JP2009082153A - 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 for detecting microorganisms includes following steps: step (1) for filtering the fluid with a filter membrane; step (2) for immersing the filter membrane in a liquid medium and cultivating the entrapped microorganisms; step (3) for concentrating the cultivated microorganisms; step (4) for extracting the DNA of the cultivated microorganisms from the concentrated culture; step (5) for performing PCR by using the extracted DNA as a template; and step (6) for detecting the obtained PCR product. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for detecting a microorganism.

  In recent years, due to rapid progress in tissue engineering, products using living cells such as cultured skin have been developed. Such products require confirmation that the cells are free of microbial contamination as well as the safety of the constituent cells. Since products using living cells such as cultured skin cannot be sterilized, sufficient care must be taken to prevent contamination of microorganisms in the manufacturing process. It may also be necessary to ensure the sterility of the final product.

  Cultured skin is manufactured by culturing a small amount of cells collected from a burned patient, etc., and transplanted to the patient from whom the cells were collected (autologous skin transplant) or transplanted to another person (same species) 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 caused by Candida albicans has become a medical problem as an infectious disease that causes a high rate of susceptible patients. Pseudomonas aeruginosa (Pseudomonas aeruginosa), etc., caused opportunistic infections in hosts with reduced resistance, causing cutaneous suppuration, urinary tract infections, respiratory infections, and sepsis, causing nosocomial infections. Has the problem of being easy.

  As an old method for detecting fungi, a method has been used in which a clinical specimen is cultured on, for example, a Petri dish, and the fungus contained in the clinical specimen is grown to detect the formed fungal colonies. However, since fungi grew slowly, it took a long time to form colonies. For this reason, the malfunction that a detection result was not obtained before applying cultured skin to a patient arose.

  As a bacteria / fungus negative test in the 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 employed. However, even in the case of the sterility test of cultured skin by this method, the test result cannot be obtained before the cultured skin is applied to a patient.

  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. .

  Many methods for directly detecting fungal DNA have been published as methods for solving the problem of taking 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 the fungus as a primer. Such a method was able to detect fungi more rapidly than the above-described method, and further improved the detection sensitivity. In particular, in Patent Document 1, for example, by using an oligonucleotide that specifically hybridizes with the nucleic acid sequence of the 18S rRNA gene of Candida albicans as a primer, the gene is amplified, and the amplified product is subjected to electrophoresis or dots. A method for detection by blot hybridization or the like is disclosed. By this method, it has become possible to finish the detection of fungi, which conventionally required several days to several weeks, within one day. However, in the method disclosed in Patent Document 1, the conventional PCR method is applied, and only about 1 to 2 ml of sample can be used. When this method is applied to a liquid such as a tissue or a tissue equivalent immersion liquid in which the culture supernatant reaches several hundred milliliters, only a small part of the obtained culture supernatant is used. Therefore, not only the detection sensitivity of microorganisms is insufficient, but also problems such as a decrease in reliability of test results occur.

  For other fungal detection methods using PCR, the detection sensitivity is not sufficient. In addition, no fungal detection method using a soaking solution of tissue equivalent such as cultured skin as a sample was disclosed at all.

Furthermore, as a rapid detection method of bacteria, a method of detecting CO ₂ produced by metabolism using glucose labeled with ¹⁴ C as a carbon source of a medium is also known (Non-patent Document 1). However, this method has a problem that fungi and bacteria cannot be detected separately. In addition, for a product containing living cells (for example, tissue equivalent), the living cells themselves produce CO ₂ , so that the method cannot be applied.

  Therefore, a method for detecting microorganisms including fungi and bacteria that are useful in tissues or tissue equivalents, particularly cultured skin, has been desired.

  As a method of detecting microorganisms in the air in a controlled environment, a certain amount of air is sucked 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 numbers are known (Non-Patent Documents 2 and 3). However, in order to be able to visually confirm colonies, it is necessary to culture for at least 2 days even for cells with a high growth rate, and for 4 days or more for cells with a low growth rate. In addition, since the shape of the colony of yeast-like fungi and bacteria is circular, it is difficult to distinguish both from the shape of the colony. When identifying the fungus, DNA is extracted from the fungus body, etc. There was a problem that it took more time. In addition, for example, since it is necessary to change the agar medium every time a certain amount of air is sucked, a large amount of agar medium is required, and the culture period on the agar medium is 5 days or more according to the Japanese Pharmacopoeia. In order to confirm the detection of bacterial cells as soon as possible, it is necessary to observe the presence or absence of colonies during the culture period. In addition to the large number of samples, there is a problem in that it takes time to observe the agar medium.

JP-A-8-89254

Edited by Japan Air Cleaners Association, “Construction and Maintenance of Clean Room Environment”, First Edition, Ohm Co., Ltd., July 20, 2000, p. 65-75 Edited by the Japanese Pharmacopoeia Editorial Committee, “14th revised Japanese Pharmacopoeia Commentary”, Tokyo Yodogawa Shoten, June 27, 2001, p. F-140 ~ 162 Edited by Kenji Sato et al., “Sterilized Medical Device GMP / Introduction to Validation”, Medical Device Center, February 21, 2002, p. 87-107

  In view of the problems of the prior 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 rapidly and highly sensitively detecting microorganisms contained in a liquid such as a tissue or tissue equivalent immersion liquid. 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.

  As a result of repeated research on a method for detecting microorganisms quickly and with high sensitivity, filtration of a liquid such as a tissue or tissue equivalent immersion liquid through a filtration membrane, pre-culture using the filtration membrane, and PCR are combined into a liquid. It was found that the contained microorganisms were detected quickly and with high sensitivity. Furthermore, the present inventors have found that not only liquid but also microorganisms contained in gas can be detected quickly and with high sensitivity, and the present invention has been completed.

That is, the present invention is a method for detecting a microorganism,
(1) A step of 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) a step of extracting microbial DNA from the concentrated culture,
The present invention relates to a method for detecting a microorganism comprising a step of performing PCR using extracted DNA as a template, and a step of detecting a PCR product obtained.

  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 can be determined before transplantation of cultured skin or the like. Can be confirmed. In the conventional method, about 1 ml of sample is used, and the detection sensitivity is 100 cells / ml or more, but if the method of the present invention is used, about 150 ml of sample can be processed. It is possible to detect one microorganism contained in the sample. In addition, bacteria and fungi have been detected so far 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 solution as a sample. PCR method. The former has a problem that a long period of 7 to 14 days is necessary. In the latter case, the period is as short as 1 to 2 days, but only about 1 to 2 ml of sample can be used, so that it can be obtained when applied to tissue or tissue equivalent immersion liquids reaching several hundred milliliters. In addition, only a small part of the culture supernatant was used, and there was a problem that not only the detection sensitivity of microorganisms was insufficient, but also the reliability of test results was lowered. However, in the method for detecting a microorganism of the present invention, when a liquid such as a tissue or tissue equivalent immersion liquid is collected in the afternoon, a test result can be obtained in the afternoon of the next day (about 24 hours). Very good.

  Furthermore, the microorganism detection method of the present invention can also be applied to gases. In the conventional method, when there is a large amount of gas to be filtered, it is necessary to replace the agar medium installed in the air sampler every time a certain amount of gas is sucked. There was a problem that the processed agar medium had to be processed. However, the amount of waste can be reduced by using the microorganism detection method of the present invention.

  When the method for detecting a microorganism of the present invention is used, the detection result of the microorganism can be obtained as well as whether the detected microorganism is a fungus or a bacterium, depending on the type of primer used. In addition, by analyzing the base sequence of a DNA fragment (PCR product) amplified by PCR using a DNA sequencer, it is possible to identify the bacterial species in a short time after collecting the fluid. Furthermore, if an oligonucleotide that hybridizes with a sequence specific to a bacterial species is used as a primer, it is possible to simultaneously identify the bacterial species.

  Hereinafter, the present invention will be described in more detail.

  In this specification, “filtration” means passing a liquid or gas through a porous substance such as a filtration membrane.

  As used herein, the term “fluid” means liquid or gas.

  The term “liquid” as used herein means a liquid that may contain microorganisms, and examples thereof include, but are not limited to, tissue or tissue equivalent immersion fluid, organ immersion fluid, or blood. It is not a thing.

  The term “tissue” in the present specification means various tissues extracted from a living body. Examples of tissue include, but are not limited to, skin, cornea, cartilage, adipose tissue, muscle, blood vessel, nerve, amniotic membrane, placenta, or lymph node.

  The term “tissue equivalent” herein 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 and cultured cornea, cultured cartilage, cultured bone, cultured kidney, cultured liver, cultured retina, cultured nerve, cultured heart or cultured consisting of living cells and biopolymers or living cells only There are inner ears and the like, and the structure is not limited to these as long as it is a structure containing living cells.

  Examples of the cultured skin include cultured epidermis, cultured dermis, and complex cultured skin. Cultured epidermis is, for example, an epidermal cell sheet produced by culturing and proliferating 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 with good cell adhesion such as polyurethane, polystyrene and polyacrylate. Cultured dermis refers to, for example, culturing and proliferating fibroblasts obtained from mammalian dermis and increasing the living body such as collagen, chitin, chitosan, chondroitin sulfate, hyaluronic acid, laminin, polylactic acid, polyglycolic acid, fibrin, and gelatin It is a cultured dermis sheet incorporated into a molecular material and / or a non-biodegradable synthetic polymer material with good cell adhesion, such as a synthetic polymer such as polyurethane, polystyrene, and 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.

  The cultured cornea is, for example, a sheet-like structure produced by culturing and proliferating corneal epithelial cells, parenchymal cells and / or endothelial cells obtained from a mammalian cornea, or collagen, chitin, chitosan, chondroitin sulfate , Hyaluronic acid, laminin, polylactic acid, polyglycolic acid, fibrin, gelatin and other biopolymer materials and / or synthetic polymers such as polyurethane, polystyrene, polyacrylate and other non-biodegradable synthetic materials with good cell adhesion 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.

  The cultured cartilage is an artificial cartilage made of cells or a structure in which cells are incorporated into a base material, which is produced by a well-known method. Examples of cultured cartilage include structures produced by incorporating chondrocytes and / or mesenchymal stem cells into a three-dimensional structure carrier made of a biocompatible material such as chitosan, collagen and / or calcium phosphate. be able to.

  As used herein, the term “tissue or tissue equivalent immersion liquid” refers to (i) a fluid used when a tissue piece is taken from a patient and then transported to a tissue or tissue equivalent supply (eg, manufacturer). (Ii) a liquid used in the manufacturing process of the tissue equivalent, or (iii) a liquid used for storing the manufactured tissue equivalent.

  “Liquid used when a tissue piece is collected from a patient and then transported to a tissue or tissue equivalent supplier” is a liquid in which the tissue piece is immersed while the collected tissue piece is transported to a destination. As long as there is no particular limitation. “Liquid used when a piece of tissue is collected from a patient and then transported to a tissue or tissue equivalent supplier” includes, for example, the liquid or skin used to store skin collected from a mammal. The liquid recovered after rinsing with a buffer solution is included.

  The “liquid used in the production process of 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. “Liquid used in the production process of 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 the trypsin treatment to disperse the cells, and the culture medium of the tissue equivalent are collected when replaced. Liquids and the like are also included in the liquid used in the manufacturing process of the tissue equivalent.

  The “liquid used for storage of the manufactured tissue equivalent” is not particularly limited as long as it is a liquid recovered after being added to the tissue equivalent between completion and actual application.

  Specific examples of what can be used for the “immersion solution of tissue or tissue equivalent” include Dulbecco's modified Eagle basic medium (DMEM), phosphate buffer solution, Ringer solution, Ringer-Rock solution, Tyrode solution, Earl solution, Hanks Solution, lock solution, Eagle's minimum essential culture solution, Ham synthetic culture solution F12, Green culture solution, Leibovitz L-15 culture solution, and serum-free culture solutions MCDB153, MCDE151, MCDE104, MCDB131, MCDB402, MCDB201, MCDB302, MCDB105, MCDB110, etc. are mentioned. In addition, when the tissue equivalent is cryopreserved after production, a cryopreservation solution in which a cryoprotectant is added to the culture solution or buffer described above can be used. Examples of cryoprotective agents include glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, sucrose, carboxymethyl cellulose salt, carboxymethyl cellulose (CMC), and disaccharides (such as trehalose). One or more types can be selected and added to the culture medium or buffer.

  The term “organ” in the present specification means various organs removed from a living body. Examples of organs include, but are not limited to, liver, pancreas, kidney or heart.

  The term “organ soaking solution” in the present specification includes a liquid used for temporarily immersing and storing an organ when the extracted organ is transported between hospitals during organ transplantation or the like.

  Specific examples of what can be used for “organ soaking solution” include DMEM, phosphate buffer solution, Ringer solution, Ringer-Lock solution, Tyrode solution, Earl solution, Hanks solution, Lock solution, Eagle's minimum essential culture solution Ham's synthetic culture solution F12, Green culture solution, Leibovitz's L-15 culture solution, and serum-free culture solutions MCDB153, MCDE151, MCDE104, MCDB131, MCDB402, MCDB201, MCDB302, MCDB105, and MCDB110. In addition, a cryopreservation solution in which a cryoprotectant is added to the culture solution or buffer described above can also be used. Examples of the cryoprotectant include glycerol, DMSO, propylene glycol, ethylene glycol, sucrose, carboxymethylcellulose salt, CMC, and disaccharides (trehalose, etc.). Alternatively, it can be added to the buffer.

  Furthermore, microbial contamination can also be detected using the microorganism detection method of the present invention for the collected blood itself from which the cells have not been removed, and the liquid components from which the cells have been removed from the blood. Examples of the liquid component from which cells are removed from blood include serum and plasma. Serum is obtained, for example, by leaving blood collected using a dry syringe and removing blood clots containing blood cells and some blood clotting factors (such as fibrinogen or prothrombin) from the blood. Plasma is obtained, for example, by adding a blood coagulation inhibitor to collected blood and removing the precipitated blood cells from the blood by centrifuging the blood.

  The term “gas” as used herein means a gas that may contain microorganisms, and examples thereof include, but are not limited to, air containing at least oxygen.

  The term “microorganism” as used herein includes, for example, fungi and / or bacteria that cause contamination before and during use of a tissue or tissue equivalent, or when an organ is removed.

  Examples of the fungi include fungi belonging to the genus Candida, Hansenula, Saccharomyces, Trichosporon, Cryptococcus, Aspergillus, Penicillium, Blastmyces, Coccidioides, Nyumocystis, Malassezia or Debariomyces . More specifically, such fungi include, for example, Candida albicans, Candida lusitaniae, Candida krusei, Candida glabrata, Cryptococcus neoformans. ), Aspergillus fumigatus or Pneumocistis carinii.

  Examples of bacteria include bacteria belonging to the genus Bacillus, Streptococcus, Pseudomonas, Escherichia, Staphylococcus, and the like. More specifically, such bacteria include, for example, Bacillus subtilis, Streptococcus pyogenes, Salmonella typhimurium, Salmonella bongori, Salmonella enteritidis. These include Salmonella enteritidis, Escherichia coli or Staphylococcus epidermidis, Staphylococcus aureus and Pseudomonas aeruginosa.

  In the detection method of the present invention, in order to increase the reliability of the detection result, when the object to be filtered is a liquid, the entire liquid is used as a sample. All the target liquid is filtered using a filtration membrane, thereby capturing microorganisms contained in the liquid. When the object to be filtered is a gas, a filtration membrane is applied to an air sampler, and the microorganisms contained in the gas are captured by sucking the gas with the air sampler.

  In the present specification, the “air sampler” is not particularly limited as long as it is a device capable of capturing microorganisms by causing the microorganisms in the air to collide with the filtration membrane. For example, an air sampler set so that microorganisms collide with the agar medium can be used. When using an air sampler in which an agar medium is set, a microorganism is contained in the gas by placing a filtration membrane on the agar medium and sucking the gas with the air sampler. When the filtration membrane is placed on the agar medium, the filtration membrane becomes wet. When a dry filter membrane is used, it is preferable to use a wet filter membrane because microorganisms may not be collected quantitatively on the filter membrane due to the influence of static electricity.

  In the present invention, the filtration membrane used for filtration preferably has a pore size of 0.2 to 0.45 μm. When the pore size of the filtration membrane is less than 0.2 μm, cell debris other than the target microorganism remains on the filtration membrane and the filtration membrane tends to be clogged. When the pore size of the filtration membrane is larger than 0.45 μm, the target microorganism tends to pass through the filtration membrane. In order to prevent clogging of the filtration membrane, a filtration membrane having a larger pore size (for example, a pore size of 8 μm) may be stacked. In addition, when a liquid that may cause clogging is included in the liquid because the liquid contains a large amount of cell debris, it is preferable to divide the liquid and filter each with a separate filtration membrane. When one sample is filtered through a plurality of filtration membranes, the plurality of filtration membranes may be collected in the following culture step, and the method of the present invention may be applied independently after the filtration step.

  Fluids such as tissue or tissue equivalent immersion fluids may contain antibiotics to prevent microbial growth. In such a case, antibiotics are adsorbed on the filtration membrane after the immersion liquid is filtered, which adversely affects the subsequent culture process. Therefore, in the method for detecting a microorganism of the present invention, it is preferable to rinse the filtration membrane after filtering a liquid such as a tissue or tissue equivalent immersion liquid. By rinsing the filtration membrane, the antibiotic adsorbed on the filtration membrane can be washed away, and the growth of the microorganism in the subsequent culturing process is not inhibited, so that the microorganism can be detected more reliably. Such filter membrane rinses include, 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 a buffer solution obtained by adding lecithin or polysorbate to the medium or the buffer solution. For rinsing the filtration membrane when detecting fungi, use GP liquid medium, SCD liquid medium, potato dextrose liquid medium, Sabouraud dextrose liquid medium, or a medium in which lecithin or polysorbate is added to these mediums. It is preferable to do. For rinsing the filtration membrane when bacteria are targeted for detection, an SCD liquid medium, a TG medium, a plain heart infusion liquid medium, a nutrient liquid medium, or a medium obtained by adding lecithin or polysorbate to these mediums is used. It is preferable.

  In the microorganism detection method of the present invention, the microorganisms captured on the filtration membrane are cultured for a certain period of time. By culturing microorganisms captured by the filtration membrane, several microorganisms contained in the fluid can be detected.

  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 cause problems when at least tissue equivalent is used as a biomaterial, and can be appropriately selected by those skilled in the art. For example, when detecting fungi and bacteria, 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 / glucose 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.

  In the microorganism detection method of the present invention, the culture method is preferably shaking culture or stirring culture.

  In the microorganism detection method of the present invention, the microorganism culture time 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 5 to 24 hours. When fungi are targeted for detection, the culture time is preferably 8 to 24 hours. In the case of culture for less than 5 hours in the case of bacteria and less than 8 hours in the case of fungi, the microorganisms tend not to grow to a sufficient extent for detection. When the culture time exceeds 24 hours, the detection sensitivity tends to be unchanged as compared with the culture for 24 hours.

  In the microorganism detection method of the present invention, the culture temperature of the microorganism can be set to, for example, 20 to 37 ° C. In bacteria, the culture temperature is preferably 30 to 35 ° C, and in fungi, the culture temperature is preferably 20 to 25 ° C. However, the culture temperature is not limited to these, and the optimum culture temperature for the microorganism to be detected can be appropriately set by those skilled in the art.

  The culture rate of microorganisms is different, and fungi tend to have a slower growth rate than bacteria. Therefore, the detection sensitivity may not be sufficient only by using a part of the culture. Therefore, after completion of the culture, it is preferable to collect all 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 DNA is extracted from the precipitated culture. As a DNA extraction method, any conventional method such as a DNA extraction method using a surfactant such as Triton X-100 or sodium dodecyl sulfate (SDS) can be applied. The extracted DNA may be purified by phenol treatment or chloroform treatment and then ethanol precipitation or isopropanol precipitation.

  For PCR in the microorganism detection method of the present invention, a normal PCR method can be applied, using a DNA extracted through the above-mentioned steps as a template and a primer capable of detecting the DNA sequence of the target microorganism. As long as it is not particularly limited.

  PCR is a repeated cycle of denaturation of double-stranded DNA into single strands, annealing of single-stranded DNA and primers, and synthesis of complementary DNA by DNA polymerase. The annealing temperature is usually set to be equal to or lower than the melting temperature (Tm) of the oligonucleotide used as a primer. The formula for obtaining Tm is known to those skilled in the art. For example, Tm can be calculated by the following formula (cell engineering separate volume “Bio-Experiment Illustrated” (3) PCR, 13-43 pages, 1996, (Published by Shujunsha Co., Ltd.).

Tm (° C.) = 4 × (% GC) + 2 × (% AT) −2n
(% GC): GC content in oligonucleotide (%)
(% AT): AT content in oligonucleotide (%)
n: Length of oligonucleotide

  In the microorganism detection method of the present invention, PCR can be carried out separately for fungi and bacteria, for example. Alternatively, PCR may be performed using two or more sets of primers, for example, a primer for the target fungus and a primer for the target bacterium, simultaneously in one PCR reaction solution.

  The primer sequences used for the PCR are designed by selecting, for example, a sequence that is conserved in fungi or bacteria and can amplify a specific gene or a part thereof. As a specific gene conserved in fungi or bacteria, an rRNA gene is preferred. The most suitable sequence as a primer can be appropriately selected by a survey based on a generally available database (for example, DDBJ (DNA Data Bank of Japan) or RDP (Ribosomal Database Project)). In addition, the following combinations are known as primers for amplifying rRNA.

  A primer capable of detecting a fungus is preferably designed based on the sequence of the 18S rRNA gene or a part thereof. Such primer combinations include a combination of a primer consisting of 5′-ACTTTCGATGGTAGGATAG-3 ′ (SEQ ID NO: 1) and a primer consisting of 5′-TGATCGTCTTCGATCCCCTA-3 ′ (SEQ ID NO: 2), and 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 25 species and 78 strains of 18S rRNA including fungi causing clinically important infections such as Candida, Saccharomyces, Aspergillus and Hansenula. When two kinds 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 kinds 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.

  As a primer capable of detecting bacteria, a primer (Tsuguo Sasaki, et al. (1996), PDA Journal of Pharmaceutical Sciences & Technology, 51, 242-247) consisting of 5′-GTGCCAGCAGCCGCGGTA-3 ′ (SEQ ID NO: 4) Combination with a primer (Kui Chen, et al. (1989), FEMS Microbiology Letters, 57, 19-24) consisting of 5′-ACGTCCATCCCCACTTCTCTC-3 ′ (SEQ ID NO: 5), and a primer consisting of SEQ ID NO: 4 and 5 ′ -A combination with a primer consisting of AGGCCCGGGAACGTATTCAC-3 '(SEQ ID NO: 6) (Kui Chen, et al., Supra) is known and can be used in the detection method of the present invention. These primer sequences are at least 100% identical between Bacillus subtilis, Streptococcus pyogen, Pseudomonas aeruginosa, Salmonella tyhummurium, Salmonella bongoli, Escherichia coli and Streptococcus epidermidis, 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); WG Weisburg et al. , J. Bacteriol., 173, 697 (1991); and DA Relman et al., Mol. Microbiol., 6, 1801 (1992)), 5′-TCAAAKGAATTGACGGGGGC-3 ′ (SEQ ID NO: 8) (K is dT or d G. DA Relman et al., N. Engl. J. Med., 327, 293 (1992); and DA Relman et al., N. Engl. J. Med., 323, 1573 (1990)), 5′-GAGGAAGGTGGGGATGACGGT-3 ′ (SEQ ID NO: 9) (supra DA Relman et al., N. Engl. J. Med., 323, 1573 (1990); and K. Chen et al., FEMS Microbiol. Lett. 48 , 19 (1989)) and the like, and 5'-GGACTACCAGGGTATCTAA as a reverse primer for detecting bacteria -3 ′ (SEQ ID NO: 10) (supra DA Relman et al., N. Engl. J. Med., 327, 293 (1992); and KH Wilson et al, J. Clin. Microbiol. 28, 1942 (1990) ) 5'-GGTTACCTTTGTTACGACTT-3 '(SEQ ID NO: 11) (above PA Eden et al., Supra WG Weisburg et al .; supra DA Relman et al., Mol. Microbiol., 6, 1801 (1992)) It has been known.

  The homology between the bacterial primer sequence and the human 18S rRNA gene sequence corresponding to the bacterial 16S rRNA gene is as low as 60% or less. If appropriate PCR conditions are selected, the human 18S rRNA gene fragment is amplified. Never happen. Even when the fungal primer is used, the human 18S rRNA gene fragment is not amplified if appropriate PCR conditions are selected. PCR conditions can be appropriately set by those skilled in the art.

  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 PCR product detection method, for example, electrophoresis using an agarose gel or the like, a hybridization assay using a labeled probe for the PCR product, or a known detection method in this field such as a PCR-ELISA method can be applied. By analyzing the base sequence of the DNA fragment (PCR product) amplified by PCR using a DNA sequencer, it is considered possible to identify the bacterial species.

  In the case of a hybridization assay, a labeled probe consisting of an oligonucleotide complementary to the PCR product or a partial sequence thereof and a detectable label is used. As the label, any label usually used in the art can be used. 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 and the labeled probe are hybridized, the enzyme is reacted with its substrate (chromogenic substance, fluorescent substance, luminescent substance, etc.), and then the substrate is colored. The PCR product can be detected by detecting fluorescence or luminescence.

  In the case of the PCR-ELISA method, for example, a PCR product can be detected by incorporating a base labeled with digoxigenin (DIG) during PCR amplification and then performing an ELISA using an antibody against DIG.

  When a PCR product is detected as described above, it means that a tissue, tissue equivalent, organ, blood, gas or the like is contaminated with microorganisms.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited to these Examples.

  Reagents such as GP liquid medium, LP diluent, and SCD liquid medium described below and instruments such as test tubes, microtubes, and tweezers were all autoclaved (121 ° C., 20 minutes) in advance. As the jumbo conical tube, catalog number FALCON 2075 manufactured by Nippon Becton Dickinson Co., Ltd. was used. Regarding potassium acetate, in Example 1, Fluka Co., Ltd., catalog number 60035, and in Examples 2-4, Wako Pure Chemical Industries, Ltd., catalog number 166-03545 were used. As isopropanol and ethanol, Wako Pure Chemical Industries, Ltd., catalog number 166-04836, and Wako Pure Chemical Industries, Ltd., catalog number 057-00456 were used, respectively. Regarding TE (pH 8.0), in Example 1, Nippon Gene Co., Ltd., catalog number 316-90025, and in Examples 2-4, Nippon Gene Co., Ltd., catalog number 0559J were used. Regarding the filtration membrane, in Example 1, a filtration membrane having a pore diameter of 0.2 μm (manufactured by Sartorius Co., Ltd., catalog number 13107-47-ACN), and in Examples 2 to 4, a filtration membrane having a pore diameter of 0.2 μm (Pole ( (Catalog No. 4803), in Example 5, a filtration membrane having a pore diameter of 0.45 μm (manufactured by Sartorius, catalog number 13106-47-ACN), and in Example 6, a filtration membrane having a pore diameter of 0.45 μm ( Sartorius Co., Ltd., catalog number 13106-47-ACN) and 0.2 μm pore size filter membrane (Pall Corp., catalog number 4803), Example 7 pore size 0.22 μm membrane filter (Millipore Corp.) And catalog number ATSMTTD60). The filter holder and the manifold used were Advantech Toyo Co., Ltd., catalog number KGS-47TF and Advantech Toyo Co., Ltd., model number KM-3. As the disposable loop (gamma-sterilized), Catalog No. GI-0457-05 manufactured by AS ONE was used. Milliflex (official name: Twinhead Milliflex Sensor II suction filtration) and adapter (official name: MFPP adapter improved type) are manufactured by Millipore Corporation, catalog number MXPS20015, and Paul Corporation, catalog number Y- 0705-2 was used. As an air sampler, M Air T Millipore Tester (manufactured by Millipore Corporation, catalog number ATAS PLR01) was used, and as a micropore sampling plate, Millipore Corporation, catalog number ATAH EAD01 was used. The PCR thermal cycler MP was manufactured by Takara Shuzo Co., Ltd., model name TP3000, and the polaroid camera was manufactured by 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). In Examples 2 to 4 and 6, SYBR Green (manufactured by BMA, catalog number 50513). Was used.

Example 1
<Preparation of bacterial solution>
(A) 10 ml of GP liquid medium (glucose, peptone liquid medium (powder) (Nippon Pharmaceutical Co., Ltd., catalog number 397-00225) 28.5 g / ultra pure water 1 l) was dispensed into one test tube. Next, one colony of Candida albicans (Accession No. IFO 1594 strain at the Institute for Fermentation) 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.

(B) 9 ml of GP liquid medium was dispensed into 10 test tubes. 1 ml of the bacterial solution prepared in (A) above was added to one of the test tubes into which the GP liquid medium was dispensed using a micropipette and mixed to prepare a 10-fold diluted solution. The chip was changed, and thereafter, dilution was repeated in the same manner until a 10 ^10- fold diluted bacterial solution was obtained. Hereinafter, in Example 1, a 10 ⁶ to 10 ¹⁰ times diluted bacterial solution was used.

(C) Each of six jumbo conical tubes with a capacity of 225 ml was charged with 150 ml of Green medium (Green + 3% FBS) containing 3% fetal bovine serum (manufactured by JRH Biosciences, Inc.), and Antibiotic consisting of streptomycin, penicillin and amphotericin B (manufactured by Lifetech Oriental Co., catalog number 15240-096, final concentration: streptomycin 100 μg / ml, penicillin 100 units / ml, amphotericin B 0.25 μg / ml) 1.5 ml Was added to each tube and mixed. Next, 1.0 ml of each 10 ⁶ to 10 ^10- fold diluted bacterial solution prepared in the above (B) was added and mixed. A negative control was prepared by adding 1.0 ml of GP liquid medium instead of the diluted bacterial solution.

<Filtrate filtration>
A filtration membrane having a pore diameter of 0.2 μm was set on the filter holder, and then the filter holder was set on the manifold. Next, 50 ml of the sample prepared in the above (C) was put into a funnel using a 50 ml pipette, and all were suction filtered. Suction filtration was repeated two more times and all samples were suction filtered.

<Rinse of filtration membrane>
LP Diluent (LP Diluent “DAIGO” (Nippon Pharmaceutical Co., Ltd., Catalog No. 397-00281) 2 / ultra pure water 2 l, final concentration: polypeptone 1 g / l, lecithin 0.7 g / l, polysorbate 80 20 g / L, pH 7.0 to 7.4) 50 ml was added to the funnel, followed by suction filtration to rinse the inner wall of the funnel. The same rinse was repeated twice more.

<Culture of cells captured by filtration membrane>
After suction filtration, remove the filtration membrane with tweezers, immerse the filtration membrane in a 50 ml centrifuge tube containing 30 ml of GP liquid medium, and then shake culture at room temperature (25 ° C) overnight (15-20 hours). did.

<Recovering bacterial cells>
After overnight culture, the filtration membrane was aseptically removed from the GP liquid medium with 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 1 ° C. (15000 g, 10 minutes), and the supernatant was gently removed.

<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 to suspend the cells. The cells were stirred for 5 seconds with a vortex mixer, and then incubated at 100 ° C. for 15 minutes. 100 μl of 2.5 M potassium acetate solution was added to each microtube, mixed, and then incubated for 60 minutes on ice. 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 DNA precipitate. Each microtube was centrifuged (15000 rpm, 10 minutes), and the supernatant was gently removed. Next, 0.5 ml of 99% ethanol was added, mixed, and centrifuged at 4 ° C. (15000 rpm, 10 minutes) to remove the supernatant. To the precipitate, 0.5 ml of 70% ethanol was added, mixed, and then centrifuged at 4 ° C. (15000 rpm, 10 minutes) to remove the supernatant. After lightly drying the precipitate on a paper towel, 50 μl of TE (pH 8.0) was added to dissolve the DNA.

<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 is 5 μl (10 ×) PCR buffer (attached to Taq polymerase), 4 μl dNTP mixed solution (attached to Taq polymerase), each primer 0.3 μM, 2.5 μl template DNA, 0.25 μl Taq polymerase (5 U / μl) (Takara Shuzo Co., Ltd., catalog number R001A) and ultrapure water. As 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, the Candida albicans IFO 1594 strain DNA prepared in <Preparation of PCR template> 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.

  Each PCR tube was set in the PCR thermal cycler MP, and PCR was performed with 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 72 ° C. for 10 minutes. It was carried out by incubation. The annealing temperature was set with reference to the melting temperature of the primer calculated by the above formula.

<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 make 2000 ml.

  To 50 μl of the PCR product, 10 μl of loading buffer (bromophenol blue 0.25% (weight / volume), glycerol 25% (volume / volume), ultrapure water) was added and mixed. Next, 20 μl of these samples were loaded on an agarose gel (Agarose S (Nippon Gene Co., Ltd., Catalog No. 312-1193) 1.2 g / 1 × TAE 100 ml), and electrophoresis was performed at 100 V for 25 minutes.

<Staining of PCR products>
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 a staining solution and allowed to stand in the dark for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a polaroid camera.

As a result of staining the PCR product, DNA of Candida albicans IFO 1594 strain was detected from the 10 ⁶ -fold diluted bacterial solution and the 10 ⁷ -fold diluted bacterial solution. As described in the following test examples, the number of viable bacteria in the 10 ⁷ -fold diluted bacterial solution was 1.

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, 1 ml of the 10 ⁵ to 10 ^10- fold diluted bacterial solution was dispensed into a 90 mm diameter petri dish (n = 2). In each petri dish, Sabouraud glucose agar medium maintained at 45 ° C. after autoclaving (Sabouraud glucose agar medium (powder) (Nippon Pharmaceutical Co., Ltd., Catalog No. 390-01011) 32.5 g / 500 ml of ultrapure water) Was dispensed in 15 ml portions, gently stirred, and allowed to stand until the agar solidified. After the agar solidified, each petri dish was inverted and cultured in an incubator at 25 ° C. The number of formed colonies was visually counted after 4 and 5 days from the culture.

As a result of the measurement, there was one Candida albicans IFO 1594 strain contained in the 10 ⁷ -fold diluted bacterial solution.

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.

(B) 9 ml of GP liquid medium was dispensed into 10 test tubes. One test tube into which a GP liquid medium was dispensed was inoculated with 2 loops of the disposable loop of the colony cultured in the above (A), and the bacterium was suspended with a 10 ml pipette to prepare a 10-fold diluted solution. Using a micropipette, 1 ml of the obtained bacterial solution was added to one of the test tubes into which the GP liquid medium was dispensed to prepare a 10 ² -fold diluted solution. The chip was replaced, and thereafter, dilution was repeated in the same manner until a 10 ⁸ -fold diluted bacterial solution was obtained. Hereinafter, in Example 2, a 10 ⁵ to 10 ⁸ times diluted bacterial solution was used.

(C) One jumbo conical tube having a capacity of 225 ml was charged with 180 ml of GP medium, and further 50 mg / ml gentamicin (manufactured by Invitrogen Corporation, catalog number 15750-060) and 250 μg / ml amphotericin B (Invitrogen ( 900 μl of each of catalog number 15290-018) manufactured by the same company was added and mixed.

<Filtrate filtration>
An adapter was set on the Milliflex, and then a funnel equipped with a filtration membrane having a pore diameter 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 1.0 ml of 10 ⁵ -fold diluted bacterial solution prepared in (B) were mixed in a funnel, and suction filtered. The same operation was performed for 10 ⁶ to 10 ⁸ times diluted bacterial solution. A negative control was prepared by adding 1.0 ml of GP liquid medium instead of the diluted bacterial solution.

<Rinse of filtration membrane>
The inner wall of the funnel was rinsed by adding 50 ml of LP diluted solution (prepared in the same manner as in Example 1) to the funnel and then suction filtration. The same rinse was repeated twice more.

<Culture of cells captured by filtration membrane>
After suction filtration, the filtration membrane was taken out with tweezers, the filtration membrane was immersed in a 50 ml centrifuge tube containing 25 ml of GP liquid medium, and then shake culture was performed at room temperature (25 ° C.) for 20 hours.

<Recovering bacterial cells>
After culture, the filtration membrane was aseptically removed from the GP liquid medium with 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.

<Preparation of template for PCR>
Preparation of the template for PCR was carried out using the cells recovered in <Recovering bacterial cells> of Example 2 instead of the cells recovered in <Recovering bacterial cells> of Example 1. The same procedure as in <Preparation of PCR template> in Example 1 was performed.

<PCR>
PCR was carried out except that the template DNA prepared in <Preparation of PCR template> in Example 2 was used in place of the template DNA prepared in <Preparation of PCR template> in Example 1. The same conditions as in <PCR> in Example 1 were used.

<Agarose electrophoresis>
<Agarose electrophoresis> in Example 1 except that the PCR product amplified in <PCR> in Example 2 is used in place of the PCR product amplified in <PCR> in Example 1. It carried out like.

<Staining of PCR products>
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 a staining solution and allowed to stand in the dark for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a polaroid camera.

As a result of staining of the PCR product, Candida albicans IFO 1594 strain DNA was detected from the 10 ⁵ fold, 10 ⁶ fold and 10 ⁷ fold diluted bacterial solutions.

Test example 2
The number of viable bacteria in the diluted bacterial solution prepared in the preparation (B) of the bacterial solution of Example 2 was measured by the following pour method. That is, 1 ml of the 10 ⁵ to 10 ⁸ times diluted bacterial solution was dispensed into a petri dish having a diameter of 90 mm (for 10 ⁵ times, 10 ⁶ times and 10 ⁸ times diluted bacterial solutions, n = 2 and 10 ⁷ times diluted bacterial solutions) n = 4). Each petri dish was dispensed with 14 ml of Sabouraud glucose agar medium (prepared in the same manner as in Example 1) maintained at 50 ° C. after autoclaving, and the mixture was gently stirred and allowed to stand until the agar solidified. After the agar solidified, each petri dish was inverted and cultured in an incubator at 25 ° C. The number of formed colonies was visually counted after 4 and 5 days from the culture.

As a result of the measurement, the viable cell counts contained in the 10 ⁵ times, 10 ⁶ times and 10 ⁷ times diluted bacterial solutions were 111, 9.5 and 2.5, respectively.

Example 3
<Preparation of bacterial solution>
(A) One colony of Saccharomyces cerevisiae (Accession No. ATCC 9763 strain in American Type Culture Collection) was collected and Sabouraud glucose agar medium (prepared in the same manner as in Test Example 1) And smeared at 32 ° C. for 24 hours.

(B) 9 ml of GP liquid medium was dispensed into 10 test tubes. One test tube into which a GP liquid medium was dispensed was inoculated with 2 loops of the disposable loop of the colony cultured in the above (A), and the bacterium was suspended with a 10 ml pipette to prepare a 10-fold diluted solution. Using a micropipette, 1 ml of the obtained bacterial solution was added to one of the test tubes into which the GP liquid medium was dispensed to prepare a 10 ² -fold diluted solution. The chip was replaced, and thereafter, dilution was repeated in the same manner until a 10 ⁸ -fold diluted bacterial solution was obtained. Hereinafter, in Example 3, a 10 ⁴ to 10 ⁸ times diluted bacterial solution was used.

(C) One jumbo conical tube having a capacity of 225 ml was charged with 180 ml of GP medium, and further 50 mg / ml gentamicin (manufactured by Invitrogen Corporation, catalog number 15750-060) and 250 μg / ml amphotericin B (Invitrogen ( 900 μl of each of catalog number 15290-018) manufactured by the same company was added and mixed.

<Filtrate filtration>
An adapter was set on the Milliflex, and then a funnel equipped with a filtration membrane having a pore diameter 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 1.0 ml of 10 ⁴ -fold diluted bacterial solution prepared in (B) were mixed in a funnel, and all were subjected to suction filtration. The same operation was performed for 10 ⁵ to 10 ⁸ times diluted bacterial solution. A negative control was prepared by adding 1.0 ml of GP liquid medium instead of the diluted bacterial solution.

<Rinse of filtration membrane>
About 100 ml of polysorbate washing solution “Digo” (manufactured by Nippon Pharmaceutical Co., Ltd., catalog number 395-01561) was added to the funnel by decantation, and then the inner wall of the funnel was rinsed by suction filtration. The same operation was further repeated twice, and the filtration membrane was rinsed with a total of 300 ml of washing solution.

<Culture of cells captured by filtration membrane>
After suction filtration, the filtration membrane was taken out with tweezers, the filtration membrane was immersed in a 50 ml centrifuge tube containing 30 ml of GP liquid medium, and then shake culture was performed at room temperature (25 ° C.) for 20 hours.

<Recovering bacterial cells>
After culture, the filtration membrane was aseptically removed from the GP liquid medium with 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 1 ° C. (15000 g, 10 minutes), and the supernatant was gently removed.

<Preparation of template for PCR>
Preparation of the template for PCR was carried out using the cells recovered in <Recovering bacterial cells> of Example 3 instead of the cells recovered in <Recovering bacterial cells> of Example 1. The same procedure as in <Preparation of PCR template> in Example 1 was performed.

<PCR>
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 PCR template> in Example 1. The same conditions as in <PCR> in Example 1 were used.

<Agarose electrophoresis>
<Agarose electrophoresis> in Example 1 except that the PCR product amplified in <PCR> in Example 3 is used in place of the PCR product amplified in <PCR> in Example 1. It carried out like.

<Staining of PCR products>
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 a staining solution and allowed to stand in the dark for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a polaroid camera.

As a result of staining the PCR product, DNA of Saccharomyces cerevisiae ATCC 9763 strain was detected from the 10 ⁴ fold, 10 ⁵ fold, 10 ⁶ fold and 10 ⁷ fold diluted bacterial solutions. No DNA of Saccharomyces cerevisiae ATCC 9763 strain was detected from the 10 ⁸ -fold diluted bacterial solution.

Test example 3
The number of viable bacteria in the diluted bacterial solution prepared in the preparation (B) of the bacterial solution of Example 3 was measured by the following pour method. That is, 1 ml of the 10 ⁴ to 10 ⁸ times diluted bacterial solution was dispensed into a 90 mm diameter petri dish (n = 3). Each petri dish was dispensed with 14 ml of Sabouraud glucose agar medium (prepared in the same manner as in Example 1) maintained at 50 ° C. after autoclaving, and the mixture was gently stirred and allowed to stand until the agar solidified. After the agar solidified, each petri dish was inverted and cultured in an incubator at 32.5 ° C. Four days after the culture, the number of formed colonies was visually counted.

As a result of the measurement, the number of viable bacteria contained in the 10 ⁴ fold, 10 ⁵ fold, 10 ⁶ fold and 10 ⁷ fold dilutions was 100 or more, 42.3, 2.3 and 0.3, respectively. Met. No colonies were detected for the 10 ⁸ -fold diluted bacterial solution.

Example 4
<Preparation of bacterial solution>
(A) 10 ml of GP liquid medium (prepared in the same manner as in Example 1) was dispensed into one test tube. Next, spores and fungi of Aspergillus fumigatus (Accession No. IFO 33022 strain at the Fermentation Research Institute) were inoculated into the GP liquid medium for one loop of Dispo Loop, and suspended in the GP liquid medium. This bacterial solution was cultured at 25 ° C. for 20 hours.

(B) 9 ml of GP liquid medium was dispensed into five test tubes. 1 ml of the bacterial solution prepared in (A) above was added to one of the test tubes into which the GP liquid medium was dispensed using a 5 ml pipette and mixed to prepare a 10-fold diluted solution. The pipette was changed, and thereafter dilution was repeated in the same manner until a 10 ³ -fold diluted bacterial solution was obtained. Hereinafter, in Example 4, the bacterial solution (stock solution) prepared in the above (A) to 10 ³ -fold diluted bacterial solution was used.

<Filtrate filtration>
An adapter was set on the Milliflex, and then a funnel equipped with a filtration membrane having a pore diameter of 0.2 μm was set on the adapter. Next, using a 25 ml pipette, 30 ml of GP liquid medium and 1.0 ml of the bacterial solution (stock solution) prepared in (B) were mixed in a funnel, and all were suction filtered. The same operation was performed for the 10 ¹ to 10 ³ times diluted bacterial solution. A negative control was prepared by adding 1.0 ml of GP liquid medium instead of the diluted bacterial solution.

<Rinse of filtration membrane>
In Example 4, no antibiotic was used, so the inner wall of the funnel was not rinsed.

<Culture of cells captured by filtration membrane>
After suction filtration, the filtration membrane was taken out with tweezers, the filtration membrane was immersed in a 50 ml centrifuge tube containing 30 ml of GP liquid medium, and then cultured with shaking at 27 ° C. for 20 hours.

<Recovering bacterial cells>
After culture, the filtration membrane was aseptically removed from the GP liquid medium with 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.

<Preparation of template for PCR>
Preparation of the template for PCR was performed using the cells recovered in <Recovering bacterial cells> of Example 4 in place of the cells recovered in <Recovering bacterial cells> of Example 1. The same procedure as in <Preparation of PCR template> in Example 1 was performed.

<PCR>
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 PCR template> in Example 1. The same conditions as in <PCR> in Example 1 were used.

<Agarose electrophoresis>
<Agarose electrophoresis> in Example 1 except that the PCR product amplified in <PCR> in Example 4 is used instead of the PCR product amplified in <PCR> in Example 1. It carried out like.

<Staining of PCR products>
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 a staining solution and allowed to stand in the dark for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a polaroid camera.

Results of staining of PCR products, 1-fold, 10-fold, 10 ^2-fold and 10 ³ times dilutions bacterial suspension of Aspergillus fumigatus IFO 33,022 strains DNA was detected.

Test example 4
Since the Aspergillus fumigatus IFO 33022 strain is a fungus having a mycelium and forming a spore, the detection sensitivity of the diluted bacterial solution of the bacterial cell cannot be indicated by the number of colonies (cfu). Therefore, the detection sensitivity of the diluted bacterial solution of Aspergillus fumigatus IFO 33022 strain was expressed not by the number of colonies (cfu) but by the amount of DNA. That is, the PCR template (DNA) of 1 ×, 10 ×, 10 ² × and 10 ³ × diluted bacterial solutions was extracted (same as in Example 1), 8 μl of this template was put into a microtube, mixed with 72 μl of TE and 10 Diluted twice. With respect to TE, the absorbance at 260 nm of the template was measured using a UV-visible spectrophotometer (manufactured by Beckman Coulter, model number DU640). The formula for determining 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, pages 61-62 1995, published by Shujunsha Co., Ltd.).

  Double-stranded DNA (μg / μl) = (260 nm absorbance) × 0.05 × dilution factor

Results of the measurement of absorbance at 260nm, 1-fold, 10-fold, 10 ^double and 10 ^triple each DNA of Aspergillus fumigatus IFO thirty-three thousand and twenty-two strain contained in diluted bacterial solution 0.9μg, 0.67μg, 68.3ng and It was 4.25 ng. From this, it was revealed that if the DNA amount of Aspergillus fumigatus IFO 33022 strain is 4.25 ng, it can be detected by the same method.

Example 5
<Preparation of bacterial solution>
(D) 10 ml of SCD liquid medium (Soybean casein digest powder medium (manufactured by Nippon Pharmaceutical Co., Ltd., catalog number 396-00991) 8.5 g / 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.

(E) 9 ml of SCD liquid medium was dispensed into 10 test tubes. One ml of the SCD liquid medium was added to 1 ml of the bacterial solution prepared in (D) above using a micropipette and mixed to prepare a 10-fold diluted solution. The chip was replaced, and thereafter, dilution was repeated in the same manner until a 10 ⁸ -fold diluted bacterial solution was obtained. Hereinafter, in Example 5, a 10 ⁵ to 10 ⁸ times diluted bacterial solution was used.

(F) 150 ml of Green + 3% FBS was put into each of 8 jumbo conical tubes having a capacity of 225 ml, and gentamicin (catalog number 15750-060 manufactured by Invitrogen) and amphotericin B (trade names “fungizone”, Invitrogen ( Co., Ltd., catalog number 15290-018) was added to a concentration of 50 μg / ml and 0.25 μg / ml, respectively. Subsequently, 1.0 ml each of the 10 ⁵ to 10 ⁸ times diluted bacterial solution prepared in (E) was added and mixed. A negative control was prepared by adding 1.0 ml of SCD liquid medium instead of the diluted bacterial solution.

<Filtrate filtration>
A filtration membrane having a pore diameter of 0.45 μm was set in the filter holder, and then the filter holder was set in the manifold. Next, 50 ml of the sample prepared in the above (F) was put into a funnel using a 50 ml pipette, and all were suction filtered. Suction filtration was repeated two more times and all samples were suction filtered.

<Rinse of filtration membrane>
The inner wall of the funnel was rinsed by adding 50 ml of LP diluted solution (prepared in the same manner as in Example 1) to the funnel and then suction filtration. The same rinse was repeated twice more.

<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 then shake culture was performed at 35 ° C. overnight (12 to 18 hours).

<Recovering bacterial cells>
After overnight culture, the filtration membrane was aseptically removed from the SCD liquid medium with tweezers. 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 1 ° C. (15000 g, 10 minutes), and the supernatant was gently removed.

<Preparation of template for PCR>
0.5 ml of each microtube suspension was transferred to a 1.5 ml Eppendorf tube and centrifuged at 4 ° C. (15000 g, 10 minutes). After centrifugation, the supernatant is removed, and the precipitate is 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. Each sample was then treated at 100 ° C. for 20 minutes and allowed to stand on ice.

<PCR>
50 μl of the reaction mixture was prepared in a 0.2 ml PCR tube and kept on ice. As 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 of Bacillus subtilis IFO 3134 prepared in <Preparation of PCR template> in Example 5 was used. The composition of the reaction mixture is the same as that of the reaction mixture of Example 1 except that 0.5 μM of primer and 2.0 μl of template DNA were used.

  Each PCR tube was set in the PCR thermal cycler MP, and PCR was performed with the following program. In this example, PCR is performed 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 then at 72 ° C. for 1 minute. It was carried out by incubation. The annealing temperature was set with reference to the melting temperature of the primer calculated by the above formula.

<Agarose electrophoresis>
10 μl of loading buffer was added to 50 μl of the PCR product and mixed. Next, 15 μl of these samples were loaded on an agarose gel (2.9 g of agarose S / 1 × TAE 100 ml), and electrophoresis was performed at 100 V for 30 minutes.

<Staining of PCR products>
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 a staining solution and allowed to stand in the dark for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a polaroid camera.

As a result of staining the PCR product, DNA of Bacillus subtilis IFO 3134 was detected from all 10 ⁵ to 10 ⁸ times diluted bacterial solutions. As described in Test Example 5 below, the numbers of Bacillus subtilis contained in the 10 ⁶ fold, 10 ⁷ fold, and 10 ⁸ fold diluted bacterial solutions were 30, 4, and 0.5, respectively.

Test Example 5
The number of viable bacteria in the diluted bacterial solution prepared in the preparation (E) of the bacterial solution of Example 5 was measured by the following pour method. That is, 1 ml of the 10 ⁵ to 10 ⁸ times diluted bacterial solution was dispensed into a 90 mm diameter petri dish (n = 2). 15 ml of SCD agar medium (SCD agar medium (manufactured by Nippon Pharmaceutical Co., Ltd., catalog number 396-00991) 8.0 g / 200 ml of ultrapure water) kept at 45 ° C. after autoclaving was dispensed to each petri dish. The mixture was gently stirred and allowed to stand until the agar solidified. After the agar solidified, each petri dish was inverted and cultured in an incubator at 35 ° C. The number of formed colonies was visually counted after 4 and 5 days from the culture.

As a result of the measurement, Bacillus subtilis IFO 3134 contained in the 10 ⁵ fold, 10 ⁶ fold, 10 ⁷ fold, and 10 ⁸ fold dilutions was 200, 30, 4, and 0.5, respectively. .

Example 6
<Preparation of bacterial solution>
(D) One colony of Pseudomonas aeruginosa (Accession No. IFO 13275 strain at the Institute for Fermentation) was collected, smeared on an SCD agar medium (prepared in the same manner as in Test Example 5), and 24 hours at 32.5 ° C. Cultured.

(E) 9 ml of SCD liquid medium (produced 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 (D), and the bacteria were suspended with a 10 ml pipette. Using the micropipette, 1 ml of the obtained bacterial solution was added to one of the test tubes into which the SCD liquid medium was dispensed to prepare a 10-fold diluted solution. The chip was changed, and thereafter, dilution was repeated in the same manner until a 10 ^10- fold diluted bacterial solution was obtained. Hereinafter, in Example 6, a 10 ⁶ to 10 ^10- fold diluted bacterial solution was used.

(F) Each of six jumbo conical tubes having a capacity of 225 ml was charged with 150 ml of SCD liquid medium, and further 50 mg / ml gentamicin (manufactured by Invitrogen Corp., catalog number 15750-060) and 250 μg / ml amphotericin B ( 150 μl of Invitrogen Corp. catalog number 15290-018) was added and mixed. Then, 1.0 ml of each 10 ⁶ to 10 ^10- fold diluted bacterial solution prepared in (E) was added and mixed. A negative control was prepared by adding 1.0 ml of SCD liquid medium instead of the diluted bacterial solution.

<Filtrate filtration>
A filtration membrane having a pore diameter of 0.45 μm was set in the filter holder, and then the filter holder was set in the manifold. Next, 50 ml of the sample prepared in the above (F) was put into a funnel using a 50 ml pipette, and all were suction filtered. Suction filtration was repeated two more times and all samples were suction filtered.

<Rinse of filtration membrane>
The inner wall of the funnel was rinsed by adding 50 ml of LP diluted solution (prepared in the same manner as in Example 1) to the funnel and then suction filtration. The same rinse was repeated twice more.

<Culture of cells captured by filtration membrane>
After suction filtration, the filtration membrane was taken out with tweezers, the filtration membrane was immersed in a 50 ml centrifuge tube containing 30 ml of SCD liquid medium, and then cultured with shaking at 35 ° C. for 19 hours.

<Recovering bacterial cells>
After culturing, the filtration membrane was aseptically removed from the SCD liquid medium with tweezers. 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.

<Preparation of template for PCR>
Preparation of the template for PCR was carried out using the cells recovered in <Recovering bacterial cells> of Example 6 instead of the cells recovered in <Recovering bacterial cells> of Example 5. The same procedure as in <Preparation of PCR template> in Example 5 was performed.

<PCR>
PCR was carried out except that the template DNA prepared in <Preparation of PCR template> in Example 6 was used in place of the template DNA prepared in <Preparation of template for PCR> in Example 5. The same procedure as in <PCR> of Example 5 was performed.

<Agarose electrophoresis>
<Agarose electrophoresis> in Example 5 except that the PCR product amplified in <PCR> in Example 6 is used in place of the PCR product amplified in <PCR> in Example 5. It carried out like.

<Staining of PCR products>
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 a staining solution and allowed to stand in the dark for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a polaroid camera.

As a result of staining the PCR product, DNA of Pseudomonas aeruginosa IFO 13275 was detected from the 10 ⁶ fold, 10 ⁷ fold and 10 ⁸ fold diluted bacterial solutions. No DNA of Pseudomonas aeruginosa IFO 13275 was detected from the 10 ⁹ -fold and 10 ¹⁰ -fold diluted bacterial solutions.

Test Example 6
The number of viable bacteria in the diluted bacterial solution prepared in the preparation (E) of the bacterial solution of Example 6 was measured by the following pour method. That is, 1 ml of the 10 ⁶ to 10 ^10- fold diluted bacterial solution was dispensed into a 90 mm diameter petri dish (n = 3). Each petri dish was dispensed with 15 ml of SCD agar medium (produced in the same manner as in Test Example 5) maintained at 50 ° C. after autoclaving, gently stirred, and allowed to stand until the agar solidified. After the agar solidified, each petri dish was inverted and cultured in an incubator at 35 ° C. The number of formed colonies was visually counted after 3 and 5 days from the culture.

As a result of the measurement, the numbers of viable bacteria contained in the 10 ⁶ fold, 10 ⁷ fold and 10 ⁸ fold diluted bacterial solutions were 100 or more, 34 and 2.7, respectively. No colonies were detected for the 10 ⁹ -fold and 10 ¹⁰ -fold diluted bacterial solutions. In addition, although the same experiment was repeated 4 times, all were able to be detected even if the number of viable bacteria was 1 to several.

  Furthermore, when the same experiment was conducted using a 0.2 μm pore size filtration membrane instead of a 0.45 μm pore size membrane, Pseudomonas aeruginosa IFO 13275 DNA was also detected.

  From the results of Examples 1-3, 5 and 6 and Test Examples 1-3, 5 and 6, it was found that the presence of bacterial cells could be detected even when the number of viable bacteria before filtration was 1 to several. In addition, from the results of Examples 1-3, 5 and 6, even when antibiotics are contained in the culture medium, if 1 to several cells are present before filtration, the presence of the cells can be detected, From the results of Example 4, it was found that even when antibiotics were not contained, the presence of the bacterial cells could be detected if several bacterial cells were present. This means that the presence of bacterial cells can be detected regardless of whether antibiotics are included or not if there are a small number of bacterial cells in the tissue or tissue equivalent immersion fluid, organ immersion fluid, or blood. means.

  Regarding the pore size of the filtration membrane, it was found from the results of Example 6 that the presence of bacteria can be detected by using either the filtration membrane having a pore size of 0.2 μm or 0.45 μm. Moreover, 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.

Example 7
<Gas filtration>
M Air T Millipore tester is filled with M Air T cassette TSA medium (hereinafter, TSA medium is referred to as SCD agar medium), then the lid of SCD agar medium is opened and sterilized on the SCD agar medium using tweezers. A filtration membrane having a pore diameter of 0.22 μm was placed. Next, a micropore sampling plate was set on the M Air T Millipore tester and the lid was capped. Next, 200 liters of air in a non-clean area (Menicon BIO Center, 2nd floor machine room) that seems to contain microorganisms was sucked using a M Air T Millipore tester. The filtration membrane was cut into a size of 70 mm in diameter and sterilized under high pressure steam (121 ° C., 20 minutes).

<Culture of cells captured by filtration membrane>
After suction filtration, the microporous sampling plate was removed, the filtration membrane was taken out with tweezers, and 50 ml of SCD liquid medium (manufactured by Nippon Pharmaceutical Co., Ltd., catalog number 396-00991) 8.5 g / distilled water 500 ml) The filter membrane was immersed in a sterilized container (Assist Corporation, Catalog No. 75.9922.414S), and then shake culture was performed at 30 ° C. for 22 hours. In addition, what put 50 ml of SCD liquid culture media in the container was made into negative control, and the shaking culture was implemented at 30 degreeC for 22 hours.

<Recovering bacterial cells>
After incubation, each culture was transferred to a 50 ml centrifuge tube, 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 1 ° C. (15000 g, 10 minutes), and the supernatant was gently removed.

<Preparation of template for PCR>
100 μl of TE (produced in the same manner as in Example 5) containing Triton X-100 was added to each microtube, and the cells were suspended. Each sample was then treated at 100 ° C. for 20 minutes and allowed to stand on ice.

<PCR>
50 μl of the reaction mixture was prepared in a 0.2 ml PCR tube and kept on ice. As 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 <Preparation of PCR template> in Example 7 was used. The composition of the reaction mixture was 0.5 μM primer having the sequence shown in SEQ ID NO: 4, 1 μM primer having the sequence shown in SEQ ID NO: 5, 0.5 μl Taq polymerase (5 U / μl) and 2.0 μl template. The composition is the same as that of the reaction mixture of Example 1 except that DNA was used.

  Each PCR tube was set in the PCR thermal cycler MP, and PCR was performed with the same program as <PCR> in Example 5.

<Agarose electrophoresis>
<Agarose electrophoresis> in Example 5 except that the PCR product amplified in <PCR> in Example 7 was used in place of the PCR product amplified in <PCR> in Example 5. It carried out like.

<Staining of PCR products>
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 a staining solution and allowed to stand in the dark for 2 hours. Then, the agarose gel was irradiated with UV (λ = 254 nm), and a photograph was taken using a polaroid camera.

  As a result of staining the PCR product, a band was detected in the vicinity of 680 bp from the culture medium in which the membrane was put. No band was detected from the negative control. Since a band was detected in the vicinity of 680 bp, it was presumed that the bacteria were detected by two kinds of primers having the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5.

Comparative Example 1
Set M Air T Millipore tester with M Air T cassette TSA medium filled (hereinafter TSA medium is called SCD agar medium), then open SCD agar medium lid, set micropore sampling plate, cover Did. Next, as in Example 7, 200 liters of air in a non-clean area (Menicon BIO center, 2nd floor machine room) that seems to contain microorganisms was sucked using a M Air T Millipore tester. Air suction was performed almost simultaneously with Example 7. The microporous sampling plate was removed, the agar medium was capped, and cultured in a 27 ° C. incubator. The number of colonies formed was visually counted after 1 day, 2 days, 3 days, 4 days, and 7 days after culturing.

  As a result of measurement, the numbers of colonies detected after 1 day, 2 days, 3 days, 4 days, and 7 days were 0, 4, 6, 8, and 8, respectively. Therefore, it can be seen that the method of Comparative Example 1 requires at least two days from the capture of the bacterial cells to the detection of the microorganisms.

  On the other hand, if the method of Example 7 is used, when the cells are collected in the afternoon, the test result can be obtained in the afternoon of the next day (about 30 hours). In addition, when the method of Example 7 is used, since the bacterial cells are captured on the filtration membrane, every time a certain amount of gas is sucked into the agar medium installed in the air sampler, even when the amount of gas to be filtered is large. By exchanging the filter membrane without exchanging it, it is possible to detect microorganisms using only one agar medium. Furthermore, by analyzing the base sequence of a DNA fragment (PCR product) amplified by PCR with a DNA sequencer, it is possible to identify the bacterial species.

SEQ ID NO: 1: Forward primer for detecting fungal 18S ribosomal RNA gene SEQ ID NO: 2: Reverse primer for detecting fungal 18S ribosomal RNA gene SEQ ID NO: 3: Forward primer for detecting fungal 18S ribosomal RNA gene SEQ ID NO: 4: Bacteria Forward primer for detection of 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: Bacterial 16S ribosomal RNA gene SEQ ID NO: 8: Forward primer sequence for detecting bacterial 16S ribosomal RNA gene SEQ ID NO: 9: Forward primer sequence number for detecting bacterial 16S ribosomal RNA gene 10: bacterial 16S ribosomal RNA gene of the detection reverse primers SEQ ID NO: 11: reverse primer for the detection of 16S ribosomal RNA gene of bacteria

Claims (9)

A method for detecting microorganisms in an immersion liquid of a tissue or tissue equivalent containing an antibiotic, an immersion liquid of an organ, and blood,
(1) A step of filtering a tissue-containing tissue equivalent immersion solution, an organ immersion solution, and blood using a filtration membrane;
(2) rinsing the obtained filtration membrane;
(3) immersing the filtration membrane in a liquid medium and culturing the microorganisms captured by the filtration membrane;
(4) a step of concentrating the culture,
(5) a step of extracting microbial DNA from the concentrated culture,
(6) A method for detecting a microorganism comprising a step of performing PCR using the extracted DNA as a template, and (7) a step of detecting the obtained PCR product. The method according to claim 1, wherein the culture time in the step (3) is 5 hours to 24 hours. The method according to claim 1 or 2, wherein the microorganism is a fungus or a bacterium. The method according to claim 1, 2 or 3, wherein the pore size of the filtration membrane is 0.2 to 0.45 μm. The method according to claim 1, 2, 3 or 4, wherein the PCR in the step (6) is carried out using two kinds of primers each having a base sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2. The PCR in the step (6) is performed by using two types of primers consisting of the base sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively, or two types of primers consisting of the base sequences shown in SEQ ID NO: 4 and SEQ ID NO: 6, respectively. The method according to claim 1, 2, 3 or 4, wherein The method according to claim 1, 2, 3, 4, 5 or 6, wherein the detection in the step (7) is performed by electrophoresis or hybridization assay using an agarose gel. The method according to claim 1, 2, 3, 4, 5, 6 or 7, wherein the tissue equivalent is cultured skin or cultured cornea. The method according to claim 8, wherein the cultured skin is cultured epidermis, cultured dermis, or complex cultured skin.