WO2015163449A1 - Procédé d'extraction d'acides nucléiques fongiques - Google Patents

Procédé d'extraction d'acides nucléiques fongiques 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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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.

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Abstract

　L'invention concerne un procédé d'extraction efficace d'acides nucléiques de champignon à partir d'un échantillon biologique contenant un champignon.　Plus spécifiquement, ce procédé d'extraction d'acides nucléiques fongiques à partir d'un échantillon biologique contenant un champignon se caractérise en ce que, après avoir mis en contact et mélangé une solution d'échantillon contenant un champignon avec des billes de 0,8 à 3mm de diamètre, les acides nucléiques sont séparés.
PCT/JP2015/062537 2014-04-24 2015-04-24 Procédé d'extraction d'acides nucléiques fongiques WO2015163449A1 (fr)

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