JPH1189567A - System for sterilizing microorganism and gene recovery system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To subject Cryptosporidium, Pseudomonas aeruginosa, legionella or the like to disinfection treatment by simple operation without using medicine by feeding a microorganism to electrification means having a porous film coated with a metallic film and applying electric current to the microorganism. SOLUTION: A microorganism is fed to electrification means having a porous film, e.g. flat sheet membrane of a resin made of porous polypropylene, coated with a metallic film, and electric current is applied from electric source to the microorganism by using the electrification means as an electrode. Thereby, membrane structure of the microorganism is broken and a gene is selectively extracted from the resultant content in the microorganism and the nucleic acid is subjected to generic analysis to identify Escherichia coliform bacillus and infection rout can be determined thereby.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a system for killing microorganisms and a system for recovering genes from specific microorganisms.

[0002]

2. Description of the Related Art In recent years, the adverse effects of pathogenic microorganisms have been widely discussed. The first problem is the presence of microorganisms that contaminate human water. Protozoa "Cryptosporidium", which is transmitted through water and food and causes severe diarrhea and abdominal pain in the human body, contaminates the water source of tap water. Unfortunately, this protozoa is ineffective in chlorine-based disinfection. As a water source, it is not possible to use an agent that exceeds the bactericidal effect of a chlorine-based bactericide.

[0003] The second is Pseudomonas aeruginosa, which causes nosocomial and opportunistic infections. The Pseudomonas aeruginosa also reduces the contamination of the stored water in the building, and may infect the human body via the air conditioner and drinking water in the building. Pseudomonas aeruginosa is resistant to drugs.

[0004] The third is Legionella bacteria, which is a source of pollution in home 24-hour baths. Legionella bacteria cause pneumonia. In order to suppress the growth of this bacterium, the use of ultraviolet rays has been sought, but it cannot be said to be a sufficient sterilization method.

[0005] Fourth, pathogenic Escherichia coli causing food poisoning, particularly O-157. Identifying the source of food poisoning is
It is essential for its prevention. O-
157 genetic analyzes are required. Regarding gene analysis, for example, Japanese Patent Application Laid-Open No. 9-178752 exists.

[0006]

With respect to microorganisms contaminating living water, chlorine-based disinfection is mainly used since it is necessary to prevent adverse effects on the human body. However, "Cryptosporidium" and the like are resistant to this.

[0007] The reason why Legionella bacteria are to be sterilized with ultraviolet rays is also to prevent the use of a drug from adversely affecting the human body. However, there is a problem that the microorganisms described above cannot be sufficiently sterilized unless a drug having a high sterilizing effect is used.

On the other hand, regarding the detection of bacteria by genetic analysis, in the above-described conventional example, since the chemical analysis method using an enzyme is used, impurities are mixed in the analysis system, and the analysis accuracy is inferior. In addition, it takes time for the chemical treatment.

[0009] The applicant of the present application has proposed in Japanese Patent Application Laid-Open No. 9-37763 that the cells of microorganisms are destroyed without using a chemical component such as an enzyme and the gene in the cell is recovered. However, in order to quickly determine the route of infection, fast and accurate genetic analysis methods for food poisoning bacteria that require quick identification of genes
There is no.

[0010] Therefore, an object of the present invention is to provide a system capable of removing the adverse effects of various pathogenic microorganisms by a simple operation. For more information,
An object of the present invention is to provide an effective disinfection system for microorganisms contaminating living water without using chemicals. Another object is to provide a system for killing Pseudomonas aeruginosa and Legionella bacteria without using drugs. It is still another object of the present invention to provide a nucleic acid detection system for a rapid and highly accurate gene analysis method for the causative bacteria of food poisoning.

[0011]

In order to achieve this object, the present invention provides an energizing means having a metal film coated on a porous film, a power source for the energizing means, and supplying a microorganism to the energizing means. Supply means for supplying water to the living water, wherein the microorganisms are a source of contamination of living water, and the system is a system for disinfecting the microorganisms by supplying electricity to the microorganisms from a power supply using the electricity supply means as an electrode.

This microorganism is, in particular, the aforementioned "Cryptosporidium", Pseudomonas aeruginosa, Legionella, and pathogenic Escherichia coli.

The porous membrane is a commercially available hollow fiber or a flat membrane in which the hollow fiber is formed into a flat membrane, particularly one made of a porous resin, a hollow ceramic tube, a sintered tube, or the like. In order to coat the porous resin with a metal, the method described in JP-A-9-37763 may be used. As described in this publication, the porous resin and the metal are preferably chemically bonded. By doing so, a larger amount of metal can be coated on the porous resin.

In the present invention, energization is essential.
It is considered that the reason why the sterilizing effect is higher than that in the case where the metal plate is used as the electrode is that the porous plate is energized while the microorganisms are captured. There is a marked difference in the germicidal rate between when energized and when not energized.

In this sterilization system, the membrane structure of the microorganism is destroyed in the course of energization, and it is possible to obtain a nucleic acid which is a cell content. If this nucleic acid is used for gene analysis, the pathogenic E. coli gene can be identified,
As a result, it becomes possible to analyze the infection route. In the above-mentioned conventional Japanese Patent Application Laid-Open No. Hei 9-178752, an enzyme is used in order to destroy the membrane structure of a cell, which causes a problem that contaminants are mixed in an analysis system and that analysis requires time. .

As a form of energization, there are pulse energization, DC energization, AC energization, and impulse energization. It is apparent from the examples described later that the direct current is effective. Also,
The fact that pulsed current is effective in destroying bacterial cells is as described in JP-A-9-37763.

[0017]

Next, a sterilization system for Pseudomonas aeruginosa according to a first embodiment of the present invention will be described.

System configuration: A flat membrane made of porous polypropylene resin (P120UA-04F, Tonen Tapils Co., Ltd.)

Japanese Patent Application Laid-Open No. 9-37763

Silver was coated by applying the above method. As shown in FIG. 1, this was cut into a square of 4 cm, and a conductive wire was fixed to this conductive flat film with low melting point solder and epoxy resin. Next, 3 ml of distilled water was placed, and 3000 rpm,
The membrane was washed for 10 minutes.

This conductive flat film was prepared by
After washing for 0 minutes and then for 10 minutes in 100 ml of distilled water, and after drying, the open side of the container was capped and fixed. The electric resistance of this conductive flat film was 0.3 ohm.

Microorganism used: Pseudomonas aerugi
nosa) and Legionella pneumophila serogr
A sterilization test was performed using oup 1). The Legionella bacterium used in the experiment was separated from the cooling water of the building air conditioner, and was confirmed to be type 1 Legionella bacterium by biochemical properties and serologic tests.

Sterilization method: 1 ml of a bacterial solution adjusted to a certain number of bacteria was placed on a conductive flat membrane, and the bacteria were captured on the membrane by centrifugation.
Then, in order to prevent drying, physiological saline was further added dropwise.
A current was applied for 10 minutes. After energization is completed, the membrane is inverted,
1 ml of physiological saline was placed on the back side and centrifuged, and the bacteria captured on the membrane were forcibly removed from the membrane and collected. As a control, a flat membrane in which only current was not applied was prepared, and the number of bacteria recovered from this membrane was counted.

Experimental Results: FIG. 2 shows the experimental results. Also,
FIG. 3 shows the steps of the experimental procedure. Each table in FIG. 2 shows the bactericidal effect at the applied current value. Also no
“current” means that only the energization process is not performed after the bacterial solution is captured on the membrane, and the respective energization values are shown in the table for those subjected to the energization process. The number of bacteria is the number of bacteria in the bacterial solution on the membrane, the number of bacteria in the filtrate is the number of bacteria that passed through the membrane, and the value obtained by subtracting the number of bacteria in the filtrate from the initial number of bacteria is the number of captured bacteria. It is. The effect of energization can be calculated from the number of recovered bacteria in the table. The number of recovered bacteria is the number of bacteria in a liquid obtained by capturing bacteria on the membrane, inverting the membrane, placing a physiological saline solution on the back side, performing centrifugation, and forcibly removing bacteria in the membrane.

Preparation of Pseudomonas aeruginosa was performed as follows. Preculture overnight in ampoules (3 ml). Wash twice using a centrifuge. The suspension was resuspended in 3 ml of physiological saline to prepare a Pseudomonas aeruginosa suspension (10 ⁸ -10 ⁹ CFU / ml).

From the results shown in FIG. 2, it can be seen that the effect of energizing Pseudomonas aeruginosa is only about 25% lower than that of the group not energizing when the applied DC current value is between 750 mA, but the current value is 1 A. It was found that the number of bacteria decreased to 0.03% as compared with the non-energized group. These results revealed that Pseudomonas aeruginosa, which had not been easily killed by drugs such as antibiotics and various chemotherapeutic agents, can be sterilized in a short time without using these drugs. Therefore, the amount of current is preferably 750 mA or more.

(Second embodiment) Sterilization against Legionella bacteria. Next, a bactericidal test against Legionella was performed in the same manner as in the first example.

FIG. 4 shows the results of the sterilization test. From this test result, after capturing 10 ¹² bacteria, only about 10 ⁹ bacteria can be recovered even if the bacteria are forcibly recovered using centrifugation. And high capture performance. Because the conductive flat membrane has this high trapping property,
Energization of bacteria is effectively performed. And since the number of collect | recovered bacteria is further reduced to about 1/100 by an electricity supply process, it turns out that Legionella bacteria could be sterilized by the electroconductive treatment. Conventionally, it has been difficult to sterilize Pseudomonas aeruginosa and Legionella bacteria, but according to the present system, these bacteria are effectively sterilized.

(Third Embodiment)

Japanese Patent Application Laid-Open No. 9-37763 discloses

−

The pathogenic Escherichia coli (Escher
From ichia coli serotype O-157), a gene was extracted.

Next, the obtained gene was amplified by a PCR method using Progene manufactured by Techne. The obtained gene was subjected to electrophoresis to obtain an electrophoresis pattern. FIG. 5 shows the pattern.

In FIG. 5, is a gene marker (gene for finding verotoxin). Is a target gene (verotoxin producing gene). Shows a gene pattern obtained by a chemical extraction method using BIO-RAD DNA purification / recovery reagent “Instagene” at 10 ⁸ Cell / ml. Is the 1/10 chemical extraction method (10 ⁷ cell / ml)
It is. Is an extraction method using no current. ,,,
Are 100 mA, 100 mA, and 300 m, respectively.
A, 500mA (however, 10 ⁸ ce for both non-energized and energized)
It is the result of the gene extracted by the energization method under the condition of (11s / mL target). The band that looks white in the white line is the verotoxin gene.

The contents of the chemical extraction method are as follows.
(1) Wash and concentrate the sample with phosphate buffered saline.
(2) Resuspend the sample in distilled water. (3) Add Instagene to the sample and incubate at 56 ° C for 30 minutes. At this stage, cell enzymatic destruction and gene aggregation occur. (4) Heat treatment at 100 ° C. for 8 minutes. Thermal denaturation of enzymes and other proteins and gene aggregation occur. (5) Mix well to obtain a PCR sample.

As is clear from FIG. 5, no band appeared in the non-energized one, whereas the band appeared in the energized one, and the O-157 gene was certainly isolated. Can be confirmed. In the chemical extraction method, a pattern of contaminants appears above the band (target gene). This does not appear in energized ones. Therefore, it is possible to extract genes in a more pure state in the case of energization as compared with the chemical extraction method.

FIG. 6 is a characteristic diagram comparing the time until the gene extraction by the energization method and the time until the gene recovery by the chemical extraction method. In the former case, the gene extraction is completed in approximately half the time in the latter case. That is,
In both cases, the same time is required for pre-culture of bacteria and collection of cells, but for destruction of cells, the former takes about 12 minutes, whereas the latter takes 1 hour or more.

There is also a remarkable difference between the two in the time required for preculture for gene extraction. That is, in the former method, the number of required bacterial cells may be 1/10 or less of the latter, because the method is a method for collecting and extracting genes having high purity and high yield. In the latter case, a sample in which the number of cells is about (10 ⁸ cells / mL) in the pre-culture for 16 hours is required. On the other hand, since the former is less than 1/10 of this, the pre-culture may be less than 1 hour. That is, in the latter method, it takes about 17 hours from the preculture of the bacterium to the extraction of the gene, whereas in the former method,
It takes less than 2 hours.

In short, the former method, unlike the latter chemical extraction method, is a gene extraction method that is simple and quick in operation, and has high purity and high yield. In addition, since less chemical components are used than in the latter case, the amount of other substances to be mixed is small, and a gene with higher purity can be obtained.

In the first and second embodiments, the flat membrane is used. However, a hollow fiber membrane coated with metal may be used.

[0038]

As described above, according to the present invention, it is possible to provide a system capable of removing the adverse effects of various pathogenic microorganisms by a simple operation. That is, the present invention provides an effective disinfection system for microorganisms contaminating living water without using a chemical.
In addition, there is provided a system for killing Pseudomonas aeruginosa and Legionella bacteria without using a drug. Still another object of the present invention is to provide a nucleic acid detection system for a rapid and highly accurate gene analysis method for the causative bacteria of food poisoning.

[Brief description of the drawings]

FIG. 1 is a manufacturing process diagram of a metal-coated flat membrane used in the present invention.

FIG. 2 is a characteristic diagram showing the results of sterilization of Pseudomonas aeruginosa.

FIG. 3 is a process chart of Example 1.

FIG. 4 is a characteristic diagram showing test results of Legionella bacteria.

FIG. 5 is a characteristic diagram of an electrophoresis pattern based on the results of a gene recovery test from pathogenic Escherichia coli.

FIG. 6 is a process chart for comparing required times required for the chemical extraction method of the gene and the extraction method by the system of the present invention.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Nobuo Katsuura 2-323 Nishihashimoto, Sagamihara City, Kanagawa Prefecture Inside the R & D Center (Nikko Industries Co., Ltd.) (72) Osamu Igarashi 2-23, Nishihashimoto, Sagamihara City, Kanagawa Prefecture No. 3 in the R & D center of Nippon Kogyo Co., Ltd. (72) Inventor Atsushi Nakayama 2-3-3 Nishihashimoto, Sagamihara-shi, Kanagawa Pref.

Claims (11)

[Claims] 1. An electric power supply having a metal film coated on a porous film, a power supply for the electric power supply, and a supply means for supplying microorganisms to the electric power supply. A system for disinfecting microorganisms by supplying electricity to the microorganisms from the power supply using the current supply means as an electrode. 2. The method according to claim 1, wherein the microorganism is Cryptosporidium,
The system according to claim 1, wherein the system is at least one of Pseudomonas aeruginosa, Legionella, and pathogenic Escherichia coli. 3. An electricity supply means having a metal film coated on a porous film, a power supply for the electricity supply means, and a supply means for supplying microorganisms to the electricity supply means, wherein the microorganisms are Pseudomonas aeruginosa, Legionella A system for sterilizing microorganisms by supplying electricity to the microorganisms from the power source using the current supply means as an electrode, wherein the microorganisms are at least one kind of bacteria or pathogenic Escherichia coli. 4. The system according to claim 1, wherein said energizing means applies a direct current to said microorganism. 5. The system according to claim 1, wherein the energizing means is means for capturing the microorganism on a porous membrane and energizing the porous membrane. 6. The system according to claim 1, wherein the metal is chemically bonded to a porous resin forming the porous membrane. 7. An electricity supply means having a metal film coated on a porous film, a power supply for the electricity supply means, and a supply means for supplying pathogenic Escherichia coli to the electricity supply means, wherein the electricity supply means is used as an electrode. A gene recovery system, wherein a gene is selectively extracted from microbial contents caused by the destruction of the E. coli cells by energizing the microorganism from a power supply. 8. A porous membrane coated with a metal,
Apply microorganisms that are a source of pollution to domestic water,
A method for disinfecting the microorganisms by energizing the metal film from a power supply. 9. The method according to claim 8, wherein the microorganism is Cryptosporidium,
The method according to claim 8, which is at least one of Pseudomonas aeruginosa, Legionella, and pathogenic Escherichia coli. 10. The system according to claim 7, wherein said porous film is a porous resin film. 11. The method according to claim 8, wherein said porous film is a porous resin film.