JPH02261372A - Collector for lytic component - Google Patents

Abstract

PURPOSE:To readily collect many samples at a time with the subject small-sized device by passing a suspension containing microorganisms through a column and then a coarse and a fine filters using suction force and separating insoluble solid substances and the microorganisms. CONSTITUTION:A plurality of a hole 11 for a polymerase chain reaction(PCR) method and holes 12 and 13 are arranged in a heating block 3 placed in a vacuum vessel and liquid feed pipes 14 are inserted into the respective holes 12. A filter device 4 is inserted into the hole of a lid unit 2 of the vacuum vessel in a pressed state and the first filter of a coarse mesh and the second filter 34 of a fine mesh are arranged. Water-insoluble solid substances and microorganisms are separated respectively from a sample suspension placed in the filter device.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a lytic component collecting device, and particularly to a lytic component collecting device for collecting lytic components from a suspension containing bacteria.

[Conventional technology]

In the fields of clinical diagnosis and research, in order to identify the type of bacteria in biological samples and analyze their constituent components, bacteria have traditionally been lysed using various methods and subjected to various analytical treatments. There is.

Among biological samples, the conventional method for obtaining lytic bacterial components from samples containing water-insoluble solids such as feces (food residue, exfoliated intestinal cells, etc.) involves placing a suspension of the sample in a medium and culturing it for several days. The resulting colony is collected and centrifuged to separate and collect the bacterial cells. Then, a lysed component is obtained by lysing this in a liquid using enzymes, surfactants, or caustic soda, destroying bacteria in a liquid by ultrasonic waves, or destroying bacteria with glass beads and a mill.

[Problem to be solved by the invention]

In the conventional structure for obtaining a lytic component, since culturing takes a long time, it is difficult to obtain a lytic component quickly.

Therefore, in Japanese Patent Application No. 1-10161, the inventor of the present application proposed a method using a filter device with a relatively large pore size of about 5 cm in diameter and a separately configured filter device with a relatively small pore size of about 5 cm in diameter. , proposes a method for obtaining bacterial components by separating water-insoluble solids and bacteria in a sample and dissolving the separated bacteria in a lysis solution.

However, in this configuration, two independent filter devices with a diameter of about 5 cm are used, so the filter device required for one sample has to be large. difficult to process.

SUMMARY OF THE INVENTION An object of the present invention is to provide a lytic component collection device that is small and can easily process a large number of samples at once.

[Means to solve the problem]

(1) The lytic component collection device according to the first invention is a device for collecting lytic components from a suspension containing bacteria.

This device includes a coarse first filter for separating insoluble solids from a suspension, and a fine second filter for separating bacteria from a suspension, and both filters are arranged at a distance from each other. the column containing the column and the second
A suction device is provided on the filter side and suctions the inside of the column so that the suspension passes from the first filter to the second filter.

(2) The lytic component collection device according to the second invention is a device for collecting lytic components from a suspension containing bacteria.

This device includes a coarse first filter for separating insoluble solids from a suspension, and a fine second filter for separating bacteria from a suspension, and both filters are arranged at a distance from each other. the column containing the column and the second
It includes a heat block disposed adjacent to the filter side opening and a control section for controlling the temperature of the heat block.

[Function] (1) In the lysate component collection device according to the first invention, a suspension containing bacteria is passed through a column, and insoluble solids are separated by a coarse first filter placed in the column. At the same time, bacteria are separated from the suspension by a fine second filter.

The separation operation in this case is performed by suction using a suction device.

In this case, since two types of filters are arranged in one column to separate insoluble solids and bacteria at the same time, the apparatus can be made more compact. Furthermore, since the apparatus becomes smaller, it becomes easier to process a large number of samples at once using a large number of apparatuses.

(2) In the lysate component collection device according to the second invention, a suspension containing bacteria is passed through a column, and insoluble solids are separated by a coarse first filter disposed within the column. At the same time, bacteria are separated from the suspension by a fine second filter.

Next, the lysate is passed through a second filter to obtain a soluble surface component. In this case, a heat block is arranged adjacent to the second filter side opening of the column. The temperature of this heat block is controlled by a controller, and the filter is heated by the heat block. This increases bacteriolytic efficiency.

In this case, since two types of filters are arranged in one column to separate insoluble solids and bacteria at the same time, the apparatus can be made more compact. Furthermore, the lysis efficiency can be increased by a heat block placed adjacent to the column. Therefore, using this apparatus, it is possible to easily process a large number of samples at once.

〔Example〕

An embodiment of the present invention is shown in FIG.

In FIG. 1, the lysate component collection device includes a vacuum container 1,
It mainly includes a lid 2 for closing the upper end of the vacuum container 1, a heat block 3 disposed inside the vacuum container 1, and a large number of filter devices 4 held by the lid 2 and protruding into the vacuum container 1. are doing.

The reduced pressure container 1 is a box-shaped member with an open upper end. A suction pump 6 is connected to the reduced pressure container 1 via a trap 5, so that the inside of the reduced pressure container 1 can be brought into a reduced pressure state.

The lid body 2 is placed on the reduced pressure container 1 and can close the upper end opening of the reduced pressure container 1 in an airtight state.

Both sides of the lid 2 are supported by a pair of drive mechanisms 7, which drive the lid 2 in the vertical direction and in the horizontal direction in FIG. As shown in Figure 2,
A large number of holes 8 arranged in a plurality of rows are formed in the lid body 2, and the filter device 4 is inserted into the holes 8 in a press-fit state to be held therein.

The heat block 3 is a cast product made of aluminum, for example, and has a temperature control water channel (not shown) inside. A temperature control device 10 is connected to the temperature control waterway via a pipe 9. The temperature of the heat block 3 can be controlled by changing the water temperature and circulating the water by the temperature control device 10. In the heat block 3, in order from the left side of FIG. 1, there are arranged a large number of sets of a PCR method 11, a pipe insertion hole 12 penetrating vertically, and a cleaning hole 13 penetrating vertically. These holes 11. Hole 12.13 is
As shown in FIG. 2, a large number of filter devices are also arranged in the direction of each row of the filter device 4. That is, hole 11. hole 12.13
are arranged in a matrix in the heat block 3. A liquid supply pipe I4 penetrating from the bottom to the top is inserted into each hole 12, respectively. The liquid supply pipe 14 penetrates the side wall surface of the reduced pressure container 1, is led out, and is connected to a syringe-shaped pump 15.

Two reagent bottles 17 , 18 are connected to the syringe-like pump 15 via an electromagnetic switching valve 16 . The switching valve 16 is operated so that only one of these reagent bottles 17, 18 is connected to the pump 15 side. Reagent bottle 1
For example, an endo-N-acetylmuraminidase solution is stored in the container 7. Further, in the reagent [18], for example, a Na OH solution is stored. ' As shown in FIG. 3, the filter device 4 protrudes downward from the lid 2 and has holes 11 . It can be inserted into any of the holes 12.13 from above. The details of this filter device 4 are shown in FIG.

In FIG. 4, the filter device 4 includes, in order from the top, a Teflon tube 31 and a coarse first filter 3.
2, a Teflon tube 33, a fine second filter 34, a Teflon tube 35, and a Teflon heat shrink tube 36 that covers them from the outer circumferential side. Note that FIG. 4 is an exploded view, and the tube 31 and the like are actually housed inside the heat-shrinkable tube 36. Furthermore, due to the contraction of the heat-shrinkable tube 36, the tubes 31 and the like are integrally fixed. Here, the contents of the tube 31 etc. have a diameter of, for example, 5.
mm, and the inner diameter of the heat shrinkable tube is, for example, 6 mm before shrinkage.

The tubes 31, 33, and 35 are made by cutting long Teflon tubes into rounds.

The upper filter 37 of the first filter 32 is
For example, it is an 80 μm mesh filter. The intermediate member is a disc-shaped spacer 38 made of coarse mesh. The lower filter 39 is, for example, a 10 μm mesh filter. This first filter 32 is
It is necessary that the pore size is large enough to allow passage of at least one bacterial body (cell). The pore diameter of the filter 39 is preferably 0.45 to 10 μm, 0°45 μm.
If it is less than m, it becomes substantially difficult to pass the bacterial cells in the suspension. In this first filter 32, two types of filters 37 and 39 are arranged with a spacer 38 in between, so clogging does not easily occur.The filters 37 and 39 are made of Tetron mesh, glass wool, etc. It will be done. The second filter 34 is constructed from three layers of glass fiber filter paper 40, 41, 42.

The upper filter paper 40 has a pore diameter of, for example, 2.711 m. The intermediate filter paper 41 has a pore diameter of, for example, 1.6 μm. The lower filter paper 42 has a thickness of, for example, 1.2 μm.
It has a pore size of As the lower filter paper 42, 0.2
Those with a pore size of ~1.6 μm are preferred, and 0.2 ~
More preferred are those with a pore size of 1.2 μm. Filter paper 4
If the pore size of No. 2 is less than 0.2 μm, the efficiency of filtering and removing contaminant components will decrease. Moreover, when the pore size exceeds 1.6 μm, the bacteria retention efficiency of the filter decreases.

In FIG. 1, a cleaning liquid injection nozzle 45 is arranged above the lid 2. As shown in FIG. The nozzle 45 is configured to be able to discharge the cleaning liquid downward, and is connected to the nozzle moving device #1.
46, it is movable in the horizontal direction and in the vertical direction. A reagent bottle 48 is connected to the nozzle 45 via a syringe-type pump 47 . Inside the reagent bottle 48,
For example, TES is stored. Here, TBS refers to 10mM-TrisHCf (pH 8,0), 1mM
- Refers to a mixed solution of EDTA and 10mM NaCl (pH 8°0).

Furthermore, the lytic component collecting device according to this embodiment is equipped with a microcomputer 50 as shown in FIG. The microcomputer 50 includes a CPU 51. RAM52.
It is equipped with a ROM 53 and the like, and has an I10 port 54 for external connection. ■10 The above-mentioned pump 6 and drive mechanism 7 are connected to the microcomputer 50 via the boat 54.
, temperature control device 10, pump 15, switching valve 16, moving mechanism 46, and pump 47 are connected. Therefore, these components are driven and controlled by the microcomputer 50.

Next, the operation of the above-mentioned lytic component sampling device and its usage will be explained.

First, the filter device 4 is assembled. In this case,
Tubes 31, 33 of three different heights as shown in FIG.
．． Prepare a filter 37.39, a spacer 38, and a filter paper 40.41.4 having the same diameter as the outer diameter of the filter 35.
Prepare 2. Further, a heat shrink tube 36 having approximately the same height as the height when these tubes are stacked is prepared. The tubes 31, 33, 35, filter 37, etc. are inserted into the heat shrink tube 36 and stacked one above the other in the order shown in FIG. 1.Next, the heat shrink tube 36 is heated with a heating device (for example, a dryer). to deflate.

As a result, the tube 31,
33, 35, filter 37, etc. are integrally fixed,
The filter device 4 is now assembled.

Next, the sample suspension is placed in the filter device 4. Typical samples are biological samples such as feces, but other samples containing at least relatively large water-insoluble solids and bacteria can also be used, such as blood, urine, and vomit. etc. can be used as a sample. Furthermore, samples other than medical diagnosis, such as foods, can also be used as targets. Here, as the aqueous medium for preparing the 0M suspension in which the sample suspension is first prepared, a non-lytic medium is suitable, such as diluted sodium chloride water or neutral phosphate buffer. can be given. This medium may contain a surfactant to the extent that it does not lyse the bacteria in order to improve the separation and dispersibility of the bacteria and solid content.As for the amount of sample to be suspended, about 2 mg is sufficient. . Note that the suspension is injected into the Teflon tube 31 from above.

The filter device 4 into which the sample is injected is inserted into each hole 8 of the lid body 2.
It is inserted and held in its normal size.

The lid 2 is placed in the position shown in FIG. 1 by the drive mechanism 7, and the filter device 4 is placed in the hole 13 of the heat block 3 in the vacuum container 1.

Next, the pump 6 is driven to bring the inside of the reduced pressure container 1 into a reduced pressure state. As a result, the suspension in the filter device 4 passes through the first filter 32 and the second filter 33 and is discharged into the vacuum container 1 .

Next, the nozzle 45 is sequentially arranged above each filter device 4 by the moving mechanism 46, and the TES in the reagent bottle 48 is injected into the filter device 4 from above by driving the pump 47. Since the inside of the reduced pressure container 1 is in a reduced pressure state, this TES passes through the first filter 32 and the second filter 33 and is discharged into the reduced pressure container 1.

This means that the inside of the filter device 4 has been cleaned.

Next, the inside of the reduced pressure container 1 is returned to normal pressure, the lid body 2 is moved by the drive mechanism 7, and the filter device 4 is fitted into the vibrator 14. That is, as shown in FIG. 6, the upper part of the vibrator 14 is fitted into the lower end of the filter device 4, and the upper end opening of the vibrator 14 is disposed directly below the second filter 34.

In this state, the lid body 2 closes the upper end opening of the decompression container 1. Next, the switching valve 16 connects the reagent bottle 17 to the pump 15 side, and by driving the pump 15, the end-N-acetyl Apply muraminidase solution to vibe 1
4 to the second filter 34. This makes the second filter 34 wet with the endo-N-acetylmuraminidase solution. At this time, since the tube 33 is present, the first filter 32
The endo-N-acetyl muraminidase solution does not reach the filter device 4. At this time, by heating the filter device 4 with the heat block 3, the action efficiency of the enzyme can be increased.

Next, the lid body 2 is moved again by the drive mechanism 7, and the first
The state shown in the figure and Fig. 3 is established. Then, by driving the pump 6, the pressure inside the vacuum container 1 is reduced, and the endo-N-acetylmuraminidase solution that has soaked into the second filter 34 is removed. As a result, the outer periphery (cell wall, etc.) of the bacteria captured by the second filter 34 is dissolved, and the DNA can be easily eluted. On the other hand, by switching the switching valve 16, the reagent [18 containing the NaOH solution and the pump 15 are brought into communication. Then, by operating the pump 15, the endo-N-acetylmuraminidase solution remaining in the vibrator 14 is discharged, and the vibrator 14 is filled with NaOH solution instead.

Next, the reduced pressure container 1 is returned to normal pressure, and the lid body 2 is again set in the position shown in FIG. 6 by the drive mechanism 7. Next, the pump 15
is driven to transfer Na from the vibrator 14 to the second filter 34.
Supply OH solution. As a result, the second filter 3
4 becomes wet with the NaOH solution. On the other hand, the first
The NaOH solution does not reach the filter 32. By wetting the second filter 34 with this NaOH solution, the DNA in the bacteria becomes ready to be eluted. At this time, the lysis efficiency is improved by heating the filter device 4 with the heat block 3. Next, the lid body 2 is driven by the drive mechanism 7, and the filter device 4 is inserted into the PCR method usage hole l. . The state at this time is shown in Figure 7.
In the illustrated state, the lid 2 closes the upper end of the reduced pressure container 1. Next, the pump 6 is driven to bring the inside of the reduced pressure container 1 into a reduced pressure state.

As a result, a lytic component containing bacterial DNA is accumulated in the hole 1 for PCR method use.

Next, the reduced pressure container 1 is returned to normal pressure, and the lid 2 is removed. Then, Tr 1sHcj! for the solution containing the lytic component placed in the PCR method hole 1. (LM.

pH8,0), HC/! Neutralize using (IM) solution.

The obtained solution containing the bacteriolytic component is subjected to a polymerase chain reaction method (PCR method) by controlling the temperature of the heat block 3 with the temperature control device 10 to amplify specific DNA of a specific country. Now, the DN of the target image.
By selecting primers that specifically hybridize to A and proceeding with the reaction, only the target DNA can be amplified. Therefore, according to the PCR method, if there are about 1 to 100 target images initially, it is possible to detect the target images by amplifying the DNA.

The lysate in which the target DNA has been amplified is, for example, EtB.
It is detected by electrophoresis using r2% agarose gel. Note that this detection can also be performed using other known methods. In addition, a method of confirmation using a DNA probe can also be adopted.

As explained above, in the above-mentioned embodiment, the filter device 4 that is integrally configured with two types of filters using a heat-shrinkable tube is used, so that the filter device 4 can be miniaturized. Therefore, the entire lysate component collection device can be downsized, and a large number of samples can be easily processed at one time. Furthermore, by providing the heat block 3 in the reduced pressure container 1, it is possible to increase the bacteriolytic efficiency. Moreover, since the extraction of the lytic component and the implementation of the PCR method can be performed continuously, the lytic component sampling device can be made more compact.

In the above embodiment, the enzyme and the alkali were supplied separately in the lysis step, but it is also possible to lyse the bacteria at once using a mixed solution of the enzyme and the surfactant.

〔Effect of the invention〕

According to the first invention, since two types of filters with different roughness are housed in one column and combined with a suction device, it is possible to downsize the lysate component collection device. Furthermore, since a portion of the filter is made smaller, it becomes easier to process a large number of samples at once.

According to the second invention, since two types of filters with different roughness are housed in one column and a heat block is provided, the lytic components can be collected efficiently, and the lytic components collection device is compact as a whole. is obtained. In addition, a portion of the filter becomes compact, making it easier to process a large number of samples at once.

[BRIEF DESCRIPTION OF THE DRAWINGS] Fig. 1 is a schematic configuration diagram of an embodiment of the present invention, Fig. 2 is a partially cutaway plan view of the reduced pressure container and lid portion, and Fig. 3 is a schematic diagram of the embodiment of the present invention. 4 is an exploded perspective view of the filter device, FIG. 5 is a block diagram of the control section, and FIGS. 6 and 7 are views corresponding to FIG. 3 for explaining the operating state. . 1... Decompression container, 2... Lid, 3... Heat block, 4... Filter device, 6... Pump, IO
... Temperature control device, 11 ... PCR method hole, 32.
...first filter, 34...second filter,
36... Heat shrink tube, 50... Microcomputer. nij, 7″, -1st figure

Claims (2)

[Claims] (1) A lytic component collection device for collecting lytic components from a suspension containing bacteria, the device comprising: a rough first tube for separating insoluble solids from the suspension;
a filter; a fine second filter for separating bacteria from the suspension; a column accommodating both of the filters spaced apart from each other; and a column disposed on the second filter side of the column. , a suction device that suctions the inside of the column so that the suspension passes from a first filter to a second filter, and a lysate component collecting device. (2) A lytic component collection device for collecting lytic components from a suspension containing bacteria, the device comprising: a rough first tube for separating insoluble solids from the suspension;
a filter; a fine second filter for separating bacteria from the suspension; a column for accommodating both of the filters spaced apart from each other; and a column adjacent to the opening on the second filter side of the column. A lysate component collection device comprising: a heat block arranged in a manner that the temperature of the heat block is low; and a control unit for controlling the temperature of the heat block.