HK40050284B - Cell collection method - Google Patents

Description

Bacterial collection method

Technical Field

This invention relates to a method for collecting microorganisms.

Background Technology

Microbial contamination is a crucial management indicator for reagents used in food, pharmaceuticals, and regenerative medicine. Therefore, methods such as autoclaving or filtration are known to prevent microbial contamination. However, in cases where sterilization is difficult, aseptic treatment is necessary to ultimately check for the absence of microbial contamination. Furthermore, even after sterilization, it is essential to confirm its effectiveness. On the other hand, even trace amounts of extremely small microorganisms are difficult to detect accurately. Moreover, testing all samples with large quantities is impractical. Therefore, a method for effectively collecting or isolating microorganisms is needed.

For example, as a simple and efficient method for collecting cells, it is known to use a protein called MDP1, which binds to sugars on the cell surface, as an aggregation promoter, which can precipitate cells without centrifugation. Furthermore, methods for adding solid supports such as particles immobilized with antibodies have also been reported (e.g., see Patent Document 1).

In addition, there are known methods for separating bacterial cells from bacterial suspensions by adding microbial extracted proteins and adjusting the pH of the cell suspension to 1 to 5 to cause bacterial aggregation (for example, see Patent Document 2).

However, in the method of Patent Document 1, MDP1 is a protein of Mycobacterium bovis (BCG), and the purification process for MDP1 is complex. Furthermore, in the method of Patent Document 2, the extracted microbial protein may contain various proteins (including enzymes such as DNase and RNase), as well as impurities such as DNA and RNA, which could potentially affect the microbial detection after collection. Additionally, the process of further adjusting the pH of the cell suspension is also quite complex.

Therefore, a method for efficiently collecting microorganisms without complicated procedures is sought.

Existing technical documents

Patent documents

Patent Document 1: Japanese Patent Application Publication No. 2010-104250

Patent Document 2: Japanese Patent Application Publication No. 50-135275

Summary of the Invention

The technical problem to be solved by the present invention

Therefore, the technical problem of the present invention is to provide a simple and efficient means for collecting microorganisms.

Technical means to solve technical problems

In view of this situation, the inventors of this application have repeatedly and carefully studied in order to solve the aforementioned technical problem. As a result, they discovered that by adding five specific, readily available proteins with well-defined structures to the specimen, and preferably by further adding a water-insoluble carrier, one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi can efficiently form aggregates, thereby enabling bacterial collection. This led to the completion of the present invention.

That is, the present invention relates to the following [1] to [10].

[1] A method for collecting microorganisms selected from the group consisting of Gram-positive bacteria (excluding mycoplasma), Gram-negative bacteria and fungi in a specimen, characterized in that one or more proteins selected from the group consisting of albumin, casein, hydrolyzed casein, milk protein and gelatin are added to the specimen, and the resulting aggregates are recovered.

[2] The method described in [1] is a method for collecting Gram-positive bacteria (excluding mycoplasma) and/or Gram-negative bacteria.

[3] According to the method described in [1] or [2], wherein the pH value after adding the protein to the sample is greater than 5.0 and less than 11.0.

[4] The method according to any one of [1] to [3], wherein the specimen is a liquid specimen selected from the group consisting of sterile water, physiological saline, liquid culture medium, biological reagent, culture supernatant, buffer solution and Ringer's solution.

[5] The method according to any one of [1] to [4] is a method for collecting Gram-positive bacteria.

[6] The method according to any one of [1] to [5], wherein the Gram-positive bacteria is selected from one or more of the group consisting of Staphylococcus, Enterococcus, Streptococcus, Clostridium, Corynebacterium and Bacillus.

[7] The method according to any one of [1] to [5], wherein the Gram-positive bacteria is selected from one or more of the group consisting of Staphylococcus aureus, Bacillus subtilis and Clostridium sporogenes.

[8] The method according to any one of [1] to [7], wherein a water-insoluble carrier is further added to the specimen.

[9] A method for determining one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi, characterized in that the microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi that have been collected by any one of the methods described in [1] to [8] are subjected to polymerase chain reaction.

[10] A method for determining one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi, characterized in that the microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi that have been collected by the method described in any one of [1] to [8] are enriched.

Invention Effects

Because the method of the present invention adds a protein selected from five specific, readily available, and known proteins, it can collect microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi with extremely high efficiency compared to when no protein is added. Furthermore, by using the method of the present invention, it is possible to effectively collect microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi from a specimen, which are test strains requiring validation in the aseptic test method specified in Japanese Pharmacopoeia (17th Revision) 4.06.

Furthermore, the method of the present invention does not require adjusting the microorganisms according to the detection date, preparing microbial proteins or extracting microbial proteins, and does not require confirmation that the solvents and reagents used to prepare these microorganisms are not contaminated with more than one microorganism selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi, thus making it very convenient.

Furthermore, since the method of the present invention can use water-insoluble carriers such as latex particles, even if a small number of microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi are separated, the aggregates are not easily diffused and are easily identified by the naked eye, thereby making it easy to remove the supernatant after the bacteria are precipitated by collecting bacteria.

Furthermore, since the method of the present invention has a low probability of damaging bacteria, the collected bacteria can be cultured, and the cultured colonies can be used for other tests.

Furthermore, the method of the present invention does not introduce DNA, RNA, DNA hydrolases, RNA hydrolases, or other proteins from cells, thus simplifying the nucleic acid amplification reaction after bacterial collection.

Detailed Implementation

The present invention will now be described in detail with a focus on preferred embodiments. However, the present invention is not limited to these embodiments in any way.

In this invention, "a collection of microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi" refers to the isolation and concentration of one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi contained in a specimen. By collecting one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi, subsequent aseptic tests can be easily performed.

The present invention provides a method for collecting microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi, which is a method for recovering aggregates formed by adding one or more proteins selected from the group consisting of albumin, casein, hydrolyzed casein, milk protein, and gelatin to a specimen.

In this invention, Gram-positive bacteria refer to bacteria that can be Gram-stained and turn indigo blue or deep purple. Gram-positive bacteria are typically bacteria with thick cell walls, no outer membrane, and a thick peptidoglycan layer. Based on their morphology, Gram-positive bacteria can be divided into Gram-positive cocci and Gram-positive bacilli. Additionally, although mycoplasma is classified as a Gram-positive bacterium for convenience, it lacks a cell wall and cannot be Gram-stained; therefore, it is not included in the Gram-positive bacteria discussed in this invention.

Gram-positive cocci include Staphylococcus (e.g., Staphylococcus aureus), Enterococcus (e.g., Enterococcus faecalis), and Streptococcus (e.g., Streptococcus pneumoniae), but are not limited to these.

Gram-positive bacilli include Clostridium (e.g., Clostridium sporogenes), Corynebacterium (e.g., Corynebacterium renale), and Bacillus (e.g., Bacillus subtilis), but are not limited to these.

Among these Gram-positive bacteria, one or more selected from Staphylococcus aureus, Bacillus subtilis, and Clostridium sporogenes are preferred. These bacteria are the test strains that need to be confirmed in the sterility test method specified in Japanese Pharmacopoeia (17th Revision) 4.06. By implementing the bacterial collection method of the present invention, the sterility of the reagents used for sterility testing can be ensured.

In this invention, Gram-negative bacteria refer to bacteria whose staining caused by crystal violet is washed away during Gram staining. Gram-negative bacteria typically have an outer membrane covered by lipopolysaccharides and a cell wall with a thin layer of peptidoglycan. Gram-negative bacteria can be classified into Gram-negative cocci and Gram-negative bacilli based on their morphology.

Gram-negative cocci include, but are not limited to, Neisseria species (e.g., Neisseria meningitis) and Veillonella species (e.g., Veillonella parvula).

Gram-negative bacilli include Escherichia coli (e.g., Echerichia coli), Salmonella (e.g., Salmonella enteritidis), Enterobacteriaceae (e.g., Enterobacter cloacae, Enterobacter aerogenes), Bacteroides (e.g., Bacteroides fragilis), and Pseudomonas (e.g., Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas putida), but are not limited to these.

The fungi of the present invention include Candida (e.g., Candida albicans) and Aspergillus (e.g., Aspergillus brasiliensis), but are not limited thereto.

In this invention, Gram-negative bacteria and fungi can be collected even without the addition of protein, but the method of adding the protein according to this invention can further improve the collection efficiency. Therefore, it is possible to collect Staphylococcus aureus, Bacillus subtilis, Clostridium sporogenes, Pseudomonas aeruginosa, Candida albicans, and Aspergillus brasiliensis, which are test strains to be confirmed in sterility tests, from the sample, which can be used to ensure the sterility of the reagent.

In this invention, a specimen refers to a sample used to test whether an organism is infected with one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi, preferably in liquid form. Examples of specimens include water (sterile water, distilled water, etc.), physiological saline, liquid culture media (e.g., culture media for culturing eukaryotic cells, such as RPMI 1640, DMEM, α-MEM, HANKS, etc.), biological reagents, culture supernatant, buffer solutions (e.g., Tris-HCl buffer, HEPES buffer, MOPS buffer, HEPPS buffer, TAPS buffer, phosphate-buffered saline (PBS), etc.) adjusted to a specified pH value), Ringer's solution, etc. The specimen can also be a cell suspension supernatant or a culture supernatant after cell culture.

The proteins used in this invention are selected from one or more of the following: albumins (e.g., bovine serum albumin (BSA), human serum albumin, primate serum albumin, rabbit serum albumin, rodent serum albumin, equine serum albumin, goat serum albumin, sheep serum albumin, dog serum albumin, guinea pig serum albumin, chicken serum albumin, and porcine serum albumin), casein, hydrolyzed casein, milk proteins (e.g., trade name: Block Ace (manufactured by DS Pharma Biomedical Co., Ltd.)), and gelatin. From the perspective of avoiding contamination during PCR (polymerase chain reaction) of one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi, sterile proteins are preferred. These proteins can be used individually or in combination of two or more.

The present invention relates to a method for collecting microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi, in which the protein is added to a sample and the resulting aggregates are recovered. Specifically, the sample and the protein are first added to a centrifuge tube. To ensure aggregation, the mixture can be stirred or left to stand for any time (e.g., 1 second to 60 minutes). The recovered aggregates can be recovered, for example, by centrifugation. During centrifugation, the centrifugal force is not particularly limited, for example, 3,000 to 30,000 G, preferably 5,000 to 28,000 G, more preferably 14,000 to 25,000 G. The centrifugation time is not particularly limited, for example, 10 seconds to 120 minutes, preferably 1 minute to 60 minutes, more preferably 10 minutes to 30 minutes. The temperature during centrifugation is not particularly limited, for example, 4 to 35°C, preferably 7 to 30°C, more preferably 10 to 25°C. Centrifugation separates microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi, which then precipitate at the bottom of the centrifuge tube. The supernatant is then removed, yielding an aggregate of microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi as residue. This centrifugation and supernatant removal process can be repeated two or more times as needed.

In addition, to obtain the aggregates, the specimen can be subjected to any physical and/or chemical treatment. Examples of physical treatments include heat treatment, ultrasonic irradiation, freezing, and melting. Examples of chemical treatments include treatment with chemical reagents, such as modification treatment using digestive enzymes, surfactants, lysozymes, etc.

The amount of protein used is not particularly limited as long as it is sufficient to cause sufficient aggregation of one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi. For example, it is preferably 0.1 to 500 mg relative to 1 ml of sample, more preferably 0.2 to 100 mg, and even more preferably 2 to 20 mg.

The pH value after adding the protein to the sample is not particularly limited. When the aim is to obtain viable bacteria, it is preferably within a range where one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi do not undergo modification, for example, greater than 5.0 and less than 11.0, more preferably 5.1 to 10.0, further preferably in the range of 6.0 to 8.0, and even more preferably in the range of 6.8 to 7.4. Furthermore, for example, the addition of protein and the recovery of aggregates of one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi can be carried out at conditions of 4 to 35°C, preferably 7 to 30°C, more preferably 10 to 25°C.

In this invention, in addition to the protein, a water-insoluble carrier can also be added. The water-insoluble carrier is not particularly limited to any substance capable of aggregating one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi by being used simultaneously with the protein. Examples include latex particles (e.g., organic polymer latex particles) of polystyrene, styrene-methacrylic acid copolymer, styrene-(meth)acrylate glycidyl ester copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile butadiene styrene copolymer, vinyl chloride-acrylate copolymer, polyvinyl acetate acrylate, etc.; latex particles processed by physical adsorption or by chemical bonding via carboxyl groups; colored cellulose particles; silica particles; gold colloidal particles, etc. Regarding the aggregation of one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi, latex particles of the above-mentioned water-insoluble carrier are preferred, and organic polymer latex particles are particularly preferred. By adding a water-insoluble carrier, it is possible to distinguish the aggregates of bacteria after centrifugation, thus preventing the removal of bacteria when removing the supernatant.

The shape of the water-insoluble carrier is not particularly limited, but from the perspective of enabling sufficient aggregation of one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi, a spherical or near-spherical shape is preferred.

The particle size of the water-insoluble carrier is not particularly limited, but for the sake of visibility and ease of experimentation, an average particle size of 10–2,000 nm is preferred, more preferably 50–1,500 nm, and even more preferably 100–1,000 nm. The particle size of the water-insoluble carrier can be determined, for example, by electron microscopy (TEM).

The amount of water-insoluble carrier used is not particularly limited as long as it is sufficient to allow one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi to aggregate sufficiently. For example, it is 0.0005 to 10 mg relative to 1 ml of sample, preferably 0.001 to 1 mg, and more preferably 0.002 to 0.1 mg.

Regarding the weight ratio of the protein to the water-insoluble carrier, starting from the point of aggregation of one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi, for example, the protein:water-insoluble carrier ratio is preferably 1:1 to 50,000:1, more preferably 10:1 to 10,000:1, and even more preferably 200:1 to 1,000:1.

The present invention also relates to a method for performing polymerase chain reaction (PCR) on residue containing one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi, which have been collected by the above method, thereby determining one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi.

This method utilizes residues containing aggregates of one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi, obtained through the aforementioned methods. DNA is extracted using commercially available kits (e.g., bacterial DNA extraction kits manufactured by Mitsui Chemicals, Inc.) via known methods. The microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi are then detected using known PCR methods or their variations (e.g., real-time PCR) under known conditions. Furthermore, by using primers and probes targeting conserved regions of one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi, it is possible to detect one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi. Alternatively, primers and probes targeting regions specific to the microorganism to be detected can be used to identify one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi.

The present invention also relates to a kit for collecting microorganisms containing the aforementioned protein, preferably further containing a water-insoluble carrier, and selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi. The protein and water-insoluble carrier contained in this kit are as described above. A manual explaining the above operations may also be appropriately added to the kit.

This invention further enables the collection of microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi while maintaining their proliferative capacity. Therefore, the collected microorganisms can be used for various tests. Furthermore, the collected microorganisms can be inoculated onto agar plates, liquid culture media, etc., for enrichment, and used for identification tests such as quantity determination, biochemical analysis, or drug susceptibility testing of the collected microorganisms.

Example

The present invention will be described in more detail below with reference to the embodiments listed, but the present invention is not limited to these embodiments in any way.

Example 1: The bacterial collection effect brought about by protein and water-insoluble carrier

Using sheep blood agar medium (trade name: Nissui plate sheep blood agar medium, manufactured by Nissui Pharmaceutical Co., Ltd.), pre-cultured Staphylococcus aureus was suspended in physiological saline to prepare a bacterial suspension with a turbidity of 1.0 McFarland 1.0. Then, using the diluted bacterial suspension ( ^10³ times dilution) as the diluted bacterial solution, 0.1 ml of the diluted bacterial solution was added to 24.9 ml of physiological saline. 350 μl of 30% (w/v) BSA solution (BSA solution derived from bovine serum, fatty acid-free, manufactured by Wako Pure Chemical Corporation) and 2.5 μl of 10% (w/v) latex solution (polystyrene latex particles with a particle size of 315 nm (IMMUTEX (registered trademark, manufactured by JSR Life Sciences, LLC)) were added. After stirring the solution, centrifuge (20,000 G, 15 min, 10 °C), remove the supernatant, and obtain 1 ml of test solution. Dilute the test solution 10-fold to obtain bacterial suspension, and inoculate 10 μl of this bacterial suspension onto sheep blood agar medium (the same as the sheep blood agar medium described above) and incubate overnight (37 °C, aerobic conditions). As a comparison, a bacterial suspension was diluted ^10⁶ times and inoculated at 100 μl onto sheep blood agar medium (the same as the sheep blood agar medium described above), without bacterial collection.

In addition, DNA was extracted from the test solution using a bacterial DNA extraction kit (manufactured by Mitsui Chemicals, Inc.), and real-time PCR was performed using Yeast-made Taq DNA polymerase (manufactured by Mitsui Chemicals, Inc.), forward primer: agagtttgatcMtggctcag (SEQ ID NO:1), reverse primer: ctttacgcccaRtRaWtccg (SEQ ID NO:2), and probe: 6FAM-tNttaccgcggctgctggcacg-BHQ (SEQ ID NO:3). The reaction was carried out at 95°C for 5 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds.

The results of the culture experiment are shown in Table 1.

[Table 1]

As shown in Table 1, the addition of BSA alone, and the combination of BSA and latex, resulted in colony counts similar to those without bacterial collection, compared to the addition of latex alone or physiological saline. Therefore, adding BSA can effectively collect Staphylococcus aureus, a Gram-positive bacterium. Furthermore, adding latex to BSA allows for the identification of precipitates during centrifugation, facilitating the removal of the supernatant.

Next, the PCR results are shown in Table 2.

[Table 2]

As shown in Table 2, in PCR, the Cq values of BSA added alone and in combination with latex were similar to those of the uncollected bacteria. Therefore, adding and concentrating BSA can effectively collect Staphylococcus aureus, a Gram-positive bacterium. On the other hand, adding physiological saline or adding latex alone resulted in increased Cq values, indicating insufficient bacterial collection.

Example 2: Relationship between Gram staining properties and bacterial collection effect

Using sheep blood agar medium (trade name: Nissui Plate sheep blood agar medium, manufactured by Nissui Pharmaceutical Co., Ltd.) or chocolate agar medium (trade name: Nissui Plate chocolate agar medium EXII, manufactured by Nissui Pharmaceutical Co., Ltd.), pre-cultured Corynebacterium renaleris, Neisseria meningitidis, and Escherichia coli were suspended in physiological saline to prepare a bacterial suspension with a turbidity of 1.0 McFarland unit. Then, using a ^10³- fold dilution as the diluted bacterial suspension, 0.1 ml of the diluted bacterial suspension was added to 24.9 ml of physiological saline. 350 μl of 30% (w/v) BSA solution (fatty acid-free, manufactured by Wako Pure Chemical Corporation) was then added. After stirring the solution, centrifuge (20,000 G, 15 min, 10 °C), remove the supernatant, and obtain 1 ml of test solution. Inoculate the bacterial suspension (using the test solution for Corynebacterium nephritis) diluted 10-fold onto sheep blood agar (the same as the sheep blood agar medium) or chocolate agar (the same as the chocolate agar medium) and incubate overnight (37 °C, aerobic conditions). For Neisseria meningitidis, incubate at 37 °C using Anaero Pack· _CO2 (manufactured by SUGIYAMA-GEN Co., Ltd.). As a comparison, a bacterial suspension diluted 10⁶ ^- fold was inoculated at 100 μl onto sheep blood agar (the same as the sheep blood agar medium), without bacterial collection.

In addition, DNA was extracted from the test solution using a bacterial DNA extraction kit (manufactured by Mitsui Chemicals, Inc.), and real-time PCR was performed using Yeast-made Taq DNA polymerase (manufactured by Mitsui Chemicals, Inc.), forward primer: agagtttgatcMtggctcag (SEQ ID NO:1), reverse primer: ctttacgcccaRtRaWtccg (SEQ ID NO:2), and probe: 6FAM-tNttaccgcggctgctggcacg-BHQ (SEQ ID NO:3). The reaction was carried out at 95°C for 5 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds.

The results of the culture experiment are shown in Table 3.

[Table 3]

As shown in Table 3, Corynebacterium nephritis, a Gram-positive bacillus, failed to be collected in physiological saline, but collection was achieved by adding BSA. On the other hand, Neisseria meningitidis, a Gram-negative coccus, and Escherichia coli, a Gram-negative bacillus, could be collected even without the addition of BSA, but the collection rate was improved by adding BSA.

The PCR results are shown in Table 4 below.

[Table 4]

As shown in Table 4, in PCR, the Cq value of Corynebacterium nephritis, a Gram-positive bacillus, was lower when BSA was added than when physiological saline was added. On the other hand, the Cq values of Neisseria meningitidis, a Gram-negative coccus, and Escherichia coli, a Gram-negative bacillus, were slightly lower when BSA was added than when physiological saline was added and when no bacteria were collected.

Example 3: Microbial collection effect based on protein type

Using sheep blood agar medium (trade name: Nissui Plate sheep blood agar medium, manufactured by Nissui Pharmaceutical Co., Ltd.), pre-cultured Staphylococcus aureus was suspended in physiological saline to prepare a bacterial suspension with a turbidity of 1.0 McFarland unit. Then, using a ^10³- fold dilution as the diluted bacterial suspension, 0.1 ml of the diluted bacterial suspension was added to 24.9 ml of physiological saline. 350 μl of 30% (w/v) BSA solution (fatty acid-free, manufactured by Wako Pure Chemical Corporation), gelatin, or milk protein (trade name: Block Ace, manufactured by DS Pharma Biomedical Co., Ltd.) was added to each solution. After stirring, each solution was centrifuged (20,000 G, 15 min, 10 °C), and the supernatant was removed to obtain 1 ml of the test solution. The bacterial suspension, diluted 10-fold, was inoculated onto sheep blood agar medium (the same as the sheep blood agar medium described above) and incubated overnight (37°C, aerobic conditions). As a comparison, a bacterial suspension diluted ^10⁶ -fold was inoculated at 100 μl onto sheep blood agar medium (the same as the sheep blood agar medium described above), without bacterial collection.

In addition, DNA was extracted from the test solution using a bacterial DNA extraction kit (manufactured by Mitsui Chemicals, Inc.), and real-time PCR was performed using Yeast-made Taq DNA polymerase (manufactured by Mitsui Chemicals, Inc.), forward primer: agagtttgatcMtggctcag (SEQ ID NO:1), reverse primer: ctttacgcccaRtRaWtccg (SEQ ID NO:2), and probe: 6FAM-tNttaccgcggctgctggcacg-BHQ (SEQ ID NO:3). The reaction was carried out at 95°C for 5 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds.

The results of the culture experiment are shown in Table 5.

[Table 5]

As shown in Table 5, compared with the addition of physiological saline, the addition of BSA alone, the addition of milk protein (Block Ace) alone, and the addition of gelatin alone resulted in colony counts similar to those of the uncollected bacteria.

The PCR results are shown in Table 6.

[Table 6]

As shown in Table 6, gelatin exhibited the same Cq value as BSA in PCR. Therefore, as a protein, gelatin can effectively target Staphylococcus aureus, a Gram-positive bacterium, not only when using BSA but also when using gelatin.

Example 4 uses a water-insoluble carrier for bacterial collection testing.

Using sheep blood agar medium (trade name: Nissui Plate sheep blood agar medium, manufactured by Nissui Pharmaceutical Co., Ltd.), pre-cultured Corynebacterium renalii and Enterococcus faecalis were separately suspended in physiological saline to prepare 1.0 McFarland unit bacterial suspensions. Then, using a ^10³ -fold dilution as the diluted bacterial suspension, 0.1 ml of the diluted bacterial suspension was added to 24.9 ml of physiological saline. 350 μl of 30% (w/v) BSA solution and 2.5 μl of 10% (w/v) latex solution were added as a water-insoluble carrier. After stirring, the solution was centrifuged (20,000 G, 15 min, 10 °C), and the supernatant was removed to obtain 1 ml of the test solution. The test solution was diluted 10-fold and inoculated at 90 μl onto sheep blood agar medium (the same as the sheep blood agar medium described above), and incubated overnight (37°C, aerobic conditions). As a comparison, the bacterial suspension was diluted 10.5 ^- fold and inoculated at 100 μl onto sheep blood agar medium (the same as the sheep blood agar medium described above), without bacterial collection.

In addition, DNA was extracted from the test solution using a bacterial DNA extraction kit (manufactured by Mitsui Chemicals, Inc.), and real-time PCR was performed using Yeast-made Taq DNA polymerase (manufactured by Mitsui Chemicals, Inc.), forward primer: agagtttgatcMtggctcag (SEQ ID NO:1), reverse primer: ctttacgcccaRtRaWtccg (SEQ ID NO:2), and probe: 6FAM-tNttaccgcggctgctggcacg-BHQ (SEQ ID NO:3). The reaction was carried out at 95°C for 5 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds.

The results of the culture experiment are shown in Table 7.

[Table 7]

As shown in Table 7, when collecting *Corynebacterium nephritis*, a Gram-positive bacillus, the recovery rate was 97% of the theoretical value without collection when BSA and latex were added in combination. On the other hand, when physiological saline was added, the recovery rate was 0% of the theoretical value. Therefore, the collection rate of *Corynebacterium nephritis* was extremely high when BSA and latex were added in combination.

Furthermore, when collecting Enterococcus faecalis, a Gram-positive coccus, the recovery rate was 90% of the theoretical value without collection when BSA and latex were added in combination. On the other hand, the recovery rate was only 10% of the theoretical value when physiological saline was added. Therefore, it can be seen that the collection rate of Enterococcus faecalis is extremely high due to the combination of BSA and latex.

Furthermore, in any experiment, by adding latex to BSA, the precipitate during centrifugation can be identified, and the supernatant can be easily removed.

The results of the PCR are shown in Table 8 below.

[Table 8]

As shown in Table 8, in PCR, when BSA and latex were added together for bacterial collection, the Cq value was similar to the theoretical value without bacterial collection, indicating sufficient collection rate. On the other hand, when only physiological saline was added, the Cq value increased, indicating insufficient bacterial collection.

Example 5: The effect of pH value during bacterial collection

Using sheep blood agar medium (trade name: Nissui Plate sheep blood agar medium, manufactured by Nissui Pharmaceutical Co., Ltd.), pre-cultured Staphylococcus aureus was suspended in physiological saline to prepare a bacterial suspension with a turbidity of 1.0 McFarland unit. Then, using the ^10³- fold dilution as the diluted bacterial suspension, 0.1 ml of the diluted bacterial suspension was added to 24.9 ml of 0.01 M sodium phosphate buffer at pH 6.0, pH 7.0, and pH 8.0, respectively. 350 μl of 30% (w/v) BSA solution (BSA solution derived from bovine serum, fatty acid-free, manufactured by Wako PureChemical Corporation) was added to each solution. After stirring, the solution was centrifuged (20,000 G, 15 min, 10 °C), and the supernatant was removed to obtain 1 ml of the test solution. The test solution was diluted 10-fold to obtain a bacterial suspension. 10 μl of this bacterial suspension was inoculated onto sheep blood agar medium (the same as described above) and incubated overnight (37°C, aerobic conditions). As a comparison, a bacterial suspension was diluted ^10⁶ times with physiological saline and inoculated at 100 μl onto sheep blood agar medium (the same as described above), without bacterial collection.

In addition, DNA was extracted from the test solution using a bacterial DNA extraction kit (manufactured by Mitsui Chemicals, Inc.), and real-time PCR was performed using Yeast-made Taq DNA polymerase (manufactured by Mitsui Chemicals, Inc.), forward primer: agagtttgatcMtggctcag (SEQ ID NO:1), reverse primer: ctttacgcccaRtRaWtccg (SEQ ID NO:2), and probe: 6FAM-tNttaccgcggctgctggcacg-BHQ (SEQ ID NO:3). The reaction was carried out at 95°C for 5 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds.

The results of the culture experiment are shown in Table 9.

[Table 9]

In addition, the pH value was measured three times after the addition of BSA. The results and their average values are shown in Table 10.

[Table 10]

As shown in Table 9, the addition of BSA allows for the collection and cultivation of Staphylococcus aureus, a Gram-positive coccus, within any pH range of 6.0, 7.0, and 8.0. Furthermore, the results in Table 10 confirm that the pH value remained almost unchanged after the addition of BSA.

The results of the PCR are shown in Table 11 below.

[Table 11]

As shown in Table 11, adding BSA to the sodium phosphate buffer solutions at pH 6.0, pH 7.0, and pH 8.0 resulted in Cq values similar to those without bacterial collection. On the other hand, adding the sample solution without BSA resulted in a higher Cq value than without bacterial collection, indicating insufficient bacterial collection.

Example 6 Results of Clostridium sporogenes

Using sheep blood agar medium (trade name: Nissui Plate sheep blood agar medium, manufactured by Nissui Pharmaceutical Co., Ltd.), pre-cultured *C. sporogenes* were suspended in physiological saline to prepare a bacterial suspension with a turbidity of 1.0 McFarland unit. Then, using a ^10³- fold dilution as the diluted bacterial suspension, 0.1 ml of the diluted bacterial suspension was added to 24.9 ml of physiological saline. 350 μl of 30% (w/v) BSA solution (BSA solution derived from bovine serum, fatty acid-free, manufactured by Wako Pure Chemical Corporation) was added. After stirring the solution, centrifugation (20,000 G, 15 min, 10 °C) was performed, and the supernatant was removed to obtain 1 ml of the test solution. The test solution was diluted 10-fold to obtain a bacterial suspension. 10 μl of this bacterial suspension was inoculated onto sheep blood agar medium (the same as described above) and cultured overnight (37°C) using Aneromate-P “Nissui” (manufactured by Nissui Pharmaceutical Co., Ltd.). As a comparison, a bacterial suspension was diluted ^10⁶ times with physiological saline and inoculated at 100 μl onto sheep blood agar medium (the same as described above), without bacterial collection.

In addition, DNA was extracted from the test solution using a bacterial DNA extraction kit (manufactured by Mitsui Chemicals, Inc.), and real-time PCR was performed using Yeast-made Taq DNA polymerase (manufactured by Mitsui Chemicals, Inc.), forward primer: agagtttgatcMtggctcag (SEQ ID NO:1), reverse primer: ctttacgcccaRtRaWtccg (SEQ ID NO:2), and probe: 6FAM-tNttaccgcggctgctggcacg-BHQ (SEQ ID NO:3). The reaction was carried out at 95°C for 5 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds.

The results of the culture experiment are shown in Table 12.

[Table 12]

As shown in Table 12, when collecting Clostridium sporogenes, a Gram-positive bacillus, the recovery rate was 151% of the theoretical value without collection when BSA was added. On the other hand, the recovery rate was 57% of the theoretical value when physiological saline was added. Therefore, the collection rate of Clostridium sporogenes was extremely high when BSA was added.

The results of the PCR are shown in Table 13 below.

[Table 13]

As shown in Table 13, in PCR, adding BSA for bacterial collection resulted in a Cq value close to the theoretical value without bacterial collection, indicating sufficient bacterial collection. On the other hand, adding only physiological saline resulted in an increased Cq value, indicating insufficient bacterial collection.

Example 7: The effect of pH in the acidic range

Using sheep blood agar medium (trade name: Nissui Plate sheep blood agar medium, manufactured by Nissui Pharmaceutical Co., Ltd.), pre-cultured Staphylococcus aureus was suspended in physiological saline to prepare a bacterial suspension with a turbidity of 1.0 McFarland unit. Then, using the ^10³- fold dilution as the diluted bacterial suspension, 0.1 ml of the diluted bacterial suspension was added to 24.9 ml of 0.01 M sodium citrate buffer (pH 4.3). 350 μl of 30% (w/v) BSA solution (BSA solution derived from bovine serum, fatty acid-free, manufactured by Wako Pure Chemical Corporation) was added to the diluted solution. After stirring, the solution was centrifuged (20,000 G, 15 min, 10 °C), and the supernatant was removed to obtain 1 ml of the test solution. The test solution was diluted 10-fold to obtain a bacterial suspension. 10 μl of this bacterial suspension was inoculated onto sheep blood agar medium (the same as described above) and incubated overnight (37°C, aerobic conditions). As a comparison, a bacterial suspension was diluted ^10⁶ times with physiological saline and inoculated at 100 μl onto sheep blood agar medium (the same as described above), without bacterial collection.

In addition, DNA was extracted from the test solution using a bacterial DNA extraction kit (manufactured by Mitsui Chemicals, Inc.), and real-time PCR was performed using Yeast-made Taq DNA polymerase (manufactured by Mitsui Chemicals, Inc.), forward primer: agagtttgatcMtggctcag (SEQ ID NO:1), reverse primer: ctttacgcccaRtRaWtccg (SEQ ID NO:2), and probe: 6FAM-tNttaccgcggctgctggcacg-BHQ (SEQ ID NO:3). The reaction was carried out at 95°C for 5 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds.

The results of the culture experiment are shown in Table 14.

[Table 14]

In addition, the pH value was measured three times after the addition of BSA. The results and their average values are shown in Table 15.

[Table 15]

Table 14 shows that Staphylococcus aureus, a Gram-positive coccus, can be collected within a pH range of 4.3, regardless of whether BSA is added. Furthermore, Table 15 confirms that the pH value hardly changes after adding BSA.

The results of the PCR are shown in Table 16 below.

[Table 16]

As shown in Table 16, regardless of whether BSA was added, the sodium citrate buffer at pH 4.3 exhibited a Cq value similar to that without bacterial collection.

Example 8: Collection of Candida albicans

Two BioBall550 syringes (trade name: BioBall550, manufactured by bioMérieux Japan Ltd.) were added to 25 ml of physiological saline to prepare a bacterial suspension equivalent to 1100 CFU/25 ml. Then, 350 μl of 30% (w/v) BSA solution (BSA solution derived from bovine serum, fatty acid-free, manufactured by Wako Pure Chemical Corporation) and 2.5 μl of 10% (w/v) latex solution were added as a water-insoluble carrier. After stirring the solution, centrifugation was performed (20,000 G, 15 min, 10 °C), and the supernatant was removed to obtain 1 ml of test solution. 100 μl of this test solution was inoculated onto sheep blood agar medium (trade name: Nissui Plate sheep blood agar medium, manufactured by Nissui Pharmaceutical Co., Ltd.) and incubated overnight (37 °C, aerobic conditions).

The results of the culture experiment are shown in Table 17.

[Table 17]

As shown in Table 17, when collecting Candida albicans, a Gram-positive fungus, the recovery rate was 80.5% of the theoretical value when BSA and latex were added, which was better than the 65.9% when physiological saline was added.

<110> Nissui Pharmaceutical Co., Ltd.

<120> Collection of bacteria

<130> KHP212110207.6

<150> JP2018-200476

<151> 2018-10-25

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic oligonucleotides

<400> 1

agagtttgat cmtggctcag 20

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<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic oligonucleotides

<400> 2

ctttacgccc artrawtccg 20

<210> 3

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic oligonucleotides

<220>

<221> Unclassified features (misc_feature)

<222> (1)..(1)

<223> 6FAM-dt

<220>

<221> Unclassified features (misc_feature)

<222> (2)..(2)

<223> n is a, c, g, or t

<220>

<221> Unclassified features (misc_feature)

<222> (22)..(22)

<223> dg-BHQ

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Claims (5)

1. A method for collecting microorganisms, characterized in that one or more proteins selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and fungi are added to a liquid sample selected from the group consisting of sterile water, physiological saline, liquid culture medium, culture supernatant, buffer, and Ringer's solution, along with a water-insoluble carrier with an average particle size of 10-2000 nm, and the resulting aggregates are recovered. The Gram-positive bacteria do not include mycoplasma, and the pH of the sample after adding the protein is greater than 5.0 and less than 11.0. The Gram-positive bacteria are selected from Staphylococcus aureus , Clostridium sporogenes , Corynebacterium renale , and Enterococcus faecalis , and the Gram-negative bacteria are selected from Neisseria meningitis and Escherichia coli. The fungus is * Candida albicans *, and the water-insoluble carrier is latex particles. 2. The bacterial collection method according to claim 1, wherein the bacterial collection method is for Gram-positive bacteria and/or Gram-negative bacteria, and the Gram-positive bacteria do not include mycoplasma. 3. The bacterial collection method according to claim 1 or 2, wherein it is a bacterial collection method for Gram-positive bacteria. 4. A method for determining one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi, characterized in that the microorganisms are collected according to the method of any one of claims 1 to 3, and then subjected to polymerase chain reaction. 5. A method for determining one or more microorganisms selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and fungi, characterized in that the microorganisms are collected according to the method of any one of claims 1 to 3, and then enriched.