and the two PDMSs 210 are bonded to each other by overlapping them. The bonding strength is sufficient to cause the PDMS 210 to break when the bonding site is peeled off. One of them may be bonded to silicon or glass as PDMS210. The PDMS molding method is not limited to the above-described method, and can be processed by, for example, extrusion molding.

  A more detailed structure of the bacterial spore processing chip 101 will be described with reference to FIGS. FIG. 3 is a cross-sectional view of the bacterial spore processing chip 101. The bacterial spore processing chip 101 is obtained by bonding two PDMS surfaces that have been surface-modified by plasma processing. First, the PDMS first layer 131 is formed with a through-hole serving as the sample inlet / waste liquid tank 102. The volume of the sample inlet / waste liquid tank 102 is 10 to 100 μL and is open to the atmosphere. The PDMS second layer 132 is patterned with microchannels 110 serving as various reagent vessels (111 to 116). The microchannel 110 was formed with a cross-sectional shape of 0.1 mm in length and 0.5 mm in width. The cross-sectional shape is not particularly limited, but is preferably horizontal / vertical 10 or less. If the horizontal / vertical ratio is 10 or more, the PDMS first layer 131 may bend and the rectangular structure of the microchannel 110 may be destroyed.

  Further, the PDMS second layer 132 is formed with a chip port 120 that communicates the micro flow path 110 and the flow path of the external device, and a through hole that becomes the gene elution tank 103 and the reaction tank 104. The volume of the liquid containing bacterial spores and germination promoters introduced into the gene elution tank 103 is 10 to 30 μL. Moreover, the volume of the liquid containing the gene introduced into the reaction vessel 104 is 10 to 40 μL. In particular, by setting the volume of the reaction vessel 104 to 10 to 20 μL, the thermal responsiveness is improved and the temperature control of the reaction vessel 104 is speeded up. Thereby, reaction can be advanced on optimal conditions, changing the temperature of the reaction tank 104 per second. Further, by reducing the volume of the reaction tank 104, the mixing operation can be facilitated because the eluent and the gene amplification reagent in the reaction tank 104 can be mixed in a short time (eg, about 1 second). The reaction tank 104 has a larger area as viewed from the thickness direction than the sample inlet 102. In the prior art disclosed in Patent Document 1, mixing is performed using an ultrasonic element or the like. However, according to the present embodiment, it is conceivable not to use a mixing operation for simplification. Or it can be simple to use.

  In addition, since the gene elution tank 103 and the reaction tank 104 are through holes, naturally, a bottom plate is necessary. As will be described later, since the bottom plate 140 of the bacterial spore processing chip 101 also serves as a medium for transferring heat from the temperature control mechanism to the gene elution tank 103 and the reaction tank 104, it is preferably a material having good thermal conductivity. Furthermore, if the surface of the bottom plate 140 is a mirror surface, the fluorescence in the reaction vessel 104 is reflected by the bottom plate 140, so that the gene detection sensitivity is increased. Preferred as the bottom plate 140 is chromium, and most preferred is silicon. This is because silicon has good thermal conductivity and can be easily joined to PDMS only by oxygen plasma treatment.

  The bacterial spore processing chip 101 contains five reagent tanks (germination promoter storage tank 111, lysate storage tank 112, washing liquid storage tank 114, eluent storage tank 115, gene amplification reagent storage tank 116). . Any reagent tank preferably has a flow channel shape. In order to send the reagent in the reagent tank, fluid (air or water) is sent from behind the reagent tank to the reagent tank. At this time, if the reagent tank is not in the shape of a flow path, the reagent is pushed out only at a part where fluid can easily pass through (part with good liquid wettability), and the reagent at other parts remains in the reagent tank. In order to reduce the amount of reagent to be consumed, it is effective to make the reagent tank into a channel shape.

  Here, the germination promoter storage tank 111 has a volume of 10 to 20 μL, the dissolution liquid storage tank 112 has a volume of 10 to 20 μL, the cleaning liquid storage tank 114 has a volume of 10 to 30 μL, and the eluent storage tank 115 has a volume of 5 to 10 μL. The volume of the gene amplification reagent storage tank 116 is preferably 5 to 10 μL. In particular, when the sum of the eluent (including the gene) and the gene amplification reagent is 10 μL or less, the mixing of the eluent and the gene amplification reagent in the reaction tank 104 is accelerated and the reaction becomes uniform as described above. Is optimal. Thus, micronizing the volume of the reagent tank or reaction tank not only reduces the amount of reagent and lowers the cost, but also provides advantages such as rapid temperature control, rapid mixing, and uniform reaction.

  Quartz wool, glass wool, glass fiber, and glass beads are applicable as the gene binding carrier to be filled in the gene extraction area 113. When glass beads are applied, the bead size is preferably 20 to 50 μm, and 20 to 30 μm is optimal in order to increase the contact area.

  Moreover, in order to keep the gene holding carrier, it is preferable that one or more flow paths constituting the area are narrowed.

  For example, in order to hold the gene-binding carrier in the gene extraction area 113, it is preferable to narrow the channel width at two locations to 10 μm in the microchannel of the gene extraction area 113. That is, the narrowed flow path becomes a weir for the gene-binding carrier. If the flow path is less than 10 μm, fluid resistance is large and fluid control becomes difficult. Therefore, the channel width as the weir is preferably 10 to 20 μm.

(Configuration of bacterial spore treatment equipment)
FIG. 4 shows a cross-sectional view of an apparatus for setting the bacterial spore processing chip 101. The analysis apparatus is roughly divided into three parts: a fluid system, a temperature control system, and an optical detection system. The substrate 100 on which the bacterial spore processing chip 101 is set has an adsorption groove 150 for adsorbing the bacterial spore processing chip 101, an in-device flow path 162 communicating with a port of the bacterial spore processing chip 101, a gene elution tank 103, and a reaction tank. A temperature control mechanism 170 for optimizing the temperature of 104 is incorporated. The apparatus includes a control mechanism for performing each control. The apparatus includes a pump control mechanism 165 for controlling the pump 160, a valve control mechanism 166 for controlling the valve 161, a light source control mechanism 185 for controlling the light source 180, a photodetector control mechanism 186 for controlling the photodetector 181 and a photodetector. An optical signal converter 187 for converting the above signals and a data display screen 188 for displaying the converted optical signals are mounted.

  The bacterial spore processing chip 101 is adsorbed to the substrate 100 by placing the bacterial spore processing chip 101 on the substrate 100 and evacuating the adsorption groove 150 of the substrate 100. By performing the vacuum chuck in this manner, the chip port 120 and the in-device flow path 162 are securely connected to prevent fluid leakage, while the bacterial spore processing chip 101 can be easily attached to and detached from the substrate 100. Become. In order to make the bacterial spore processing chip 101 disposable, a fixing method of the bacterial spore processing chip 101 using a vacuum chuck is extremely practical.

  As the temperature control mechanism 170, various heating elements can be applied. For example, a Peltier is preferable. When a Peltier is used, the temperature raising / cooling operation can be easily performed by simply changing the direction of the applied current.

  The in-device flow paths 162 are each connected to the pump 160 via a valve 161. The pump 160 is more preferably capable of switching between blowing and suction, and more preferably a plurality of pumps. When it is desired to send a reagent in a certain reagent tank, the valve 161 is switched to blow air only to a port communicating with the reagent tank.

  Thus, it is preferable to provide the valve 161 for controlling the fluid not on the inside of the bacterial spore processing chip 101 but on the analyzer side. As a result, the bacterial spore processing chip 101 has no mechanical parts, and can be downsized and made disposable.

  The light detection system includes a light source 180 that irradiates a gene in the reaction tank 104 with excitation light and a light detector 181 that measures fluorescence in the reaction tank 104. The light source 180 can be used in various wavelength ranges, but when a common ethidium bromide is used as a fluorescent dye, it is preferable to use an ultraviolet lamp or an ultraviolet laser. The light detector 181 is arranged so that the light receiving surface of the light detector 181 is directly above the reaction vessel 104. As the photodetector 181, a CCD camera, a photomultiplier tube, a photodiode, or the like can be used, but a photodiode is preferable for downsizing the apparatus.

  In the present invention, it is possible to provide a small and portable analyzer in which a small bacterial spore processing chip that does not contain mechanical parts is placed on a substrate and a simple photodetector is combined.

(Analysis procedure)
An analysis procedure using the bacterial spore processing chip 101 will be described with reference to FIGS. 4, 5, and 6. FIG. 5 is a flowchart showing the procedure of the analysis method. FIG. 6 is a diagram showing a fluid handling profile of Example 1. FIG.

The analysis procedure can mainly include the following procedures.
First, a germination promoting agent is supplied to the bacterial spores of the sample to germinate the bacterial spores.
Then, the lysate that breaks the cell wall of the sample is mixed with the germinated sample.
Next, the mixed solution of the lysate and the sample is sent to a channel filled with a gene holding carrier.
Then, a washing solution for washing proteins and the like contained in the sample is sent to the channel filled with the gene holding carrier, and the waste solution is sent to the tank in which the sample was originally held.

Next, an eluent for eluting the gene adsorbed on the gene holding carrier is sent to a flow path filled with the gene holding carrier, and further sent to a reaction tank for detecting the gene.
Thereafter, the presence or absence of the gene to be analyzed is detected. An example will be specifically described below.

  First, five types of reagents, namely germination promoter, cell membrane lysate, washing solution, gene eluent, and gene amplification reagent, are germination promoter storage tank 111, lysate storage tank 112, cleaning liquid storage tank 114, and eluent storage, respectively. The bacterial spore processing chip 101 that is built in the tank 115 and the gene amplification reagent storage tank 116 and has been cryopreserved is thawed at room temperature. By providing the bacteria spore processing chip 101 with a reagent for one test in advance and providing it to the user, even if the bacteria spore processing chip 101 is used up for one test, the reagent is not wasted and the economy is improved. In addition, the user can save the trouble of dispensing the reagent into each reagent storage tank, and not only the time is shortened but also contamination can be prevented. Furthermore, the bacterial spore processing chip 101 is provided to the user in a frozen state, and the user keeps the bacterial spore processing chip 101 frozen at 0 ° C., so that the activity of the reagent is maintained for one month. In addition, if stored frozen at −20 ° C., the reagent activity can be maintained for more than half a year. As described above, a reagent for only one test is built in the disposable bacterial spore processing chip 101 in advance, and the bacterial spore processing chip 101 is provided to the user in a refrigerated or frozen state, thereby creating a simple analysis environment. (Step 311).

Next, the bacterial spore processing chip 101 is placed on the substrate 100, and after confirming that the chip port 120 and the in-device flow path 162 communicate with each other, the adsorption groove 150 is decompressed. In this way, vacuuming is performed, and the bacterial spore processing chip 101 is fixed to the substrate 100 by a vacuum chuck (step 312).
Then, 10 μL of the sample is dispensed into the sample inlet / waste liquid tank 102. The sample is a powder of bacteria in which spores are formed or a suspension liquid (step 313).

  Next, the valve 161 in the analyzer is switched to allow the fluid to flow from the pump 160 only to the germination promoter port 121. (Port 121: Open, Other ports 122 to 128: Closed) The fluid used here may be water, alcohol, air, or the like that does not impair the activity of the reagent when in contact with the reagent.

  20 μL of the germination accelerator in the germination accelerator storage tank 111 is injected into the sample inlet / waste liquid tank 102 by a fluid and mixed with the sample in the sample inlet / waste liquid tank 102. Here, as the germination promoter, bouillon containing alanine, adenosine and glucose is preferable. In particular, a bouillon containing 1 mM to 10 mM of L-alanine is optimal (step 314).

  Next, the valve 160 in the analyzer is switched, and the pump 160 is aspirated to depressurize the inside of the chip from the chip port A122. (Ports 121, 122: open, other ports 123-128: closed, port 121 may be closed.) Thus, the sample and germination promoter in the sample inlet / waste liquid tank 102 are transferred to the gene elution tank 103. Moving. Then, when all the liquid in the sample inlet / waste liquid tank 102 is transferred to the gene elution tank 103, the pump 160 is stopped. Furthermore, the temperature control mechanism 170 provided in the lower part of the bacterial spore processing chip 101 is driven to control the temperature of the reaction tank 104 to 25 to 40 ° C. via the bottom plate 140. More preferred for germinating bacterial spores is 35-40 ° C, and most preferred is 35-37 ° C. The bacterial spores begin to germinate after 10 minutes and all of the bacterial spores germinate after 60 minutes. Thus, since the germination process of bacterial spores is performed in a closed space in the chip, it is suitable for treatment of anthrax and Bacillus cereus that have a negative effect on the human body once germinated (step 315).

  Next, the fluid is allowed to flow from the pump 160 only to the solution port 123. (Ports 121, 123: open, other ports 122, 124-128: closed, port 121 may be closed.) 20 μL of cell membrane lysate in lysate storage tank 112 is injected into gene elution tank 103 by fluid. , Mixed with germinated bacteria. Thereby, the bacterial cell membrane is destroyed, and the bacterial gene is released to the outside of the cell. Here, the cell membrane lysis solution is preferably a solution containing chaotropic ions such as guanidine thiocyanate, guanidine hydrogen chloride, sodium iodide, potassium bromide (step 316).

  Next, the pump 160 is aspirated so as to depressurize the inside of the chip from the chip port B124 by switching the valve 161 in the analyzer. (Ports 121-124: open, other ports 121, 125-128: closed, port 121 may be closed.) As a result, the gene suspension in gene elution tank 103 moves to gene extraction area 113. . Then, when all the gene suspension in the gene elution tank 103 has moved to the gene extraction area 113, the pump 160 is stopped. As a result, the gene is captured on the gene binding carrier in the gene extraction area 113 (step 317).

  Next, the valve 161 in the analyzer is switched to close the ports 122 to 124, and the fluid is allowed to flow from the pump 160 only to the cleaning liquid port 125. (Ports 121 to 123, 125: open, other ports 124, 126 to 128: closed, and the lysate port 121 may be closed.) 20 μL of the cleaning solution in the cleaning solution storage tank 114 is sent to the gene extraction area 113 by the fluid. To be liquidated. Here, TRIS hydrochloric acid can be used as the cleaning liquid, and high concentration ethanol of 50% or more is more preferable. This washing solution removes germination promoters, proteins, and chaotropic ions remaining in the gene extraction area 113. Then, the cleaning solution that has passed through the gene extraction area 113 is allowed to flow to the gene elution tank 103 and further to the sample injection port 102 side. For example, the pump 160 is stopped at the stage where the cleaning liquid that has cleaned the gene extraction area 113 has moved to the sample inlet / waste liquid tank 102. By making the waste liquid tank also serve as the sample injection port as in this embodiment, the size of the bacterial spore processing chip 101 can be reduced, which is more suitable for a disposable application. At this time, the used cleaning liquid may be introduced into the germination promoter storage tank 111 or the solution storage tank 112 in addition to or instead of the sample injection port 102. As a result, waste liquid can be stored without increasing the size of the sample inlet 102. Alternatively, it is possible to effectively suppress leakage of waste liquid from the sample injection port to the outside. At this time, the germination promoter storage tank 111, the sample inlet / waste liquid tank 102, the gene extraction area 113, and the eluent storage tank 115 are arranged in series, or the solution storage tank 112 and the sample inlet / waste liquid tank. Since 102, the gene extraction area 113, and the eluent storage tank 115 are arranged in series, the fluid control procedure is the simplest and the analysis time is the shortest (step 318).

  Next, the valve 161 in the analyzer is switched to close the ports 122, 123, and 125, and the elution port 126 and the chip port C128 are opened. Then, the pump 160 is driven to flow the fluid to the elution port 126. (Ports 121, 126, 128: open, other ports 122-125, 127: closed, and the lysate port 121 may be closed.) 5 μL of the eluent in the eluent storage tank 115 is transferred to the gene extraction area 113 by the fluid. The liquid is sent to Here, as the eluent, sterilized distilled water, buffer solutions such as TRIS-EDTA and TRIS-acetate can be used. With this eluent, the gene captured on the gene binding carrier in the gene extraction area 113 is eluted. Then, when the eluent containing the gene reaches the end of the gene extraction area 113, another pump 160 is aspirated to depressurize the reaction tank 104 from the chip port C128. As a result, the eluent containing the gene is guided to the reaction tank 104 without being sent to the sample inlet / waste liquid tank 102. Then, when all of the eluent has been transferred to the reaction tank 104, the pump 160 is stopped. As a result, sample pretreatment, that is, gene extraction is completed (step 319).

Here, the extracted sample is detected by a gene detection device.
The following shows an example of a gene detection procedure. The eluent port 126 and the chip port C128 are closed by switching the valve 161 in the analyzer, and the fluid flows from the pump 160 only to the gene amplification reagent port 127. (Gene amplification reagent port 127: open, other ports 121-126, 128: closed.) 5 μL of gene amplification reagent in the gene amplification reagent storage tank 116 is injected into the reaction tank 104 by a fluid, and the gene in the reaction tank 104 Mixed. Here, the gene amplification reagent is 4 types of dNTP (dATP, dCTP, dGTP, dTTP) at a concentration of 2.5 mM, a buffer (100 mM concentration TRIS hydrochloric acid, 500 mM concentration KCl, 15 mM concentration MgCl2), two types of primer, DNA synthase. (Any of Taq DNA polymerase, Tth DNA polymerase, Vent DNA polymerase, and thermosequenase) and a fluorescent dye (any of ethidium bromide or SYBR GREEN (manufactured by Molecular Probe)) (step 320).

  Next, the temperature control mechanism 170 provided in the lower part of the bacterial spore processing chip 101 is driven, and a temperature cycle is applied so that the temperature of the reaction tank 104 reciprocates the following two set values via the bottom plate 140 (step) 321).

As an example of the temperature cycle, the following degree is carried out.
“90 to 95 ° C., 10 to 30 seconds⇔65 to 70 ° C., 10 to 30 seconds” × 30 to 45 times As a preferred example, the following temperature cycle is carried out.
“94 ℃, 30 seconds ６８ 68 ℃, 30 seconds” x 45 times

  While applying a temperature cycle, the reaction vessel 104 is irradiated with excitation light from the light source 180 above the bacterial spore processing chip 101. When the gene has a fluorescent dye intercalated inside the double strand, it passes the energy of the absorbed light source 180 light to the fluorescent dye (energy transfer). As a result, the fluorescent dye is excited and emits fluorescence. That is, when the target gene is present in the sample, the amount of fluorescence emitted increases as the gene is amplified. Therefore, by monitoring the amount of fluorescence in the reaction tank 104 by the photodetector 181 during the temperature cycle, the presence or absence of the target gene can be detected in real time as shown in FIG. 7 (step 322).

  Then, after the suction force of the suction groove 150 of the substrate 100 is lowered, the chip is taken out from the substrate. For example, when the analysis is completed, the bacterial spore processing chip 101 is taken out from the analyzer and discarded (step 323). Since no post-treatment of the sample or reagent is required, and no washing operation of the reaction detection unit is necessary, simple and rapid analysis can be provided.

  By using the bacterial spore processing chip of the present invention, the process of extracting a gene from bacterial spore is automated in a small chip. Anyone can safely extract genes from bacterial spores because the germination process of bacterial spores does not involve any human intervention. Further, since the bacterial spore processing chip does not include mechanical parts such as a valve and the space saving is realized such that the waste tank also serves as a reagent injection port, a bacterial spore processing chip suitable for disposable use can be provided. In addition, as a result of microfabrication of reaction vessels and channels and miniaturization of the volume, not only the amount of reagent is reduced and the cost is reduced, but also advantages such as rapid temperature control, rapid mixing, and uniform reaction are obtained. . Furthermore, by disposing a reagent for only one test in advance in a disposable bacterial spore processing chip and providing the user with the bacterial spore processing chip in a refrigerated / frozen state, extremely simple and rapid gene detection becomes possible.

  In addition, the reaction tank 104 of the chip | tip demonstrated in the present Example can be a form provided with the wall which prevents communication of air | atmosphere between air | atmosphere. On the other hand, from the viewpoint of manufacturing, the reaction vessel 104 region can be open to the atmosphere. At this time, if the reaction vessel 104 is covered with quartz glass or the like that transmits ultraviolet rays, evaporation of the reaction solution can be prevented.

  In addition, the number of reaction vessels is one. However, a plurality may be used from the viewpoint of depending on the object to be inspected.

  In addition, with this configuration, the fluid in the analysis chip is controlled by the fluid equipment (pumps, valves) outside the chip, so the pump and many valves are placed in the chip. It is possible to make a simple chip configuration that can suppress this.

  This makes it easy and quick to extract and analyze genes from a sample containing bacterial spores, and can provide an analysis chip that can store a reagent in advance and dispose of it together with the reagent after the analysis, and an analyzer equipped with the chip.

  Further, in this embodiment, the cross-sectional area of the meandering channel-like liquid storage tank and the narrow tube of the gene extraction area is larger than the communication channel section connecting these storage tanks and other areas (for example, the inlet and the reaction tank). By forming, the pressure loss at the time of discharging fluids, such as a reagent, from a storage tank etc. can be made small.

  On the other hand, it is preferable that the cross-sectional area of the narrow tube of the storage tank part in which the liquid is stored is too small to suppress the generation of liquid residue when the liquid is discharged. For example, it is conceivable to make the cross-sectional area of the communication channel portion about 10 times or less. Alternatively, from the viewpoint of reducing the expansion / contraction loss during the liquid flow between the storage tank and the communication channel, for example, it is conceivable to make it about 5 times or less.

  Although the above is defined as a cross-sectional area, the height of the narrow tube is the same or not much different between the area of the storage tank etc. and the communication channel, and the width is changed to be larger than the above-mentioned height difference from the viewpoint of manufacturing. It is easy and preferable. For example, the numerical value defined as the cross-sectional area can be defined as the width.

  Further, between the storage tank or the gene extraction area and the corresponding port, having a region narrower than the cross-sectional area of the thin tube of the storage tank or the gene extraction area can prevent leakage of stored liquid, etc. It is preferable in terms of suppression.

[Example 2]
Although this embodiment can basically have the form described in Embodiment 1, in this embodiment, the sample inlet / waste liquid tank 102 of the bacterial spore processing chip 101 is not open to the atmosphere, and at least the sample is supplied. After dispensing into the sample inlet / waste liquid tank 102, the sample inlet 102 is provided with a cover such as a wall that prevents the atmosphere from communicating. For example, as shown in FIG. 8, a sample inlet / waste liquid tank cover 105 such as a glass thin plate (for example, a cover glass for a microscope) having good adhesion to the resin is placed on the sample inlet / waste liquid tank 102, and the sample inlet / outlet is also used. The waste liquid tank 102 can also be sealed. The step of covering the sample inlet / waste liquid tank 102 may be performed manually, but it is more preferable that a mechanism for mounting the sample inlet / waste liquid tank cover 105 is provided on the analyzer side. Note that it is efficient from an operational point of view that these covers are preliminarily covered to prevent communication with the atmosphere.

  Accordingly, FIG. 9 shows an example of a fluid handling profile in the second embodiment. The steps 311 to 314 in the first embodiment are the same in the second embodiment. Steps 315 and after will be described. The valve 161 in the analyzer is switched to open the chip port A122, and the fluid is allowed to flow from the pump 160 to the germination promoter port 121. (Ports 121 to 122: open, other ports 123 to 128: closed.) Thereby, the sample and germination promoter in the sample inlet / waste liquid tank 102 move to the gene elution tank 103. Then, when all the liquid in the sample inlet / waste liquid tank 102 has moved to the gene elution tank 103, the pump 160 is stopped (step 315).

  Next, the valve 161 in the analyzer is switched to close the chip port A122, and the fluid flows through the lysate port 123 while the germination promoter port 121 remains open. (The germination promoting liquid port 121, the washing liquid port 123: open, and the other ports 122, 124 to 128: closed.) The cell membrane lysate in the lysate storage tank 112 is injected into the gene elution tank 103 by the fluid, and germinated bacteria and Mixed (step 316).

  Next, the solution 161 is closed by switching the valve 161 in the analyzer, the chip port B124 is opened, and the fluid flows through the germination promoter port 121. (Ports 121, 124: open, other ports 122-123, 125-128: closed.) Thereby, the gene suspension in the gene elution tank 103 moves to the gene extraction area 113. Then, the pump 160 is stopped when the gene suspension in the gene elution tank 103 has been transferred to the gene extraction area 113 (step 317).

  Next, the valve 161 in the analyzer is switched to close the chip port B124, and the fluid flows through the washing liquid port 125 while the germination promoter port 121 remains open. (Ports 121, 125: open, other ports 122-124, 126-128: closed.) The cleaning liquid in the cleaning liquid storage tank 114 is sent to the gene extraction area 113 by a fluid. Then, the pump 160 is stopped at the stage where all of the washing solution for washing the gene extraction area 113 has moved to the gene elution tank 103 and further to the sample injection port 102 side (step 318).

  Next, the valve 161 in the analyzer is switched to close the germination promoter port 121 and the cleaning liquid port 125, and the eluent port 126 and the chip port C128 are opened. (Ports 126, 128: open, other ports 121-125, 127: closed.) Then, the pump 160 is driven to flow fluid to the elution port 126. The eluent in the eluent storage tank 115 is sent to the gene extraction area 113 by a fluid. By this eluent, the gene captured on the gene binding carrier in the gene extraction area 113 is eluted and guided to the reaction tank 104 in which the chip port C128 is open. Then, when all of the eluent has been transferred to the reaction tank 104, the pump 160 is stopped (step 319).

  Thus, by sealing the sample inlet / waste liquid tank 102, the processing can be effectively performed by inflow operation of the fluid (here, for example, air) by the pump. It may be considered that the suction operation as in the first embodiment is not required. Further, it is possible to prevent the contamination from the atmosphere and the leakage of reagents, which may occur because the sample inlet / waste liquid tank 102 is open to the atmosphere.

[Example 3]
In this example, the form described in Example 1 can be basically used. In this example, gene amplification is performed while keeping the temperature constant.

The process until the gene is extracted from the sample is the same as in Example 1. In this case, the components of the gene amplification reagent are different. That is, the gene amplification reagent is composed of 4 types of dNTP (dATP, dCTP, dGTP, dTTP) at 10 mM concentration, buffer (2 mM concentration MgSO4), 4 types of primer, 100 mM concentration MgSO ₄ , 4M concentration BETAINE, DNA synthase (4Unit / ΜL of Bst polymerase) and a fluorescent dye (either ethidium bromide or SYBR GREEN (manufactured by Molecular Probe)). The temperature adjustment of the reaction tank 104 by the temperature control mechanism 170 is set to a range of 60 to 65 ° C. When the target gene is present, the amount of fluorescence increases about 1 hour after the start of temperature control. Further, instead of performing fluorescence detection, white turbidity due to magnesium pyrophosphate as a by-product may be measured with an absorptiometer.

  Although it is somewhat difficult to design four types of primers, the temperature can be adjusted with a simpler heater than Peltier because no temperature cycle is required. It has the advantage that the components of the analyzer can be simplified.

[Example 4]
This embodiment can basically use the form described in the first embodiment, but a piezoelectric element such as a crystal resonator or a surface acoustic wave element is applied as the bottom plate 140 of the analysis chip 101. A piezoelectric element is widely used as a technique for continuously measuring a small amount of mass change in a reaction atmosphere because it quantitatively converts the weight attached on the electrode into a change in oscillation frequency. Therefore, various nucleotides whose base sequences are known in advance are fixed to the piezoelectric element as the chip bottom plate 140. The fixing method is preferably as follows. First, a glass thin film is formed on the electrode of the piezoelectric element by a method such as sputtering or vapor deposition. The glass is preferably composed mainly of SiO ₂ which has the best adhesion with chromium or titanium as electrode materials. When aminopropyltrimethoxysilane (APS) is added to the glass thin film and baked at about 120 to 160 ° C., amino groups are fixed on the surface of the glass thin film. Here, it is preferable that the thickness of an electrode and a glass thin film is respectively 0.1-1 micrometer. This is because when both thicknesses exceed 1 μm, the frequency response of the piezoelectric element is deteriorated. Furthermore, a nucleotide is applied to a glass thin film coated with an amino group, and kept at 37 ° C. and 90% humidity for 1 hour in a constant temperature and humidity chamber. Thereafter, irradiation of ultraviolet light of 60 mJ / cm ² to the piezoelectric element by using a UV cross-linker, nucleotides are firmly fixed to the piezoelectric element.

  The process until the gene is extracted from the sample is the same as in Example 1. Then, when the temperature of the gene sent to the reaction vessel 104 is raised to about 94 ° C. by the temperature control mechanism 170, the gene is thermally denatured and becomes a single strand. When the single-stranded gene and the nucleotide fixed on the bottom plate 140 are combined, the oscillation frequency of the piezoelectric element changes. Therefore, by measuring this frequency change, it is possible to read the sequence of the gene complementary to the fixed nucleotide.

  When a piezoelectric element is used in the liquid, if the liquid temperature changes by 1 ° C., the frequency changes by 15 to 30 Hz, so accurate control of the liquid temperature is essential, but in this example, a gene amplification reagent is not necessary, Since there is no need for a temperature cycle, the detection time is shortened.

It is an enlarged view which shows the structure of a bacterial spore processing chip | tip. It is a flowchart figure which shows the preparation procedure of an anti-bacterial spore processing chip | tip. 1 is a cross-sectional view of a bacterial spore processing chip of Example 1. FIG. It is sectional drawing which shows the structure of a bacterial spore processing apparatus. FIG. 3 is a flowchart showing an analysis procedure of Example 1. It is a figure which shows the profile of the fluid handling of Example 1. FIG. 2 is an example of an experimental result of Example 1. It is sectional drawing of the bacterial spore processing chip | tip of Example 2. FIG. It is a figure which shows the profile of the fluid handling of Example 2. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 ... Substrate, 101 ... Bacteria spore processing chip, 102 ... Sample inlet / waste liquid tank, 103 ... Gene elution tank / waste liquid tank, 104 ... Reaction tank, 105 ... Sample inlet / waste liquid tank cover, 110 ... Microchannel, DESCRIPTION OF SYMBOLS 111 ... Germination promoter storage tank, 112 ... Lysate storage tank, 113 ... Gene extraction area, 114 ... Cleaning liquid storage tank, 115 ... Eluent storage tank, 116 ... Gene amplification reagent storage tank, 120 ... Chip port, 121 ... Germination Accelerator port, 122 ... chip port A, 123 ... dissolution solution port, 124 ... chip port B, 125 ... washing solution port, 126 ... eluent port, 127 ... gene amplification reagent port, 128 ... chip port C, 131 ... PDMS 1 layer, 132 ... PDMS second layer, 140 ... bottom plate, 150 ... adsorption groove, 160 ... pump, 161 ... valve, 162 ... flow path in the apparatus, 16 DESCRIPTION OF SYMBOLS ... Pump control mechanism, 166 ... Valve control mechanism, 170 ... Temperature control mechanism, 180 ... Light source, 181 ... Photo detector, 185 ... Light source control mechanism, 186 ... Photo detector control mechanism, 187 ... Optical signal converter, 188 ... Data display screen

Claims (11)

An inlet to which a sample containing bacteria forming spores is supplied;
A germination promoting liquid storage unit for storing the germination promoting liquid;
A solution storage unit for storing the solution,
A gene in which a gene is eluted from the sample by mixing the sample supplied through the inlet, the germination promoting solution supplied from the germination promoting solution storage unit, and the lysing solution supplied from the lysate storage unit An elution part;
A gene extraction unit comprising a gene-binding carrier that binds to the eluted gene supplied from the gene elution unit;
A cleaning liquid storage unit for storing the cleaning liquid;
An eluent storage section for storing the eluent;
A reaction unit into which the gene eluted from the gene-binding carrier is introduced by the eluent supplied from the eluate storage unit to the gene extraction unit;
A gene amplification reagent storage unit for storing the gene amplification reagent,
The lysate storage part is constituted by a meandering flow path connected to the gene elution part,
The washing liquid storage part and the eluent storage part are constituted by meandering flow paths connected to the gene extraction part,
The gene extraction part is constituted by a meandering flow path connected to the reaction part and the gene elution part,
The gene amplification reagent storage part is constituted by a meandering flow path connected to the reaction part,
The bacterial spore treatment characterized in that the flow path constituting any one of the storage units has a maximum cross-sectional area of 10 times or less with respect to the cross-sectional area of the communication flow path connecting the storage unit and the inlet. Chip. In claim 1,
A bacterium characterized in that a sample inlet / waste liquid tank having the inlet is provided, and the germination promoting liquid storage part is constituted by a meandering flow path connected to the sample inlet / waste liquid tank. Spore processing chip. In claim 1,
Bacterial spore characterized in that the cleaning liquid introduced from the cleaning liquid storage unit into the gene extraction unit passes through the flow path formed to flow from the gene elution unit to the injection port side after passing through the gene extraction unit. Processing chip. In claim 1,
The bacterial spore processing chip, wherein the gene elution part to the injection port, the gene extraction part, and the washing liquid storage part are arranged in series via a flow path. In claim 1,
A bacterial spore treatment chip comprising a flow path that branches from between the gene elution part and the gene extraction part and communicates with the reaction part. In claim 1,
The storage part of the germination promoting liquid storage part, the lysate storage part, the washing liquid storage part or the eluent storage part has a plurality of flow paths having a longer length in the longitudinal direction than the width through the bent part. Formed and contacted
The bacterial spore processing chip, wherein the other end of the storage unit is provided with a fluid introduction unit that is introduced when the stored liquid is discharged from the storage unit. In claim 1,
The bacterial spore processing chip, wherein the cross-sectional structure of the flow path constituting any one of the storage units is 10 or less in horizontal / vertical direction. In claim 1,
The germination promotion liquid storage part is located on the side farther from the injection port than the area where the germination promotion liquid is stored, and when the germination promotion liquid is introduced into the injection hole, fluid flows into the germination promotion liquid storage part. A first fluid introduction section to be supplied;
A fluid that is located on the side farther from the injection port than the gene elution portion and is in the gene elution portion before the liquid in which the sample and the germination promoting liquid are mixed is introduced from the injection port into the gene elution portion A first fluid discharge part for discharging the outside of the gene elution part,
A fluid is supplied to the lysate storage part when the lysate is introduced into the gene elution part, located on a side farther from the gene elution part than the area where the lysate is stored in the lysate storage part A second fluid introduction section,
Second, located on the side farther from the gene elution part than the gene extraction part, and discharges the fluid in the gene extraction part outside the gene extraction part before the eluted gene is introduced into the gene extraction part A fluid discharge section of
A third fluid introduction unit to which a fluid is supplied when the cleaning solution is introduced into the inlet on the side away from the gene extraction unit from the region where the cleaning solution is stored in the cleaning solution storage unit;
A fourth fluid introduction unit to which a fluid is supplied when the eluent is introduced into the injection port on a side farther from the gene extraction unit than a region where the eluent is stored in the elution storage unit;
A fifth fluid introduction unit to which a fluid is supplied when the solution containing the eluted gene is introduced from the gene extraction unit to the reaction unit;
A bacterial spore processing chip comprising: Adding a germination promoter to the bacterial spores;
Adding a lysing solution that dissolves the cell wall after germination of bacterial spores, and eluting the gene;
A step of adsorbing the eluted gene to a silica carrier, and then adding a washing solution to wash,
Eluting the gene from the silica support by adding an eluent;
Detecting the eluted gene; and
In a bacterial spore treatment and gene detection method comprising:
A bacterial spore treatment and gene detection method using the bacterial spore treatment chip according to any one of claims 1 to 8. In claim 9 ,
Bacterial spore treatment and gene detection method, wherein the germination promoter is a broth containing alanine, adenosine and glucose. In claim 9 ,
Bacterial spore treatment and gene detection method, characterized in that after adding said germination promoter, it is held at 25 to 37 ° C for 10 to 60 minutes.

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