JP2004000200A - Method for detecting microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting microorganisms contained in fluid promptly and sensitively. <P>SOLUTION: The method includes a process (1) for filtering the fluid by using a filter membrane, a process (2) in which the filter membrane is immersed in a liquid medium, and the microorganisms entrapped by the filter membrane are cultivated, a process (3) for concentrating the cultivated microorganisms, a process (4) for extracting the DNA of the cultivated microorganisms from the concentrated culture, a process (5) for performing PCR (polymerase chain reaction) by using the extracted DNA as a template, and a process (6) for detecting the obtained PCR product. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for detecting a microorganism.
[0002]
[Prior art]
In recent years, with the rapid progress of tissue engineering, products utilizing living cells such as cultured skin have been developed. In such a product, it is required to confirm not only the safety of the constituent cells but also the absence of microbial contamination. Since products using living cells such as cultured skin cannot be terminally sterilized, it is necessary to pay sufficient attention so that microorganisms are not mixed in the production process. It may also be necessary to ensure the sterility of the final product.
[0003]
Cultured skin is produced by culturing small amounts of cells collected from patients with burns, etc., and transplanted into the patient from whom the cells were collected (autologous skin transplantation) or transplanted to another person (allogeneic). Cultured skin transplantation). For this reason, it is necessary to confirm that the collected cells are not infected with fungi, bacteria or viruses. In particular, deep (systemic) mycosis due to Candida albicans and the like has become a medical problem as an infectious disease in which highly susceptible patients are affected at a high rate. Pseudomonas aeruginosa and other chlorophylls cause opportunistic infections in hosts with reduced resistance, causing suppuration of the skin, urinary tract infections, respiratory infections, and sepsis, causing hospital-acquired infections. There is a problem that it is easy.
[0004]
As a method of detecting a fungus for a long time, a method of culturing a clinical specimen on, for example, a petri dish, growing fungi contained in the clinical specimen, and detecting a colony of the formed fungus has been performed. However, fungi in particular grew slowly, so it took a long time to form colonies. For this reason, there was a problem that a detection result could not be obtained before applying the cultured skin to a patient.
[0005]
As a negative test for bacteria and fungi in a medical device GMP, a method of culturing a filtration membrane obtained by filtering a sample for 7 to 14 days and visually confirming that the culture solution is not cloudy is generally adopted. However, even in the sterility test of the cultured skin by this method, the test result cannot be obtained before the cultured skin is applied to the patient.
[0006]
Other methods include immunological detection of fungal antigens and biochemical detection of fungal cell components or metabolites, all of which have low detection sensitivity and require a lot of time. .
[0007]
Many methods for directly detecting fungal DNA have been disclosed as methods for solving the problem that it takes a long time to obtain test results. Specifically, it is a method of detecting a fungus by amplifying a gene by PCR using an oligonucleotide that hybridizes with a specific gene sequence of a fungus as a primer. Such a method was able to detect fungi more quickly than the above-mentioned method, and the detection sensitivity was also improved. In particular, Patent Document 1 discloses that, for example, an oligonucleotide that specifically hybridizes with the nucleic acid sequence of the 18S rRNA gene of Candida albicans is used as a primer to amplify the gene, and the amplified product is subjected to electrophoresis or dot blot. A method for detection by a hybridization method or the like is disclosed. With this method, it has become possible to end the detection of fungi, which conventionally required several days to several weeks, within one day. However, in the method disclosed in Patent Document 1, a conventional PCR method is applied, and only about 1 to 2 ml of a sample can be used. If the method is applied to liquids such as immersions of tissues or tissue equivalents where the culture supernatant reaches several hundred milliliters, only a small portion of the resulting culture supernatant will be used. Therefore, not only is the detection sensitivity of the microorganism insufficient, but also disadvantages such as a decrease in the reliability of the test result occur.
[0008]
The detection sensitivity of other fungal detection methods using PCR is not sufficient. Further, there has been no disclosure of a method for detecting a fungus using an immersion liquid of a tissue equivalent such as cultured skin as a sample.
[0009]
Furthermore, as a rapid detection method for bacteria, use as a carbon source in the medium ¹⁴ CO produced by metabolism using glucose labeled with C ₂ Is also known (Non-Patent Document 1). However, this method has a problem that fungi and bacteria cannot be distinguished and detected. Also, for products containing living cells (eg, tissue equivalents), the living cells themselves are CO 2 ₂ Therefore, the method cannot be applied.
[0010]
Therefore, a method for detecting microorganisms, including fungi and bacteria, useful in tissues or tissue equivalents, particularly in cultured skin, has been desired.
[0011]
As a method for detecting microorganisms in the air in the controlled environment, a certain amount of air is suctioned using an air sampler, the microorganisms in the air collide with the agar medium, and then the agar medium is cultured and grown. Methods for measuring the number are known (Non-Patent Documents 2 and 3). However, in order to be able to visually confirm colonies, it is necessary to culture at least two days even for cells having a high growth rate, and four days or more for cells having a low growth rate. In addition, since the colonies of yeast-like fungi and bacteria are circular in shape, it is difficult to discriminate them from the shape of the colonies. And it takes more time. In addition, for example, it is necessary to change the agar medium every time a certain amount of air is sucked in, so a large amount of agar medium is required, and the culturing period on the agar medium is 5 days or more according to the Japanese Pharmacopoeia. Because it is specified, a place for culturing the agar medium is required for several days, or in order to confirm the detection of bacterial cells as soon as possible, observe the presence or absence of colonies during the culture period. However, there is a problem that it takes time and effort to observe the agar medium as well as a large number of samples.
[0012]
[Patent Document 1]
JP-A-8-89254
[Non-patent document 1]
Edited by Japan Air Purification Association, "Construction and Maintenance of Clean Room Environment", 1st edition, Ohmsha Co., Ltd., July 20, 2000, p. 65-75
[Non-patent document 2]
Edited by the Japanese Pharmacopoeia Commentary Editing Committee, “14th Revision of the Japanese Pharmacopoeia Commentary”, Tokyo Hirokawa Shoten, June 27, 2001, p. F-140-162
[Non-Patent Document 3]
Kenji Sato et al., "Sterile Medical Device GMP Validation Introductory Course", Medical Device Center, February 21, 2002, p. 87-107
[0013]
[Problems to be solved by the invention]
In view of the above-mentioned problems of the related art, an object of the present invention is to provide a method for detecting microorganisms quickly and with high sensitivity. In particular, an object of the present invention is to provide a method for quickly and highly sensitively detecting microorganisms contained in a liquid such as an immersion liquid of a tissue or a tissue equivalent. Another object of the present invention is to provide a method for detecting microorganisms contained in a gas quickly and with high sensitivity. That is, an object of the present invention is to provide a method for detecting microorganisms contained in a fluid quickly and with high sensitivity.
[0014]
[Means for Solving the Problems]
As a result of repeated research on a method for detecting microorganisms quickly and with high sensitivity, the combination of filtration of a liquid, such as a tissue or tissue equivalent immersion liquid, with a filtration membrane, pre-culture using the filtration membrane, and PCR enables the formation of a liquid. It was found that the contained microorganisms were detected quickly and with high sensitivity. Further, they have found that microorganisms contained in gas as well as liquid can be detected quickly and with high sensitivity, and have completed the present invention.
[0015]
That is, the present invention relates to a method for detecting a microorganism,
(1) filtering a fluid using a filtration membrane,
(2) immersing the filtration membrane in a liquid medium and culturing the microorganisms captured by the filtration membrane;
(3) a step of concentrating the culture,
(4) extracting microbial DNA from the concentrated culture,
(5) performing PCR using the extracted DNA as a template, and
(6) Step of detecting the obtained PCR product
And a method for detecting a microorganism containing the same.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in more detail.
[0017]
As used herein, "filtration" means passing a liquid or gas through a porous substance such as a filtration membrane.
[0018]
As used herein, the term “fluid” means a liquid or gas.
[0019]
As used herein, the term "liquid" refers to a liquid that may contain microorganisms, including, but not limited to, immersion liquid for tissues or tissue equivalents, immersion liquid for organs, or blood. Not something.
[0020]
The term “tissue” in the present specification means various tissues extracted from a living body. Examples of tissues include, but are not limited to, skin, cornea, cartilage, adipose tissue, muscle, blood vessels, nerves, amniotic membrane, placenta, or lymph nodes.
[0021]
As used herein, the term “tissue equivalent” is defined as a structure containing living cells or a structure in which living cells are incorporated into a biocompatible material. Examples of tissue equivalents include, for example, cultured skin consisting of living cells and biopolymers or living cells, cultured cornea, cultured cartilage, cultured bone, cultured kidney, cultured liver, cultured retina, cultured nerve, cultured heart or cultured The inner ear is not limited to these as long as it is a structure containing living cells.
[0022]
The cultured skin includes cultured epidermis, cultured dermis, composite cultured skin, and the like. Cultured epidermis is, for example, an epidermal cell sheet produced by culturing and growing epidermal cells obtained from mammalian epidermis such as keratinocytes, or collagen, chitin, chitosan, chondroitin sulfate, hyaluronic acid, laminin, polylactic acid, It is a structure in which epidermal cells are incorporated into a biopolymer material such as polyglycolic acid, fibrin, and gelatin, and / or a non-biodegradable synthetic polymer material having good cell adhesion such as polyurethane, polystyrene, and polyacrylate. Cultured dermis refers to, for example, culture and proliferation of fibroblasts obtained from mammalian dermis, and the growth of collagen, chitin, chitosan, chondroitin sulfate, hyaluronic acid, laminin, polylactic acid, polyglycolic acid, fibrin, gelatin, etc. It is a cultured dermis sheet incorporated in a non-biodegradable synthetic polymer material having good cell adhesion such as a molecular material and / or a synthetic polymer such as polyurethane, polystyrene, or polyacrylate. The composite cultured skin is a structure close to the skin of a living body in which epidermal cells are further incorporated into the cultured dermis-like structure.
[0023]
The cultured cornea is, for example, a sheet-like structure produced by culturing and growing corneal epithelial cells, stromal cells, and / or endothelial cells obtained from a mammalian cornea, or collagen, chitin, chitosan, chondroitin sulfate. Non-biodegradable synthesis with good cell adhesion, such as biopolymer materials such as hyaluronic acid, laminin, polylactic acid, polyglycolic acid, fibrin, gelatin, and / or synthetic polymers such as polyurethane, polystyrene and polyacrylate It is a structure in which corneal epithelial cells, parenchymal cells, and / or endothelial cells obtained from a mammalian cornea are incorporated into a polymer material.
[0024]
The cultured cartilage is an artificial cartilage made of a cell or a structure in which the cell is incorporated into a base material, which is manufactured by a known method. The cultured cartilage includes, for example, a structure produced by incorporating a chondrocyte and / or mesenchymal stem cell into a three-dimensional structure carrier made of a biocompatible material such as chitosan, collagen and / or calcium phosphate. be able to.
[0025]
As used herein, the term "immersion liquid for tissue or tissue equivalent" refers to the liquid used in (i) removing a piece of tissue from a patient and transporting it to a tissue or tissue equivalent supply organization (eg, a manufacturer). , (Ii) a liquid used in the process of manufacturing the tissue equivalent, or (iii) a liquid used to store the manufactured tissue equivalent.
[0026]
`` Liquid used in removing a piece of tissue from a patient and then transporting it to a tissue or tissue equivalent supply organization '' is a liquid in which the piece of tissue is immersed while transporting the collected piece of tissue to a destination. It is not particularly limited as long as it is possible. "Liquid used in collecting a piece of tissue from a patient and then transported to a tissue or tissue equivalent supply organization" includes, for example, the liquid or skin used in storing skin collected from a mammal. Liquids recovered after rinsing with a buffer are included.
[0027]
The “liquid used in the process of producing the tissue equivalent” is not particularly limited as long as it is a liquid added to and recovered from the tissue equivalent during the production of the tissue equivalent. The “liquid used in the process of producing a tissue equivalent” is, for example, a liquid collected after rinsing cells collected from a mammal with a buffer. Furthermore, the culture supernatant obtained after culturing the cells constituting the tissue equivalent, the liquid collected after performing trypsin treatment for dispersing the cells and the culture supernatant of the tissue equivalent are collected when the culture medium is exchanged. Liquids and the like are also included in the liquid used in the process of manufacturing the tissue equivalent.
[0028]
The "liquid used for storage of the manufactured tissue equivalent" is not particularly limited as long as it is a liquid that is recovered after being added to the tissue equivalent between completion and actual application.
[0029]
Specific examples of what can be used for the “immersion liquid for tissue or tissue equivalent” include Dulbecco's modified Eagle's basic medium (DMEM), phosphate buffer, Ringer's solution, Ringer-Rock's solution, Tyrode's solution, Earl's solution, Hanks Liquid, rock solution, Eagle's minimum essential culture, Ham's synthetic culture F12, Green culture, Leibovitz's L-15 culture, and serum-free culture MCDB153, MCDE151, MCDE104, MCDB131, MCDB402, MCDB201, MCDB302, MCDB105, MCDB110, and the like. When the tissue equivalent is cryopreserved after production, a cryopreservation solution obtained by adding a cryoprotectant to the above-mentioned culture solution or buffer solution can be used. Examples of the cryoprotectant include glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, sucrose, carboxymethylcellulose salt, carboxymethylcellulose (CMC), and disaccharides (trehalose and the like). One or more types can be selected and added to the above-mentioned culture solution or buffer solution.
[0030]
The term “organ” as used herein means various organs extracted from a living body. Examples of organs include, but are not limited to, for example, liver, pancreas, kidney or heart.
[0031]
As used herein, the term “organ immersion liquid” includes a liquid used for temporarily immersing and storing an organ when transporting the removed organ between hospitals during organ transplantation and the like.
[0032]
Specific examples of the “organ immersion liquid” that can be used include DMEM, phosphate buffer, Ringer's solution, Ringer-Rock's solution, Tyrode's solution, Earl's solution, Hanks's solution, Rock's solution, and the minimum essential culture solution of Eagle. , Ham synthetic culture medium F12, Green culture medium, Leibovitz's L-15 culture medium, and serum-free culture medium MCDB153, MCDE151, MCDE104, MCDB131, MCDB402, MCDB201, MCDB302, MCDB105, and MCDB110. Further, a cryopreservation solution obtained by adding a cryoprotectant to the above-mentioned culture solution or buffer solution can also be used. Examples of the cryoprotectant include glycerol, DMSO, propylene glycol, ethylene glycol, sucrose, carboxymethylcellulose salts, CMC, and disaccharides (trehalose and the like). Alternatively, it can be added to a buffer.
[0033]
Furthermore, microbial contamination can also be detected in the collected blood itself from which cells have not been removed, and in liquid components from which cells have been removed from blood, using the microorganism detection method of the present invention. Examples of the liquid component obtained by removing cells from blood include serum or plasma. Serum is obtained, for example, by leaving blood collected using a dry syringe and removing a blood clot containing blood cells and some blood clotting factors (such as fibrinogen or prothrombin) from the blood. Plasma is obtained, for example, by adding an anticoagulant to collected blood and centrifuging the blood to remove precipitated blood cells from the blood.
[0034]
As used herein, the term "gas" refers to a gas that may contain microorganisms, including, but not limited to, air containing at least oxygen.
[0035]
As used herein, the term “microorganism” includes, for example, fungi and / or bacteria that cause contamination before and during use of tissues or tissue equivalents, or when removing organs.
[0036]
Fungi include, for example, fungi belonging to the genus Candida, Hansenula, Saccharomyces, Trichosporon, Cryptococcus, Aspergillus, Penicillium, Blastmyces, Coccidioides, Pneumocystis, Malassezia or Debaryomyces. . More specifically, such fungi include, for example, Candida albicans, Candida lusitaniae, Candida krusei, Candida glabrata, Cryptomancos crucosco crococtus cryptococcosa cryptococcosa cryptococcosa cryptococcosa Cryptococcosa cryptococcosa cryptococcosa cryptococcosa crypococtus ), Aspergillus fumigatus or Pneumocystis carinii.
[0037]
Bacteria include, for example, bacteria belonging to the genus Bacillus, Streptococcus, Pseudomonas, Escherichia or Staphylococcus. More specifically, such bacteria include, for example, Bacillus subtilis, Streptococcus pyogenes, Salmonella typhimurium, Salmonella montella salmonella mongola salmonella mongori salmonella mongori salmonella mongori salmonella mongori ngolli monger salmonella mongori salmonella mongori ngolli mongeri bon mori monger salmonella mongori sangoni terri monger salmonella mongori sul monger simonella montmorri sul monella bon mori montero sul monella salmonella mongori sul monger simonella montero montana sul monella sul monella sul monella salmonella Includes Salmonella enteritidis, Escherichia coli or Staphylococcus epidermidis, Staphylococcus aureus and Staphylococcus aureus, including Staphylococcus epidermidis. .
[0038]
In the detection method of the present invention, when the object to be filtered is a liquid, the entire liquid is used as a sample in order to increase the reliability of the detection result. By filtering all liquids of interest using a filtration membrane, microorganisms contained in the liquid are captured. When the object to be filtered is a gas, the filtration membrane is applied to an air sampler, and the gas is sucked by the air sampler to capture microorganisms contained in the gas.
[0039]
In the present specification, the “air sampler” is not particularly limited as long as it is a device capable of capturing microorganisms by colliding microorganisms in the air with a filtration membrane. For example, an air sampler or the like set so that microorganisms collide with the agar medium can be used. When an air sampler on which an agar medium is set is used, a microorganism contained in the gas can be captured by mounting a filtration membrane on the agar medium and aspirating a gas with the air sampler. When the filtration membrane is placed on the agar medium, the filtration membrane becomes wet. When a dry filtration membrane is used, microorganisms may not be collected quantitatively on the filtration membrane due to the influence of static electricity. Therefore, it is preferable to use a wet filtration membrane.
[0040]
In the present invention, the filtration membrane used for filtration preferably has a pore diameter of 0.2 μm to 0.45 μm. When the pore size of the filtration membrane is less than 0.2 μm, cell debris other than the target microorganisms remains on the filtration membrane, and the filtration membrane tends to be clogged. If the pore size of the filtration membrane is larger than 0.45 μm, the target microorganisms tend to pass through the filtration membrane. In order to prevent clogging of the filtration membrane, a filtration membrane having a larger pore diameter (for example, a pore diameter of 8 μm) may be stacked. When a liquid that may cause clogging, such as a liquid containing a large amount of cell debris, is to be filtered, it is preferable to divide the liquid and filter each liquid with another filtration membrane. When one sample is filtered through a plurality of filtration membranes, the plurality of filtration membranes may be combined in the following culturing step, or the method of the present invention may be applied independently after the filtration step.
[0041]
Liquids, such as immersion fluids for tissue or tissue equivalents, may contain antibiotics to prevent microbial growth. In such a case, the antibiotic is adsorbed to the filtration membrane after filtering the immersion liquid, which adversely affects the subsequent culture step. Therefore, in the method for detecting microorganisms of the present invention, it is preferable to rinse a filtration membrane after filtering a liquid such as an immersion liquid of tissue or tissue equivalent. By rinsing the filtration membrane, the antibiotics adsorbed on the filtration membrane can be washed away, and the growth of microorganisms in the subsequent culture step is not inhibited, so that the microorganisms can be detected more reliably. Rinsing such a filter membrane includes, for example, lecithin, polysorbate diluent (LP diluent), glucose / peptone liquid medium (GP liquid medium), soybean casein digest liquid medium (SCD liquid medium), thioglycolic acid medium (TG medium), Sabouraud / glucose medium, glucose / peptone medium (GP medium), phosphate buffer (pH 7.2), phosphate buffered saline (pH 7.2), peptone / saline buffer (pH 7.0) ), And a medium or buffer in which lecithin or polysorbate is added to the medium or the buffer. When the fungus is to be detected, the filtration membrane is rinsed with a GP liquid medium, SCD liquid medium, potato dextrose liquid medium, Sabouraud dextrose liquid medium, or a medium containing lecithin or polysorbate added to these mediums. Is preferred. When bacteria are to be detected, the filtration membrane is rinsed with SCD liquid medium, TG medium, plain heart infusion liquid medium, nutrient liquid medium, or a medium in which lecithin or polysorbate is added to these mediums. Is preferred.
[0042]
In the method for detecting microorganisms of the present invention, microorganisms captured by the filtration membrane are cultured for a certain period of time. By culturing the microorganisms captured by the filtration membrane, several microorganisms contained in the fluid can be detected.
[0043]
The culture medium used in the culture is not particularly limited as long as it is a medium in which fungi and / or bacteria that are problematic at least when a tissue equivalent is used as a biomaterial are grown, and can be appropriately selected by those skilled in the art. For example, when fungi and bacteria are to be detected, a medium such as an SCD medium or a GP medium can be used. When only fungi are to be detected, it is preferable to use a GP liquid medium, an SCD liquid medium, a Sabouraud dextrose liquid medium, a potato dextrose liquid medium, or a Sabouraud dextrose liquid medium. When only bacteria are to be detected, it is preferable to use an SCD liquid medium, a TG liquid medium, a plain heart infusion liquid medium or a nutrient liquid medium.
[0044]
In the microorganism detection method of the present invention, the culture method is preferably shaking culture or stirring culture.
[0045]
In the method for detecting a microorganism of the present invention, the culture time of the microorganism is preferably 5 hours to 24 hours, more preferably 12 hours to 24 hours. When only bacteria are to be detected, the culture time is preferably from 5 hours to 24 hours. When a fungus is to be detected, the culture time is preferably from 8 hours to 24 hours. In cultures of less than 5 hours for bacteria and less than 8 hours for fungi, microorganisms tend not to grow to a sufficient degree for detection. When the cultivation time exceeds 24 hours, the detection sensitivity tends to be unchanged as compared with the cultivation for 24 hours.
[0046]
In the method for detecting a microorganism of the present invention, the culture temperature of the microorganism can be, for example, 20 to 37 ° C. Further, the culture temperature is preferably 30 to 35 ° C for bacteria, and the culture temperature is preferably 20 to 25 ° C for fungi. However, the culture temperature is not limited to these, and the optimal culture temperature for the microorganism to be detected can be appropriately set by those skilled in the art.
[0047]
The cultivation rate of microorganisms differs, and fungi tend to grow at a slower rate than bacteria. Therefore, the detection sensitivity may not be sufficient if only a part of the culture is used. Therefore, after completion of the culture, it is preferable to collect all of the grown microorganisms and use them in the next step. Therefore, the culture is concentrated by centrifugation or the like. The supernatant is removed by centrifugation and the DNA is extracted from the precipitated culture. As a method for extracting DNA, any conventional method such as a DNA extraction method using a surfactant such as Triton X-100 and sodium dodecyl sulfate (SDS) can be applied. The extracted DNA may be subjected to a phenol treatment or a chloroform treatment, and then purified by performing an ethanol precipitation method or an isopropanol precipitation method.
[0048]
For the PCR in the method for detecting a microorganism of the present invention, an ordinary PCR method can be applied, and DNA extracted through the above-described steps is used as a template, and a primer capable of detecting the DNA sequence of the target microorganism is used. As long as it is not particularly limited.
[0049]
PCR is a cycle of denaturation of double-stranded DNA into single-stranded DNA, annealing of single-stranded DNA with primers, and synthesis of complementary DNA by DNA polymerase. The annealing temperature is usually set to be lower than the melting temperature (Tm) of the oligonucleotide used as a primer. The formula for determining Tm is known to those skilled in the art, and for example, Tm can be calculated by the following formula (Cell Engineering Separate Volume “Bio-Experimental Illustrated” (3) Really Increased PCR, 13-43, 1996, Published by Shujunsha Co., Ltd.).
[0050]
Tm (° C.) = 4 × (% GC) + 2 × (% AT) −2n
(% GC): GC content in oligonucleotide (%)
(% AT): AT content in oligonucleotide (%)
n: length of oligonucleotide
[0051]
In the method for detecting a microorganism of the present invention, PCR can be performed separately for fungi and bacteria, for example. Alternatively, two or more sets of primers, for example, a primer for a target fungus and a primer for a target bacterium may be simultaneously used in one PCR reaction solution to carry out PCR.
[0052]
The sequences of the primers used in the PCR are, for example, selected and designed to be conserved in fungi or bacteria and to be capable of amplifying a specific gene or a part thereof. The rRNA gene is preferred as the gene that is conserved and specific in fungi or bacteria. The most suitable sequence as a primer can be appropriately selected by a search based on a generally available database (for example, DDBJ (DNA Data Bank of Japan) or RDP (Ribosomal Database Project)). The following combinations are known as primers for amplifying rRNA.
[0053]
Primers capable of detecting fungi are preferably designed based on the sequence of the 18S rRNA gene or a part thereof. Examples of such a combination of primers include a combination of a primer consisting of 5′-ACTTTCGATGGTAGGATAG-3 ′ (SEQ ID NO: 1) and a primer consisting of 5′-TGATCGTCTCTCGATCCCCCTA-3 ′ (SEQ ID NO: 2), and combinations of SEQ ID NOs: 2 and 5 A combination with '-CTGAGAAACGGCTACCACAT-3' (SEQ ID NO: 3) is known (K. Makimura, et al. (1994), J. Med. Microbiol., 40, 358-364), and the detection method of the present invention. Can be used for These primers can amplify 18S rRNA of 78 strains of 25 species, including fungi causing clinically significant infections such as Candida, Saccharomyces, Aspergillus and Hansenula. When two types of primers each having the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 are used for PCR, the PCR product is about 687 bp. When two types of primers each having the nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3 are used for PCR, the PCR product is about 600 bp.
[0054]
Examples of primers capable of detecting bacteria include primers composed of 5′-GTGCCAGCAGCGCGGGTA-3 ′ (SEQ ID NO: 4) (Tsuguo Sasaki, et al. (1996), PDA Journal of Pharmaceutical Sciences & Technology, 24, and Technol. 5'-ACGTCATCCCCACCTCTCTC-3 '(SEQ ID NO: 5), a primer (Kui Chen, et al. (1989), FEMS Microbiology Letters, 57, 19-24), and a primer consisting of SEQ ID NO: 4 and 5' Combination with a primer consisting of AGGCCCGGGGAACGTATTCAC-3 ′ (SEQ ID NO: 6) (Kui Chen, et al., Supra). It is known and can be used in the detection method of the present invention. These primer sequences are at least 100% consistent between Bacillus subtilis, Streptococcus pyogen, Pseudomonas aeruginosa, Salmonella tyhumurium, Salmonella vongori, Escherichia coli and Streptococcus epidermidis, with about 677 bp or about 876 bp. A 16S rRNA gene fragment can be amplified. In addition, 5'-AGAGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 7) (PA Eden et al., Int. J. Syst. Bacteriol. 41, 324 (1991); G. Weisburg et al., J. Bacteriol., 173, 697 (1991); and DA Relman et al., Mol. Microbiol., 6, 1801 (1992)), 5'-TCAAAKGAATTGACGGCGC'GGGGC-3 '. SEQ ID NO: 8) (K represents dT or dG. DA Relman et al., N. Engl. J. Med., 327, 293 (1992); and DA Relman et al., N. Engl.JM d., 323, 1573 (1990)), 5'-GAGGAAGGTGGGGATGACGT-3 '(SEQ ID NO: 9) (see above, DA Relman et al., N. Engl. J. Med., 323, 1573 (1990); And K. Chen et al., FEMS Microbiol. Lett. 48, 19 (1989)), and 5'-GGACTACCAGGGTATCTAAT-3 '(SEQ ID NO: 10) as a reverse primer for detecting bacteria. A. Relman et al., N. Engl. J. Med., 327, 293 (1992); and K. H. Wilson et al, J. Clin. Microbiol. 28, 1942 (1990)) 5'-GGTTACCTGTTA. CTT-3 ′ (SEQ ID NO: 11) (PA Eden et al., Supra; WG Weisburg et al., Supra; and DA Relman et al., Mol. Microbiol., 6, 1801 (supra). 1992)).
[0055]
The homology between the primer sequence of the bacterium and the human 18S rRNA gene sequence corresponding to the 16S rRNA gene of the bacterium is as low as 60% or less, and the human 18S rRNA gene fragment can be amplified by selecting appropriate PCR conditions. Never. In addition, even when the fungal primer is used, the human 18S rRNA gene fragment will not be amplified if appropriate PCR conditions are selected. PCR conditions can be appropriately set by those skilled in the art.
[0056]
In the method for detecting a microorganism of the present invention, a DNA fragment (PCR product) amplified by PCR can be detected by a method generally practiced in the art. As a method for detecting a PCR product, for example, a known detection method in this field such as electrophoresis using an agarose gel, a hybridization assay using a labeled probe for the PCR product, or a PCR-ELISA method can be applied. By analyzing the base sequence of the DNA fragment (PCR product) amplified by PCR with a DNA sequencer, it is considered that the species can be identified.
[0057]
In the case of a hybridization assay, a labeled probe comprising an oligonucleotide complementary to a sequence of a PCR product or a part thereof and a detectable label is used. As the label, any label commonly used in the art can be used, and for example, enzymes such as alkaline phosphatase, peroxidase or beta-D-galactosidase, fluorescent dyes or radioisotopes can be used. When an enzyme such as alkaline phosphatase is used as a label, the PCR product is hybridized with a labeled probe, the enzyme is reacted with its substrate (a coloring substance, a fluorescent substance or a luminescent substance, etc.), and then the substrate is colored. By detecting fluorescence, luminescence, or the like, a PCR product can be detected.
[0058]
In the case of the PCR-ELISA method, a PCR product can be detected, for example, by incorporating a base labeled with digoxigenin (DIG) during PCR amplification and then performing ELISA using an antibody against DIG.
[0059]
When a PCR product is detected as described above, it means that a tissue, a tissue equivalent, an organ, blood, gas, or the like is contaminated with a microorganism.
[0060]
Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.
[0061]
【Example】
Reagents such as the GP liquid medium, LP diluent and SCD liquid medium described below, and all instruments such as test tubes, microtubes, and tweezers, were previously autoclaved (121 ° C., 20 minutes). As the jumbo conical tube, catalog number FALCON2075 manufactured by Becton Dickinson Japan Co., Ltd. was used. Regarding potassium acetate, in Example 1, catalog number 60035, manufactured by Fluka, and in Examples 2 to 4, catalog number 166-03545, manufactured by Wako Pure Chemical Industries, Ltd. were used. For isopropanol and ethanol, catalog numbers 166-04836, manufactured by Wako Pure Chemical Industries, Ltd., and catalog number 057-00456, manufactured by Wako Pure Chemical Industries, Ltd., were used, respectively. Regarding TE (pH 8.0), in Example 1, catalog number 316-90025 manufactured by Nippon Gene Co., Ltd., and in Examples 2 to 4, catalog number 5779J manufactured by Nippon Gene Co., Ltd. was used. Regarding the filtration membrane, in Example 1, a filtration membrane having a pore diameter of 0.2 μm (manufactured by Sartorius Co., catalog number 13107-47-ACN), and in Examples 2 to 4, a filtration membrane having a pore diameter of 0.2 μm (pole ( Co., Ltd., catalog number 4803), a filtration membrane with a pore size of 0.45 μm (manufactured by Sartorius Co., catalog number 13106-47-ACN) in Example 5, and a filtration membrane with a pore size of 0.45 μm in Example 6 ( Sartorius Co., Ltd., catalog number 13106-47-ACN) and a filtration membrane having a pore size of 0.2 μm (manufactured by Pall Corporation, catalog number 4803), and a filtration membrane having a pore size of 0.22 μm in Example 7 (Millipore Corp.) Catalog number ATSMTTD60). As the filter holder and the manifold, catalog number KGS-47TF manufactured by Advantech Toyo Co., Ltd. and model number KM-3 manufactured by Advantech Toyo Co., Ltd. were used. The disposable loop (gamma sterilized) used was a product of AS ONE, catalog number GI-0457-05. Milliflex (formal name: Twinhead Milliflex Sensor II suction filtration) and adapter (formal name: MFPP adapter improved type) are manufactured by Millipore Co., Ltd., catalog number MXPS20015, and Pall Corporation, catalog number Y-, respectively. 0705-2 was used. As an air sampler, a M Air T Millipore Tester (manufactured by Millipore Co., Ltd., catalog number ATAS PLR01) was used, and as a micropore sampling plate, a catalog number ATAH EAD01 manufactured by Millipore Co., Ltd. was used. The PCR thermal cycler MP used was Takara Shuzo Co., Ltd., model number TP3000, and the Polaroid camera used was Funakoshi Co., Ltd. model number DS-300 (M). Regarding SYBR Green, in Examples 1 and 5, SYBR Green I (× 10000) (manufactured by Takara Shuzo Co., Ltd., code number F0513), and in Examples 2 to 4 and 6, SYBR Green (manufactured by BMA, catalog number 50513) Was used.
[0062]
Example 1
<Preparation of bacterial solution>
(A) To one test tube, 10 ml of a GP liquid medium (28.5 g of glucose, peptone liquid medium (powder) (manufactured by Nippon Pharmaceutical Co., Ltd., catalog No. 397-0225) / 1 l of ultrapure water) was dispensed. Next, one colony of Candida albicans (Accession No. IFO 1594 strain at the Fermentation Research Institute) was inoculated into the GP liquid medium, and the cells were sufficiently dispersed by pipetting. This bacterial solution was cultured at room temperature (about 25) ° C. for 18 hours.
[0063]
(B) 9 ml of the GP liquid medium was dispensed into 10 test tubes. To one of the test tubes into which the GP liquid medium was dispensed, 1 ml of the bacterial solution prepared in the above (A) was added using a micropipette and mixed to prepare a 10-fold diluted solution. The tip was changed, and thereafter, ¹⁰ The dilution was repeated until a double dilution bacterial solution was obtained. Hereinafter, in Example 1, 10 ⁶ -10 ¹⁰ A double dilution bacterial solution was used.
[0064]
(C) 150 ml of a Green medium (Green + 3% FBS) containing 3% of fetal bovine serum (manufactured by JRH Biosciences, Inc.) was placed in each of six jumbo conical tubes having a capacity of 225 ml. 1.5 ml of an antibiotic consisting of streptomycin, penicillin and amphotericin B (manufactured by Lifetech Oriental Co., Ltd., catalog number 15240-096, final concentrations: streptomycin 100 μg / ml, penicillin 100 units / ml, amphotericin B 0.25 μg / ml) Was added to each tube and mixed. Then, the 10 prepared in the above (B) was prepared. ⁶ -10 ¹⁰ 1.0 ml of the 1: 2 dilution was added and mixed. A negative control was prepared by adding 1.0 ml of a GP liquid medium instead of the diluted bacterial solution.
[0065]
<Filtration of bacterial solution>
A filtration membrane having a pore size of 0.2 μm was set in a filter holder, and then the filter holder was set in a manifold. Next, using a 50 ml pipette, 50 ml of the sample prepared in the above (C) was placed in a funnel, and all were subjected to suction filtration. The suction filtration was repeated twice more and all of the samples were suction filtered.
[0066]
<Rinse of filtration membrane>
2 LP diluents (LP diluent "Digo" (manufactured by Nippon Pharmaceutical Co., Ltd., catalog number 397-00281), 2 bottles / ultra pure water, final concentration: polypeptone 1 g / l, lecithin 0.7 g / l, polysorbate 80 20 g / L, pH 7.0-7.4) was added to the funnel, followed by suction filtration to rinse the inner wall of the funnel. The same rinsing was repeated twice more.
[0067]
<Culture of cells captured by filtration membrane>
After suction filtration, the filter membrane was removed with tweezers, the filter membrane was immersed in a 50 ml centrifuge tube containing 30 ml of GP liquid medium, and shaking culture was performed at room temperature (25 ° C.) overnight (15 to 20 hours). did.
[0068]
<Recovery of bacteria>
After overnight culture, the filter membrane was aseptically removed from the GP liquid medium using forceps. The GP liquid medium was centrifuged (2000 g, 10 minutes) and the supernatant was gently removed. The precipitate was suspended in 1.0 ml of PBS (-), and the suspension was transferred to a microtube. The microtube was centrifuged at 4 ° C. (15000 g, 10 minutes) and the supernatant was gently removed.
[0069]
<Preparation of template for PCR>
100 μl of a lysis solution (100 mM Tris · HCl (pH 7.5), 30 mM EDTA · 2Na, 0.5% (weight / volume) SDS) was added to each microtube, and the cells were suspended. The cells were stirred with a vortex mixer for 5 seconds, and then incubated at 100 ° C. for 15 minutes. 100 μl of a 2.5 M potassium acetate solution was added to each microtube, mixed, and then incubated on ice for 60 minutes. Each microtube was centrifuged at 4 ° C. (12000 rpm, 5 minutes), and the supernatant was transferred to a new microtube. The same amount of isopropanol as the supernatant was added to obtain a precipitate of DNA. Each microtube was centrifuged (15000 rpm, 10 minutes) and the supernatant was gently removed. Then, 0.5 ml of 99% ethanol was added, mixed, and then centrifuged at 4 ° C. (15000 rpm, 10 minutes) to remove the supernatant. After adding 0.5 ml of 70% ethanol to the precipitate and mixing, the mixture was further centrifuged at 4 ° C. (15000 rpm, 10 minutes) to remove the supernatant. After the precipitate was lightly dried on a paper towel, 50 μl of TE (pH 8.0) was added to dissolve the DNA.
[0070]
<PCR>
50 μl of the reaction mixture was prepared in a 0.2 ml PCR tube and kept on ice. The composition of the reaction mixture was 5 μl of (10 ×) PCR buffer (supplied with Taq polymerase), 4 μl of dNTP mixture (supplied with Taq polymerase), 0.3 μM each of primers, 2.5 μl of template DNA, 0.25 μl of Taq polymerase (5 U / μl) (Takara Shuzo Co., Ltd., catalog number R001A) and ultrapure water. As the primers, two kinds of primers having the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 were used. As the template DNA, DNA of Candida albicans IFO 1594 strain prepared in <Preparation of template for PCR> in Example 1 was used. The dNTP mixture used in this example is a solution containing 2.5 mM each of dATP, dTTP, dCTP and dGTP.
[0071]
Each PCR tube was set in the PCR thermal cycler MP, and PCR was performed by the following program. In this example, the PCR was incubated at 95 ° C for 5 minutes, followed by 30 cycles of 95 ° C for 45 seconds, 55 ° C for 1 minute and 72 ° C for 2 minutes, followed by 10 minutes at 72 ° C. Incubation was performed. The annealing temperature was set with reference to the melting temperature of the primer calculated by the above equation.
[0072]
<Agarose electrophoresis>
An electrophoresis buffer (1 × TAE) was prepared as follows. 9.6 g of Tris-HCl (pH 8.0), 1.48 g of EDTA and 2.28 ml of acetic acid were dissolved in 1900 ml of ultrapure water. After adjusting the solution to pH 8.1 with 1N NaOH, ultrapure water was further added to 2000 ml.
[0073]
To 50 μl of the PCR product, 10 μl of loading buffer (bromophenol blue 0.25% (weight / volume), glycerol 25% (vol / vol), ultrapure water) was added and mixed. Next, 20 μl of each of these samples was loaded on an agarose gel (Agarose S (manufactured by Nippon Gene Co., Ltd., catalog number 312-01193), 1.2 g / 1 × TAE, 100 ml), and electrophoresis was performed at 100 V for 25 minutes.
[0074]
<Staining of PCR product>
10 μl of SYBR Green I (× 10000) was added to 100 ml of 1 × TAE to prepare a staining solution. The agarose gel after electrophoresis was immersed in the staining solution, and allowed to stand in a dark place for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a Polaroid camera.
[0075]
As a result of staining the PCR product, 10 ⁶ Double dilution bacterial solution and 10 ⁷ DNA of Candida albicans IFO 1594 strain was detected from the double dilution bacterial solution. As described in the following test examples, 10 ⁷ The number of viable bacteria in the double dilution bacterial solution was 1.
[0076]
Test example 1
The number of viable bacteria in the diluted bacterial solution prepared in the preparation (B) of the bacterial solution of Example 1 was measured by the following pour method. That is, the 10 ⁵ -10 ¹⁰ 1 ml of the double-diluted bacterial solution was dispensed into a Petri dish having a diameter of 90 mm (n = 2). In each petri dish, 32.5 g of Sabouraud / Glucose agar medium (Sabouraud / Glucose agar medium (powder) (Nippon Pharmaceutical Co., Ltd., catalog number 390-01011), 32.5 g / 500 ml of ultrapure water) kept at 45 ° C. after autoclaving Was dispensed in 15 ml portions, stirred gently, and allowed to stand until the agar solidified. After the agar solidified, each petri dish was inverted and cultured in a 25 ° C. incubator. Four and five days after the culture, the number of formed colonies was visually counted.
[0077]
As a result of measurement, 10 ⁷ The number of Candida albicans IFO 1594 strains contained in the double dilution bacterial solution was one.
[0078]
Example 2
<Preparation of bacterial solution>
(A) One colony of Candida albicans IFO 1594 strain was collected, smeared on Sabouraud glucose agar medium (prepared in the same manner as in Test Example 1), and cultured at 30 ° C. for 24 hours.
[0079]
(B) 9 ml of the GP liquid medium was dispensed into 10 test tubes. One of the test tubes into which the GP liquid medium was dispensed was inoculated with two loops of the disposable loop of the colony cultured in the above (A), and the bacteria were suspended with a 10 ml pipette to prepare a 10-fold diluted solution. 1 ml of the obtained bacterial liquid was added to one of the test tubes into which the GP liquid medium was dispensed using a micropipette, and mixed. ² A two-fold dilution was prepared. The tip was changed, and thereafter, ⁸ The dilution was repeated until a double dilution bacterial solution was obtained. Hereinafter, in Example 2, 10 ⁵ -10 ⁸ A double dilution bacterial solution was used.
[0080]
(C) 180 ml of GP medium was placed in one jumbo conical tube having a capacity of 225 ml, and 50 mg / ml gentamicin (manufactured by Invitrogen Corp., catalog number 15750-060) and 250 μg / ml amphotericin B (Invitrogen ( (Catalog No. 15290-018), respectively, was added to 900 μl and mixed.
[0081]
<Filtration of bacterial solution>
The adapter was set on the Milliflex, and then a funnel equipped with a filtration membrane having a pore size of 0.2 μm was set on the adapter. Next, using a 25 ml pipette, 30 ml of the GP liquid medium prepared in the above (C) and 10 ml of the GP liquid medium prepared in the above (B) were used. ⁵ 1.0 ml of a 1: 2 dilution was added to each funnel, mixed, and suction filtered. 10 ⁶ -10 ⁸ The same operation was performed for the double dilution bacterial solution. A negative control was prepared by adding 1.0 ml of a GP liquid medium instead of the diluted bacterial solution.
[0082]
<Rinse of filtration membrane>
50 ml of an LP diluent (produced in the same manner as in Example 1) was added to the funnel, followed by suction filtration to rinse the inner wall of the funnel. The same rinsing was repeated twice more.
[0083]
<Culture of cells captured by filtration membrane>
After suction filtration, the filter membrane was removed with tweezers, the filter membrane was immersed in a 50 ml centrifuge tube containing 25 ml of GP liquid medium, and shake culture was performed at room temperature (25 ° C.) for 20 hours.
[0084]
<Recovery of bacteria>
After the culture, the filter membrane was aseptically removed from the GP liquid medium using tweezers. The GP liquid medium was centrifuged (1310 g, 10 minutes) and the supernatant was gently removed. The precipitate was suspended in 1.0 ml of PBS (-), and the suspension was transferred to a microtube. The microtube was centrifuged at 4 ° C. (16100 g, 10 minutes) and the supernatant was gently removed.
[0085]
<Preparation of template for PCR>
The template for PCR was prepared by using the cells recovered in <Recovery of cells> in Example 2 instead of the cells recovered in <Recovery of cells> in Example 1. The procedure was performed in the same manner as in <Preparation of template for PCR> in Example 1.
[0086]
<PCR>
PCR was performed in the same manner as in Example 1 except that the template DNA prepared in Example 2 <Preparation of PCR template> was used instead of the template DNA prepared in Example 2 <Preparation of template for PCR>. Performed under the same conditions as in <PCR> of Example 1.
[0087]
<Agarose electrophoresis>
Agarose electrophoresis was performed in the same manner as in <Agarose electrophoresis> of Example 1, except that the PCR product amplified in <PCR> of Example 2 was used instead of the PCR product amplified in <PCR> of Example 1. The same was done.
[0088]
<Staining of PCR product>
10 μl of SYBR Green was added to 100 ml of 1 × TAE to prepare a staining solution. The agarose gel after electrophoresis was immersed in the staining solution, and allowed to stand in a dark place for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a Polaroid camera.
[0089]
As a result of staining the PCR product, 10 ⁵ Times 10 ⁶ Double and 10 ⁷ DNA of Candida albicans IFO 1594 strain was detected from the double dilution bacterial solution.
[0090]
Test example 2
The number of viable bacteria in the diluted bacterial solution prepared in the preparation of the bacterial solution (B) of Example 2 was measured by the following pour method. That is, the 10 ⁵ -10 ⁸ 1 ml of the 1: 2 diluted bacterial solution was dispensed into a Petri dish having a diameter of 90 mm (10 ⁵ Times 10 ⁶ Double and 10 ⁸ N = 2, 10 for double dilution bacterial solution ⁷ N = 4 for a double dilution bacterial solution. To each petri dish, 14 ml of a Sabouraud glucose agar medium (prepared in the same manner as in Example 1) maintained at 50 ° C. after autoclaving was dispensed, stirred gently, and allowed to stand until the agar solidified. After the agar solidified, each petri dish was inverted and cultured in a 25 ° C. incubator. Four and five days after the culture, the number of formed colonies was visually counted.
[0091]
As a result of measurement, 10 ⁵ Times 10 ⁶ Double and 10 ⁷ The viable cell counts contained in the double dilution bacterial solution were 111, 9.5, and 2.5, respectively.
[0092]
Example 3
<Preparation of bacterial solution>
(A) One colony of Saccharomyces cerevisiae (accession number ATCC 9763 strain in the American Type Culture Collection) was collected and subjected to the same procedure as in Sabouraud glucose agar medium (prepared in Test Example 1). And cultured at 32 ° C. for 24 hours.
[0093]
(B) 9 ml of the GP liquid medium was dispensed into 10 test tubes. One of the test tubes into which the GP liquid medium was dispensed was inoculated with two loops of the disposable loop of the colony cultured in the above (A), and the bacteria were suspended with a 10 ml pipette to prepare a 10-fold diluted solution. 1 ml of the obtained bacterial liquid was added to one of the test tubes into which the GP liquid medium was dispensed using a micropipette, and mixed. ² A two-fold dilution was prepared. The tip was changed, and thereafter, ⁸ The dilution was repeated until a double dilution bacterial solution was obtained. Hereinafter, in Example 3, 10 ⁴ -10 ⁸ A double dilution bacterial solution was used.
[0094]
(C) 180 ml of GP medium was placed in one jumbo conical tube having a capacity of 225 ml, and 50 mg / ml gentamicin (manufactured by Invitrogen Corp., catalog number 15750-060) and 250 μg / ml amphotericin B (Invitrogen ( (Catalog No. 15290-018), respectively, was added to 900 μl and mixed.
[0095]
<Filtration of bacterial solution>
The adapter was set on the Milliflex, and then a funnel equipped with a filtration membrane having a pore size of 0.2 μm was set on the adapter. Next, using a 25 ml pipette, 30 ml of the GP liquid medium prepared in the above (C) and 10 ml of the GP liquid medium prepared in the above (B) were used. ⁴ 1.0 ml of the double-diluted bacterial solution was placed in each funnel and mixed, followed by suction filtration. 10 ⁵ -10 ⁸ The same operation was performed for the double dilution bacterial solution. A negative control was prepared by adding 1.0 ml of a GP liquid medium instead of the diluted bacterial solution.
[0096]
<Rinse of filtration membrane>
About 100 ml of polysorbate-added washing liquid "Digo" (manufactured by Nippon Pharmaceutical Co., Ltd., catalog number 395-01561) was added to the funnel by decantation, followed by suction filtration to rinse the inner wall of the funnel. The same operation was further repeated twice, and the filtration membrane was rinsed with a total of 300 ml of the washing solution.
[0097]
<Culture of cells captured by filtration membrane>
After suction filtration, the filter membrane was removed with tweezers, the filter membrane was immersed in a 50 ml centrifuge tube containing 30 ml of GP liquid medium, and shake culture was performed at room temperature (25 ° C.) for 20 hours.
[0098]
<Recovery of bacteria>
After the culture, the filter membrane was aseptically removed from the GP liquid medium using tweezers. The GP liquid medium was centrifuged (2000 g, 10 minutes) and the supernatant was gently removed. The precipitate was suspended in 1.0 ml of PBS (-), and the suspension was transferred to a microtube. The microtube was centrifuged at 4 ° C. (15000 g, 10 minutes) and the supernatant was gently removed.
[0099]
<Preparation of template for PCR>
The template for PCR was prepared by using the cells recovered in <Recovery of cells> in Example 3 instead of the cells recovered in <Recovery of cells> in Example 1. The procedure was performed in the same manner as in <Preparation of template for PCR> in Example 1.
[0100]
<PCR>
The PCR was performed except that the template DNA prepared in <Preparation of PCR template> in Example 3 was used instead of the template DNA prepared in <Preparation of template for PCR> in Example 1. Performed under the same conditions as in <PCR> of Example 1.
[0101]
<Agarose electrophoresis>
The agarose electrophoresis was performed in the same manner as in Example 1 except that the PCR product amplified in <PCR> of Example 3 was used instead of the PCR product amplified in <PCR> of Example 1. The same was done.
[0102]
<Staining of PCR product>
10 μl of SYBR Green was added to 100 ml of 1 × TAE to prepare a staining solution. The agarose gel after electrophoresis was immersed in the staining solution, and allowed to stand in a dark place for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a Polaroid camera.
[0103]
As a result of staining the PCR product, 10 ⁴ Times 10 ⁵ Times 10 ⁶ Double and 10 ⁷ DNA of Saccharomyces cerevisiae ATCC 9763 strain was detected from the double dilution bacterial solution. 10 ⁸ No DNA of Saccharomyces cerevisiae ATCC 9763 strain was detected from the double dilution bacterial solution.
[0104]
Test example 3
The number of viable bacteria in the diluted bacterial solution prepared in Preparation of bacterial solution (B) of Example 3 was measured by the following pour method. That is, the 10 ⁴ -10 ⁸ 1 ml of the double-diluted bacterial solution was dispensed into a Petri dish having a diameter of 90 mm (n = 3). To each petri dish, 14 ml of a Sabouraud glucose agar medium (prepared in the same manner as in Example 1) maintained at 50 ° C. after autoclaving was dispensed, stirred gently, and allowed to stand until the agar solidified. After the agar solidified, each dish was inverted and cultured in an incubator at 32.5 ° C. Four days after the cultivation, the number of formed colonies was counted visually.
[0105]
As a result of measurement, 10 ⁴ Times 10 ⁵ Times 10 ⁶ Double and 10 ⁷ The viable cell count contained in the double dilution bacterial solution was 100 or more, 42.3, 2.3, and 0.3, respectively. 10 ⁸ No colonies were detected for the double dilution bacterial solution.
[0106]
Example 4
<Preparation of bacterial solution>
(A) 10 ml of a GP liquid medium (prepared in the same manner as in Example 1) was dispensed into one test tube. Next, one loop of disposable loop was inoculated with spores and fungi of Aspergillus fumigatus (accession number: IFO 33022 strain at the Fermentation Research Institute) into the GP liquid medium, and suspended in the GP liquid medium. This bacterial solution was cultured at 25 ° C. for 20 hours.
[0107]
(B) 9 ml of the GP liquid medium was dispensed into five test tubes. To one of the test tubes into which the GP liquid medium was dispensed, 1 ml of the bacterial solution prepared in the above (A) was added using a 5 ml pipette and mixed to prepare a 10-fold diluted solution. Replace the pipette and repeat ³ The dilution was repeated until a double dilution bacterial solution was obtained. Hereinafter, in Example 4, the bacterial liquid (stock solution) prepared in the above (A) to 10 ³ A double dilution bacterial solution was used.
[0108]
<Filtration of bacterial solution>
The adapter was set on the Milliflex, and then a funnel equipped with a filtration membrane having a pore size of 0.2 μm was set on the adapter. Next, using a 25 ml pipette, 30 ml of the GP liquid medium and 1.0 ml of the bacterial solution (stock solution) prepared in (B) were each mixed in a funnel, and all were suction-filtered. 10 ¹ -10 ³ The same operation was performed for the double dilution bacterial solution. A negative control was prepared by adding 1.0 ml of a GP liquid medium instead of the diluted bacterial solution.
[0109]
<Rinse of filtration membrane>
In Example 4, the inner wall of the funnel was not rinsed because no antibiotic was used.
[0110]
<Culture of cells captured by filtration membrane>
After suction filtration, the filter membrane was taken out with tweezers, the filter membrane was immersed in a 50 ml centrifuge tube containing 30 ml of GP liquid medium, and shake culture was performed at 27 ° C. for 20 hours.
[0111]
<Recovery of bacteria>
After the culture, the filter membrane was aseptically removed from the GP liquid medium using tweezers. The GP liquid medium was centrifuged (1310 g, 10 minutes) and the supernatant was gently removed. The precipitate was suspended in 1.0 ml of PBS (-), and the suspension was transferred to a microtube. The microtube was centrifuged at 4 ° C. (16100 g, 10 minutes) and the supernatant was gently removed.
[0112]
<Preparation of template for PCR>
The template for PCR was prepared by using the cells recovered in <Recovery of cells> in Example 4 instead of the cells recovered in <Recovery of cells> in Example 1. The procedure was performed in the same manner as in <Preparation of template for PCR> in Example 1.
[0113]
<PCR>
The PCR was carried out except that the template DNA prepared in <Preparation of PCR template> in Example 4 was used instead of the template DNA prepared in <Preparation of template for PCR> in Example 1. Performed under the same conditions as in <PCR> of Example 1.
[0114]
<Agarose electrophoresis>
The agarose electrophoresis of Example 1 was different from the PCR product of Example 1 except that the PCR product amplified in <PCR> was used instead of the PCR product amplified in <PCR> of Example 1. Was performed in the same manner as described above.
[0115]
<Staining of PCR product>
10 μl of SYBR Green was added to 100 ml of 1 × TAE to prepare a staining solution. The agarose gel after electrophoresis was immersed in the staining solution, and allowed to stand in a dark place for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a Polaroid camera.
[0116]
As a result of staining of the PCR product, ² Double and 10 ³ DNA of Aspergillus fumigatus IFO 33022 strain was detected from the double dilution bacterial solution.
[0117]
Test example 4
Aspergillus fumigatus IFO 33022 strain is a mold having hyphae and forming spores. Therefore, the detection sensitivity of a diluted bacterial solution of cells cannot be indicated by the number of colonies (cfu). Therefore, the detection sensitivity of the diluted bacterial solution of Aspergillus fumigatus IFO 33022 was indicated not by the number of colonies (cfu) but by the amount of DNA. That is, 1 time, 10 times, 10 times ² Double and 10 ³ A PCR template (DNA) of the double-diluted bacterial solution was extracted (as in Example 1), and 8 μl of the template was placed in a microtube, mixed with 72 μl of TE, and diluted 10-fold. For TE, the absorbance at 260 nm of this template was measured using a UV-visible spectrophotometer (manufactured by Beckman Coulter, model number DU640). The formula for calculating the amount of DNA is known to those skilled in the art. For example, the amount of DNA can be calculated by the following formula (Cell Engineering Separate Volume “Bio-Experiment Illustrated” (1) Basics of Molecular Biology, pp. 61-62) 1995, published by Shujunsha Co., Ltd.).
[0118]
Double-stranded DNA (μg / μl) = (260 nm absorbance) × 0.05 × dilution factor
[0119]
As a result of measuring the absorbance at 260 nm, 1 ×, 10 ×, 10 × ² Double and 10 ³ The DNA amounts of Aspergillus fumigatus IFO 33022 contained in the double dilution bacterial solution were 0.9 μg, 0.67 μg, 68.3 ng and 4.25 ng, respectively. From this, it became clear that detection was possible by the same method as long as the DNA amount of Aspergillus fumigatus IFO 33022 strain was 4.25 ng.
[0120]
Example 5
<Preparation of bacterial solution>
(D) 10 ml of SCD liquid medium (8.5 g of soybean casein digest powder medium (manufactured by Nippon Pharmaceutical Co., Ltd., catalog number 396-0091) / 500 ml of ultrapure water) was dispensed into one test tube. Next, one colony of Bacillus subtilis (Accession No. IFO 3134 at the Fermentation Research Institute) was inoculated into the SCD liquid medium, and the cells were sufficiently dispersed by pipetting.
[0121]
(E) 9 ml of the SCD liquid medium was dispensed into 10 test tubes. To one of the test tubes into which the SCD liquid medium was dispensed, 1 ml of the bacterial solution prepared in the above (D) was added using a micropipette and mixed to prepare a 10-fold diluted solution. The tip was changed, and thereafter, ⁸ The dilution was repeated until a double dilution bacterial solution was obtained. Hereinafter, in Example 5, 10 ⁵ -10 ⁸ A double dilution bacterial solution was used.
[0122]
(F) 150 ml of Green + 3% FBS was placed in each of eight jumbo conical tubes having a capacity of 225 ml, and gentamicin (catalog number 15750-060, manufactured by Invitrogen Corp.) and amphotericin B (trade name “Fangisone”, Invitrogen ( Co., Ltd., Catalog No. 15290-018) was added so as to have a concentration of 50 μg / ml and 0.25 μg / ml, respectively. Then, the 10 prepared in the above (E) was prepared. ⁵ -10 ⁸ 1.0 ml of the 1: 2 dilution was added and mixed. A negative control was prepared by adding 1.0 ml of SCD liquid medium instead of the diluted bacterial solution.
[0123]
<Filtration of bacterial solution>
A filtration membrane having a pore size of 0.45 μm was set in a filter holder, and then the filter holder was set in a manifold. Next, using a 50 ml pipette, 50 ml of the sample prepared in the above (F) was placed in a funnel, and all were subjected to suction filtration. The suction filtration was repeated twice more and all of the samples were suction filtered.
[0124]
<Rinse of filtration membrane>
50 ml of an LP diluent (produced in the same manner as in Example 1) was added to the funnel, followed by suction filtration to rinse the inner wall of the funnel. The same rinsing was repeated twice more.
[0125]
<Culture of cells captured by filtration membrane>
After suction filtration, the filter membrane was taken out with tweezers, the filter membrane was immersed in a 50 ml centrifuge tube containing 30 ml of SCD liquid medium, and shaking culture was performed overnight at 35 ° C (12 to 18 hours).
[0126]
<Recovery of bacteria>
After overnight culture, the filter membrane was aseptically removed from the SCD liquid medium using forceps. The SCD liquid medium was centrifuged (2000 g, 10 minutes) and the supernatant was gently removed. The precipitate was suspended in 1.0 ml of PBS (-), and the suspension was transferred to a microtube. The microtube was centrifuged at 4 ° C. (15000 g, 10 minutes) and the supernatant was gently removed.
[0127]
<Preparation of template for PCR>
0.5 ml of the suspension in each microtube was transferred to a 1.5 ml Eppendorf tube and centrifuged at 4 ° C. (15000 g, 10 minutes). After centrifugation, the supernatant was removed, and the precipitate was suspended in 50 μl of TE (10 mM Tris · HCl (pH 8.0), 1 mM EDTA, 1.0% (weight / volume) Triton X-100) containing Triton X-100. did. Then, each sample was treated at 100 ° C. for 20 minutes and left on ice.
[0128]
<PCR>
50 μl of the reaction mixture was prepared in a 0.2 ml PCR tube and kept on ice. As the primers, two kinds of primers having the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5 were used. As the template DNA, Bacillus subtilis IFO 3134 DNA prepared in <Preparation of template for PCR> of Example 5 was used. The composition of the reaction mixture was the same as that of Example 1 except that 0.5 μM and 2.0 μl of the template DNA were used for each primer.
[0129]
Each PCR tube was set in the PCR thermal cycler MP, and PCR was performed by the following program. In this example, the PCR was incubated at 94 ° C. for 2 minutes, followed by 30 cycles of 94 ° C. for 1 minute, 61 ° C. for 1 minute and 72 ° C. for 1 minute, and further at 72 ° C. for 1 minute. Incubation was performed. The annealing temperature was set with reference to the melting temperature of the primer calculated by the above equation.
[0130]
<Agarose electrophoresis>
10 μl of loading buffer was added to 50 μl of the PCR product and mixed. Then, 15 μl of these samples were loaded on an agarose gel (agarose S 2.9 g / 1 × TAE 100 ml) and electrophoresed at 100 V for 30 minutes.
[0131]
<Staining of PCR product>
10 μl of SYBR Green I (× 10000) was added to 100 ml of 1 × TAE and mixed to prepare a staining solution. The agarose gel after electrophoresis was immersed in the staining solution, and allowed to stand in a dark place for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a Polaroid camera.
[0132]
As a result of staining the PCR product, 10 ⁵ -10 ⁸ Bacillus subtilis IFO 3134 DNA was detected in all of the double dilution bacterial solutions. As described in Test Example 5 below, 10 ⁶ Times 10 ⁷ Double, and 10 ⁸ The number of Bacillus subtilis contained in the double dilution bacterial solution was 30, 4, and 0.5, respectively.
[0133]
Test example 5
The number of viable bacteria in the diluted bacterial solution prepared in Preparation of bacterial solution (E) of Example 5 was measured by the following pour method. That is, the 10 ⁵ -10 ⁸ 1 ml of the double-diluted bacterial solution was dispensed into a Petri dish having a diameter of 90 mm (n = 2). To each petri dish, 15 ml of SCD agar medium (8.0 g of SCD agar medium (manufactured by Nippon Pharma Co., Ltd., catalog number 396-00991) / 200 ml of ultrapure water) kept at 45 ° C. after autoclaving was dispensed. Gently stirred and allowed to stand until the agar solidified. After the agar solidified, each dish was inverted and cultured in an incubator at 35 ° C. Four and five days after the culture, the number of formed colonies was visually counted.
[0134]
As a result of measurement, 10 ⁵ Times 10 ⁶ Times 10 ⁷ Double, and 10 ⁸ The number of Bacillus subtilis IFO 3134 contained in the double dilution bacterial solution was 200, 30, 4, and 0.5, respectively.
[0135]
Example 6
<Preparation of bacterial solution>
(D) One colony of Pseudomonas aeruginosa (Accession No. IFO 13275 at the Fermentation Research Institute) was collected, smeared on an SCD agar medium (prepared in the same manner as in Test Example 5), and then incubated at 32.5 ° C. for 24 hours. Cultured.
[0136]
(E) 9 ml of SCD liquid medium (prepared in the same manner as in Example 5) was dispensed into 11 test tubes. One of the test tubes into which the SCD liquid medium was dispensed was inoculated with 2 loops of the disposable loop of the colony cultured in the above (D), and the bacteria were suspended with a 10 ml pipette. 1 ml of the obtained bacterial solution was added to one of the test tubes into which the SCD liquid medium was dispensed using a micropipette and mixed to prepare a 10-fold diluted solution. The tip was changed, and thereafter, ¹⁰ The dilution was repeated until a double dilution bacterial solution was obtained. Hereinafter, in Example 6, 10 ⁶ -10 ¹⁰ A double dilution bacterial solution was used.
[0137]
(F) 150 ml of SCD liquid medium was placed in each of six jumbo conical tubes having a capacity of 225 ml, and 50 mg / ml of gentamicin (manufactured by Invitrogen Corporation, catalog number 15750-060) and 250 μg / ml of amphotericin B ( 150 μl each of Invitrogen Corporation, catalog No. 15290-018) was added and mixed. Then, the 10 prepared in the above (E) was prepared. ⁶ -10 ¹⁰ 1.0 ml of the 1: 2 dilution was added and mixed. A negative control was prepared by adding 1.0 ml of SCD liquid medium instead of the diluted bacterial solution.
[0138]
<Filtration of bacterial solution>
A filtration membrane having a pore size of 0.45 μm was set in a filter holder, and then the filter holder was set in a manifold. Next, using a 50 ml pipette, 50 ml of the sample prepared in the above (F) was placed in a funnel, and all were subjected to suction filtration. The suction filtration was repeated twice more and all of the samples were suction filtered.
[0139]
<Rinse of filtration membrane>
50 ml of an LP diluent (produced in the same manner as in Example 1) was added to the funnel, followed by suction filtration to rinse the inner wall of the funnel. The same rinsing was repeated twice more.
[0140]
<Culture of cells captured by filtration membrane>
After suction filtration, the filter membrane was removed with tweezers, the filter membrane was immersed in a 50 ml centrifuge tube containing 30 ml of SCD liquid medium, and shake culture was performed at 35 ° C. for 19 hours.
[0141]
<Recovery of bacteria>
After the culture, the filter membrane was aseptically removed from the SCD liquid medium using forceps. The SCD liquid medium was centrifuged (1310 g, 10 minutes) and the supernatant was gently removed. The precipitate was suspended in 1.0 ml of PBS (-), and the suspension was transferred to a microtube. The microtube was centrifuged at 4 ° C. (16100 g, 10 minutes) and the supernatant was gently removed.
[0142]
<Preparation of template for PCR>
The template for PCR was prepared by using the cells recovered in <Recovery of cells> in Example 6 instead of the cells recovered in <Recovery of cells> in Example 5. The procedure was the same as in <Preparation of PCR template> in Example 5.
[0143]
<PCR>
The PCR was carried out except that the template DNA prepared in Example 6, <Preparation of PCR template> was used instead of the template DNA prepared in Example 5, <Preparation of template for PCR>. Performed in the same manner as in <PCR> of Example 5.
[0144]
<Agarose electrophoresis>
Agarose electrophoresis was performed using the agarose electrophoresis of Example 5 except that the PCR product amplified in <PCR> of Example 6 was used instead of the PCR product amplified in <PCR> of Example 5. Was performed in the same manner as described above.
[0145]
<Staining of PCR product>
10 μl of SYBR Green was added to 100 ml of 1 × TAE and mixed to prepare a staining solution. The agarose gel after electrophoresis was immersed in the staining solution, and allowed to stand in a dark place for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a Polaroid camera.
[0146]
As a result of staining the PCR product, 10 ⁶ Times 10 ⁷ Double and 10 ⁸ DNA of Pseudomonas aeruginosa IFO 13275 strain was detected from the double dilution bacterial solution. 10 ⁹ Double and 10 ¹⁰ No DNA of Pseudomonas aeruginosa IFO 13275 strain was detected in the doubling dilution.
[0147]
Test Example 6
The number of viable bacteria in the diluted bacterial solution prepared in Preparation of bacterial solution (E) of Example 6 was measured by the following pour method. That is, the 10 ⁶ -10 ¹⁰ 1 ml of the double-diluted bacterial solution was dispensed into a Petri dish having a diameter of 90 mm (n = 3). 15 ml of an SCD agar medium (prepared in the same manner as in Test Example 5) maintained at 50 ° C. after autoclaving was dispensed into each petri dish, gently stirred, and allowed to stand until the agar solidified. After the agar solidified, each dish was inverted and cultured in an incubator at 35 ° C. After 3 days and 5 days from the culture, the number of formed colonies was visually counted.
[0148]
As a result of measurement, 10 ⁶ Times 10 ⁷ Double and 10 ⁸ The number of viable bacteria contained in the double dilution bacterial solution was 100 or more, 34 and 2.7, respectively. 10 ⁹ Double and 10 ¹⁰ No colonies were detected for the double dilution bacterial solution. In addition, although the same experiment was repeated 4 times, in any case, detection was possible even when the number of viable bacteria was 1 to several.
[0149]
Furthermore, when a similar experiment was performed using a filtration membrane having a pore size of 0.2 μm instead of a filtration membrane having a pore size of 0.45 μm, DNA of Pseudomonas aeruginosa IFO 13275 strain was detected.
[0150]
From the results of Examples 1 to 3, 5 and 6 and Test Examples 1 to 3, 5 and 6, it was found that the presence of cells could be detected even when the number of viable cells before filtration was 1 to several. Also, from the results of Examples 1-3, 5 and 6, even if the medium contains an antibiotic, if there is one to several cells before filtration, the presence of the cells can be detected, Also, from the results of Example 4, it was found that even when no antibiotic was contained, the presence of the cells could be detected if several cells were present. This means that the presence of small numbers of cells in tissue or tissue equivalent immersion fluids, organ immersion fluids, or blood can be detected regardless of whether antibiotics are present. means.
[0151]
Regarding the pore size of the filtration membrane, the results of Example 6 showed that the presence of bacteria could be detected using either of the filtration membranes having a pore size of 0.2 μm or 0.45 μm. In addition, since fungi are generally larger in size than bacteria, the results of Example 6 mean that the presence of fungi can be detected even when a filtration membrane having a pore size of 0.45 μm is used.
[0152]
Example 7
<Gas filtration>
The M Air T Millipore Tester was filled with the M Air T cassette TSA medium-filled (hereinafter, the TSA medium is referred to as SCD agar medium), then the lid of the SCD agar medium was opened, and sterilized on the SCD agar medium using tweezers. A filtration membrane having a pore size of 0.22 μm was mounted. Then, the micropore sampling plate was set on the M Air T Millipore Tester, and the lid was closed. Next, 200 liters of air in a non-clean area (Menicon BIO Center, 2nd floor machine room) suspected of containing microorganisms was aspirated using a M Air T Millipore Tester. The filtration membrane used was cut into a size of 70 mm in diameter and sterilized by high-pressure steam (121 ° C., 20 minutes).
[0153]
<Culture of cells captured by filtration membrane>
After suction filtration, the microporous sampling plate was removed, the filter membrane was removed with tweezers, and γ-rays containing 50 ml of SCD liquid medium (8.5 g of catalog No. 396-00991, manufactured by Nippon Pharmaceutical Co., Ltd.) / 500 ml of distilled water. The filtration membrane was immersed in a sterilized container (manufactured by Assist Co., Ltd., catalog number 75.9922.414S), followed by shaking culture at 30 ° C. for 22 hours. A container containing 50 ml of the SCD liquid medium was used as a negative control, and shaking culture was performed at 30 ° C. for 22 hours.
[0154]
<Recovery of bacteria>
After culturing, each culture was transferred to a 50 ml centrifuge tube, and then centrifuged (3000 g, 15 minutes), and the supernatant was gently removed. The precipitate was suspended in 1.0 ml of PBS (-), and the suspension was transferred to a microtube. The microtube was centrifuged at 4 ° C. (15000 g, 10 minutes) and the supernatant was gently removed.
[0155]
<Preparation of template for PCR>
100 μl of TE containing Triton X-100 (produced in the same manner as in Example 5) was added to each microtube to suspend the cells. Then, each sample was treated at 100 ° C. for 20 minutes and left on ice.
[0156]
<PCR>
50 μl of the reaction mixture was prepared in a 0.2 ml PCR tube and kept on ice. As the primers, two kinds of primers having the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5 were used. As the template DNA, the DNA prepared in Example 7 <Preparation of template for PCR> was used. The composition of the reaction mixture was as follows: 0.5 μM of the primer having the sequence shown in SEQ ID NO: 4, 1 μM of the primer having the sequence shown in SEQ ID NO: 5, 0.5 μl of Taq polymerase (5 U / μl) and 2.0 μl of the template The composition was the same as that of the reaction mixture of Example 1 except that DNA was used.
[0157]
Each PCR tube was set in the PCR thermal cycler MP, and PCR was performed by the same program as <PCR> in Example 5.
[0158]
<Agarose electrophoresis>
The agarose electrophoresis of Example 5 <Agarose electrophoresis> except that the PCR product amplified in <PCR> of Example 7 was used instead of the PCR product amplified in <PCR> of Example 5. Was performed in the same manner as described above.
[0159]
<Staining of PCR product>
10 μl of SYBR Green was added to 100 ml of 1 × TAE and mixed to prepare a staining solution. The agarose gel after electrophoresis was immersed in the staining solution, and allowed to stand in a dark place for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a Polaroid camera.
[0160]
As a result of staining of the PCR product, a band was detected at around 680 bp from the medium in which the membrane was cultured. No band was detected from the negative control. Since a band was detected at around 680 bp, it was inferred that the bacterium was detected by two types of primers having the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5.
[0161]
Comparative Example 1
The M Air T Millipore Tester is set with the M Air T cassette TSA medium-filled (hereinafter, the TSA medium is referred to as SCD agar medium), then, after opening the lid of the SCD agar medium, setting the micropore sampling plate, and setting the lid. Did. Next, as in Example 7, 200 liters of air in a non-cleaning area (Menicon BIO Center, 2nd floor machine room) that is thought to contain microorganisms was sucked using a M Air T Millipore Tester. The suction of air was performed almost simultaneously with Example 7. The micropore sampling plate was removed, the agar medium was covered, and the cells were cultured in a 27 ° C. incubator. One day, two days, three days, four days, and seven days after the culturing, the number of formed colonies was visually counted.
[0162]
As a result of the measurement, the number of colonies detected on day 1, 2, 3, 4, and 7 was 0, 4, 6, 8, and 8, respectively. Therefore, it can be seen that the method of Comparative Example 1 requires at least two days or more from the capture of bacterial cells to detection of microorganisms.
[0163]
On the other hand, by using the method of Example 7, when the cells are collected in the afternoon, the test results can be obtained in the afternoon of the next day (about 30 hours). When the method of Example 7 is used, the cells are trapped on the filtration membrane. Therefore, even when the amount of gas to be filtered is large, the agar medium installed in the air sampler is drawn every time a certain amount of gas is sucked. By exchanging the filtration membrane without exchanging the microorganism, it is possible to detect the microorganism with only one agar medium. Furthermore, by analyzing the base sequence of the DNA fragment (PCR product) amplified by PCR with a DNA sequencer, it is possible to identify the bacterial species.
[0164]
【The invention's effect】
By using the microorganism detection method of the present invention, the presence or absence of microorganisms in a fluid can be confirmed, for example, the presence or absence of microorganisms in a tissue or tissue equivalent immersion liquid before transplantation of cultured skin or the like. You can check. In the conventional method, a sample of about 1 ml was used, and the detection sensitivity was 100 cells / ml or more. However, by using the method of the present invention, a sample of about 150 ml can be processed. It is possible to detect one microorganism contained in the sample. In addition, detection of bacteria and fungi so far is performed by culturing a filtration membrane obtained by filtering a sample for 7 to 14 days, and visually confirming that the culture solution is not cloudy, and using a part of the immersion liquid as a sample. It was a PCR method. The former has a problem that a long period of 7-14 days is required. In the latter, the period is as short as 1 to 2 days, but only about 1 to 2 ml of sample can be used. Therefore, when applied to an immersion liquid of tissue or tissue equivalent reaching up to several hundred milliliters, it is obtained. Since only a small portion of the culture supernatant is used, there is a problem that not only the detection sensitivity of the microorganism is insufficient, but also the reliability of the test results is reduced. However, according to the method for detecting microorganisms of the present invention, when a liquid such as an immersion liquid of a tissue or a tissue equivalent is collected in the afternoon, a test result can be obtained in the afternoon of the next day (about 24 hours). Very good.
[0165]
Furthermore, the method for detecting microorganisms of the present invention can also be applied to gases. In the conventional method, when the amount of gas to be filtered is large, it is necessary to change the agar medium installed in the air sampler every time a certain amount of gas is sucked. There is a problem that the used agar medium must be processed. However, the amount of waste can be reduced by using the microorganism detection method of the present invention.
[0166]
When the method for detecting a microorganism of the present invention is used, not only a detection result of the microorganism can be obtained but also whether the detected microorganism is a fungus or a bacterium can be identified depending on the type of the primer used. In addition, by analyzing the base sequence of the DNA fragment (PCR product) amplified by PCR with a DNA sequencer, it is also possible to identify the bacterial species in a short time after collecting the fluid. Furthermore, if an oligonucleotide that hybridizes to a sequence specific to the strain is used as a primer, the strain can be identified at the same time.
[0167]
[Sequence List Free Text]
SEQ ID NO: 1 forward primer for detection of fungal 18S ribosomal RNA gene
SEQ ID NO: 2: Reverse primer for detection of fungal 18S ribosomal RNA gene
SEQ ID NO: 3: forward primer for detection of fungal 18S ribosomal RNA gene
SEQ ID NO: 4: forward primer for detection of bacterial 16S ribosomal RNA gene
SEQ ID NO: 5: reverse primer for detection of bacterial 16S ribosomal RNA gene
SEQ ID NO: 6: reverse primer for detection of bacterial 16S ribosomal RNA gene
SEQ ID NO: 7: forward primer for detection of bacterial 16S ribosomal RNA gene
SEQ ID NO: 8: forward primer for detection of bacterial 16S ribosomal RNA gene
SEQ ID NO: 9: forward primer for detection of bacterial 16S ribosomal RNA gene
SEQ ID NO: 10: reverse primer for detection of bacterial 16S ribosomal RNA gene
SEQ ID NO: 11: reverse primer for detection of bacterial 16S ribosomal RNA gene
[0168]
[Sequence list]

Claims (12)

A method for detecting microorganisms,
(1) filtering a fluid using a filtration membrane,
(2) immersing the filtration membrane in a liquid medium and culturing the microorganisms captured by the filtration membrane;
(3) a step of concentrating the culture,
(4) extracting microbial DNA from the concentrated culture,
(5) A method for detecting a microorganism, comprising the steps of: performing PCR using the extracted DNA as a template; and (6) detecting the obtained PCR product. The method according to claim 1, further comprising a step of rinsing the obtained filtration membrane after the step (1). The method according to claim 1 or 2, wherein the culture time in the step (2) is from 5 hours to 24 hours. The method according to claim 1, 2 or 3, wherein the microorganism is a fungus or a bacterium. 5. The method according to claim 1, wherein the filtration membrane has a pore size of 0.2 to 0.45 [mu] m. The method according to claim 1, 2, 3, 4, or 5, wherein the PCR in the step (5) is performed using two types of primers each having the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The PCR in the step (5) was carried out using two types of primers each having the nucleotide sequence shown in SEQ ID NO: 4 and SEQ ID NO: 5, or two types of primers each having the nucleotide sequence shown in SEQ ID NO: 4 and SEQ ID NO: 6, respectively. 6. The method according to claim 1, 2, 3, 4 or 5, wherein the method is implemented using. The method according to claim 1, 2, 3, 4, 5, 6, or 7, wherein the detection in the step (6) is performed by an electrophoresis or hybridization assay using an agarose gel. The method according to claim 1, 2, 3, 4, 5, 6, 7, or 8, wherein the fluid is a gas or a liquid. 10. The method of claim 9, wherein the liquid is a tissue or tissue equivalent immersion liquid, an organ immersion liquid, or blood. The method according to claim 10, wherein the tissue equivalent is cultured skin or cultured cornea. The method according to claim 11, wherein the cultured skin is cultured epidermis, cultured dermis, or complex cultured skin.