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WO2018062573A1 - Procédé de récupération de cellules - Google Patents

Procédé de récupération de cellules Download PDF

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Publication number
WO2018062573A1
WO2018062573A1 PCT/JP2017/036399 JP2017036399W WO2018062573A1 WO 2018062573 A1 WO2018062573 A1 WO 2018062573A1 JP 2017036399 W JP2017036399 W JP 2017036399W WO 2018062573 A1 WO2018062573 A1 WO 2018062573A1
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Prior art keywords
cells
bacterial
bacterial solution
cell
dna
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English (en)
Japanese (ja)
Inventor
保孝 幸
森　安義
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Eiken Chemical Co Ltd
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Eiken Chemical Co Ltd
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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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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

Definitions

  • the present invention relates to a method for recovering microbial cells or animal cells in a specimen, for example.
  • Genetic detection kits for microbial cells or animal cells such as pathogens have minimal detection sensitivity.
  • the amount of specimen used in the genetic test is several to several tens of ⁇ L per test. For this reason, for example, a sample containing 1,000 or more pathogens per mL of sample can be determined as positive, but a sample containing less than 1,000 pathogens cannot be determined as positive and cannot be detected.
  • a method of increasing the sample amount of the kit can be mentioned. However, simply increasing the amount of the sample simultaneously dilutes the reagent component and also increases the amount of the inhibitory component derived from the sample, which may cause erroneous determination in the test result.
  • the centrifugation method In order to avoid this problem, an operation for separating and concentrating microbial cells or animal cells such as pathogens from the sample components is necessary.
  • the centrifugation method is mentioned as a method for separating and concentrating microbial cells or animal cells, the centrifugation method requires expensive equipment and a use environment.
  • an inexpensive and simple method for concentrating microbial cells or animal cells is desired.
  • Patent Document 1 discloses an aliphatic solution at a concentration in which only bacterial cells are aggregated and high molecular polysaccharides are not aggregated in a culture broth of bacteria that secrete and produce polymeric polysaccharides outside the bacterial cells. Alcohol is added to agglomerate cells to separate the cells, while alcohol is added to the culture broth from which the cells are removed to precipitate and recover the polymer polysaccharide. A purification method is disclosed.
  • Patent Document 2 discloses a microorganism recovery method in which microparticles in a sample are adsorbed to the microparticles by bringing the microparticles into contact with the sample.
  • a slurry liquid is prepared by contacting water containing a microorganism and a magnetic substance or an aggregate thereof, and the slurry liquid is solid-liquid separated into an aggregate cake and water by filtering the slurry liquid.
  • a method for concentrating microorganisms in which the cake is separated into microorganisms and magnetic substances to concentrate the microorganisms.
  • conventionally there has not been known a method capable of efficiently concentrating microbial cells or animal cells by using magnetic particles and alcohol in combination.
  • an efficient method for recovering microbial cells or animal cells is desired in order to improve the detection performance of genetic test kits for microbial cells or animal cells such as pathogens.
  • an object of this invention is to provide the efficient collection
  • microbial cells or animal cells co-aggregate with the magnetic particles in the presence of magnetic particles and alcohol, and the microbial cells or animal cells can be easily recovered.
  • the invention has been completed. That is, the present invention includes the following. (1) A method for recovering cells, comprising a step of mixing cells and magnetic particles in the presence of alcohol.
  • kits according to (7), wherein the cells are microbial cells or animal cells.
  • the magnetic particles are magnetic particles containing magnetite and / or silica as a surface material.
  • the alcohol is selected from the group consisting of methanol, ethanol, 1-propanol, isopropyl alcohol, ethylene glycol, 2,2′-iminodiethanol, polyethylene glycol, and mixtures thereof.
  • a kit according to claim 1. (11) The kit according to (10), wherein the polyethylene glycol has a molecular weight of 20,000 or less.
  • a test kit comprising the cell recovery kit according to any one of (7) to (11). This specification includes the disclosure of Japanese Patent Application No. 2016-192190, which is the basis of the priority of the present application.
  • Photo b) shows the appearance of mycobacteria in a typical bacterial cell shape (gonococcal shape) stained red in the aggregate of magnetite particles.
  • Photo c) shows the mycobacteria cells in the field of view with few magnetite particles and the magnetite particles adsorbed so as to border the cells.
  • recovery protocol in the method of this invention is shown.
  • the bacterial cells in the bacterial solution or specimen are recovered as an aggregate consisting of magnetite particles and bacterial cells.
  • the collected agglomerates can then be subjected to an appropriate nucleic acid extraction method or genetic test.
  • the cell collection method according to the present invention is a method including a step of mixing cells and magnetic particles in the presence of alcohol.
  • the present invention by mixing cells and magnetic particles in the presence of alcohol, the cells and magnetic particles co-aggregate to form aggregates.
  • the aggregate formed in this way can be collected and used as an object for a test such as a genetic test.
  • Cells collected by this method are present in, for example, specimens (for example, clinical specimens such as sputum, gastric juice, stool emulsion, urine, pleural effusion, whole blood, serum, plasma).
  • the cells include animal cells such as humans and cells such as microorganisms.
  • the microorganism may be a microorganism (or pathogen) belonging to any genus, such as Mycobacterium bovis BCG strain, Mycobacterium tuberculosis (Mycobacterium tuberculosis), Gram-positive bacteria such as mycobacteria, including Mycobacterium avium and Mycobacterium intracellulare, Bordetella pertussis li, Escherichia coli, etc. Gram-negative bacteria, yeasts (Saccharomyces cerevisiae) and other fungi.
  • the magnetic particles used in this method may be any particles as long as they have magnetism, and examples thereof include particles containing (or consisting of) magnetite and / or silica as surface materials.
  • magnetic particles coated with magnetite and / or silica can be used.
  • the magnetic particles may be surface-modified with a functional group such as a carboxyl group, a methyl group, an octadecyl group, albumin, or boronic acid.
  • the shape of the magnetic particle is not particularly limited, and examples thereof include a spherical shape and an octahedron.
  • examples of the size of the magnetic particles include a diameter ( ⁇ ) of 25 nm to 2 ⁇ m. As these magnetic particles, commercially available particles can be used.
  • examples of alcohol used in the present method include methanol, ethanol (EtOH), 1-propanol, isopropyl alcohol (2-propanol, IPA), ethylene glycol, 2,2′-iminodiethanol, polyethylene glycol (PEG), and These mixtures etc. are mentioned.
  • examples of PEG include those having a molecular weight of 20,000 or less (monoethylene glycol to molecular weight 20,000).
  • 0.5 to 20 mg of magnetic particles and 1,400 ⁇ L of alcohol are added to 600 ⁇ L of a specimen containing cells, mixed, and allowed to stand for 0 to 10 minutes.
  • EtOH for example, EtOH is added to a final concentration of 40 to 90%.
  • IPA IPA is added so that the final concentration is, for example, 20 to 90%.
  • PEG molecular weight 1,000
  • PEG molecular weight 1,000
  • PEG molecular weight 1,000
  • the present invention also relates to a testing method such as genetic testing, which includes a step of extracting nucleic acid from cells collected by the method and a step of subjecting the extracted nucleic acid to testing such as genetic testing.
  • genetic testing refers to PCR (for example, JP-A-2001-286300 and JP-A-62-2000281), SDA (for example, JP-A-5-192195), NASBA (for example, JP-A-5-192181).
  • nucleic acid extraction methods generally used for nucleic acid extraction such as genomes and plasmids (for example, phenol / chloroform method and alkali extraction method) and commercially available methods are available.
  • the nucleic acid extraction method using the nucleic acid extraction kit is not particularly limited.
  • the present invention also relates to a cell collection kit containing magnetic particles and alcohol used in the present method.
  • the cell collection kit can contain, for example, reagents, containers, instruction manuals, etc. used for cell collection.
  • the cell collection kit can be included in a test kit for genetic testing or the like, and can also be provided as a kit for a pretreatment reagent.
  • the test kit can further include reagents, containers, instruction manuals, etc. used for nucleic acid extraction and genetic testing.
  • the objectives of this example are as follows: Observation of magnetite particles agglomerated in alcohol; Observation of the location of bacteria when the cells are aggregated under the condition where the cells and magnetite particles coexist. 2.
  • the materials used in this example were as follows: Mycobacterium bovis BCG Tokyo KK-12-21 strain; Magnetite particles: ferric oxide (III) iron (II) (Wako Pure Chemical Industries); Physiological saline (hereinafter referred to as “raw food”, Otsuka Pharmaceutical Factory); Magnet for collecting magnets; Aggregating solvent: ethanol (hereinafter referred to as “EtOH”; Wako Pure Chemical Industries), distilled water (hereinafter referred to as “DW”; Otsuka Pharmaceutical Factory); Optical microscope: Olympus biological microscope CH2 (OLYMPUS); Spectrophotometer: CO8000 Biowave (Biochrom Ltd.); 2.
  • the prepared bacterial solution was 10 7 CFU / mL, and a 10 6 CFU / mL bacterial solution was prepared by 10-fold dilution.
  • 2) Cell recovery in bacterial solution 2.5 mg of magnetite particles were suspended in 300 ⁇ L of saline or 10 6 CFU / mL bacterial solution. Next, 700 ⁇ L of aggregation solvent (EtOH or DW) was mixed (final concentration 70%). After mixing, magnetite particles were collected with a magnet and the supernatant was removed.
  • EtOH or DW aggregation solvent
  • the purpose of this example is to examine the conditions necessary for cell recovery by this method.
  • Materials The materials used in this example were as follows: 1) Bacterial solution preparation and cell recovery Mycobacterium bovis BCG Tokyo KK-12-21 strain; Magnetite particles: ferric oxide (III) iron (II) (Wako Pure Chemical Industries); Raw food (Otsuka Pharmaceutical Factory); Magnet for collecting magnets Aggregating solvent: EtOH (Wako Pure Chemical Industries), 2-propanol (hereinafter referred to as “IPA”; Wako Pure Chemical Industries), polyethylene glycol 1,000 (hereinafter referred to as “PEG 1,000”) Wako Pure Chemical Industries), DW (Otsuka Pharmaceutical Factory), and PEG 1,000 were prepared in 20% (w / v) aqueous solution and used in the following experiments.
  • Experiment A Method 1) DNA extracted from each concentration of bacterial solution prepared in “Bacterial solution preparation” was subjected to PURE-TB-LAMP.
  • Bacteria recovery by this method (1) 1. Purpose The purpose of this example is to estimate the cell recovery rate by this method. 2. Materials The materials used in this example were as follows: 1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 2 were used. In this experiment, EtOH was used as the aggregation solvent. 2) Gene amplification test The same material as in Example 2 was used. 3. Method 1) Bacterial Solution Preparation McFarland No. The same method as in Example 1 was used for the method for preparing the bacterial solution 1. The prepared bacterial solution was made 10 7 CFU / mL and diluted 10,000 times to prepare a 10 3 CFU / mL bacterial solution.
  • the minimum detection sensitivity was 62.5 CFU / mL.
  • the minimum concentration of the bacterial liquid that can stably obtain a positive result has been improved by 8 times, and the amount of the bacterial liquid used for the method of collecting bacterial cells has been increased by 10 times. Considering that there is a difference, it was speculated that this method can recover the cells in the bacterial solution in a considerable proportion.
  • Bacteria recovery by this method (2) 1.
  • the purpose of this example is to collect cells from a high-concentration bacterial solution and to investigate the recovery rate.
  • Materials The materials used in this example were as follows: 1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. In this experiment, EtOH was used as the aggregation solvent. 2) Gene amplification test PURE DNA extraction kit (Eiken Chemical); Commercial real-time PCR measurement apparatus: Mx3005P (Agilent Technologies); Template quantification LAMP reaction reagent prepared in house; It was used. 3. Method 1) Preparation of Bacterial Solution The same method as in Example 1 was used. 2) Cell recovery in the bacterial solution The same method as in Example 2 was used.
  • DNA recovery by this method Purpose The purpose of this example is to confirm the ability of DNA recovery by this method.
  • the obtained DNA solution was dissolved in 10 mM Tris-HCl (pH 8.0), the absorbance value of the DNA solution was measured at an absorption wavelength of 260 nm using a spectrophotometer, and the copy number was calculated from the obtained weight concentration. did. According to the calculated copy number, a 10-fold serial dilution series DNA solution of 100 to 10 5 copies / mL was prepared by saline. 2) DNA recovery from DNA solution The DNA solution prepared in Method 1) was subjected to the same method as in Example 2. The final concentration of the aggregation solvent EtOH is 70%. 3) Extraction of nucleic acid The same method as in Example 2 was used.
  • the purpose of this example is to investigate the relationship between the surface material of magnetic particles, the presence / absence of functional groups on the particle surface, the shape and particle size, and the recovery of bacterial cells.
  • Materials The materials used in this example were as follows: 1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. In this experiment, EtOH was used as the aggregation solvent. For the magnetic particle types used in this experiment, see Table 6. As magnetite particles whose surfaces were coated with silica, SiMAG-active, SiMAG-hydrophobe, and SiMAG-affinity series (Chemicell) were used.
  • Magnetite particles include ferric oxide (III) iron (II) (Wako Pure Chemical Industries), ferric oxide (III) iron (II) nanoparticles (Wako Pure Chemical Industries), and magnetite product series (Mitsui). Metal Industry Co., Ltd.) was used. 2) Gene amplification test The same material as in Example 3 was used. 3. Method 1) Bacterial solution preparation The same method as in Example 2 was used. 2) Cell recovery in the bacterial solution The same method as in Example 2 was used. The final concentration of the aggregation solvent EtOH is 70%. 3) Extraction of nucleic acid The same method as in Example 2 was used. 4) Gene amplification test The same method as in Example 2 was used. 4). Results The results are shown in Table 6.
  • n 2 in the triple measurement was negative. From 12 types of each of the particles from which the cells were collected, all obtained positive results in triplicate measurement. From these results, it was confirmed that the cells can be recovered by this method regardless of the surface material of the particles, the presence / absence of the surface-modifying functional group, the shape and the particle size within the range examined in this experiment.
  • coagulation solvent (2) 1. Purpose The purpose of this example is to investigate the optimum concentration range of alcohol as a coagulation solvent species. 2. Materials The materials used in this example were as follows: 1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. EtOH and IPA were used as the aggregation solvent. 2) Gene amplification test The same material as in Example 3 was used. 3. Method 1) Bacterial solution preparation The same method as in Example 2 was used. 2) Cell recovery in the bacterial solution The same method as in Example 2 was used. The amount of the aggregation solvent added to 600 ⁇ L of the bacterial solution was changed. See Table 9 for the concentration of the flocculating solvent at the time of mixing, that is, the range of the final concentration.
  • coagulation solvent (3) 1. Purpose The purpose of this example is to investigate the molecular weight and optimal concentration range of PEG. 2. Materials The materials used in this example were as follows: 1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. The aggregating solvents used were four kinds of PEG molecular weights of 1,000, 3,350,8,000, and 20,000, and 20% (w / v) aqueous solutions were prepared and used in Experiment A. Further, a 50% (w / v) aqueous solution of PEG 1,000 and a 30% (w / v) aqueous solution of PEG 8,000 were prepared and used in Experiment B. 2) Gene amplification test The same material as in Example 3 was used. 3.
  • Results The results are shown in Tables 10-12. From the results of this experiment A, bacterial recovery was observed at all molecular weights used. From the results of Experiment B, bacterial recovery was observed in the range of 2.5 to 30% for PEG 1,000 and 5.25 to 21% for PEG 8,000. Based on the results of this experiment and the ethylene glycol (monomer) of Example 8, it was confirmed that the molecular weight can be recovered in the range of 62.1 to 20,000. In addition, it has been clarified that there is an optimum concentration range for cell recovery even in the case of PEG.
  • Experiment B Method 2 As a result of subjecting DNA extracted from the aggregate collected by the method of “recovering bacterial cells in bacterial solution” to PURE-TB-LAMP, the minimum detection sensitivity was 15.6 CFU / mL. From these results, it was confirmed that this method can be applied to other species of the genus Mycobacterium other than Mycobacterium bovis. In Experiment A, the detection sensitivity of Mycobacterium tuberculosis used in this experiment was 8 times higher than that of Mycobacterium bovis. (IS6110) copy number difference (16 for Mycobacterium tuberculosis H37Rv KK11-291 and 2 for Mycobacterium bovis BCG Tokyo KK-12-21) It was.
  • the purpose of this example is to investigate the range of applicable bacterial species 2: gram-negative bacteria.
  • Materials The materials used in this example were as follows: 1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 2 were used. In this experiment, Bordetella pertussis Tohama strain; Magnetite particles; Aggregation solvent: Polyethylene glycol 8000 (PEG 8,000, Wako Pure Chemical Industries) and PEG 8,000 were prepared in a 15% (w / v) aqueous solution and used in the following experiments. It was used.
  • the prepared bacterial solution is 1.5 ⁇ 10 8 CFU / mL, diluted 10,000 times to prepare a 1.5 ⁇ 10 3 CFU / mL bacterial solution, and subsequently diluted 4 to 8 times. 3,750, 1,875 CFU / mL of bacterial solution was prepared. 2) Cell recovery in the bacterial solution The same method as in Example 2 was used.
  • the final concentrations of the aggregation solvent EtOH (70%) and PEG 1,000 are 70% and 10.5%, respectively.
  • 3) Extraction of nucleic acid The following treatment was performed using various bacterial solutions.
  • Treatment A Method 1) DNA was extracted from 60 ⁇ L of the bacterial solution prepared by “Preparation of bacterial solution” using the method of Example 2.
  • Process B Method 2) “Recovering bacterial cells in bacterial solution” method, and collecting aggregates collected using EtOH (70%) as an agglutination solvent. Extracted.
  • Process C Method 2) “Recovering bacterial cells in bacterial solution” method, and using PEG 1,000 (15%) as an aggregating solvent to collect and recover the aggregated mass using the nucleic acid extraction method of Example 2 Extracted.
  • the purpose of this example is to investigate biological specimen species that can be recovered by this method.
  • Materials The materials used in this example were as follows: 1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. In this experiment, EtOH was used as the aggregation solvent. 2) Specimen Sputum, gastric juice, 10% stool suspension supernatant (stool emulsion), urine, pleural effusion, and whole blood were used as biological specimens. The sputum sample used was a sample obtained by adding 1,200 ⁇ L of 0.5 M NaOH to 600 ⁇ L of the sample, mixing and dissolving. 3) Gene amplification test The same material as in Example 3 was used. 3.
  • Method 1 Bacterial solution preparation The same method as in Example 2 was used. However, in this operation, a 10 3 CFU / mL bacterial solution was prepared. 2) Sample preparation 1/10 amount of the bacterial solution of 1) was added to each biological sample to prepare various bacterial solutions of 10 2 CFU / mL. 3) Cell recovery in bacterial solution The same method as in Example 2 was used. The final concentration of the aggregation solvent EtOH is 70%. 4) Extraction of nucleic acid The same method as in Example 2 was used. 5) Gene amplification test The same method as in Example 2 was used. 4). Results The results are shown in Table 16. All samples listed above were positively detected by LAMP. From this, it was confirmed that the cells can be recovered from a wide variety of specimen species by this method.
  • the purpose of this example is to investigate the order of addition of particles and flocculating solvent.
  • Materials The materials used in this example were as follows: 1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. In this experiment, EtOH was used as the aggregation solvent. 2) Gene amplification test The same material as in Example 3 was used. 3. Method 1) Bacterial solution preparation 2) Cell recovery in bacterial solution The same method as in Example 2 was used. The final concentration of the aggregation solvent EtOH is 70%. The addition order of the magnetite particles and the solvent is shown in Table 17. 3) Extraction of nucleic acid The same method as in Example 2 was used.
  • Method 2 DNA extraction was performed with the Loopamp PURE DNA extraction kit using 60 ⁇ L of the magnetite particle suspension prepared in “Recovering cells in suspension”.
  • the DNA extract obtained by the operations of A and B was subjected to a LAMP reaction using Loopamp DNA amplification reagent D and CYP2C19 primer, and triple measurement was performed. 4). Results The results are shown in Table 20. In the cell recovery group using 300 ⁇ L of cell suspension b and the control group using 30 ⁇ L of cell suspensions a and b, all were positive for LAMP reaction in triplicate measurement. On the other hand, in the control group using 30 ⁇ L of the cell suspension c, all LAMP reactions were negative in triplicate measurement. 5).
  • microbial cells or animal cells can be collected inexpensively and easily.
  • the detection performance of the genetic test kit for microbial cells or animal cells such as pathogenic bacteria can be improved.

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Abstract

Le but de la présente invention concerne un procédé efficace de récupération de cellules, telles que des micro-organismes ou des cellules animales ; plus particulièrement, la présente invention concerne un procédé de récupération de cellules, ledit procédé comprenant une étape de mélange de cellules et de particules magnétiques en présence d'un alcool.
PCT/JP2017/036399 2016-09-29 2017-09-29 Procédé de récupération de cellules Ceased WO2018062573A1 (fr)

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JP2016-192190 2016-09-29
JP2016192190 2016-09-29

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05504095A (ja) * 1990-02-13 1993-07-01 アマーシャム・インターナショナル・ピーエルシー 高分子に特異的に結合しない磁気吸引可能なビーズを用いた高分子の単離方法
EP1621618A1 (fr) * 1999-05-14 2006-02-01 Promega Corporation Concentration de cellules et élimination de lysats au moyen de particules paramagnétiques
JP2006508668A (ja) * 2002-12-09 2006-03-16 ツィンファ・ユニバーシティ 磁性ベースの迅速な細胞分離
WO2006123781A1 (fr) * 2005-05-20 2006-11-23 Arkray, Inc. Procedes de recuperation de microorganisme et d’acide nucleique en utilisant une particule fine et necessaire a utiliser pour les procedes
JP2007252240A (ja) * 2006-03-22 2007-10-04 Hitachi Metals Ltd 細胞回収用磁気ビーズ
WO2007114758A1 (fr) * 2006-03-30 2007-10-11 Ge Healthcare Bio-Sciences Ab billes magnetiques
JP2010178750A (ja) * 2005-04-15 2010-08-19 Samsung Electronics Co Ltd 疎水性固体支持体を利用した細胞分離方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05504095A (ja) * 1990-02-13 1993-07-01 アマーシャム・インターナショナル・ピーエルシー 高分子に特異的に結合しない磁気吸引可能なビーズを用いた高分子の単離方法
EP1621618A1 (fr) * 1999-05-14 2006-02-01 Promega Corporation Concentration de cellules et élimination de lysats au moyen de particules paramagnétiques
JP2006508668A (ja) * 2002-12-09 2006-03-16 ツィンファ・ユニバーシティ 磁性ベースの迅速な細胞分離
JP2010178750A (ja) * 2005-04-15 2010-08-19 Samsung Electronics Co Ltd 疎水性固体支持体を利用した細胞分離方法
WO2006123781A1 (fr) * 2005-05-20 2006-11-23 Arkray, Inc. Procedes de recuperation de microorganisme et d’acide nucleique en utilisant une particule fine et necessaire a utiliser pour les procedes
JP2007252240A (ja) * 2006-03-22 2007-10-04 Hitachi Metals Ltd 細胞回収用磁気ビーズ
WO2007114758A1 (fr) * 2006-03-30 2007-10-11 Ge Healthcare Bio-Sciences Ab billes magnetiques

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