JPH02255074A - Apparatus for collecting lysed bacterial component, method for collecting lysed bacterial component and examination of bacteria - Google Patents

Abstract

PURPOSE:To provide a small-sized apparatus effective in quickly and easily collecting a lysed bacterial component from a specimen and composed of a column containing the 1st filter shielding the solid component in the specimen and passing the bacterial cells and a 2nd filter shielding the bacterial cells interposing a gap between the filters. CONSTITUTION:The objective apparatus is produced by stacking an introduction pipe 1, a 1st filter 2 shielding the solid component and passing bacterial cells of a specimen, a cylindrical spacer 3, a 2nd filter 4 shielding the bacterial cells and a discharge pipe 5, applying a heat-shrinkable tube 6 to the outer circumference of the assembly and heat-treating the product. A specimen is put into the introduction pipe 1 and treated by a centrifugal separator to pass the liquid component while leaving the solid component on the 1st filter 2 and the bacterial cells on the 2nd filter 4. A washing liquid is introduced through the introduction pipe 1 to wash the cells, a liquid containing a bacteriolytic agent is charged through the discharging pipe 5 from the bottom to the middle part of the spacer 3 to lyse the bacterial cells and the treated liquid is discharged to obtain the objective component of lysed bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a lytic component sampling device, a lytic component sampling method, and a bacterial testing method. In particular, the present invention relates to an apparatus and method for collecting lytic components from a suspension containing bacteria, and a bacterial testing method using the method.

[Conventional technology]

For example, in the fields of clinical diagnosis and research, in order to identify the type of bacteria in a biological sample or analyze its constituent components, bacteria have traditionally been lysed using various methods and subjected to various analytical treatments. It is being said.

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.

However, the conventional bacterial identification method is: l) Sprinkle the sample on a medium such as agar containing a certain antibiotic, separate the bacterial groups that grow as a result, and collect the separated bacterial groups. 2) latex beads are filled with antibodies specific to a certain bacterial cell and immersed in the sample; Latex agglutination method, which detects bacterial cells by chaining and aggregating beads by recognizing the cells;
3) A standard antibody is placed on the surface of a predetermined number of small pores formed in a polystyrene plate, and bacterial cells (or bacterial cell components) are applied to this to determine whether or not it binds by antigen-antibody reaction. EL that separates and identifies by sorting, then applying an enzyme-labeled specific antibody, and developing color against the label.
ISA method, and 4) DNA probe method, which uses enzyme-labeled DNA probes on specific NA fragments and hybridizes them to separate and identify them.

[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. 1-101'61, the inventor proposed a filter device with a relatively large pore diameter of about 5 cm and a separate filter device with a relatively small pore diameter of about 5 cm. The paper proposes a method for obtaining bacterial components by separating water-insoluble solids and bacteria in a sample using a microorganism, and dissolving the separated bacteria in a lysing solution.

However, in this configuration, two independent filter devices each having a diameter of about 5 cm are used, which inevitably increases the size of the device. Moreover, sufficient operability cannot be obtained.

On the other hand, among the conventional bacterial identification methods, 1) isolation culture method involves a culture step, so it takes a long time (about 1 week)
Requires. In the methods using antibodies 2) and 3), Campylobacter jejui (Campylobacter jejui)
ter Jeju) 4 X 1 0'
Detection sensitivity is poor, with only some reports of detection of up to 5 tools/g. The DNA probe method (4) requires a large amount of specimen because a normal bacterial genome DNA is one piece and detection is difficult unless there is a considerable amount of DNA.

A first object of the present invention is to provide a lytic component collection device that can be miniaturized and that can quickly and easily obtain lytic components from a sample suspension. The second object of the present invention is to
It is an object of the present invention to provide a method for collecting lytic components that can quickly and easily obtain lytic components from a sample suspension, and a bacterial testing method that can easily identify bacteria using the method.

[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. It is equipped with a column for accommodating.

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

This device includes a filter for separating bacteria from a suspension, a support member that supports the filter on both sides and has holes for passing the suspension through the filter, and a support member that covers the filter and the support member from the outside. It is equipped with heat shrink tubing that fits in the same way.

(3) A method for collecting lytic components according to the third invention is a method for collecting lytic components from a suspension containing bacteria.

This method involves passing a suspension through a filter using centrifugal force to separate the bacteria onto the filter, and then
This involves flowing the lysate through a filter using centrifugal force to obtain a lysate containing bacterial components.

(4) The bacterial testing method according to the fourth invention comprises subjecting the lysate obtained by the method according to the third invention to a polymerase chain reaction method to amplify the DNA of a desired bacterium;
This includes testing the presence or absence of desired bacteria in the suspension using NA.

[Effect]

(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.

Next, the lysate is passed through a second filter to obtain a lysed component. In this case, since two types of filters can be used to separate the insoluble solids and bacteria at the same time, the operation becomes quick and simple. Furthermore, since two types of filters are arranged in one column, the device can be made smaller.

(2) In the lytic component collection device according to the second invention, the filter is sandwiched between support members, and the filters are inserted into a heat-shrinkable tube. Then, when they are heated, the heat-shrinkable tube shrinks and the tube, support member, and filter are integrated. In this case, it is easy to form the lytic component collection device, and the lytic component can be collected quickly and easily. Furthermore, since the support member and the filter are integrated using a heat shrinkable tube, the device can be made smaller.

(3) In the method for collecting lytic components according to the third invention, bacteria are separated by a filter using centrifugal force, and lytic components of the bacteria separated by the filter are obtained using a lysate using centrifugal force. Therefore, compared to the suction operation using a conventional pump, the lysed component can be obtained more quickly and easily.

(4) Since the bacterial testing method according to the fourth invention employs the rapid and simple method for collecting lytic components according to the third invention, the bacterial testing method as a whole is quick and simple.

[Example] First, an example of a lytic component collecting device according to the present invention will be described.

As shown in Fig. 1, the lytic component collection device consists of a Teflon tube 1, a coarse first filter 2, a Teflon tube 3, a fine second filter 4, and a Teflon tube in order from the top. It has a tube 5 made of aluminum, and a heat shrinkable tube 6 made of Teflon that covers the tube from the outer circumferential side. Furthermore, Figure 1 is an exploded view of the lytic component collection device.
In reality, the tube 1 and the like are housed within the heat-shrinkable tube 6. Furthermore, due to the contraction of the heat-shrinkable tube 6, the tubes 1, etc. are in a state of being integrally fixed. Here, the contents of tube 1 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.

Tubes 1 and 3.5 are made by cutting long Teflon tubes into rounds.

The upper filter 7 of the first filter 2 is, for example, an 80 μm mesh filter. The intermediate member is a disc-shaped spacer 8 made of coarse mesh. The lower filter 9 is, for example, a 10 μm mesh filter. This first filter 2 needs to have a pore size large enough to allow one microorganism (cell) to pass through.The pore size of the filter 9 is 0.45
~10 μm is preferred. Below 0.45 μm,
It becomes substantially difficult to pass the bacterial cells in suspension. Further, when the bacterial cells are aggregated in the suspension, it is preferable to use a filter having a pore size of approximately less than 100 μm.

If it exceeds 100 μm, the water-insoluble solids in the suspension will easily pass through, and the separation between the water-insoluble solids and the bacterial cells will be significantly reduced. Furthermore, in the first filter 2, two types of filters 7.9 are arranged with the spacer 8 in between, so that clogging is less likely to occur. As the filter 7.9, Tetron mesh, glass wool, etc. are used.

The second filter 4 consists of three layers of glass fiber filter paper 10.1
1.12. The upper filter paper 10 has a pore size of, for example, 2.7 μm. The intermediate filter paper 11 has a pore size of, for example, 1.6 μm. The lower filter paper 12 has a pore diameter of, for example, 1°2 μm. The lower filter paper 12 preferably has a pore diameter of 0.2 to 1.6 .mu.m, more preferably 0.2 to 1.2 .mu.m. When the pore size of the filter paper 12 is less than 0.2 μm, the efficiency of filtering and removing contaminant components decreases.

Furthermore, when the pore size exceeds 1.6 μm, the bacteria retention efficiency of the filter decreases.

Next, a method for collecting lytic components and a bacterial testing method using the above-mentioned lytic component collecting device will be explained according to the flowchart shown in FIG.

First, assemble the lytic component collection device. In this case,
Tubes 1, 3.5 with three different heights as shown in Figure 1
A filter 7 having the same diameter as the outer diameter of the filter 7 is prepared.
．． 9. Prepare spacer 8 and filter paper 10.11.12. Further, a heat shrink tube 6 having approximately the same height as the height when these tubes are stacked is prepared. tube 1,
3.5, the filter 7, etc. are inserted into the heat shrink tube 6, and stacked one on top of the other in the order shown in FIG. Next, the heat-shrinkable tube 6 is heated by a heating bag W (for example, a dryer), and the heat-shrinkable tube 6 is shrunk. As a result, the tubes 1, 3.5, filter 7, etc. are integrally fixed by the heat-shrinkable tube 6, and the lysate component collection device is assembled.

Next, the sample suspension is placed in the lysate component collection device. The sample is typically a biological sample such as feces, but other samples containing at least relatively large water-insoluble solids and bacteria can also be used. For example, blood, urine, vomit, etc. can be used as the sample. Furthermore, samples other than medical diagnosis, such as foods, can also be used as targets. Here, a sample suspension is first prepared. Suitable aqueous media for preparing the suspension include those that do not have bacteriolytic properties, such as diluted sodium chloride water and neutral phosphate buffer. 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 bacteria and solid content. About 2 mg is sufficient as the amount of sample to be suspended. The suspension is injected into a Teflon tube 1.

The lysate component collection device into which the sample has been injected is inserted into a centrifuge tube (not shown), and centrifugal force is applied by a centrifuge.

The conditions for centrifugation include, for example, 5,000 rpm;
It is 30 seconds. By centrifugation, the suspension passes through the first filter 2 and the second filter 3 and is discharged below the lysate component collection device. The resulting filtrate is discarded.

Next, TBS is injected into tube 1. TB here
S is 10mM-TrtsHCI (pH 8,0)
, 1mM-EDTA and 10mM-NaCl (pH 8
, 0). With TBS injected, centrifugal force is applied again. The centrifugation operation may be performed, for example, in 5. OOOr
pm for 30 seconds. By performing this washing treatment, a more purified lysate can be obtained. The filtrate produced by this washing process is discarded.

After washing, a solution containing 60 μg/mQ of endo-N acetyl muraminidase is injected from the lower end of the lytic component collecting device. Due to the solution, only the second filter 4 becomes wet. Since the Teflon tube 3 is interposed between the two filters 2 and 4, the enzyme solution does not reach the first filter 2. In this state, apply centrifugal force again.

The conditions for centrifugation are, for example, 5.00 rpm and 30 seconds. The resulting filtrate is discarded. As a result, the outer periphery (cell wall, etc.) of the bacteria captured by the second filter 4 is dissolved, making it possible to easily dissolve the DNA.

Next, 0.2M NaOH is injected from below the lysate component collecting device to wet the second filter 4 with the NaOH solution. This creates a state in which the DNA within the bacteria can be eluted. Furthermore, centrifugation is performed at 5.00 rpm for 30 seconds.

The obtained filtrate contains a lytic component containing bacterial DNA. The solution containing this bacteriolytic component was subjected to Tri 5H
cI1. (LM, pH 8,0), HCl2 (IN)? I
Neutralize using liquid.

The resulting solution containing the lytic component is subjected to a polymerase chain reaction method (hereinafter referred to as PCR method) to amplify specific DNA on a specific surface. According to this PCR method, only the DNA of the target bacteria can be amplified by selecting primers that specifically hybridize to the DNA of the target bacteria and proceeding with the reaction. Therefore, according to the PCR method, if there are about 1 to 100 target bacteria at the beginning, the DNA
By amplifying the target bacteria, it becomes possible to detect the target bacteria. In this PCR method, a known device such as a commercially available dedicated machine such as a DNA thermal cycler (manufactured by Birkin Elmer Cetus) can be used. When using this PCR method, the lysate is adjusted to pH 5-9.

The obtained lysate is detected by electrophoresis using a 2% agarose gel containing EtBr.

Note that this detection can be performed using a known method such as absorbance measurement. In addition, a method of confirmation using a DNA probe can also be adopted.

As explained above, in the above-mentioned example, a lytic component collection device in which two types of filters were integrally constructed using a heat shrink tube was used, so that lytic components could be quickly and easily obtained from a sample suspension. be able to. In addition, the lytic component sampling device is much smaller than the conventional one.

In addition, the above-mentioned lytic component collection device has a simple structure using a heat shrink tube and is inexpensive.
It is easy to carry out so-called disposable use, and the fear of contamination can also be eliminated. Also,
As a result of the miniaturization of the device, it becomes possible to proceed with the work in a closed system (for example, inside a centrifuge tube), which also makes it possible to eliminate contamination. Furthermore, since the device becomes smaller, it becomes easier to test a large number of samples at once.

Furthermore, according to the above-mentioned method for collecting lytic components and the bacterial testing method using the same, there is no need to go through long-term procedures such as culturing bacteria, so lytic components can be obtained quickly and easily. , the desired bacteria can be tested.

A lytic component collecting device was prepared by sandwiching a first filter 2 and a second filter 4 between tubes 1 and 3.5 and fixing them with a heat shrink tube 6 as shown in FIG. 1. On the other hand, B
A fecal sample containing 104 to 107 Cereus/g was prepared. Place the lysate component collection device into a 0.5 ml centrifuge tube and centrifuge (5, OOOrpm, 30 seconds)
The following operations were performed, including:

22.5 μl of the sample suspension was injected into the upper part of the lysate component collection device and centrifuged. Next, 56° 5μI for washing! , T
The BS was passed through a lysate component collection device by centrifugation. Furthermore, 10μ from the bottom of the lytic component collection device! The enzyme solution was injected to wet the second filter with the enzyme solution. A lysate component collection device was placed in a centrifuge tube, and the tube was incubated at 50°C for 5 minutes and then centrifuged.

And 10 μj! The second filter was moistened from below with NaOH water (0.2M) and kept at 80°C for 7 minutes. Furthermore, a lysate component collection device was placed in a new centrifuge tube, and centrifugation was performed.

The resulting solution was neutralized, and PCR was performed using primers that allow B. Cereus to selectively create a 300 bp (base pair) DNA strand.

As a result, for samples with 106 particles/g or more, aoob
I was able to recognize the p band. That is, in this experiment, it was possible to detect feces containing 106 B. Cereus/g.

In this way, according to the above-described embodiment, even if the sample contains dangerous bacteria, the amount used is small, making it relatively safe and sanitary. In addition, the amount of sample collected can be reduced, so use a urinal sampler.

Transporters, etc. become smaller, etc. 1. It is also possible to downsize other instruments, leading to overall space savings and cost reductions. In addition, in this embodiment, centrifugal filtration is possible using a simple centrifuge such as a tabletop centrifuge.
Bacteria testing can be easily performed. Moreover, since the entire structure is fixed in one piece using heat-shrinkable tubes, there is no need to use suction bottles, pumps, cocks, etc.
There is no need to operate them.

〔Effect of the invention〕

According to the first invention, since two types of filters with different roughness are housed in one column, it is possible to downsize the lysed component collection device, and moreover, it is possible to separate insoluble solids and bacteria at once. You will be able to perform simple and quick operations.

According to the second invention, since the filter is fixed using a heat-shrinkable tube, not only can the lysed component sampling device be assembled quickly and easily, but also the device can be made smaller.

According to the third invention, since the bacteria are separated from the suspension by centrifugal force and the lysed component is obtained, there is no need to culture the bacteria, and the lysed component can be obtained quickly and easily.

In the fourth invention, since a lysate is obtained using the third invention and a polymerase chain reaction method is used, bacteria can be tested simply and quickly as in the third invention.

[Brief explanation of drawings]

FIG. 1 is an exploded perspective view of one embodiment of the present invention, and FIG. 2 is a flowchart showing the working steps of one embodiment of the present invention. 1.3.5...Tube, 2...First filter 4...Second filter, 6...Heat shrink tube. Figure 2

Claims (4)

[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 lytic component collection device comprising: a filter; a fine second filter for separating bacteria from the suspension; and a column that accommodates both of the filters spaced apart from each other. (2) A lytic component collection device for collecting lytic components from a suspension containing bacteria, comprising: a filter for separating bacteria from the suspension; A lysate component collection device comprising: a support member having a hole for passing a suspension; and a heat shrink tube that fits over the filter and the support member from the outside. (3) A method for collecting lytic components from a suspension containing bacteria, the method comprising: passing the suspension through a filter using centrifugal force;
A method for collecting lysed components, comprising: separating bacteria on a filter; and after the separation, flowing a lysate through the filter by centrifugal force to obtain a lysate containing bacterial components. (4) subjecting the lysate obtained by the method of claim (3) to a polymerase chain reaction method to amplify the DNA of a desired bacterium; A bacterial testing method comprising: testing for the presence or absence of desired bacteria;