WO2015163449A1 - Fungal nucleic acids extraction method - Google Patents

Fungal nucleic acids extraction method Download PDF

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WO2015163449A1
WO2015163449A1 PCT/JP2015/062537 JP2015062537W WO2015163449A1 WO 2015163449 A1 WO2015163449 A1 WO 2015163449A1 JP 2015062537 W JP2015062537 W JP 2015062537W WO 2015163449 A1 WO2015163449 A1 WO 2015163449A1
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nucleic acid
candida
beads
diameter
biological sample
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French (fr)
Japanese (ja)
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清仁 緒方
一乗 松田
辻　浩和
康二 野本
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株式会社ヤクルト本社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present invention relates to a method for extracting nucleic acid of fungi present in a biological sample.
  • Candida (Candida) fungi are established as permanent bacteria in the mucous membranes and skin of human digestive tract, upper respiratory tract, vagina, etc., but when immunity decreases, become. Among them, candidemia has the highest frequency of occurrence and has a very high mortality rate.
  • Candida albicans As the causative species of candidemia, four bacterial species belonging to Candida albicans and non-albicans Candida spp. (C. glabrata, C. tropicalis, C. parapsilosis and C. krusei) are widely known. It accounts for about 90%. Moreover, these Candida genus fungi are greatly different in susceptibility to antifungal agents among bacterial species, and it is an important issue to quickly identify the causative bacterial species.
  • Non-patent Document 2 A method for extraction and detection / quantification using RT-qPCR is already known (Non-patent Document 2).
  • yeast which is a kind of fungus, a method is known in which the transformant is cultured in a medium and then disrupted with about 0.45-0.55 mm glass beads (Patent Document 1). And Patent Document 2).
  • the present invention relates to providing a method for efficiently extracting fungal nucleic acids from a biological sample containing fungi.
  • the present inventors use beads having a diameter of about 0.1 to 0.5 mm, which have been conventionally used for disrupting fungal and bacterial cells.
  • fungal nucleic acids in a biological sample cannot be sufficiently extracted, so that fungi cannot be detected and quantified.
  • beads having a diameter of 0.8 mm to 3 mm are used, nucleic acids are efficiently extracted. They found that fungi could be detected and quantified accurately.
  • the present invention relates to the following 1) to 10).
  • the method of 2), wherein the Candida fungus is one or more selected from Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida crusei.
  • nucleic acid fragment is a nucleic acid fragment consisting of a base sequence represented by SEQ ID NOs: 1 to 12 or a nucleic acid fragment consisting of a base sequence complementary thereto.
  • a nucleic acid fragment that can specifically hybridize to a bead having a diameter of 0.8 mm to 3 mm and a fungal nucleic acid and may include one or more selected from a nucleic acid extraction reagent, a nucleic acid amplification reaction reagent, and a protocol, 7) or A kit for carrying out the method of 8).
  • the kit according to 9), wherein the beads have a diameter of 1 mm to 2.5 mm.
  • fungal nucleic acid can be efficiently extracted directly from a biological sample containing fungi, and the number of fungi present in the biological sample can be accurately measured.
  • nucleic acids in bacterial cells can be accurately measured directly from a biological sample without culturing the bacterial cells contained in the biological sample, diagnosis of Candidaemia and the like is possible in a short time. is there.
  • the graph which showed the detection sensitivity of the Candida genus 5 species specific primer and the Candida group specific primer The graph which shows the result of the microbial cell addition collection
  • the biological sample containing fungi is not particularly limited as long as it is a biological sample in which fungi can exist, and blood, urine, spinal fluid, semen, sputum, throat swab, cervical mucus, vaginal secretions,
  • biological samples such as ascites, tissue, conjunctival wipes, calculus, plaque, saliva, nasal discharge, alveolar lavage fluid, pleural effusion, gastric juice, gastric lavage fluid, skin lesions, feces, joint fluid, and affected area wipes.
  • blood is preferred, and human peripheral blood is more preferred.
  • fungus means the eubacteria Eumycota and is a generic name including yeast.
  • Candida genus, Issatchenkia genus, Aspergillus genus, Hansenula genus, Saccharomyces genus, Trichosporon genus, Penicillium genus, z Sporothrix genus, Absidia genus, Mucor genus, Rhizomucor genus, Rhizopus genus, Pneumocystis genus, Coccidioidomycosis genus, Histoplasma genus Histoplasma Fungi belonging to the genus Paracoccidioides (Paracoccidioides) and the like are included, among which Candida, Isachenchia, and Aspergillus are preferred, and Candida is more preferred.
  • the Candida fungi include Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei and Candida krusei, (Candida guilliermondii), Candida lucitaniae (Clavispora lusitaniae) and the like, and Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida crusei are more preferable.
  • Isachenchia examples include Issatchenkia orientalis and Issatchenkia terricola, with Isachenchia orientalis being more preferred.
  • Isachenchia Orientalis and Candida Crusei are the same bacteria, but the sexual generation is called Isachenchia Orientalis and the asexual generation is Candida Crusei.
  • Aspergillus fungi include Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and the like.
  • the nucleic acid means single-stranded or double-stranded DNA or RNA.
  • the sample solution containing fungi include a biological sample collected from a living body, or a solution obtained by suspending a sample obtained by appropriately concentrating bacterial cells from the sample in a liquid such as distilled water, buffer solution, or physiological saline. It is done.
  • a buffer solution a phosphate buffer solution (PBS), Tris-HCl buffer, lysis buffer (a mixture of RLT buffer, TE (Tris-EDTA) and ⁇ -Mercaptoethanol) or the like can be used. Is preferred.
  • the bacterial cell concentration treatment include centrifugation, filtration concentration, and the like. Centrifugation is preferable.
  • RNA stabilizer eg, RNA protect Bacterial Reagent (QIAGEN), RNAlater (Ambion), etc.
  • the biological sample used in the method of the present invention may be a sample obtained by culturing cells in advance as in the blood culture method, or a sample collected from a living body without culturing.
  • the material of the “bead” is not particularly limited as long as it satisfies the condition that the diameter is 0.8 mm or more and 3 mm or less, and may be an inorganic material or an organic material. Further, it may be porous or non-porous. Examples include glass (borosilicate glass, lime glass) beads, zirconia beads, silica beads, zirconia / silica beads (beads containing both zirconia and silica in one bead), stainless beads, polystyrene beads, and the like. Of these, glass beads and zirconia / silica beads are preferred. Such beads can be purchased from commercial products (for example, beads from Tommy Seiko Co., Ltd., BioSpec Products, etc.).
  • the diameter of the beads is preferably 0.8 mm or more, more preferably 1 mm or more, from the viewpoint of improving fungal nucleic acid extraction efficiency. Moreover, it is preferable that it is 3 mm or less, and 2.5 mm or less is more preferable. Furthermore, since beads having a large diameter are difficult to set in a sample tube used for nucleic acid extraction, 1 mm to 2.5 mm is preferable, and 1 mm is more preferable.
  • the stirring method is not particularly limited as long as the cells and the beads can be sufficiently contacted, and the container containing the sample solution may be manually reciprocally vibrated, but from the viewpoint of extraction efficiency and reproducibility, a shaker or bead crushing It is preferable to stir using a machine.
  • the stirring time may be until the cell membrane of the fungus is broken, and specifically, it is preferably about 1 to 10 minutes, more preferably about 2 to 5 minutes.
  • an enzyme or a surfactant may be added as necessary to further improve the nucleic acid extraction efficiency.
  • the enzyme in this case include protease P.
  • the surfactant include Triton X-100.
  • fungal cells can be efficiently disrupted and fungal nucleic acids can be easily released.
  • Separation of the released nucleic acid can be performed by employing a known method known as a method for separating and extracting nucleic acid, for example, a general-purpose method such as a phenol-chloroform method or a guanidine method.
  • a general-purpose method such as a phenol-chloroform method or a guanidine method.
  • phenol is added to the sample solution that has been contact-stirred with the beads and allowed to react, then chloroform / isoamyl alcohol (or phenol / chloroform / isoamyl alcohol) is added and stirred, and the supernatant is removed after centrifugation.
  • the nucleic acid can be extracted by recovering, adding a sodium chloride solution, a sodium acetate buffer solution, an ammonium acetate buffer solution, etc., and precipitating with ethanol.
  • RNA extraction it is preferable to carry out a hot phenol method in which phenol is added and reacted at 50 to 70 ° C. for 5 to 15 minutes. It is also preferable to use chloroform / isoamyl alcohol. Further, the ethanol-precipitated precipitate is preferably dissolved with Nuclease-free water. On the other hand, when the main purpose is DNA extraction, phenol / chloroform / isoamyl alcohol is preferably used, and the reaction can be carried out at room temperature. Moreover, it is preferable to dissolve the ethanol-precipitated precipitate with TE.
  • the nucleic acid thus extracted is amplified by a known nucleic acid amplification method using a nucleic acid fragment that can specifically hybridize to the target fungal nucleic acid, and the amount thereof is measured. Based on this, the amount of the fungus in the biological sample is measured. Detection and / or quantification can be performed.
  • nucleic acid amplification methods include PCR (Polymerase Chain Reaction), RT-PCR (Reverse-Transcriptase PCR), LCR (Ligase Chain Reaction), LAMP (Loop-mediated Isothermal Amplification of DNA), NASBA (Nucleic Acid Sequence). Based Amplification; Nature, 1991 Mar.
  • TMA Transcription-Mediated Amplification; Advanced Biomedical Technologies. 1998; 189-201
  • TRC Transcription Reverse Transcription Concerted Reaction; Anal Biochem, 2003) Mar. 1; 314 (1): 77-86).
  • nucleic acid fragment that can specifically hybridize to the target fungal nucleic acid can be appropriately designed by taking into account the base sequence of the fungal nucleic acid.
  • nucleic acid fragments that can specifically hybridize to Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida crusei, and Candida rRNA are represented by SEQ ID NOS: 1 to 12 described below.
  • a nucleic acid fragment consisting of a base sequence or a complementary base sequence or a nucleic acid fragment consisting of a base sequence in which one or several, preferably 1 to 10 bases of the base sequence are substituted, added or deleted, or A stringent condition with a nucleic acid fragment consisting of a base sequence having 90% or more, preferably 95% or more, more preferably 99% or more identity with the base sequence, or a DNA consisting of a base sequence complementary to the base sequence Examples thereof include a nucleic acid fragment consisting of a base sequence that hybridizes below. The identity of the base sequence is calculated by using the GENETYX (R) homology analysis program.
  • “Stringent conditions” include, for example, conditions in which 50% formamide, 5 ⁇ SSC, 5 ⁇ Denhardt's solution and 250 mg / mL salmon sperm DNA are incubated at 42 ° C. for 16 to 24 hours and hybridized. Is mentioned.
  • PCR or RT-PCR When PCR or RT-PCR is used as a nucleic acid amplification method, (1) a step of performing PCR or RT-PCR using one or more of the above nucleic acid fragments on DNA or RNA extracted from a sample solution as described above, And (2) the step of detecting the amplified DNA fragment in step (1).
  • a DNA fragment (PCR product) specific to the target fungus can be obtained by combining the nucleic acid fragment with a template DNA derived from the target fungus (cDNA when the template is RNA) and performing an amplification reaction. When the DNA thus obtained is electrophoresed, the target fungus can be specifically detected and identified from the presence or absence of a band.
  • the target fungus can be quantified.
  • a method using real-time PCR is more preferable. By observing the amount of PCR product amplified by PCR over time and specifying the number of PCR cycles when a certain amount of DNA is reached, the target fungus in the sample solution can be quantified.
  • the PCR product to be amplified can be observed over time by labeling with a fluorescent dye that is an intercalator such as SYBR (R) Green I and measuring the fluorescence intensity at each PCR stage. Since the intercalator has the property that the fluorescence intensity increases by intercalating into double-stranded nucleic acid, it accurately measures the PCR product generated by PCR reaction from the DNA of the target fungus (cDNA in the case of RNA) In particular, SYBR (R) Green I is preferably used.
  • the CT value the number of PCR cycles
  • DNA amount a certain set fluorescence intensity
  • TaqMan probe labeled with a fluorescent dye a Molecular Beacon, or the like.
  • TaqMan probe and Molecular Beacon are detection methods using a probe in which a fluorescent dye and a quencher are bound to an oligonucleotide having homology with the internal sequence of a region amplified by PCR. Since the fluorescence corresponding to the PCR amplification reaction is emitted by the interaction between the fluorescent dye and the quencher bound to the probe, the PCR product amplified over time can be observed by measuring the fluorescence intensity at each PCR stage. it can.
  • Fungi of interest in a biological sample quantitatively, or detection and identification can be determined by a separate calibration curve of logarithmic values and C T values of the fungal number measured. That is, the logarithm of the fungal number of targeted horizontal axis, and the C T value in advance to create the plotted calibration curve on the vertical axis, substituting the C T values obtained as a result of the PCR reactions calibration curve, Quantify or detect / identify the target fungus in a biological sample.
  • the nucleic acid fragment can be used as a primer in the PCR method or RT-PCR method, or can be used alone as a probe, and these can be used in combination with other known universal primers, oligonucleotides, and the like.
  • Examples of the analysis method using the nucleic acid fragment as a probe include in situ hybridization and dot blot hybridization. Among them, in situ hybridization is preferable as a rapid method, and is labeled with a fluorescent substance. FISH using the obtained nucleic acid fragment as a probe is more preferable.
  • FISH consists of (1) a step of fixing a biological sample with formaldehyde or formalin, (2) a step of smearing the fixed biological sample on a slide glass or a membrane filter, and (3) a high level by fluorescently labeled nucleic acid fragments.
  • a step of performing hybridization (4) a step of washing excess nucleic acid fragments after hybridization and non-specifically bound nucleic acid fragments, and (5) macroscopic observation of the result after hybridization using a fluorescence microscope, Or it can carry out by the process of acquiring as an image with a CCD camera etc.
  • the target fungus When the target fungus is present in the biological sample, it hybridizes with the nucleic acid fragment used, and the signal in the result after the hybridization becomes positive, so these fungi must be specifically detected and identified. Can do. Moreover, quantification is also possible by measuring the signal intensity.
  • the nucleic acid extracted by the above-described fungal nucleic acid extraction method of the present invention is subjected to a nucleic acid amplification reaction using a nucleic acid fragment that can specifically hybridize to the nucleic acid.
  • a kit for a kit in which all or part of what is necessary for carrying out all or part of the method is collected.
  • “necessary for performing the process” can be appropriately selected by taking into account the description of the present specification.
  • nucleic acid extraction reagent for example, for sample preparation
  • Buffer RNA fixing reagent
  • nucleic acid separation reagent for example, for sample preparation
  • reagent used for nucleic acid amplification reaction for example, for sample preparation
  • protocol describing the method of implementation examples include kits that may include one or more.
  • Condition E Static culture was performed in Preston medium at 37 ° C. under microaerobic conditions for 24 hours. After measuring the number of cells by the DAPI method, these cells were appropriately diluted so as to have a certain number of cells to prepare a bacterial solution.
  • lysis buffer prepared by mixing 346.5 ⁇ L RLT buffer, 100 ⁇ L TE and 3.5 ⁇ L ⁇ -Mercaptoethanol
  • 1 mm (300 mg) diameter glass Beads TOMY Seiko
  • 500 ⁇ L of water-saturated phenol was added and vortexed for 5-10 seconds.
  • a sample tube was set in a heat block at 60 ° C. and reacted for 10 minutes (hot phenol method).
  • RT-qPCR was performed using QIAGEN OneStep RT-PCR Kit (QIAGEN), and the composition of the reaction solution was 1 ⁇ QIAGEN OneStep RT-PCR Buffer, 0.5 ⁇ Q-Solution, 0.4 mM dNTP Mix, 1/25 amount QIAGEN OneStep RT-PCR Enzyme Mix, 1 / 100,000 volume of SYBR® Green I (Molecular Probes), 1 ⁇ ROX Reference Dye (Invitrogen), 0.60 ⁇ M of each primer shown in Table 2 above, and 5 ⁇ L of the above primers The reaction was carried out in a reaction solution (total volume 10 ⁇ L) containing the RNA sample (10 ⁇ 3 to 10 3 cells) prepared in 13).
  • the reaction solution was first subjected to a reverse transcription reaction at 50 ° C. for 30 minutes, and then heated at 95 ° C. for 15 minutes to inactivate the reverse transcriptase. Subsequently, 94 ° C./20 seconds, 55 ° C. or 60 ° C. (for C. albicans, C. glabrata, and C. krusei primers at 60 ° C., for C. tropicalis, C. parapsiosis, and Candida group primers) was performed at 55 ° C.) for 45 seconds at 20 seconds and 72 ° C. for 50 seconds, and the amount of amplification product was measured as the fluorescence intensity of SYBR (R) Green I for each cycle.
  • SYBR SYBR
  • the denaturation temperature of the amplified product was measured. After reacting at 94 ° C. for 15 seconds, the temperature was gradually increased from 60 ° C. to 99 ° C. at a rate of 0.2 ° C./second to prepare an amplification product denaturation curve.
  • These series of reactions were carried out by the ABI PRISM® 7900HT system (Applied Biosystems).
  • the obtained CT value was plotted on the vertical axis, and the number of sample bacteria used in the PCR reaction was plotted on the horizontal axis.
  • RT-PCR High correlation was obtained in the range of 1 ⁇ 10 ⁇ 3 to 1 ⁇ 10 3 per reaction. That is, any of the primer sets shown in Table 2 was able to detect RNA corresponding to 10 ⁇ 2 cells per RT-PCR reaction. This was estimated to be able to detect one bacterial cell when converted per 1 mL of blood.
  • the prepared Candida genus 5 species-specific primer and the Candida group-specific primer including Candida genus and its related species can specifically detect each target species.
  • Examples 1-2 and Comparative Examples 1-2 Nucleic acid extraction from bacterial cells and measurement of the number of bacteria using RT-qPCR method (1) Preparation of blood sample 1/10 volume of 3.8% sodium citrate aqueous solution was added to peripheral blood collected from healthy adults Anticoagulated. In addition, C.I. glabrata JCM3761 T , I.I. orientalis IFO1279 T and P. as a positive control. A pure culture specimen of aeruginosa ATCC10145 T was added to the collected human peripheral blood so that it would be 10 5 cells and 10 2 cells per mL. As a control, the bacterial solution was added to YM broth instead of blood.
  • RNAprotective Bacterial Reagent QIAGEN
  • RNAlater (Ambion) prepared in (1) were added to the sample tube and allowed to stand at room temperature for 5 minutes. Thereafter, the mixture was centrifuged at 13,000 g for 5 minutes, and the supernatant was removed by decantation.
  • lysis buffer prepared by mixing 346.5 ⁇ L RLT buffer, 100 ⁇ L TE and 3.5 ⁇ L ⁇ -Mercaptoethanol
  • 0.1 mm (300 mg) in diameter to the residue after removing the supernatant 0.5 mm (300 mg), 1 mm (300 mg) and 2.5 mm (10 grains, 250 to 300 mg) glass beads (TOMY Seiko, and BioSpec Products) were added in predetermined amounts.
  • the extraction operation was carried out in the same manner as in the methods described in 2) to 12) of Reference Example 1 (3).
  • RNA extraction of a standard strain for preparing a calibration curve was carried out under cell disruption conditions corresponding to the cell-added specimen.
  • Table 7 shows the results when the bacterial cell liquid was added to YM broth.
  • P. is a positive control.
  • aeruginosa the number of bacteria of the same degree as the number of added bacteria was measured in YM broth and human peripheral blood in any cell disruption using any glass beads.
  • the number of added bacteria was about 1/10 of the number of added bacteria in human peripheral blood specimens by crushing cells using 0.1 mm and 0.5 mm diameter glass beads (Comparative Example) 1 and 2).
  • cell disruption using glass beads having a diameter of 1.0 mm and 2.5 mm the number of bacteria of the same level as the number of added bacteria was measured in human peripheral blood (Examples 1 and 2).
  • Example 2 In the same manner as in Example 1, the specimen to which the bacterial cells were added was subjected to bacterial cell disruption using 1.0 mm diameter glass beads to extract nucleic acids. With respect to the extracted nucleic acid, the number of bacteria was measured by the RT-qPCR method, the number of added bacteria was plotted on the horizontal axis, and the number of measured bacteria obtained by the RT-qPCR method was plotted on the vertical axis (FIG. 2). For all the strains examined, when cells were added to human peripheral blood, linearity was observed in the range of 10 1 to 10 5 cells / mL, and the approximate equations were peripheral blood collected from different subjects and YM broth. There was almost agreement between the two. From the above results, it was considered that Candida 5 species in human peripheral blood can be accurately quantified by using 1.0 mm diameter glass beads and Candida 5 species specific primers.
  • Example 6 Bacterial addition recovery test to human peripheral blood using Candida group specific primer albicansIFO 1385 T , C.I. tropicalis JCM 1541 T , C.I. parapsilosis DSM 5784 T , C.I. glabrata JCM 3761 T , I.I.
  • Each of the pure cultured cells of orientalis IFO 1279 T was collected from 3 healthy adults into human peripheral blood (Subject A, Subject B, Subject C) and YM broth at 10 5 , 10 4 , 10 3 , 10, respectively. 2 and 10 1 cells were added (the total number of added bacteria was 5 ⁇ 10 5 , 5 ⁇ 10 4 , 5 ⁇ 10 3 , 5 ⁇ 10 2 and 5 ⁇ 10 1 cells per mL).
  • the sample to which the cells were added in the same manner as in Example 1 was subjected to cell disruption using 1.0 mm diameter glass beads, and the nucleic acid was extracted.
  • the number of bacteria was measured by the RT-qPCR method, the number of added bacteria was plotted on the horizontal axis, and the number of measured bacteria obtained by the RT-qPCR method was plotted on the vertical axis (FIG. 3).
  • FIG. 3 When cells were added to human peripheral blood, linearity was observed in the range of 10 1 to 10 5 cells / mL, and the approximate expression was almost the same between peripheral blood collected from different subjects and YM broth. . From the above results, it was considered that the genus Candida in human peripheral blood can be accurately quantified by using 1.0 mm diameter glass beads and Candida group-specific primers.

Abstract

　Provided is a method for effectively extracting nucleic acids of fungi from a biological sample that contains fungi.　The method for extracting fungal nucleic acids from a biological sample containing fungi is characterized in that, after bringing a sample solution that contains fungi into contact with beads having a diameter of 0.8-3mm and mixing the resultant solution, the nucleic acids are isolated.

Description

真菌核酸の抽出方法Method for extracting fungal nucleic acid
　本発明は、生体試料中に存在する真菌の核酸抽出方法に関する。 The present invention relates to a method for extracting nucleic acid of fungi present in a biological sample.
　Ｃａｎｄｉｄａ（カンジダ）属真菌は、ヒトの消化管、上気道、膣などの粘膜や皮膚に常在菌として定着しているが、免疫能が低下した場合において、侵襲性カンジダ感染症発症の原因となる。その中でも、カンジダ血症は、最も発症頻度が高く、その死亡率も非常に高いものとなっている。 Candida (Candida) fungi are established as permanent bacteria in the mucous membranes and skin of human digestive tract, upper respiratory tract, vagina, etc., but when immunity decreases, Become. Among them, candidemia has the highest frequency of occurrence and has a very high mortality rate.
　カンジダ血症の原因菌種として、Ｃａｎｄｉｄａ　ａｌｂｉｃａｎｓ及びｎｏｎ-ａｌｂｉｃａｎｓ　Ｃａｎｄｉｄａ　ｓｐｐ.に属する４菌種（Ｃ．ｇｌａｂｒａｔａ、Ｃ．ｔｒｏｐｉｃａｌｉｓ、Ｃ．ｐａｒａｐｓｉｌｏｓｉｓ及びＣ．ｋｒｕｓｅｉ）が広く知られており、原因菌の約９０％を占めている。また、これらのＣａｎｄｉｄａ属真菌は、菌種間で抗真菌薬への感受性が大きく異なっており、原因菌種を迅速に同定することが重要な課題となっている。 As the causative species of candidemia, four bacterial species belonging to Candida albicans and non-albicans Candida spp. (C. glabrata, C. tropicalis, C. parapsilosis and C. krusei) are widely known. It accounts for about 90%. Moreover, these Candida genus fungi are greatly different in susceptibility to antifungal agents among bacterial species, and it is an important issue to quickly identify the causative bacterial species.
　従来、血液検体からのＣａｎｄｉｄａ属真菌の定量は、（１→３）－β－Ｄ－グルカン測定法、病理組織学的検査法、血液培養法、及び血液培養法の補助的な検査法としてＰＣＲ法が使用されている。しかしながら、（１→３）－β－Ｄ－グルカン測定法、病理組織学的検査法では、菌種の同定が困難であり、培養検査法では培養工程に時間がかかるため、Ｃａｎｄｉｄａ血症の診断に時間を要するという問題がある。また、血液検体から直接真菌のＤＮＡを抽出してｑＰＣＲにより真菌を検出・定量する方法も知られている。この場合、酵素処理を用いてＤＮＡの抽出が行われる（非特許文献１）が、当該酵素処理は一般に処理や精製に時間がかかるほか、後述するビーズ法に比べ高価である。 Conventionally, quantification of Candida fungi from blood samples has been performed by PCR as an auxiliary test method for (1 → 3) -β-D-glucan measurement method, histopathological test method, blood culture method, and blood culture method. The law is used. However, in the (1 → 3) -β-D-glucan measurement method and histopathological examination method, it is difficult to identify the bacterial species, and in the culture examination method, the culturing process takes time. There is a problem that it takes time. In addition, a method for detecting and quantifying fungi by qPCR by directly extracting fungal DNA from a blood sample is also known. In this case, DNA is extracted using an enzyme treatment (Non-patent Document 1). However, the enzyme treatment generally takes time for treatment and purification, and is more expensive than the bead method described later.
　一方、血液検体中のＳｔａｐｈｙｌｏｃｏｃｃｕｓ　ａｕｒｅｕｓやＰｓｅｕｄｏｍｏｎａｓ　ａｅｒｕｇｉｎｏｓａといった細菌を検出・定量する方法として、血液検体に０．１ｍｍのガラスビーズを入れ、血液中の細菌の菌体を破砕（ビーズ法）し、ＲＮＡを抽出し、ＲＴ－ｑＰＣＲを用いて、検出・定量する方法が既に知られている（非特許文献２）。また、真菌の一種である酵母については、その形質転換体を培地にて培養し、その後、０．４５－０．５５ｍｍ程度のガラスビーズで菌体破砕する方法が知られている（特許文献１及び特許文献２）。 On the other hand, as a method for detecting and quantifying bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa in blood samples, 0.1 mm glass beads are placed in the blood sample, the bacterial cells in the blood are crushed (bead method), and RNA is collected. A method for extraction and detection / quantification using RT-qPCR is already known (Non-patent Document 2). In addition, for yeast, which is a kind of fungus, a method is known in which the transformant is cultured in a medium and then disrupted with about 0.45-0.55 mm glass beads (Patent Document 1). And Patent Document 2).
　しかしながら、生体試料から、ビーズを用いて真菌細胞を破砕し、その核酸を抽出して、真菌を検出・定量する方法はこれまでに知られていない。 However, a method for detecting and quantifying fungi has not been known so far by disrupting fungal cells from a biological sample using beads and extracting the nucleic acid.
特開昭６３－２２０９８号公報Japanese Patent Laid-Open No. 63-22098 国際公開第２０１０／０８２６４０号International Publication No. 2010/082640
　本発明は、真菌を含む生体試料から真菌核酸を効率的に抽出する方法を提供することに関する。 The present invention relates to providing a method for efficiently extracting fungal nucleic acids from a biological sample containing fungi.
　本発明者らは、血液等の生体試料から真菌の核酸を抽出する際に、従来、真菌や細菌の菌体破砕に用いられていた直径０．１～０．５ｍｍ程度のビーズを用いた場合には、生体試料中の真菌核酸を十分に抽出することができず、このため真菌を検出・定量できないが、直径が０．８ｍｍ～３ｍｍのビーズを使用した場合に、核酸が効率よく抽出され、真菌を正確に検出・定量できることを見い出した。 In the case of extracting fungal nucleic acid from a biological sample such as blood, the present inventors use beads having a diameter of about 0.1 to 0.5 mm, which have been conventionally used for disrupting fungal and bacterial cells. In this method, fungal nucleic acids in a biological sample cannot be sufficiently extracted, so that fungi cannot be detected and quantified. However, when beads having a diameter of 0.8 mm to 3 mm are used, nucleic acids are efficiently extracted. They found that fungi could be detected and quantified accurately.
　すなわち、本発明は、以下の１）～１０）に係るものである。
　１）真菌を含む生体試料から真菌核酸を抽出する方法であって、真菌を含む試料溶液を直径０．８ｍｍ～３ｍｍのビーズと接触させて撹拌した後、核酸を分離することを特徴とする、真菌核酸の抽出方法。
　２）真菌がカンジダ属真菌である、１）の方法。
　３）カンジダ属真菌が、カンジダ・アルビカンス、カンジダ・グラブラータ、カンジダ・パラプシローシス、カンジダ・トロピカリス及びカンジダ・クルーセイから選ばれる１以上である、２）の方法。
　４）ビーズがガラスビーズ又はジルコニア／シリカビーズである、１）～３）のいずれかの方法。
　５）ビーズの直径が１ｍｍ～２．５ｍｍである、１）～４）のいずれかの方法。
　６）生体試料がヒト血液である、１）～５）のいずれかの方法。
　７）１）～６）のいずれかの方法により抽出された核酸に特異的にハイブリダイズし得る核酸断片を用いて核酸増幅反応を行う、生体試料中の真菌の検出及び／又は定量方法。
　８）核酸断片が、配列番号１～１２で示される塩基配列からなる核酸断片又はそれと相補的な塩基配列からなる核酸断片である、７）の方法。
　９）直径０．８ｍｍ～３ｍｍのビーズ及び真菌核酸に特異的にハイブリダイズし得る核酸断片を含み、核酸抽出試薬、核酸増幅反応試薬及びプロトコールから選ばれる１種以上を含んでもよい、７）又は８）の方法を実施するためのキット。
　１０）ビーズの直径が１ｍｍ～２．５ｍｍである、９）のキット。 That is, the present invention relates to the following 1) to 10).
1) A method for extracting fungal nucleic acid from a biological sample containing fungi, wherein the sample solution containing fungus is contacted with beads having a diameter of 0.8 mm to 3 mm and stirred, and then the nucleic acid is separated. Extraction method of fungal nucleic acid.
2) The method according to 1), wherein the fungus is a Candida fungus.
3) The method of 2), wherein the Candida fungus is one or more selected from Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida crusei.
4) The method according to any one of 1) to 3), wherein the beads are glass beads or zirconia / silica beads.
5) The method according to any one of 1) to 4), wherein the beads have a diameter of 1 mm to 2.5 mm.
6) The method according to any one of 1) to 5), wherein the biological sample is human blood.
7) A method for detecting and / or quantifying fungi in a biological sample, wherein a nucleic acid amplification reaction is performed using a nucleic acid fragment that can specifically hybridize to the nucleic acid extracted by any one of the methods 1) to 6).
8) The method according to 7), wherein the nucleic acid fragment is a nucleic acid fragment consisting of a base sequence represented by SEQ ID NOs: 1 to 12 or a nucleic acid fragment consisting of a base sequence complementary thereto.
9) A nucleic acid fragment that can specifically hybridize to a bead having a diameter of 0.8 mm to 3 mm and a fungal nucleic acid, and may include one or more selected from a nucleic acid extraction reagent, a nucleic acid amplification reaction reagent, and a protocol, 7) or A kit for carrying out the method of 8).
10) The kit according to 9), wherein the beads have a diameter of 1 mm to 2.5 mm.
　本発明の方法によれば、真菌を含む生体試料から、直接真菌核酸を効率的に抽出することができ、生体試料中に存在する真菌数を正確に測定することができる。また、本発明によれば、生体試料中に含まれる菌体を培養すること無く、生体試料から直接菌体中の核酸を正確に測定できることから、短時間でＣａｎｄｉｄａ血症等の診断が可能である。 According to the method of the present invention, fungal nucleic acid can be efficiently extracted directly from a biological sample containing fungi, and the number of fungi present in the biological sample can be accurately measured. In addition, according to the present invention, since nucleic acids in bacterial cells can be accurately measured directly from a biological sample without culturing the bacterial cells contained in the biological sample, diagnosis of Candidaemia and the like is possible in a short time. is there.
Ｃａｎｄｉｄａ属５菌種特異的プライマー及びＣａｎｄｉｄａグループ特異的プライマーの検出感度を示したグラフ。The graph which showed the detection sensitivity of the Candida genus 5 species specific primer and the Candida group specific primer. Ｃａｎｄｉｄａ属５菌種特異的プライマーを用いたヒト末梢血への菌体添加回収試験の結果を示すグラフ。The graph which shows the result of the microbial cell addition collection | recovery test to the human peripheral blood using the Candida genus 5 species specific primer. Ｃａｎｄｉｄａグループ特異的プライマーを用いたヒト末梢血への菌体添加回収試験の結果を示すグラフ。The graph which shows the result of the microbial cell addition collection | recovery test to the human peripheral blood using the Candida group specific primer.
　本発明において、真菌を含む生体試料としては、真菌が存在し得る生体サンプルであれば特に限定されず、血液、尿、髄液、精液、喀痰、咽頭ぬぐい液、子宮頸管粘液、膣分泌物、腹水、組織、結膜ぬぐい液、歯石、歯垢、唾液、鼻汁、肺胞洗浄液、胸水、胃液、胃洗浄液、皮膚病巣、糞便、関節液、患部ぬぐい液等の生体試料が挙げられる。このうち、血液が好適であり、ヒト末梢血がより好適である。 In the present invention, the biological sample containing fungi is not particularly limited as long as it is a biological sample in which fungi can exist, and blood, urine, spinal fluid, semen, sputum, throat swab, cervical mucus, vaginal secretions, Examples include biological samples such as ascites, tissue, conjunctival wipes, calculus, plaque, saliva, nasal discharge, alveolar lavage fluid, pleural effusion, gastric juice, gastric lavage fluid, skin lesions, feces, joint fluid, and affected area wipes. Of these, blood is preferred, and human peripheral blood is more preferred.
　本発明において、「真菌」とは、真正菌類Ｅｕｍｙｃｏｔａを意味し、酵母を含む総称である。例えば、カンジダ（Candida）属、イサチェンキア（Issatchenkia）属、アスペルギルス（Aspergillus）属、ハンセヌラ（Hansenula）属、サッカロマイセス（Saccharomyces）属、トリコスポロン（Trichosporon）属、ペニシリウム（Penicillium）属、マラセチア（Malassezia）属、スポロトリックス（Sporothrix）属、アブシジア（Absidia）属、ムーコル（Mucor）属、リゾムーコル（rhizomucor）属、リゾプス（rhizopus）属、ニューモシスチス（Pneumocystis）属、コクシジオイデス（coccidioidomycosis）属、ヒストプラスマ（Histoplasma）属、パラコクシジオイデス（Paracoccidioides）属などに属する真菌が含まれ、このうちカンジダ属、イサチェンキア属、アスペルギルス属真菌が好適であり、カンジダ属真菌がより好適である。 In the present invention, “fungus” means the eubacteria Eumycota and is a generic name including yeast. For example, Candida genus, Issatchenkia genus, Aspergillus genus, Hansenula genus, Saccharomyces genus, Trichosporon genus, Penicillium genus, z Sporothrix genus, Absidia genus, Mucor genus, Rhizomucor genus, Rhizopus genus, Pneumocystis genus, Coccidioidomycosis genus, Histoplasma genus Histoplasma Fungi belonging to the genus Paracoccidioides (Paracoccidioides) and the like are included, among which Candida, Isachenchia, and Aspergillus are preferred, and Candida is more preferred.
　カンジダ属真菌としては、カンジダ・アルビカンス（Candida albicans）、カンジダ・グラブラータ（Candida glabrata）、カンジダ・パラプシローシス（Candida parapsilosis）、カンジダ・トロピカリス（Candida tropicalis）、カンジダ・クルーセイ（Candida krusei）、カンジダ・ギリエルモンジイ（Candida guilliermondii）、カンジダ・ルシタニアエ（Clavispora lusitaniae）等が挙げられ、カンジダ・アルビカンス、カンジダ・グラブラータ、カンジダ・パラプシローシス、カンジダ・トロピカリス、カンジダ・クルーセイがより好ましい。 The Candida fungi include Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei and Candida krusei, (Candida guilliermondii), Candida lucitaniae (Clavispora lusitaniae) and the like, and Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida crusei are more preferable.
　イサチェンキア属真菌としては、イサチェンキア・オリエンタリス（Issatchenkia orientalis）、イサチェンキア・テリコラ(Issatchenkia terricola)等が挙げられ、イサチェンキア・オリエンタリスがより好ましい。
　なお、イサチェンキア・オリエンタリスとカンジダ・クルーセイは同一の菌であるが、有性世代をイサチェンキア・オリエンタリス、無性世代をカンジダ・クルーセイという。
　アスペルギルス属真菌としては、アスペルギルス・フミガタス（Aspergillus fumigatus）、アスペルギルス・フラバス（Aspergillus flavus）、アスペルギルス・ニガー（Aspergillus niger）等が挙げられる。 Examples of the fungus belonging to the genus Isachenchia include Issatchenkia orientalis and Issatchenkia terricola, with Isachenchia orientalis being more preferred.
In addition, Isachenchia Orientalis and Candida Crusei are the same bacteria, but the sexual generation is called Isachenchia Orientalis and the asexual generation is Candida Crusei.
Examples of Aspergillus fungi include Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and the like.
　本発明において、核酸とは、１本鎖又は２本鎖のＤＮＡ又はＲＮＡを意味する。
　また、真菌を含む試料溶液は、生体から採取された生体試料、又は当該試料から適宜菌体を濃縮処理したサンプルを、蒸留水、緩衝液、生理食塩水等の液体に懸濁した溶液が挙げられる。ここで、緩衝液としては、リン酸緩衝液（ＰＢＳ）、トリス塩酸バッファー、溶菌バッファー（ＲＬＴ　ｂｕｆｆｅｒ、ＴＥ（Ｔｒｉｓ－ＥＤＴＡ）及びβ‐Ｍｅｒｃａｐｔｏｅｔｈａｎｏｌを混合したもの）等を用いることができ、溶菌バッファーが好適である。
　菌体濃縮処理としては、例えば遠心分離、ろ過濃縮等が挙げられるが、遠心分離が好適である。また、当該処理に際しては、ＲＮＡ安定化剤（例えば、ＲＮＡｐｒｏｔｅｃｔ　Ｂａｃｔｅｒｉａｌ　Ｒｅａｇｅｎｔ（QIAGEN）、ＲＮＡｌａｔｅｒ（Ambion）等）を添加して行うのが好ましい。
　尚、本発明の方法において用いられる生体試料は、血液培養法のように予め培養して菌体を増殖させた試料であっても、培養せずに生体から採取した試料であってもよい。 In the present invention, the nucleic acid means single-stranded or double-stranded DNA or RNA.
Examples of the sample solution containing fungi include a biological sample collected from a living body, or a solution obtained by suspending a sample obtained by appropriately concentrating bacterial cells from the sample in a liquid such as distilled water, buffer solution, or physiological saline. It is done. Here, as the buffer solution, a phosphate buffer solution (PBS), Tris-HCl buffer, lysis buffer (a mixture of RLT buffer, TE (Tris-EDTA) and β-Mercaptoethanol) or the like can be used. Is preferred.
Examples of the bacterial cell concentration treatment include centrifugation, filtration concentration, and the like. Centrifugation is preferable. In addition, the treatment is preferably performed by adding an RNA stabilizer (eg, RNA protect Bacterial Reagent (QIAGEN), RNAlater (Ambion), etc.).
The biological sample used in the method of the present invention may be a sample obtained by culturing cells in advance as in the blood culture method, or a sample collected from a living body without culturing.
　斯かる真菌を含む試料溶液とビーズとの接触は、当該試料溶液中にビーズを添加し撹拌する（接触撹拌処理）ことにより行われる。
　ここで、「ビーズ」は、直径が０．８ｍｍ以上３ｍｍ以下であるという条件を満たすものであれば、その材質は特に限定されず、無機材料でも有機材料であってもよい。また、多孔性又は非多孔性の何れでもよい。例えば、ガラス（ホウケイ酸ガラス、石灰ガラス）ビーズ、ジルコニアビーズ、シリカビーズ、ジルコニア／シリカビーズ（１ビーズ中にジルコニアとシリカの両方を成分として含むビーズ）、ステンレスビーズ、ポリスチレンビーズ等が挙げられ、このうち、ガラスビーズ、ジルコニア／シリカビーズが好ましい。斯かるビーズは市販品（例えば、株式会社トミー精工、BioSpec Products社等のビーズ）を購入して使用することができる。 Contact between the sample solution containing such fungi and the beads is performed by adding the beads to the sample solution and stirring (contact stirring treatment).
Here, the material of the “bead” is not particularly limited as long as it satisfies the condition that the diameter is 0.8 mm or more and 3 mm or less, and may be an inorganic material or an organic material. Further, it may be porous or non-porous. Examples include glass (borosilicate glass, lime glass) beads, zirconia beads, silica beads, zirconia / silica beads (beads containing both zirconia and silica in one bead), stainless beads, polystyrene beads, and the like. Of these, glass beads and zirconia / silica beads are preferred. Such beads can be purchased from commercial products (for example, beads from Tommy Seiko Co., Ltd., BioSpec Products, etc.).
　ビーズの直径は、真菌核酸の抽出効率を向上する点から、０．８ｍｍ以上であるのが好ましく、１ｍｍ以上であるのがより好ましい。また、３ｍｍ以下であるのが好ましく、２．５ｍｍ以下がより好ましい。さらに、直径の大きなビーズは、核酸抽出時に使用するサンプルチューブへのセットが困難になるため、１ｍｍ～２．５ｍｍが好ましく、１ｍｍがより好ましい。 The diameter of the beads is preferably 0.8 mm or more, more preferably 1 mm or more, from the viewpoint of improving fungal nucleic acid extraction efficiency. Moreover, it is preferable that it is 3 mm or less, and 2.5 mm or less is more preferable. Furthermore, since beads having a large diameter are difficult to set in a sample tube used for nucleic acid extraction, 1 mm to 2.5 mm is preferable, and 1 mm is more preferable.
　撹拌方法は、菌体とビーズが十分に接触できれば特に限定されず、試料溶液を入れた容器を手動で往復振動させることでも良いが、抽出効率及び再現性の点から、振とう機やビーズ破砕機を用いて攪拌することが好ましい。この場合、攪拌時間は、真菌の細胞膜が破壊されるまででよく、具体的には１～１０分間程度が好ましく、２～５分間程度がより好ましい。 The stirring method is not particularly limited as long as the cells and the beads can be sufficiently contacted, and the container containing the sample solution may be manually reciprocally vibrated, but from the viewpoint of extraction efficiency and reproducibility, a shaker or bead crushing It is preferable to stir using a machine. In this case, the stirring time may be until the cell membrane of the fungus is broken, and specifically, it is preferably about 1 to 10 minutes, more preferably about 2 to 5 minutes.
　また、ビーズによる接触撹拌処理においては、必要に応じて、核酸の抽出効率を更に向上させるために、酵素や界面活性剤を添加してもよい。この場合の酵素としては、例えばプロテアーゼＰが挙げられる。また、界面活性剤としては、例えばＴｒｉｔｏｎＸ－１００が挙げられる。 In the contact stirring treatment with beads, an enzyme or a surfactant may be added as necessary to further improve the nucleic acid extraction efficiency. Examples of the enzyme in this case include protease P. Examples of the surfactant include Triton X-100.
　斯くして、上記ビーズによる接触撹拌処理によれば、真菌細胞を効率良く破砕することができ、真菌核酸を容易に遊離させることができる。 Thus, according to the contact stirring treatment with the beads, fungal cells can be efficiently disrupted and fungal nucleic acids can be easily released.
　遊離した核酸の分離は、核酸の分離抽出法として知られている公知の方法、例えば、フェノール－クロロホルム法やグアニジン法等の汎用法を採用することにより行うことができる。
　例えば、前記ビーズにより接触撹拌処理された試料溶液に、フェノールを加えて、反応させた後、クロロホルム／イソアミルアルコール（またはフェノール／クロロホルム／イソアミルアルコール）を加えて撹拌し、遠心分離後その上清を回収し、塩化ナトリウム溶液、酢酸ナトリウム緩衝液、酢酸アンモニウム緩衝液等を添加して、エタノール沈殿することにより、核酸を抽出することができる。尚、ＲＮＡの抽出を主目的とする場合は、フェノールを加えた際に５０～７０℃で５～１５分間反応させるホットフェノール法を行うことが好ましい。また、クロロホルム／イソアミルアルコールを使用することが好ましい。さらに、エタノール沈殿させた沈殿物は、Ｎｕｃｌｅａｓｅ－ｆｒｅｅ　ｗａｔｅｒで溶解させることが好ましい。一方、ＤＮＡの抽出を主目的とする場合は、フェノール／クロロホルム／イソアミルアルコールを使用することが好ましく、反応は常温で行うことができる。また、エタノール沈殿させた沈殿物は、ＴＥで溶解させることが好ましい。 Separation of the released nucleic acid can be performed by employing a known method known as a method for separating and extracting nucleic acid, for example, a general-purpose method such as a phenol-chloroform method or a guanidine method.
For example, phenol is added to the sample solution that has been contact-stirred with the beads and allowed to react, then chloroform / isoamyl alcohol (or phenol / chloroform / isoamyl alcohol) is added and stirred, and the supernatant is removed after centrifugation. The nucleic acid can be extracted by recovering, adding a sodium chloride solution, a sodium acetate buffer solution, an ammonium acetate buffer solution, etc., and precipitating with ethanol. When the main purpose is RNA extraction, it is preferable to carry out a hot phenol method in which phenol is added and reacted at 50 to 70 ° C. for 5 to 15 minutes. It is also preferable to use chloroform / isoamyl alcohol. Further, the ethanol-precipitated precipitate is preferably dissolved with Nuclease-free water. On the other hand, when the main purpose is DNA extraction, phenol / chloroform / isoamyl alcohol is preferably used, and the reaction can be carried out at room temperature. Moreover, it is preferable to dissolve the ethanol-precipitated precipitate with TE.
　斯くして抽出された核酸を、目的の真菌核酸に特異的にハイブリダイズし得る核酸断片を用い、公知の核酸増幅法により増幅してその量を測定し、これに基づき生体試料中の真菌の検出及び／又は定量を行うことができる。
　斯かる核酸増幅法としては、例えば、ＰＣＲ（Polymerase Chain Reaction）、ＲＴ－ＰＣＲ（Reverse-Transcriptase PCR）、ＬＣＲ（Ligase Chain Reaction）、ＬＡＭＰ（Loop-mediated Isothermal Amplification of DNA）、ＮＡＳＢＡ（Nucleic Acid Sequence Based Amplification；Nature, 1991 Mar. 7;350(6313);91-2）、ＴＭＡ（Transcription-Mediated Amplification；Advanced Biomedical Technologies. 1998; 189-201）、ＴＲＣ（Transcription Reverse Transcription Concerted Reaction；Anal Biochem, 2003 Mar. 1;314(1):77-86）等が挙げられる。 The nucleic acid thus extracted is amplified by a known nucleic acid amplification method using a nucleic acid fragment that can specifically hybridize to the target fungal nucleic acid, and the amount thereof is measured. Based on this, the amount of the fungus in the biological sample is measured. Detection and / or quantification can be performed.
Examples of such nucleic acid amplification methods include PCR (Polymerase Chain Reaction), RT-PCR (Reverse-Transcriptase PCR), LCR (Ligase Chain Reaction), LAMP (Loop-mediated Isothermal Amplification of DNA), NASBA (Nucleic Acid Sequence). Based Amplification; Nature, 1991 Mar. 7; 350 (6313); 91-2), TMA (Transcription-Mediated Amplification; Advanced Biomedical Technologies. 1998; 189-201), TRC (Transcription Reverse Transcription Concerted Reaction; Anal Biochem, 2003) Mar. 1; 314 (1): 77-86).
　目的の真菌核酸に特異的にハイブリダイズし得る核酸断片の設計は、当該真菌核酸の塩基配列を参酌することにより適宜設計することができる。
　例えば、カンジダ・アルビカンス、カンジダ・トロピカリス、カンジダ・パラプシローシス、カンジダ・グラブラータ、カンジダ・クルーセイ及びカンジダ属のｒＲＮＡに特異的にハイブリダイズし得る核酸断片としては、後述する配列番号１～１２で示される塩基配列若しくはそれと相補的な塩基配列からなる核酸断片、或いは当該塩基配列のうちの１又は数個、好ましくは１乃至１０個の塩基が置換、付加又は欠失した塩基配列からなる核酸断片、或いは当該塩基配列と９０％以上、好ましくは９５％以上、より好ましくは９９％以上の同一性を有する塩基配列からなる核酸断片、或いは当該塩基配列と相補的な塩基配列からなるＤＮＡとストリンジェントな条件下でハイブリダイズする塩基配列からなる核酸断片が挙げられる。
　尚、塩基配列の同一性は、ＧＥＮＥＴＹＸ（Ｒ）のホモロジー解析プログラムを用いることにより算出される。
　また、「ストリンジェントな条件」としては、例えば、５０％ホルムアミド、５×ＳＳＣ、５×デンハルト溶液及び２５０ｍｇ／ｍＬサケ精子ＤＮＡを含む溶液に４２℃で１６～２４時間恒温し、ハイブリダイズさせる条件が挙げられる。 The design of a nucleic acid fragment that can specifically hybridize to the target fungal nucleic acid can be appropriately designed by taking into account the base sequence of the fungal nucleic acid.
For example, nucleic acid fragments that can specifically hybridize to Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida crusei, and Candida rRNA are represented by SEQ ID NOS: 1 to 12 described below. A nucleic acid fragment consisting of a base sequence or a complementary base sequence, or a nucleic acid fragment consisting of a base sequence in which one or several, preferably 1 to 10 bases of the base sequence are substituted, added or deleted, or A stringent condition with a nucleic acid fragment consisting of a base sequence having 90% or more, preferably 95% or more, more preferably 99% or more identity with the base sequence, or a DNA consisting of a base sequence complementary to the base sequence Examples thereof include a nucleic acid fragment consisting of a base sequence that hybridizes below.
The identity of the base sequence is calculated by using the GENETYX (R) homology analysis program.
“Stringent conditions” include, for example, conditions in which 50% formamide, 5 × SSC, 5 × Denhardt's solution and 250 mg / mL salmon sperm DNA are incubated at 42 ° C. for 16 to 24 hours and hybridized. Is mentioned.
　核酸増幅法としてＰＣＲあるいはＲＴ－ＰＣＲを用いる場合、（１）上記のように試料溶液より抽出したＤＮＡ又はＲＮＡに対して、上記核酸断片の１以上を用いてＰＣＲあるいはＲＴ－ＰＣＲを行う工程、及び（２）工程（１）による増幅されたＤＮＡ断片を検出する工程により行うことができる。目的の真菌由来の鋳型ＤＮＡ（鋳型がＲＮＡの場合はｃＤＮＡ）に上記核酸断片を組み合わせ、増幅反応を行うことにより、目的の真菌に特異的なＤＮＡ断片（ＰＣＲ産物）を得ることができる。このようにして得られたＤＮＡを電気泳動すれば、バンドの有無から目的の真菌を特異的に検出、同定することができる。
　また、鋳型のＤＮＡ又はＲＮＡ（ｃＤＮＡ）を段階的に希釈しＰＣＲを行えば、目的の真菌の定量化も可能である。ＰＣＲを用いて定量を行う際は、上記方法の他、リアルタイムＰＣＲを用いる方法がより好ましい。ＰＣＲにより増幅されるＰＣＲ産物量を経時的に観察し、一定のＤＮＡ量に達した時のＰＣＲサイクル数を特定することにより、試料溶液中の目的の真菌の定量が可能となる。 When PCR or RT-PCR is used as a nucleic acid amplification method, (1) a step of performing PCR or RT-PCR using one or more of the above nucleic acid fragments on DNA or RNA extracted from a sample solution as described above, And (2) the step of detecting the amplified DNA fragment in step (1). A DNA fragment (PCR product) specific to the target fungus can be obtained by combining the nucleic acid fragment with a template DNA derived from the target fungus (cDNA when the template is RNA) and performing an amplification reaction. When the DNA thus obtained is electrophoresed, the target fungus can be specifically detected and identified from the presence or absence of a band.
In addition, if the template DNA or RNA (cDNA) is diluted stepwise and PCR is performed, the target fungus can be quantified. When performing quantification using PCR, in addition to the above method, a method using real-time PCR is more preferable. By observing the amount of PCR product amplified by PCR over time and specifying the number of PCR cycles when a certain amount of DNA is reached, the target fungus in the sample solution can be quantified.
　増幅されるＰＣＲ産物の経時的な観察は、ＳＹＢＲ（Ｒ）Ｇｒｅｅｎ　Ｉ等のインターカレーターである蛍光色素により標識し、各ＰＣＲ段階での蛍光強度を測定することにより行うことができる。インターカレーターは二本鎖核酸にインターカレーションすることで蛍光強度が増加する性質を有することから、標的真菌のＤＮＡ（ＲＮＡの場合はｃＤＮＡ）からＰＣＲ反応により生成するＰＣＲ産物を正確に測定することができ、特にＳＹＢＲ（Ｒ）Ｇｒｅｅｎ　Ｉが好適に用いられる。任意に設定された一定の蛍光強度（ＤＮＡ量）に達した時のＰＣＲサイクル数（以下ＣＴ値とする）を特定することにより、生体試料中の標的真菌の定量、或いは検出・同定が可能となる。また、蛍光色素により標識したＴａｑＭａｎプローブやＭｏｌｅｃｕｌｅｒ　Ｂｅａｃｏｎ等を使用することにより行うこともできる。ＴａｑＭａｎプローブやＭｏｌｅｃｕｌｅｒ　Ｂｅａｃｏｎは、ＰＣＲにより増幅される領域の内部配列と相同性を有するオリゴヌクレオチドに蛍光色素とクエンチャーを結合させたプローブを用いた検出法である。プローブに結合した蛍光色素とクエンチャーの相互作用でＰＣＲ増幅反応に応じた蛍光を発するため、各ＰＣＲ段階での蛍光強度を測定することにより増幅されるＰＣＲ産物の経時的な観察を行うことができる。 The PCR product to be amplified can be observed over time by labeling with a fluorescent dye that is an intercalator such as SYBR (R) Green I and measuring the fluorescence intensity at each PCR stage. Since the intercalator has the property that the fluorescence intensity increases by intercalating into double-stranded nucleic acid, it accurately measures the PCR product generated by PCR reaction from the DNA of the target fungus (cDNA in the case of RNA) In particular, SYBR (R) Green I is preferably used. By specifying the number of PCR cycles (hereinafter referred to as the CT value) when a certain set fluorescence intensity (DNA amount) is reached, the target fungus in the biological sample can be quantified or detected / identified. It becomes. It can also be carried out by using a TaqMan probe labeled with a fluorescent dye, a Molecular Beacon, or the like. TaqMan probe and Molecular Beacon are detection methods using a probe in which a fluorescent dye and a quencher are bound to an oligonucleotide having homology with the internal sequence of a region amplified by PCR. Since the fluorescence corresponding to the PCR amplification reaction is emitted by the interaction between the fluorescent dye and the quencher bound to the probe, the PCR product amplified over time can be observed by measuring the fluorescence intensity at each PCR stage. it can.
　生体試料中の目的の真菌の定量、或いは検出・同定は、別途計測した真菌数の対数値とＣＴ値の検量線により求めることができる。すなわち、標的とする真菌数の対数値を横軸に、ＣＴ値を縦軸にプロットした検量線を予め作成し、ＰＣＲ反応の結果得られたＣＴ値を該検量線に代入して、生体試料中の目的の真菌の定量、或いは検出・同定を行う。 Fungi of interest in a biological sample quantitatively, or detection and identification can be determined by a separate calibration curve of logarithmic values and C T values of the fungal number measured. That is, the logarithm of the fungal number of targeted horizontal axis, and the C T value in advance to create the plotted calibration curve on the vertical axis, substituting the C T values obtained as a result of the PCR reactions calibration curve, Quantify or detect / identify the target fungus in a biological sample.
　上記核酸断片は、上記のＰＣＲ法又はＲＴ－ＰＣＲ法においてプライマーとして用いる他、単独でもプローブとして使用でき、これらは他の公知のユニバーサルプライマー、オリゴヌクレオチド等と組み合わせて用いることもできる。 The nucleic acid fragment can be used as a primer in the PCR method or RT-PCR method, or can be used alone as a probe, and these can be used in combination with other known universal primers, oligonucleotides, and the like.
　上記核酸断片をプローブとして用いる分析方法としては、例えばインサイチュハイブリダイゼーション（ｉｎ　ｓｉｔｕ　ｈｙｂｒｉｄｉｚａｔｉｏｎ）、ドットブロットハイブリダイゼーション（ｄｏｔ　ｂｌｏｔｈｙｂｒｉｄｉｚａｔｉｏｎ）等が挙げられ、中でもインサイチュハイブリダイゼーションは迅速な手法として好ましく、蛍光物質により標識した核酸断片をプローブとして用いるＦＩＳＨがより好ましい。 Examples of the analysis method using the nucleic acid fragment as a probe include in situ hybridization and dot blot hybridization. Among them, in situ hybridization is preferable as a rapid method, and is labeled with a fluorescent substance. FISH using the obtained nucleic acid fragment as a probe is more preferable.
　ＦＩＳＨは、具体的には、（１）生体試料をホルムアルデヒド或いはホルマリンにより固定する工程、（２）固定した生体試料をスライドグラス又はメンブレンフィルターに塗抹する工程、（３）蛍光標識した核酸断片によりハイブリダイゼーションを行う工程、（４）ハイブリダイズ後の余分な核酸断片及び非特異的に結合した核酸断片を洗浄する工程、及び（５）ハイブリダイズ後の結果について蛍光顕微鏡を用いて肉眼的に観察、或いはＣＣＤカメラ等により画像として取得する工程により行うことができる。 Specifically, FISH consists of (1) a step of fixing a biological sample with formaldehyde or formalin, (2) a step of smearing the fixed biological sample on a slide glass or a membrane filter, and (3) a high level by fluorescently labeled nucleic acid fragments. A step of performing hybridization, (4) a step of washing excess nucleic acid fragments after hybridization and non-specifically bound nucleic acid fragments, and (5) macroscopic observation of the result after hybridization using a fluorescence microscope, Or it can carry out by the process of acquiring as an image with a CCD camera etc.
　生体試料中に目的の真菌が存在する場合は、用いた上記核酸断片とハイブリダイズし、上記ハイブリダイズ後の結果におけるシグナルが陽性となるので、これらの真菌を特異的に検出し、同定することができる。また、シグナル強度を測定することにより、定量化も可能となる。 When the target fungus is present in the biological sample, it hybridizes with the nucleic acid fragment used, and the signal in the result after the hybridization becomes positive, so these fungi must be specifically detected and identified. Can do. Moreover, quantification is also possible by measuring the signal intensity.
　上記生体試料中の真菌の検出及び／又は定量は、上述した本発明の真菌核酸の抽出方法により抽出された核酸について、これに特異的にハイブリダイズし得る核酸断片を用いて核酸増幅反応を行うためのキットにより行うことができる。ここで、斯かる方法を実施するためのキットとしては、当該方法の全部又は一部の工程を行うのに必要なものの全部又は一部を集めたものが挙げられる。ここで、「工程を行うのに必要なもの」は、本明細書の記載を参酌することにより、適宜選択することができる。例えば、（１）直径０．８ｍｍ～３ｍｍのビーズ及び（２）真菌核酸に特異的にハイブリダイズし得る核酸断片を含み、（３）核酸抽出のための各種試薬（例えば、試料調製のための緩衝液、ＲＮＡの固定試薬、核酸分離試薬）（核酸抽出試薬）、（４）核酸増幅反応に用いる試薬（核酸増幅反応試薬）、及び（５）実施方法を記載したプロトコール（プロトコール）から選ばれる１種以上を含んでもよいキットが挙げられる。 In the detection and / or quantification of the fungus in the biological sample, the nucleic acid extracted by the above-described fungal nucleic acid extraction method of the present invention is subjected to a nucleic acid amplification reaction using a nucleic acid fragment that can specifically hybridize to the nucleic acid. Can be carried out with a kit for Here, as a kit for carrying out such a method, a kit in which all or part of what is necessary for carrying out all or part of the method is collected. Here, “necessary for performing the process” can be appropriately selected by taking into account the description of the present specification. For example, (1) including beads having a diameter of 0.8 mm to 3 mm and (2) a nucleic acid fragment capable of specifically hybridizing to fungal nucleic acid, and (3) various reagents for nucleic acid extraction (for example, for sample preparation) Buffer, RNA fixing reagent, nucleic acid separation reagent) (nucleic acid extraction reagent), (4) reagent used for nucleic acid amplification reaction (nucleic acid amplification reaction reagent), and (5) protocol describing the method of implementation (protocol) Examples include kits that may include one or more.
　次に実施例を挙げて本発明を詳細に説明する。 Next, the present invention will be described in detail with reference to examples.
［１］使用菌株
　株式会社ヤクルト本社中央研究所にて保存していた、表１に示す菌株を使用した。各菌株の初発菌数は、１×１０５ｃｅｌｌｓ程度となるように調整した。
　各菌株の培養条件を表１に示した。培養条件Ａ～Ｅの詳細は以下の通りである。
　条件Ａ：ＹＭブロスにて、２６℃、好気条件下で２４～４８時間の振とう培養を行った。
　条件Ｂ：１％グルコース加変法ＧＡＭブロスにて、３７℃、嫌気条件下で１８～７２時間の静置培養を行った。
　条件Ｃ：ブレインハートインフュージョンブロスにて、３７℃、好気条件下で１８時間の静置培養を行った。
　条件Ｄ：ＭＲＳブロスにて、３７℃、嫌気条件下で２４時間静置培養を行った。
　条件Ｅ：プレストン培地にて、３７℃、微好気条件下で２４時間静置培養を行った。
　これらの菌体は、ＤＡＰＩ法により菌数を測定した後、一定の菌数となるように適宜希釈し、菌液を調製した。 [1] Strains used The strains shown in Table 1 which were stored at Yakult Central Research Laboratory, Inc. were used. The initial bacterial count of each strain was adjusted to be about 1 × 10 5 cells.
The culture conditions for each strain are shown in Table 1. Details of the culture conditions A to E are as follows.
Condition A: Shaking culture was performed in YM broth under aerobic conditions at 26 ° C. for 24 to 48 hours.
Condition B: Static culture was performed for 18 to 72 hours under anaerobic conditions at 37 ° C. in GAM broth supplemented with 1% glucose.
Condition C: static culture was performed in a brain heart infusion broth at 37 ° C. under aerobic conditions for 18 hours.
Condition D: Static culture was performed in MRS broth at 37 ° C. under anaerobic conditions for 24 hours.
Condition E: Static culture was performed in Preston medium at 37 ° C. under microaerobic conditions for 24 hours.
After measuring the number of cells by the DAPI method, these cells were appropriately diluted so as to have a certain number of cells to prepare a bacterial solution.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
参考例１　Ｃａｎｄｉｄａ属５菌種特異的プライマー及びＣａｎｄｉｄａグループ特異的プライマーの調製
　Ｃａｎｄｉｄａ属の検出・定量をＲＴ－ＰＣＲ法で行う際に使用するプライマーの調製を行った。
（１）使用したプライマー
　使用したプライマーを表２に示す。 Reference Example 1 Preparation of Candida genus 5 species-specific primer and Candida group-specific primer Primers used for detection and quantification of Candida genus by RT-PCR were prepared.
(1) Used primers Table 2 shows the used primers.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（２）プライマーの設計
　Ｃａｎｄｉｄａ属菌種及びその近縁菌種（以下、Ｃａｎｄｉｄａグループ）の１８Ｓ又は２６ＳｒＲＮＡ遺伝子配列をデータベースより入手した。Ｃｌｕｓｔａｌ　Ｘソフトウェア（Thompson, J. D., Higgins, D. G., and Gibson, T. J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680）を用いて得られた配列の多重整列を行い、近隣接合法による系統樹を作成した。整列配列の比較から、Ｃ．ａｌｂｉｃａｎｓ、Ｃ．ｔｒｏｐｉｃａｌｉｓ、Ｃ．ｐａｒａｐｓｉｌｏｓｉｓ、Ｃ．ｇｌａｂｒａｔａ、Ｃ．ｋｒｕｓｅｉ及びＣａｎｄｉｄａ属及びその近縁種にそれぞれ特異的な配列を同定し、それらの配列情報をもとに菌種特異的プライマー及びＣａｎｄｉｄａグループ特異的プライマーを設計した。 (2) Primer design The 18S or 26S rRNA gene sequences of Candida spp. And related strains (hereinafter Candida group) were obtained from the database. Clustal X software (Thompson, JD, Higgins, DG, and Gibson, TJ 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673 -4680) was used for multiple alignment of the sequences, and a phylogenetic tree was created by the neighbor joining method. From comparison of aligned sequences, C.I. albicans, C.I. tropicalis, C.I. parapsilosis, C.I. glabrata, C.I. Specific sequences were identified for each of the genus krusei and Candida and related species, and based on the sequence information, species-specific primers and Candida group-specific primers were designed.
（３）プライマーの検出感度の検討
　表２に示した全てのプライマーセットについて、それぞれの標準菌株を純培養した菌体よりＲＮＡを抽出し、ＲＴ－ｑＰＣＲにより検量線を作成した。具体的には以下の方法を行った。
　　１）サンプルチューブに、上記［１］使用菌株で調製した各標準菌株の菌液２００μＬ及びＲＮＡｌａｔｅｒ（Ａｍｂｉｏｎ）４００μＬを添加し、５分間室温にて静置した。その後、１３，０００ｇにて５分間遠心分離し、デカンテーションにより上清を除去した。上清を除去した後の残渣に溶菌バッファー　４５０μＬ（１サンプルあたり３４６．５μＬのＲＬＴ　ｂｕｆｆｅｒ、１００μＬのＴＥ及び３.５μＬのβ―Ｍｅｒｃａｐｔｏｅｔｈａｎｏｌを混合して調製する）及び直径が１ｍｍ（３００ｍｇ）のガラスビーズ（ＴＯＭＹ精工）を添加した。
　　２）振とう機（ＳｈａｋｅＭａｓｔｅｒ）にサンプルチューブをセットした後、５分間振とうし、菌体を破砕した。
　　３）５００μＬ水飽和Ｐｈｅｎｏｌを加え、ボルテックスにより５～１０秒間撹拌した。
　　４）６０℃のヒートブロックにサンプルチューブをセットし、１０分間反応させた（ホットフェノール法）。
　　５）１００μＬ　Ｃｈｌｏｒｏｆｏｒｍ／Ｉｓｏａｍｙｌ　ａｌｃｈｏｌ（２４：１）を加え、ボルテックスにより５～１０秒間撹拌した。
　　６）遠心分離後（１３，０００ｇ×５分）、上清４７０μＬを新しい蓋付マイクロチューブ（１．５ｍＬ）に移した。
　　７）４７０μＬ　Ｃｈｌｏｒｏｆｏｒｍ／Ｉｓｏａｍｙｌ　ａｌｃｈｏｌ（２４：１）を加え、ボルテックスにより５～１０秒間撹拌した。
　　８）遠心分離後（１３，０００ｇ×５分）、上清４００μＬを新しい蓋付マイクロチューブ（１．５ｍＬ）に移した。
　　９）３Ｍ　酢酸Ｎａ（ｐＨ５．４）４０μＬ及びＩｓｏｐｒｏｐａｎｏｌ　４００μＬを加え、転倒混和した。
　　１０）遠心分離（２０，０００ｇ×１０分）を行った。
　　１１）デカンテーションにて上清を除いた後、８０％Ｅｔｈａｎｏｌ　５００μＬを加えた。
　　１２）遠心分離後（２０，０００ｇ×２分）、デカンテーションにて上清を除いた。
　　１３）風乾（口を上にして約２０分間）した後、ＤＡＰＩ法による菌数測定に基づき、２×１０８ｃｅｌｌｓ／ｍＬとなるようにＮｕｃｌｅａｓｅ－ｆｒｅｅ　ｗａｔｅｒ（Ａｍｂｉｏｎ）を加えて、撹拌して均一に溶解させた。さらにＮｕｃｌｅａｓｅ－ｆｒｅｅ　ｗａｔｅｒにより１０倍段階希釈を実施し、２×１０－１～２×１０１ｃｅｌｌｓ／ｍＬの範囲の希釈したサンプルを次の１４）記載のＲＮＡサンプルとして使用し、ＲＴ－ｑＰＣＲ反応に供試した。
　　１４）ＲＴ－ｑＰＣＲは、ＱＩＡＧＥＮ　ＯｎｅＳｔｅｐ　ＲＴ-ＰＣＲ　Ｋｉｔ（ＱＩＡＧＥＮ）を用いて実施し、反応液組成は、１ｘＱＩＡＧＥＮ　ＯｎｅＳｔｅｐ　ＲＴ-ＰＣＲ　Ｂｕｆｆｅｒ、０．５ｘＱ－Ｓｏｌｕｔｉｏｎ、０．４ｍＭ　ｄＮＴＰ　Ｍｉｘ、１／２５量のＱＩＡＧＥＮ　ＯｎｅＳｔｅｐ　ＲＴ－ＰＣＲ　Ｅｎｚｙｍｅ　Ｍｉｘ、１／１００，０００量のＳＹＢＲ（Ｒ）Ｇｒｅｅｎ　Ｉ（Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅｓ）、１ｘＲＯＸ　Ｒｅｆｅｒｅｎｃｅ　Ｄｙｅ（Ｉｎｖｉｔｒｏｇｅｎ）、０．６０μＭの表２に示した各プライマー、及び５μＬの上記１３）で調製したＲＮＡサンプル（１０－３～１０３ｃｅｌｌｓ）を含む反応液（総量１０μＬ）で行った。
　　１５）反応液はまず５０℃で３０分間逆転写反応を行い、その後逆転写酵素を失活させるため９５℃で１５分間加熱した。引き続いて、９４℃・２０秒、５５℃又は６０℃（Ｃ．ａｌｂｉｃａｎｓ、Ｃ．ｇｌａｂｒａｔａ及びＣ．ｋｒｕｓｅｉの各プライマーについては６０℃で行い、Ｃ．ｔｒｏｐｉｃａｌｉｓ、Ｃ．ｐａｒａｐｓｉｌｏｓｉｓ及びＣａｎｄｉｄａ　ｇｒｏｕｐの各プライマーについては５５℃で行った）・２０秒、７２℃・５０秒を４５サイクル行い、増幅産物の量をサイクルごとにＳＹＢＲ（Ｒ）Ｇｒｅｅｎ　Ｉの蛍光強度として測定した。引き続いてＰＣＲの特異性を検定するため、増幅産物の変性温度の測定を行った。９４℃で１５秒間反応後、６０℃から９９℃にかけて０．２℃／秒の速度で緩やかに温度を上昇させ、増幅産物の変性曲線を作製した。これらの一連の反応は、ＡＢＩ　ＰＲＩＳＭ（Ｒ）７９００ＨＴシステム（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）により行った。得られたＰＣＲ曲線について、蛍光強度のベースラインおよび閾値を設定し、PCR曲線と閾値が交差するサイクル数（Ｔｈｒｅｓｈｏｌｄ　ｃｙｃｌｅ:ＣＴ値）を求めた。得られたＣＴ値を縦軸に、ＰＣＲ反応に供試したサンプル菌数を横軸にプロットした。これらの解析には、Ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍ（ＳＤＳ）ソフトウェア（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）を用いた。
　　１６）ＤＡＰＩ法により測定しＣａｎｄｉｄａ属５菌種の菌数をｘ軸に、それに対応するＲＴ－ｑＰＣＲにより得たＣＴ値をｙ軸にプロットし、決定係数（Ｒ２）が０．９９を上回る値が得られる範囲を決定した。
　その結果を図１に示した。ＲＴ－ｑＰＣＲの結果より、ｓ－Ｃａｌｂ－Ｆ／Ｒを用いた場合、ＲＴ－ＰＣＲ一反応あたり１×１０－２から１×１０３個の範囲で高い相関が得られた。また、ｓ－Ｃｇｌａｂ－Ｆ／Ｒ、ｓ－Ｃｋｒｕ－Ｆ／Ｒ、ｓ－Ｃｔｒｐ－Ｆ／Ｒ、ｓ－Ｃｐａｒａ－Ｆ／Ｒ、及びｇ－Ｃａｎｄ－Ｆ／Ｒを用いた場合、ＲＴ－ＰＣＲ一反応あたり１×１０－３から１×１０３個の範囲で高い相関が得られた。すなわち、表２に示したプライマーセットは、いずれにおいても、ＲＴ－ＰＣＲ反応あたり１０－２cells相当のＲＮＡを検出可能であった。これは、血液１ｍＬあたりに換算すると、１個の菌体を検出可能であると推定された。
　検出感度の確認に使用した図１は、前述の通り、ＲＴ－ＰＣＲ一反応あたり１×１０－２から１×１０３の範囲で、決定係数（Ｒ２）が０．９９を上回る値を示していることから、後述するプライマーの特異性検討、及び菌数の測定において標準曲線として使用した。 (3) Examination of Primer Detection Sensitivity For all the primer sets shown in Table 2, RNA was extracted from cells obtained by pure culture of each standard strain, and a calibration curve was prepared by RT-qPCR. Specifically, the following method was performed.
1) 200 μL of the bacterial solution of each standard strain prepared in the above [1] strain used and 400 μL of RNAlater (Ambion) were added to the sample tube and allowed to stand at room temperature for 5 minutes. Thereafter, the mixture was centrifuged at 13,000 g for 5 minutes, and the supernatant was removed by decantation. After removing the supernatant, 450 μL of lysis buffer (prepared by mixing 346.5 μL RLT buffer, 100 μL TE and 3.5 μL β-Mercaptoethanol) and 1 mm (300 mg) diameter glass Beads (TOMY Seiko) were added.
2) After setting the sample tube on a shaker (ShakeMaster), the sample was shaken for 5 minutes to disrupt the cells.
3) 500 μL of water-saturated phenol was added and vortexed for 5-10 seconds.
4) A sample tube was set in a heat block at 60 ° C. and reacted for 10 minutes (hot phenol method).
5) 100 μL Chloroform / Isoamyl alchol (24: 1) was added and vortexed for 5-10 seconds.
6) After centrifugation (13,000 g × 5 minutes), 470 μL of the supernatant was transferred to a new microtube (1.5 mL) with a lid.
7) 470 μL Chloroform / Isoamyl alchol (24: 1) was added and vortexed for 5-10 seconds.
8) After centrifugation (13,000 g × 5 minutes), 400 μL of the supernatant was transferred to a new microtube with a lid (1.5 mL).
9) 40 μL of 3M Na acetate (pH 5.4) and 400 μL of Isopropanol were added and mixed by inversion.
10) Centrifugation (20,000 g × 10 minutes) was performed.
11) After removing the supernatant by decantation, 500 μL of 80% Ethanol was added.
12) After centrifugation (20,000 g × 2 minutes), the supernatant was removed by decantation.
13) After air-drying (about 20 minutes with the mouth facing up), add Nuclease-free water (Ambion) to 2 × 10 8 cells / mL based on the count of bacteria by DAPI method, and stir It was dissolved uniformly. Furthermore, 10-fold serial dilution was performed with Nuclease-free water, and the diluted sample in the range of 2 × 10 −1 to 2 × 10 1 cells / mL was used as the RNA sample described in 14) below, and RT-qPCR reaction I tried it.
14) RT-qPCR was performed using QIAGEN OneStep RT-PCR Kit (QIAGEN), and the composition of the reaction solution was 1 × QIAGEN OneStep RT-PCR Buffer, 0.5 × Q-Solution, 0.4 mM dNTP Mix, 1/25 amount QIAGEN OneStep RT-PCR Enzyme Mix, 1 / 100,000 volume of SYBR® Green I (Molecular Probes), 1 × ROX Reference Dye (Invitrogen), 0.60 μM of each primer shown in Table 2 above, and 5 μL of the above primers The reaction was carried out in a reaction solution (total volume 10 μL) containing the RNA sample (10 −3 to 10 3 cells) prepared in 13).
15) The reaction solution was first subjected to a reverse transcription reaction at 50 ° C. for 30 minutes, and then heated at 95 ° C. for 15 minutes to inactivate the reverse transcriptase. Subsequently, 94 ° C./20 seconds, 55 ° C. or 60 ° C. (for C. albicans, C. glabrata, and C. krusei primers at 60 ° C., for C. tropicalis, C. parapsiosis, and Candida group primers) Was performed at 55 ° C.) for 45 seconds at 20 seconds and 72 ° C. for 50 seconds, and the amount of amplification product was measured as the fluorescence intensity of SYBR (R) Green I for each cycle. Subsequently, in order to test the specificity of PCR, the denaturation temperature of the amplified product was measured. After reacting at 94 ° C. for 15 seconds, the temperature was gradually increased from 60 ° C. to 99 ° C. at a rate of 0.2 ° C./second to prepare an amplification product denaturation curve. These series of reactions were carried out by the ABI PRISM® 7900HT system (Applied Biosystems). The resulting PCR curve, to set the baseline and threshold the fluorescence intensity, the number of cycles PCR curve and the threshold is crossed: was obtained (Threshold cycle C T value). The obtained CT value was plotted on the vertical axis, and the number of sample bacteria used in the PCR reaction was plotted on the horizontal axis. For these analyses, Sequence Detection System (SDS) software (Applied Biosystems) was used.
16) The number of bacteria measured genus Candida 5 species by DAPI method x-axis, the C T values obtained by RT-qPCR corresponding thereto plotted on the y-axis, the coefficient of determination (R 2) of 0.99 The range over which values were obtained was determined.
The results are shown in FIG. From the results of RT-qPCR, when s-Calb-F / R was used, high correlation was obtained in the range of 1 × 10 −2 to 1 × 10 3 per RT-PCR reaction. When s-Cglab-F / R, s-Ckru-F / R, s-Ctrp-F / R, s-Cpara-F / R, and g-Cand-F / R are used, RT-PCR High correlation was obtained in the range of 1 × 10 −3 to 1 × 10 3 per reaction. That is, any of the primer sets shown in Table 2 was able to detect RNA corresponding to 10 −2 cells per RT-PCR reaction. This was estimated to be able to detect one bacterial cell when converted per 1 mL of blood.
FIG. 1 used for confirming the detection sensitivity shows a value with a coefficient of determination (R 2 ) exceeding 0.99 in the range of 1 × 10 −2 to 1 × 10 3 per RT-PCR reaction as described above. Therefore, it was used as a standard curve in the primer specificity examination and the bacterial count measurement described below.
（４）プライマーの特異性の検討
　Ｃａｎｄｉｄａグループに属する３０株（表１－１）及びヒト腸内細菌及び腸管感染症起因細菌２６株（表１－２）について、ＲＴ－ｑＰＣＲ法により合成したプライマーの特異性の検討を行った。ＲＴ－ＰＣＲ一反応に対して、各菌株から抽出された全ＲＮＡの１０５ｃｅｌｌｓ相当を供試した。各菌株より得られたＣＴ値を図１の標準曲線に代入し、菌数が１０４ｃｅｌｌｓ以上のものを陽性（＋）、１００ｃｅｌｌｓ以下のものを陰性（－）、１００－１０４ｃｅｌｌｓのものを（±）と判定した。 (4) Examination of primer specificity Primers synthesized by RT-qPCR method for 30 strains belonging to Candida group (Table 1-1) and human intestinal bacteria and 26 strains caused by intestinal infection (Table 1-2) The specificity of was examined. For one RT-PCR reaction, 10 5 cells equivalent of total RNA extracted from each strain were used. The C T values obtained from each strain was substituted for the standard curve of Figure 1, positive ones bacteria count of 10 4 cells or (+), 10 0 cells following are negative (-), 10 0 -10 Those of 4 cells were determined as (±).
　（ａ）各プライマーのＣａｎｄｉｄａグループに対する特異性
　Ｃａｎｄｉｄａ属５菌種特異的プライマー及びＣａｎｄｉｄａグループ特異的プライマーのＣａｎｄｉｄａグループに対する特異性を検討した。その結果、各菌株より表３に示すＣＴ値が得られた。得られたＣＴ値を対応するプライマーごとに図１のグラフＡ～Ｆに代入して（＋）、（－）、（±）を判定した。
　その結果、Ｃａｎｄｉｄａ属５菌種特異的プライマーでは、それぞれの菌種のみ（＋）となり、他のＣａｎｄｉｄａ属菌種とは反応しなかった。また、Ｃａｎｄｉｄａグループ特異的プライマーでは、すべての菌種で（＋）となり、高い反応性を有していることが分かった。 (A) Specificity of each primer for Candida group Specificity of Candida genus 5 species-specific primer and Candida group-specific primer for Candida group was examined. As a result, C T value shown in Table 3 from each strain was obtained. The obtained CT value was assigned to the graphs A to F of FIG. 1 for each corresponding primer, and (+), (−), and (±) were determined.
As a result, with the Candida genus 5 species-specific primer, only each species was (+) and did not react with other Candida species. In addition, it was found that the Candida group-specific primer is (+) in all bacterial species and has high reactivity.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
（ｂ）各プライマーのヒト腸内細菌及び腸管感染症起因細菌に対する特異性
　Ｃ．ａｌｂｉｃａｎｓ、Ｃ．ｔｒｏｐｉｃａｌｉｓ、Ｃ．ｐａｒａｐｓｉｌｏｓｉｓ、Ｃ．ｇｌａｂｒａｔａ、Ｃ．ｋｒｕｓｅｉ及びＣａｎｄｉｄａグループ特異的プライマーについて、ヒト腸内細菌及び腸管感染症起因細菌２６株との特異性を検討した。その結果、各菌株より表４に示すＣＴ値が得られた。また、プライマーと細菌が反応しなかったため、ＣＴ値が決定できなかったものは、ＵＤ（ｕｎｄｅｔｅｒｍｉｎｅｄ）と示した。得られたＣＴ値を対応するプライマーごとに図１のグラフＡ～Ｆに代入して（＋）、（－）、（±）を判定した。
　その結果、表４に示す通り、すべて陰性（－）となった。なお、ＵＤはプライマーと細菌が反応しないことから、陰性と判定した。
　よって、検討したいずれのプライマーにおいても、交差反応は認められなかった。 (B) Specificity of each primer for human intestinal bacteria and intestinal infection-causing bacteria. albicans, C.I. tropicalis, C.I. parapsilosis, C.I. glabrata, C.I. About the krusei and Candida group specific primer, the specificity with human intestinal bacteria and 26 intestinal infection origin bacteria was examined. As a result, C T value shown in Table 4 from each strain was obtained. Further, since the primer and bacteria have not reacted, which C T value can not be determined, it showed a UD (undetermined). The obtained CT value was assigned to the graphs A to F of FIG. 1 for each corresponding primer, and (+), (−), and (±) were determined.
As a result, as shown in Table 4, all were negative (-). UD was determined to be negative because the primer and bacteria did not react.
Therefore, no cross reaction was observed with any of the primers examined.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　すなわち、作製したＣａｎｄｉｄａ属５菌種特異的プライマー及びＣａｎｄｉｄａ属とその近縁種を含むＣａｎｄｉｄａグループ特異的プライマーは、それぞれの標的菌種を特異的に検出可能であることが確認された。 That is, it was confirmed that the prepared Candida genus 5 species-specific primer and the Candida group-specific primer including Candida genus and its related species can specifically detect each target species.
実施例１～２及び比較例１～２
　菌体からの核酸抽出並びにＲＴ－ｑＰＣＲ法を用いた菌数の測定
（１）血液サンプルの調製
　健常成人から採血した末梢血液に１／１０量の３．８％クエン酸ナトリウム水溶液を添加して抗凝固処理した。これに、Ｃ．ｇｌａｂｒａｔａ　ＪＣＭ３７６１Ｔ、Ｉ．ｏｒｉｅｎｔａｌｉｓ　ＩＦＯ１２７９Ｔ及び陽性コントロールとしてＰ．ａｅｒｕｇｉｎｏｓａ　ＡＴＣＣ１０１４５Ｔの純培養検体を、１ｍＬあたり１０５ｃｅｌｌｓ及び１０２ｃｅｌｌｓとなるように、採取したヒト末梢血に添加した。対照として、血液の代わりにＹＭブロスへ菌液を添加した。各菌液を添加した血液検体及び対照検体１ｍＬに、２ｍＬのＲＮＡｐｒｏｔｅｃｔ　Ｂａｃｔｅｒｉａｌ　Ｒｅａｇｅｎｔ（ＱＩＡＧＥＮ）を添加し、５分間室温にて静置した。その後、１４,０００ｇにて１０分間遠心分離し、デカンテーションにより上清を除去した後、残渣をＲＮＡ抽出用サンプルとして使用した。 Examples 1-2 and Comparative Examples 1-2
Nucleic acid extraction from bacterial cells and measurement of the number of bacteria using RT-qPCR method (1) Preparation of blood sample 1/10 volume of 3.8% sodium citrate aqueous solution was added to peripheral blood collected from healthy adults Anticoagulated. In addition, C.I. glabrata JCM3761 T , I.I. orientalis IFO1279 T and P. as a positive control. A pure culture specimen of aeruginosa ATCC10145 T was added to the collected human peripheral blood so that it would be 10 5 cells and 10 2 cells per mL. As a control, the bacterial solution was added to YM broth instead of blood. 2 mL of RNAprotective Bacterial Reagent (QIAGEN) was added to 1 mL of the blood sample and the control sample to which each bacterial solution was added, and allowed to stand at room temperature for 5 minutes. Thereafter, the mixture was centrifuged at 14,000 g for 10 minutes, the supernatant was removed by decantation, and the residue was used as a sample for RNA extraction.
（２）菌体からの核酸抽出
　下記手順に従い、ＲＮＡ抽出操作を行った。
　　１）サンプルチューブに、（１）で調製したＲＮＡ抽出用サンプル２００μＬ及びＲＮＡｌａｔｅｒ（Ａｍｂｉｏｎ）４００μＬを添加し、５分間室温にて静置した。その後、１３，０００ｇにて５分間遠心分離し、デカンテーションにより上清を除去した。上清を除去した後の残渣に溶菌バッファー　４５０μＬ（１サンプルあたり３４６．５μＬのＲＬＴ　ｂｕｆｆｅｒ、１００μＬのＴＥ及び３.５μＬのβ―Ｍｅｒｃａｐｔｏｅｔｈａｎｏｌを混合して調製する）及び直径が０．１ｍｍ（３００ｍｇ）、０．５ｍｍ（３００ｍｇ）、１ｍｍ（３００ｍｇ）及び２．５ｍｍ（１０粒、２５０～３００ｍｇ）のガラスビーズ（ＴＯＭＹ精工、及びＢｉｏＳｐｅｃ　Ｐｒｏｄｕｃｔｓ社）を所定量添加した。
　　２）前記参考例１（３）の２）～１２）記載の方法と同様に抽出操作を行った。
　　３）風乾（口を上にして約２０分間）した後、Ｎｕｃｌｅａｓｅ－ｆｒｅｅ　ｗａｔｅｒを加えて、撹拌して均一に溶解させた。 (2) Extraction of nucleic acid from bacterial cells RNA extraction was performed according to the following procedure.
1) 200 μL of the RNA extraction sample prepared in (1) and 400 μL of RNAlater (Ambion) prepared in (1) were added to the sample tube and allowed to stand at room temperature for 5 minutes. Thereafter, the mixture was centrifuged at 13,000 g for 5 minutes, and the supernatant was removed by decantation. 450 μL of lysis buffer (prepared by mixing 346.5 μL RLT buffer, 100 μL TE and 3.5 μL β-Mercaptoethanol) and 0.1 mm (300 mg) in diameter to the residue after removing the supernatant 0.5 mm (300 mg), 1 mm (300 mg) and 2.5 mm (10 grains, 250 to 300 mg) glass beads (TOMY Seiko, and BioSpec Products) were added in predetermined amounts.
2) The extraction operation was carried out in the same manner as in the methods described in 2) to 12) of Reference Example 1 (3).
3) After air-drying (about 20 minutes with the mouth facing up), Nuclease-free water was added and stirred to dissolve evenly.
（３）菌数の測定
　（２）で得られた核酸抽出液について、ＲＴ－ｑＰＣＲ法を用いて、菌数を測定した。ＲＴ－ｑＰＣＲは、前記参考例１（３）の１４）、１５）記載の方法と同様に行った。プライマーとして表２に示す配列番号３～６及び表５に示す配列番号１３、１４を使用した。ＲＴ－ｑＰＣＲ解析において、検量線作成のための標準菌株のＲＮＡ抽出は、菌体添加検体と対応する菌体破砕条件により実施した。 (3) Measurement of the number of bacteria The number of bacteria was measured for the nucleic acid extract obtained in (2) using the RT-qPCR method. RT-qPCR was performed in the same manner as described in 14) and 15) of Reference Example 1 (3). As primers, SEQ ID NOs: 3 to 6 shown in Table 2 and SEQ ID NOs: 13 and 14 shown in Table 5 were used. In RT-qPCR analysis, RNA extraction of a standard strain for preparing a calibration curve was carried out under cell disruption conditions corresponding to the cell-added specimen.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　結果を表６に示した。また、ＹＭブロスへ菌体液を添加した場合の結果を表７に示す。 The results are shown in Table 6. Table 7 shows the results when the bacterial cell liquid was added to YM broth.
（４）結果 (4) Results
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　陽性コントロールであるＰ．ａｅｒｕｇｉｎｏｓａは、いずれのガラスビーズを用いた菌体破砕でも、ＹＭブロス及びヒト末梢血において、添加した菌数と同程度の菌数が測定された。Ｃａｎｄｉｄａ菌種の場合は、ヒト末梢血検体において、０．１ｍｍ及び０．５ｍｍ径のガラスビーズを用いた菌体破砕では、添加菌数の１/１０程度の菌数が測定された（比較例１及び２）。一方で、１．０ｍｍ及び２．５ｍｍ径のガラスビーズを用いた菌体破砕では、ヒト末梢血において、添加菌数と同程度の菌数が測定された（実施例１及び２）。また、ＹＭブロスでは、いずれのビーズ径を用いた場合でも、添加菌数と同程度の菌数が測定された。以上より、ヒト血液において真菌であるＣａｎｄｉｄａ菌種の菌体破砕に１．０～２．５ｍｍ径のガラスビーズを用いた場合に、特異的に菌体破砕効率が上がることが分かった。 P. is a positive control. As for aeruginosa, the number of bacteria of the same degree as the number of added bacteria was measured in YM broth and human peripheral blood in any cell disruption using any glass beads. In the case of Candida spp., The number of added bacteria was about 1/10 of the number of added bacteria in human peripheral blood specimens by crushing cells using 0.1 mm and 0.5 mm diameter glass beads (Comparative Example) 1 and 2). On the other hand, in cell disruption using glass beads having a diameter of 1.0 mm and 2.5 mm, the number of bacteria of the same level as the number of added bacteria was measured in human peripheral blood (Examples 1 and 2). In addition, in YM broth, the number of bacteria of the same level as the number of added bacteria was measured regardless of the bead diameter. From the above, it was found that the cell crushing efficiency is specifically increased when glass beads having a diameter of 1.0 to 2.5 mm are used for crushing cells of Candida species that are fungi in human blood.
実施例３～４及び比較例３
　ガラスビーズをジルコニア／シリカビーズ（ＴＯＭＹ精工及びＢｉｏＳｐｅｃ　Ｐｒｏｄｕｃｔｓ社）に換え、実施例１～２及び比較例１～２と同様にして、菌体からの核酸抽出並びにＲＴ－ｑＰＣＲ法を用いた菌数の測定を行った。結果を表８及び表９に示した。 Examples 3 to 4 and Comparative Example 3
Replacing glass beads with zirconia / silica beads (TOMY Seiko and BioSpec Products), in the same manner as in Examples 1-2 and Comparative Examples 1-2, the number of bacteria using nucleic acid extraction from cells and RT-qPCR method Was measured. The results are shown in Tables 8 and 9.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　実施例１～２及び比較例１～２と同様、Ｐ. aｅｒｕｇｉｎｏｓａのＹＭブロス及びヒト末梢血への添加検体並びにＣａｎｄｉｄａ菌種のＹＭブロスへの添加検体では、添加した菌数と同程度の菌数が測定されたが、Ｃａｎｄｉｄａ菌種のヒト末梢血への添加検体では、０．５ｍｍのジルコニア／シリカビーズを使用した際には、添加した菌数の１／３程度の菌数しか測定されず（比較例３）、一方で、１．０ｍｍ及び２．５ｍｍ径のジルコニア／シリカビーズを用いた菌体破砕では、添加菌数と同程度の菌数が測定された（実施例３及び４）。以上より、ヒト血液において真菌であるＣａｎｄｉｄａ菌種の菌体破砕に１．０～２．５ｍｍ径のジルコニア／シリカビーズを用いた場合にも、特異的に菌体破砕効率が上がることが分かった。 As in Examples 1 and 2 and Comparative Examples 1 and 2, in the specimens added to YM broth of P. aeruginosa and human peripheral blood and specimens added to YM broth of Candida spp. The number of Candida species added to human peripheral blood was measured only about 1/3 of the number of added bacteria when 0.5 mm zirconia / silica beads were used. (Comparative Example 3) On the other hand, in the bacterial cell disruption using 1.0 mm and 2.5 mm diameter zirconia / silica beads, the same number of bacteria as the number of added bacteria was measured (Examples 3 and 4). ). From the above, it was found that even when zirconia / silica beads with a diameter of 1.0 to 2.5 mm are used for disrupting Candida species, which are fungi in human blood, the efficiency of disrupting the cells is specifically increased. .
実施例５　Ｃａｎｄｉｄａ属５菌種特異的プライマーを用いたヒト末梢血への菌体添加回収試験
　Ｃ．ａｌｂｉｃａｎｓ　ＩＦＯ　１３８５Ｔ、Ｃ．ｔｒｏｐｉｃａｌｉｓ　ＪＣＭ　１５４１Ｔ、Ｃ．ｐａｒａｐｓｉｌｏｓｉｓ　ＤＳＭ　５７８４Ｔ、Ｃ．ｇｌａｂｒａｔａＪＣＭ　３７６１Ｔ、Ｉ．ｏｒｉｅｎｔａｌｉｓ　ＩＦＯ　１２７９Ｔ及び実験の陽性コントロールとしてのＰ．ａｅｒｕｇｉｎｏｓａ　ＡＴＣＣ１０１４５Ｔ純培養菌体を、健常成人３名（Blood A, Blood B, Blood C）より採取したヒト末梢血及びＹＭブロスへ１ｍＬあたり１０５、１０４、１０３、１０２、１０１ｃｅｌｌｓとなるように添加した。実施例１と同様に、菌体を添加した検体について、１．０ｍｍ径のガラスビーズを用いた菌体破砕を行い、核酸を抽出した。抽出した核酸について、ＲＴ－ｑＰＣＲ法により菌数を測定し、横軸に添加菌数を、縦軸にＲＴ－ｑＰＣＲ法により得た測定菌数をプロットした（図２）。検討した全ての菌株について、ヒト末梢血に菌体を添加した場合において、１０１から１０５ｃｅｌｌｓ/ｍＬの範囲で直線性が認められ、その近似式は異なる被験者より採取した末梢血及びＹＭブロス間でほぼ一致していた。以上の結果より、１．０ｍｍ径のガラスビーズ及びＣａｎｄｉｄａ属５菌種特異的プライマーを用いることにより、ヒト末梢血中のＣａｎｄｉｄａ属５菌種を正確に定量可能であると考えられた。 Example 5 Cell Addition and Recovery Test to Human Peripheral Blood Using Candida Genus 5 Species Specific Primer albicans IFO 1385 T , C.I. tropicalis JCM 1541 T , C.I. parapsilosis DSM 5784 T , C.I. glabrata JCM 3761 T , I.I. orientalis IFO 1279 T and P. as a positive control for the experiment. 10 5 , 10 4 , 10 3 , 10 2 , 10 1 cells per mL of aeruginosa ATCC 10145 T pure cultured cells to human peripheral blood and YM broth collected from 3 healthy adults (Blood A, Blood B, Blood C) It added so that it might become. In the same manner as in Example 1, the specimen to which the bacterial cells were added was subjected to bacterial cell disruption using 1.0 mm diameter glass beads to extract nucleic acids. With respect to the extracted nucleic acid, the number of bacteria was measured by the RT-qPCR method, the number of added bacteria was plotted on the horizontal axis, and the number of measured bacteria obtained by the RT-qPCR method was plotted on the vertical axis (FIG. 2). For all the strains examined, when cells were added to human peripheral blood, linearity was observed in the range of 10 1 to 10 5 cells / mL, and the approximate equations were peripheral blood collected from different subjects and YM broth. There was almost agreement between the two. From the above results, it was considered that Candida 5 species in human peripheral blood can be accurately quantified by using 1.0 mm diameter glass beads and Candida 5 species specific primers.
実施例６　Ｃａｎｄｉｄａグループ特異的プライマーを用いたヒト末梢血への菌体添加回収試験
　Ｃ．ａｌｂｉｃａｎｓＩＦＯ　１３８５Ｔ、Ｃ．ｔｒｏｐｉｃａｌｉｓＪＣＭ　１５４１Ｔ、Ｃ．ｐａｒａｐｓｉｌｏｓｉｓ　ＤＳＭ　５７８４Ｔ、Ｃ．ｇｌａｂｒａｔａ　ＪＣＭ　３７６１Ｔ、Ｉ．ｏｒｉｅｎｔａｌｉｓ　ＩＦＯ　１２７９Ｔの各純培養菌体を、健常成人３名より採取したヒト末梢血（被験者Ａ、被験者Ｂ、被験者Ｃ）及びＹＭブロスへ、それぞれ１ｍＬあたり１０５、１０４、１０３、１０２、１０１ｃｅｌｌｓとなるように添加した（添加した総菌数は１ｍＬあたり、５×１０５、５×１０４、５×１０３、５×１０２、５×１０１ｃｅｌｌｓとなる）。菌体を添加した検体から実施例１と同様に菌体を添加した検体について、１．０ｍｍ径のガラスビーズを用いた菌体破砕を行い、核酸を抽出した。抽出した核酸について、ＲＴ－ｑＰＣＲ法により菌数を測定し、添加菌数を横軸に、ＲＴ－ｑＰＣＲ法により得た測定菌数を縦軸にプロットした（図３）。ヒト末梢血に菌体を添加した場合において、１０１から１０５ｃｅｌｌｓ/ｍＬの範囲で直線性が認められ、その近似式は異なる被験者より採取した末梢血及びＹＭブロス間でほぼ一致していた。以上の結果より、１．０ｍｍ径のガラスビーズ及びＣａｎｄｉｄａグループ特異的プライマーを用いることにより、ヒト末梢血中のＣａｎｄｉｄａ属を正確に定量可能であると考えられた。 Example 6 Bacterial addition recovery test to human peripheral blood using Candida group specific primer albicansIFO 1385 T , C.I. tropicalis JCM 1541 T , C.I. parapsilosis DSM 5784 T , C.I. glabrata JCM 3761 T , I.I. Each of the pure cultured cells of orientalis IFO 1279 T was collected from 3 healthy adults into human peripheral blood (Subject A, Subject B, Subject C) and YM broth at 10 5 , 10 4 , 10 3 , 10, respectively. 2 and 10 1 cells were added (the total number of added bacteria was 5 × 10 5 , 5 × 10 4 , 5 × 10 3 , 5 × 10 2 and 5 × 10 1 cells per mL). From the sample to which the cells were added, the sample to which the cells were added in the same manner as in Example 1 was subjected to cell disruption using 1.0 mm diameter glass beads, and the nucleic acid was extracted. With respect to the extracted nucleic acid, the number of bacteria was measured by the RT-qPCR method, the number of added bacteria was plotted on the horizontal axis, and the number of measured bacteria obtained by the RT-qPCR method was plotted on the vertical axis (FIG. 3). When cells were added to human peripheral blood, linearity was observed in the range of 10 1 to 10 5 cells / mL, and the approximate expression was almost the same between peripheral blood collected from different subjects and YM broth. . From the above results, it was considered that the genus Candida in human peripheral blood can be accurately quantified by using 1.0 mm diameter glass beads and Candida group-specific primers.

Claims (10)

  1. 　真菌を含む生体試料から真菌核酸を抽出する方法であって、真菌を含む試料溶液を直径０．８ｍｍ～３ｍｍのビーズと接触させて撹拌した後、核酸を分離することを特徴とする、真菌核酸の抽出方法。 A method for extracting fungal nucleic acid from a biological sample containing fungi, wherein the nucleic acid is separated after contacting the sample solution containing fungi with beads having a diameter of 0.8 mm to 3 mm and stirring. Extraction method.
  2. 　真菌がカンジダ属真菌である、請求項１記載の方法。 The method according to claim 1, wherein the fungus is a Candida fungus.
  3. 　カンジダ属真菌が、カンジダ・アルビカンス、カンジダ・グラブラータ、カンジダ・パラプシローシス、カンジダ・トロピカリス及びカンジダ・クルーセイから選ばれる１以上である、請求項２記載の方法。 The method according to claim 2, wherein the Candida fungus is one or more selected from Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida crusei.
  4. 　ビーズがガラスビーズ又はジルコニア／シリカビーズである、請求項１～３のいずれか１項記載の方法。 The method according to any one of claims 1 to 3, wherein the beads are glass beads or zirconia / silica beads.
  5. 　ビーズの直径が１ｍｍ～２．５ｍｍである、請求項１～４のいずれか１項記載の方法。 The method according to any one of claims 1 to 4, wherein the beads have a diameter of 1 mm to 2.5 mm.
  6. 　生体試料がヒト血液である、請求項１～５のいずれか１項記載の方法。 The method according to any one of claims 1 to 5, wherein the biological sample is human blood.
  7. 　請求項１～６のいずれか１項記載の方法により抽出された核酸に特異的にハイブリダイズし得る核酸断片を用いて核酸増幅反応を行う、生体試料中の真菌の検出及び／又は定量方法。 A method for detecting and / or quantifying a fungus in a biological sample, wherein a nucleic acid amplification reaction is performed using a nucleic acid fragment that can specifically hybridize to the nucleic acid extracted by the method according to any one of claims 1 to 6.
  8. 　核酸断片が、配列番号１～１２で示される塩基配列からなる核酸断片又はそれと相補的な塩基配列からなる核酸断片である、請求項７記載の方法。 The method according to claim 7, wherein the nucleic acid fragment is a nucleic acid fragment consisting of a base sequence represented by SEQ ID NOs: 1 to 12 or a nucleic acid fragment consisting of a base sequence complementary thereto.
  9. 　直径０．８ｍｍ～３ｍｍのビーズ及び真菌核酸に特異的にハイブリダイズし得る核酸断片を含み、核酸抽出試薬、核酸増幅反応試薬及びプロトコールから選ばれる１種以上を含んでもよい、請求項７又は８記載の方法を実施するためのキット。 9. A bead having a diameter of 0.8 mm to 3 mm and a nucleic acid fragment capable of specifically hybridizing to a fungal nucleic acid, and may include one or more selected from a nucleic acid extraction reagent, a nucleic acid amplification reaction reagent, and a protocol. Kit for carrying out the described method.
  10. 　ビーズの直径が１ｍｍ～２．５ｍｍである、請求項９記載のキット。
          The kit according to claim 9, wherein the beads have a diameter of 1 mm to 2.5 mm.
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