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WO2019188552A1 - Procédé de mesure de cellules de micro-organismes et/ou de virus - Google Patents

Procédé de mesure de cellules de micro-organismes et/ou de virus Download PDF

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Publication number
WO2019188552A1
WO2019188552A1 PCT/JP2019/011355 JP2019011355W WO2019188552A1 WO 2019188552 A1 WO2019188552 A1 WO 2019188552A1 JP 2019011355 W JP2019011355 W JP 2019011355W WO 2019188552 A1 WO2019188552 A1 WO 2019188552A1
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Prior art keywords
cells
measurement
viruses
test sample
microorganisms
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English (en)
Japanese (ja)
Inventor
隆志 副島
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Morinaga Milk Industry Co Ltd
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Morinaga Milk Industry Co Ltd
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Priority to JP2020510728A priority Critical patent/JP7350716B2/ja
Publication of WO2019188552A1 publication Critical patent/WO2019188552A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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

Definitions

  • the present invention relates to a method for measuring microbial cells and / or viruses in a test sample.
  • Non-Patent Documents 1 to 4 dairy products and other foods to which microorganisms such as lactic acid bacteria are added are widely accepted by consumers.
  • Non-Patent Documents 1 to 4 it is necessary to measure the content of microorganisms in the food by a shipping inspection of the food or the like or an acceptance inspection of the recipient.
  • Patent Document 1 a method is known in which a test sample is filtered through a membrane filter, cultured in an appropriate medium, and grown colonies are observed.
  • Non-patent Document 5 a method using a modified NBB medium as a lactic acid bacteria detection medium
  • Non-patent Document 6 a method using a KOT medium
  • Non-patent Document 7 a method using chemiluminescence by luciferin-luciferase reaction using ATP in bacterial cells
  • Non-patent Document 8 a method using chemiluminescence by luciferin-luciferase reaction using ATP in bacterial cells
  • a capturing method (Non-Patent Document 9) is also known.
  • detection and quantification of viruses are widely performed for the purpose of detection of contamination and detection of infection by viruses.
  • Patent Document 2 a method using an antigen-antibody reaction between an antibody against a virus and a virus nucleoprotein is known for the purpose of detecting and quantifying influenza virus.
  • an object of the present invention is to provide a technique capable of measuring microorganism cells and viruses in a test sample quickly and accurately.
  • the present invention for solving the above problems
  • a measurement sample preparation step for preparing a measurement sample from a test sample containing cells of microorganisms and / or viruses;
  • Amplification product measurement step of amplifying a target region of a microorganism cell and / or virus-specific DNA or RNA in the measurement sample by a digital PCR method,
  • the measurement sample preparation step does not include an operation of extracting DNA or RNA from microbial cells and / or viruses
  • the measurement sample preparation step includes a dispersion operation that continuously repeats ultrasonic treatment and intermittent treatment on the cells and / or viruses of the microorganisms contained in the test sample.
  • the present invention it is possible to accurately measure microorganism cells and / or viruses in a test sample. Moreover, since the operation of extracting DNA from the cells of the microorganism is not performed, the cells of the microorganism in the test sample can be rapidly measured.
  • the cell is a living cell.
  • the dispersion operation is repeated 20 to 100 times.
  • the cells and / or viruses of the microorganisms in the test sample can be measured with higher accuracy.
  • the ultrasonic treatment is ultrasonic treatment under conditions of an output of 10 W to 100 W and a treatment time of 0.1 second to 1 second.
  • the ultrasonic treatment conditions By setting the ultrasonic treatment conditions to an output of 10 W to 100 W and a treatment time of 0.1 seconds to 1 second, it is possible to more accurately measure the cells of microorganisms and / or viruses in the test sample.
  • the method of the present invention is a method for measuring the cells and / or viruses of microorganisms in a test sample by a digital PCR method.
  • the method of the present invention is not limited to a method of determining the amount of microbial cells and / or viruses, but also includes a method of detecting the presence of microbial cells and / or viruses together with a measured value of the amount.
  • the cells of the microorganism to be measured are not particularly limited as long as the DNA or RNA of the microorganism can be amplified by the digital PCR method, and examples thereof include cells such as bacteria, filamentous fungi, and yeast.
  • Gram-positive bacteria include bacteria of the genus Lactobacillus, bacteria of the genus Orsenella, bacteria of the genus Carnobacterium, bacteria of the genus Weissella, bacteria of the genus Enterococcus, or Bifidobacterium ( Bifidobacterium) genus bacteria and the like can be mentioned.
  • the method of the present invention can be applied to Lactobacillus and / or Bifidobacteria. It is preferable to apply to the measurement of cells of the genus Bifidobacterium.
  • Lactobacillus examples include Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus gasseri, and Lactobacillus L.
  • the bacteria belonging to the genus Bifidobacterium include Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium breve, Bifidobacterium Bifidobacterium adolescentis) and Bifidobacterium infantis (reclassified to Bifidobacterium infantitis, Bifidobacterium longum subspecies infantis).
  • the virus is not particularly limited as long as the DNA or RNA of the virus can be amplified by a digital PCR method.
  • poxviridae, herpesviridae, adenoviridae, papillomaviridae, polyomaviridae, parvovirus Family Picornaviridae, Caliciviridae, Astroviridae, Coronaviridae, Togaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Bornaviridae, Arena Viridae, Bunyaviridae, Reoviridae, Retroviridae, hepatitis virus and the like can be mentioned. Further, the presence or absence of the outermost envelope is not limited.
  • test sample which can be used for the method of this invention.
  • food, a biological sample, a vaccine formulation, drinking water, industrial water, environmental water, drainage, soil, or a wipe sample etc. are mentioned. Can do.
  • a food as a test sample from the viewpoint of the usefulness of measuring cells of microorganisms having physiological activity advantageous to a living body.
  • Beverages such as soft drinks, carbonated drinks, nutrient drinks, fruit juice drinks, lactic acid bacteria drinks (including concentrated concentrates and powders for preparation of these drinks); ice cream, ice sherbet, shaved ice and other frozen desserts; chocolate, caramel, Candy, cakes, biscuits, cookies and other confectionery; milk, processed milk, milk drinks, fermented milk, butter and other dairy products; enteral nutritional fluids such as enteral nutrition foods, child-rearing milk, sports beverages; And functional foods such as health supplements.
  • the test sample may be the aforementioned food, biological sample, vaccine preparation, drinking water, industrial water, environmental water, wastewater, soil, wiped sample, or the like itself, which is diluted or concentrated.
  • any other pretreatment Preferred examples of the pretreatment include heat treatment, filtration, and centrifugation.
  • the cells of the microorganism to be measured and / or cells other than viruses, protein colloid particles, fats and carbohydrates, etc. present in the test sample are removed by treatment with an enzyme having an activity of decomposing them. Or you may reduce.
  • an enzyme having an activity of decomposing them you may reduce.
  • the test sample is milk, dairy products, milk or foods made from milk or dairy products, bovine leukocytes and mammary epithelial cells, etc. as microbial cells and / or cells other than viruses present in the test sample. Can be mentioned.
  • the enzyme is not particularly limited as long as it can decompose impurities, and examples thereof include lipolytic enzymes, proteolytic enzymes, and carbohydrases.
  • the enzyme one type of enzyme may be used alone, or two or more types of enzymes may be used in combination. Among them, it is preferable to use both lipolytic enzyme and proteolytic enzyme, or all of lipolytic enzyme, proteolytic enzyme, and carbohydrase.
  • lipolytic enzyme examples include lipase and phosphatase.
  • proteolytic enzymes include serine protease, cysteine protease, proteinase K, and pronase (registered trademark).
  • saccharide-degrading enzyme examples include amylase, cellulase, N-acetylmuramidase and the like.
  • the test sample it is more preferable to dilute the test sample.
  • the cells and / or viruses of the microorganisms contained in the test sample can be more efficiently dispersed by the dispersion operation described later.
  • the dilution rate of the test sample is preferably 3 to 35 times, more preferably 5 to 30 times.
  • the dilution rate of the test sample is within the above range, the cells and / or viruses of the microorganisms contained in the test sample can be more efficiently dispersed by the dispersion operation described later.
  • Measurement sample preparation process involves adding or processing necessary reagents to the test sample, and providing a measurement sample (nucleic acid amplification reaction) for measurement of amplification products using the digital PCR method. Liquid).
  • the measurement sample preparation step includes a dispersion operation in which the microbial cells and / or viruses contained in the test sample are continuously repeated by ultrasonic treatment and intermittent treatment.
  • a dispersion operation that repeats ultrasonic treatment and intermittent treatment continuously, it is possible to prevent dispensing of aggregates containing multiple cells in one well of a digital PCR chip, and to improve quantitativeness. Can be made.
  • the time per ultrasonic treatment is preferably 0.1 second to 1 second, more preferably 0.3 second to 0.9 second, still more preferably 0.4 second to 0.8 second. Particularly preferred is 0.45 seconds to 0.75 seconds.
  • the time per intermittent treatment is preferably 0.1 seconds to 0.7 seconds, more preferably 0.25 seconds to 0.55 seconds.
  • the output of ultrasonic treatment is preferably 10 W to 100 W, more preferably 25 W to 45 W, and still more preferably 30 W to 40 W.
  • the number of repetitions of the dispersion operation is preferably 20 to 100 times, more preferably 35 to 80 times.
  • the number of repetitions of the dispersion operation is preferably 30 to 90 times, more preferably 40 to 60 times. It is.
  • the number of repetitions of the dispersion operation is preferably 25 to 85 times, more preferably 30 to 50 times. is there.
  • the number of repetitions of the dispersion operation is preferably 20 to 80 times, more preferably 25 to 45 times. is there.
  • the total processing time of ultrasonic treatment (time per ultrasonic treatment ⁇ number of repetitions) is preferably 2 to 100 seconds, more preferably 12 to 41 seconds.
  • the microbial cell and virus in a test sample can be measured more rapidly and more accurately.
  • An ultrasonic device can be used for the dispersion operation.
  • the various conditions described above can be adjusted by changing the settings of the ultrasonic apparatus to be used.
  • the dispersing operation can be performed at any stage before the addition of various reagents and drugs, which will be described later, and after the addition of various reagents and drugs.
  • the extraction of microbial cells and / or viral DNA or RNA contained in the test sample is not performed.
  • the microorganism cells and / or virus DNA or RNA contained in the test sample can be measured more rapidly.
  • extracting DNA or RNA means an operation of collecting or purifying nucleic acid by actively destroying or lysing cells. That is, letting components necessary for nucleic acid amplification such as primers flow into the cell without substantially destroying or lysing the cell, allowing a part of the amplification product to remain in the cell or to flow out of the cell, It is not included in “extracting DNA or RNA”.
  • reagents usually used in the digital PCR method are added. Specifically, in addition to a primer for amplifying a target region, which will be described later, and a probe for measuring an amplification product, a reagent used in ordinary digital PCR such as a dNTP mixed solution and a DNA polymerase is added.
  • a reagent other than the primer and the probe for example, when using a digital PCR apparatus described later, QuantStudio (registered trademark) 3D digital PCR Master Mix v2 ( ⁇ 2) (Thermo Fisher Scientific) can be used.
  • the measurement sample preparation step it is preferable from the viewpoint of improving the quantitativeness to add a drug that suppresses the action of the nucleic acid amplification inhibitor to the test sample.
  • a drug that suppresses the action of a nucleic acid amplification inhibitor is added to the test sample. It is effective to improve the quantitativeness.
  • the “nucleic acid amplification inhibitor” is a substance that inhibits a nucleic acid amplification reaction or a nucleic acid extension reaction.
  • a positive charge inhibitor that adsorbs to a DNA template, or a nucleic acid synthase (DNA polymerase, etc.).
  • negative charge inhibitory substances examples include calcium ions, polyamines, and heme.
  • examples of the negative charge inhibitor include phenol, a phenol compound, heparin, and a cell wall outer membrane of Gram-negative bacteria. Foods and clinical specimens are said to contain many substances that inhibit such nucleic acid amplification reactions.
  • Drugs that suppress the action of the nucleic acid amplification inhibitor as described above include albumin, dextran, T4 gene 32 protein, acetamide, betaine, dimethyl sulfoxide, formamide, glycerol, polyethylene glycol, soybean trypsin inhibitor, ⁇ 2-macroglobulin, tetra From methylammonium chloride and lysozyme, hydrophilic drugs such as phosphorylase and lactate dehydrogenase can be exemplified. These hydrophilic drugs may be used alone or in combination of two or more.
  • polyethylene glycol 400 or polyethylene glycol 4000 can be preferably exemplified as polyethylene glycol.
  • betaine include trimethylglycine and its derivatives.
  • phosphorylase and lactate dehydrogenase include rabbit muscle-derived glycogen phosphorylase and lactate dehydrogenase.
  • glycogen phosphorylase glycogen phosphorylase b is preferable. In particular, it is preferable to use albumin, dextran, T4 gene 32 protein, and lysozyme.
  • albumin typified by BSA bovine serum albumin
  • BSA bovine serum albumin
  • T4 gene 32 protein is a single-stranded DNA-binding protein, and prevents the template from being degraded by nucleolytic enzymes by pre-binding to the single-stranded DNA that is the template in the nucleic acid amplification process. It is thought that inhibition of nucleic acid amplification may be reduced by binding to a nucleic acid amplification inhibitor similar to BSA (Abu Al-Soud, W. et al, Journal of Clinical Microbiology, 38: 4463 -4470, 2000)).
  • BSA, T4 Gene 32 protein, and proteinase inhibitor can reduce proteolytic activity by binding to proteinase and maximize the function of nucleic acid synthase. Sex has been suggested. In fact, there may be proteolytic enzymes remaining in milk and blood, so that the addition of BSA or proteolytic enzyme inhibitors (soybean trypsin inhibitor or ⁇ 2-macroglobulin) prevents the nucleic acid synthase from being degraded. Cases in which the nucleic acid amplification reaction progressed well have been introduced (Abu Al-Soud et al.).
  • Dextran is a polysaccharide generally synthesized by lactic acid bacteria using glucose as a raw material.
  • mucin adheres to the intestinal mucosa (Ruas-Madiedo, P., Applied and Environmental Microbiology, 74: 1936-1940, 2008). It is presumed that there is a sufficient possibility of binding to an inhibitory substance (adsorbed on a nucleic acid synthase) or a positive charge inhibitory substance (adsorbed on a nucleic acid) in advance.
  • lysozyme adsorbs with nucleic acid amplification inhibitors contained in a large amount in milk (AbuSoAl-Soud et al.).
  • hydrophilic drugs represented by albumin, T4 gene 32 protein, dextran, and lysozyme are drugs that suppress the action of nucleic acid amplification inhibitors.
  • albumin examples include bovine serum albumin, ovalbumin, milk albumin, human serum albumin and the like. Of these, bovine serum albumin (BSA) can be preferably exemplified. Albumin may be a purified product or may be used in combination with other components such as globulin as long as the effects of the present invention are not impaired. The albumin may be a fraction.
  • BSA bovine serum albumin
  • the concentration of albumin in the measurement sample is, for example, usually 0.0001 to 1% by mass, preferably 0.01 to 1% by mass, and more preferably 0.2 to 0.00%. 6% by mass.
  • dextran examples include dextran 40 and dextran 500, with dextran 40 being particularly preferred.
  • concentration of dextran in the measurement sample is, for example, usually 1 to 8%, preferably 1 to 6%, more preferably 1 to 4%.
  • the T4 gene 32 protein As the T4 gene 32 protein, a commercially available product (for example, Roche's: also called gp32) may be used.
  • the concentration of the T4 gene 32 protein in the measurement sample is usually 0.01 to 1%, preferably 0.01 to 0.1%, more preferably 0.01 to 0. 0.02%. In the measurement sample preparation step, it is preferable to add T4 gene 32 protein to the test sample so that the concentration of T4 gene 32 protein in the measurement sample falls within the above range.
  • lysozyme lysozyme derived from egg white can be preferably mentioned.
  • concentration of lysozyme in the measurement sample is, for example, usually 1 to 20 ⁇ g / mL, preferably 6 to 15 ⁇ g / mL, and more preferably 9 to 13 ⁇ g / mL.
  • lysozyme is preferably added to the test sample so that the concentration of lysozyme in the measurement sample falls within the above range.
  • lysozyme and polyethylene glycol are preferable to use as a drug that suppresses the action of the nucleic acid amplification inhibitor. Since lysozyme and polyethylene glycol inhibit a protein-derived nucleic acid amplification inhibitor contained in food, the quantification of microorganism cells and / or viruses can be improved by adding them to a test sample.
  • lysozyme and polyethylene glycol as a drug that suppresses the action of the nucleic acid amplification inhibitor. Since lysozyme and polyethylene glycol act in concert with nucleic acid amplification inhibitors to alter the surface structure of the nucleic acid amplification inhibitors, the combination of these results in more efficient use of protein-derived nucleic acid amplification inhibitors in the test sample. Can be inhibited.
  • a magnesium salt, an organic acid salt, a phosphate salt, or the like it is preferable to add a magnesium salt, an organic acid salt, a phosphate salt, or the like to the test sample or a solution of DNA or RNA extracted from the test sample.
  • magnesium salt examples include magnesium chloride, magnesium sulfate, magnesium carbonate and the like. Extracted from the test sample or the test sample so that the concentration of the magnesium salt in the measurement sample (nucleic acid amplification reaction solution) is, for example, 1 to 10 mM, preferably 2 to 6 mM, more preferably 2 to 5 mM. It is preferable to add a magnesium salt to the DNA or RNA solution.
  • organic acid salt examples include salts of citric acid, tartaric acid, propionic acid, butyric acid and the like.
  • the salt include sodium salt and potassium salt.
  • pyrophosphate etc. can be mentioned as a phosphate. These may be used alone or in combination of two or more.
  • the concentration of the organic acid salt or phosphate in the measurement sample is, for example, 0.1 to 20 mM in total, preferably 1 to 10 mM, more preferably 1 to 5 mM. It is preferable to add an organic acid salt or phosphate to a test sample or a solution of DNA or RNA extracted from the test sample.
  • nucleic acid amplification using the above-described primer and probe-containing digital PCR method nucleic acid amplification using the above-described primer and probe-containing digital PCR method, reagents for measuring amplification products, agents that suppress the action of nucleic acid amplification inhibitors, magnesium salts, and organic
  • the order of addition of the acid salt or phosphate is not limited, and they may be added simultaneously (including a form to be mixed in advance).
  • the amplification product measurement step is a step of amplifying the DNA or RNA target region of the cell in the measurement sample prepared in the measurement sample preparation step by the digital PCR method and measuring the amplification product. is there.
  • a sample containing DNA or RNA to be measured is distributed to a chip having a large number of wells, nucleic acid amplification is performed for each well, and the presence or absence of amplification in each well is detected, This is a method of directly calculating the number of wells with a target as the number of copies of the target.
  • Digital PCR can use a commercially available digital PCR device.
  • the digital PCR for example, it is preferable to use Quant Studio (registered trademark) 3D digital PCR (Thermo Fisher Scientific) that performs analysis using a hydrophobic chip having 20000 wells.
  • the conditions for the nucleic acid amplification reaction are not particularly limited, and can be appropriately set in consideration of the length of the target region of DNA or RNA, the TM value of the primer, and the like.
  • the presence or absence of nucleic acid amplification in each well can be determined by hybridizing a probe labeled with a fluorescent molecule or the like to the amplification product. That is, a signal derived from the probe is observed in the well where the nucleic acid amplification reaction has occurred.
  • a probe labeled with a fluorescent molecule is used, the fluorescence emitted from the well can be detected by a device such as a chemirmiphotometer.
  • the “target region of DNA or RNA” is a region targeted for amplification by digital PCR in the DNA or RNA of the microorganism and / or virus to be measured.
  • the target region of DNA or RNA may contain a sequence specific to the microorganism and / or virus to be measured. It is preferable to set to. Further, depending on the purpose, it may have a sequence common to a plurality of types of microorganisms and / or viruses.
  • the target region of DNA or RNA may be single or plural.
  • Primers used for nucleic acid amplification can be appropriately set based on the principle of nucleic acid amplification, and are not particularly limited as long as they can specifically amplify the target region of DNA or RNA.
  • Examples of preferred DNA or RNA target regions are various specific genes such as 5S rDNA gene, 16S rDNA gene, 23S rDNA gene, tDNA gene, and pathogenic gene. One or a part of these genes may be targeted, and a region spanning two or more genes may be targeted.
  • the primer set shown in SEQ ID NO: 1 and SEQ ID NO: 2 and the probe shown in SEQ ID NO: 3 can be used (see Table 3).
  • This primer set can specifically amplify a part of the 16S rDNA gene of Lactobacillus paracasei.
  • the primer sets shown in SEQ ID NO: 4 and SEQ ID NO: 5 and the probe shown in SEQ ID NO: 6 can be used (see Table 7).
  • This primer set can specifically amplify a part of the 16S rDNA gene of Bifidobacterium breve.
  • the cells and / or viruses of the plurality of types of microorganisms in the test sample can be measured.
  • cells and / or viruses of the specific microorganism in the test sample can be measured.
  • the heating step in the present invention may be a step in which the thermal cycle of PCR at the time of amplification product measurement also serves as the heating step, or may be a step in which heat is separately applied to the test sample or the measurement sample.
  • the cell membrane is damaged without outflow of viable DNA, and components such as primers, DNA polymerase, and probes enter the cell. Therefore, cells of a specific microorganism in the test sample and / or Alternatively, viruses can be measured with higher accuracy.
  • the heating temperature in the heating step is preferably 80 ° C. or higher, more preferably 85 ° C. or higher, more preferably 90 ° C. or higher, and still more preferably 95 ° C. or higher.
  • the heating time in the heating step is preferably 5 minutes or more, more preferably 7 minutes or more, and further preferably 9 minutes or more.
  • the heating time in the heating step is preferably 20 minutes or less, more preferably 15 minutes or less, and even more preferably 12 minutes or less.
  • Various conditions in the heating process can be adjusted by changing the settings of the digital PCR device to be used.
  • the measuring step is a step of measuring the number of microbial cells and / or viruses based on the measurement result of the amplification product measuring step.
  • the method for calculating the quantitative value of the cells and / or viruses of the microorganisms in the test sample based on this information is not particularly limited, and examples thereof include a method of analyzing in conformity with a Poisson distribution model.
  • the calculation for calculating the quantitative value of the microbial cell and / or virus from the information about the positive or negative well in the nucleic acid amplification reaction can also be performed using a dedicated cloud analysis software.
  • Test Example 1 Influence of Dispersion Conditions on Measurement Results In Test Example 1, the influence of dispersion conditions on the measurement results was examined.
  • test sample Lactobacillus paracasei dead cells were added to 10 ml of a clinical food so that the cell concentration would be 2.0 ⁇ 10 8 cells / ml. Then, it diluted 20 times using the dilution solvent shown in Table 5, and was set as the test sample.
  • cDBC (abbreviation of concentrated direct component) in Table 1 is a drug that suppresses the action of a nucleic acid amplification inhibitor.
  • CDBC is bovine serum albumin (Sigma, hereinafter referred to as BSA), trisodium citrate dihydrate (Kanto Chemical Co., hereinafter referred to as TSC), magnesium chloride hexahydrate (Nacalai Tesque, hereinafter referred to as MgCl 2), egg white lysozyme (Wako pure Chemical, hereinafter simply referred to as lysozyme), Brij 58 (TM: the sigma) by mixing to a concentration shown in Table 2 were prepared.
  • BSA bovine serum albumin
  • TSC trisodium citrate dihydrate
  • MgCl 2 magnesium chloride hexahydrate
  • MgCl 2 egg white lysozyme
  • TM the sigma
  • Table 3 shows the sequences of the forward primer, reverse primer, and TaqMan (registered trademark) probe used.
  • the number of wells emitting green fluorescence and the intensity of the fluorescence were measured using a chip reader dedicated to the kit, and amplification products were measured. Based on the measurement result of the amplified product, the number of copies of the Lactobacillus paracasei-specific gene contained in the test sample subjected to digital PCR according to the Poisson distribution was calculated using a dedicated cloud type analysis software.
  • the number of repetitions of the dispersion operation is 100 times or less, so that the cells of microorganisms in the test sample can be accurately measured. I found out that I can do it.
  • the number of repetitions of the dispersion operation is large, it is considered that the quantification accuracy of the cells of the microorganism in the test sample is lowered due to the collapse of the cell membrane and the outflow of DNA.
  • Test Example 2 Effect of selection of bacterial species and viability of bacterial cells on measurement results
  • Test Example 2 the effect of selection of bacterial species and viability of bacterial cells on measurement results was examined.
  • test sample (1-1) Preparation of viable cell suspension Bifidobacterium breve MCC1274 (B-3 strain) was anaerobically cultured in MRS broth containing L-Cystein for 16 hours did. After culturing, the cells were washed and mixed with the same volume of 0.1% Tween 80-PBS to prepare a viable cell suspension. The concentration of viable bacteria suspension prepared was measured using a bacterial counting chamber, was 1.6 ⁇ 10 10 cells / ml.
  • test sample The suspension containing each bacterial cell prepared in (1-1) and (1-2) was adjusted to a concentration of 2.0 ⁇ 10 8 cells / ml. It was added to commercially available milk (manufactured by Morinaga Milk Industry Co., Ltd.). Then, a test sample was prepared by diluting milk containing the bacterial cells 20-fold with 0.1% Tween 80-PBS.
  • Table 7 shows the sequences of the forward primer, reverse primer, and TaqMan (registered trademark) probe used.
  • Test Example 3 Influence of selection of test sample and dilution rate of test sample on measurement result In Test Example 3, influence of selection of test sample and dilution rate of test sample on measurement result Study was carried out.
  • test sample As shown in Table 9, bacterial cells were added to food. Thereafter, a test sample was prepared by diluting the bacterial cell-containing food with 0.1% Tween80-PBS so that the dilution ratio shown in Table 9 was obtained.
  • the cells of the microorganisms in the test sample can be accurately obtained by performing a dispersion operation that repeats ultrasonic treatment and intermittent treatment continuously regardless of the dilution rate of the test sample. It was found that it can be measured well. Further, from the results of Table 9, regardless of the type of the test sample, the cells of the microorganisms in the test sample are accurately measured by performing a dispersion operation that continuously repeats the ultrasonic treatment and the intermittent process. I found out that I could do it.
  • Test Example 4 Effect of the presence of cells other than the measurement target on the measurement result
  • Test Example 4 the effect of the presence of the cell other than the measurement target on the measurement result was examined.
  • test sample Lactobacillus paracasei was added to 10 g of food containing microbial cells other than Lactobacillus paracasei so that the cell concentration shown in Table 10 was obtained. Thereafter, a test sample was prepared by diluting into food using 0.1% Tween 80-PBS so that the dilution ratios shown in Table 10 were obtained.
  • the cells of microorganisms in the test sample can be obtained by performing a dispersion operation that continuously repeats ultrasonic treatment and intermittent treatment. It was found that can be measured with high accuracy.
  • the present invention can be applied to the measurement of bacterial cell content and the quantification of viruses in the shipping inspection of foods and the like and the acceptance inspection of the recipient.

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Abstract

La présente invention aborde le problème consistant à fournir un procédé de quantification précise de cellules de micro-organismes et/ou de virus dans un échantillon d'essai. Le moyen pour résoudre le problème de la présente invention est un procédé de mesure de cellules de micro-organismes et/ou de virus dans un échantillon d'essai, le procédé étant caractérisé en ce qu'il comprend : une étape de préparation d'échantillon de mesure servant à préparer un échantillon de mesure à partir d'un échantillon d'essai comprenant des cellules de micro-organismes et/ou de virus; une étape de mesure de produit amplifié servant à amplifier, au moyen d'un procédé de PCR numérique, une région cible d'ADN ou d'ARN qui est intrinsèque à des cellules de micro-organismes et/ou de virus dans l'échantillon de mesure, et servant à mesurer le produit amplifié; et une étape de mesure servant à mesurer le nombre de cellules des micro-organismes et/ou des virus sur la base du résultat de mesure de l'étape de mesure de produit amplifié, l'étape de préparation d'échantillon de mesure ne comprenant pas la mise en œuvre d'une extraction d'ADN ou d'ARN contenu dans des cellules de micro-organismes et/ou de virus, et l'étape de préparation d'échantillon de mesure comprenant la mise en œuvre d'une dispersion qui répète en continu un traitement par ultrasons et un traitement intermittent sur des cellules des micro-organismes et/ou des virus inclus dans l'échantillon d'essai.
PCT/JP2019/011355 2018-03-27 2019-03-19 Procédé de mesure de cellules de micro-organismes et/ou de virus Ceased WO2019188552A1 (fr)

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JPH02203799A (ja) * 1988-11-17 1990-08-13 Becton Dickinson & Co エステラーゼの色原体基質及びそれを用いるイムノアッセイ
WO2011010740A1 (fr) * 2009-07-24 2011-01-27 森永乳業株式会社 Procédé et trousse pour la détection de microorganismes
WO2014021351A1 (fr) * 2012-08-03 2014-02-06 森永乳業株式会社 Procédé de détection de micro-organismes et trousse de détection de micro-organismes
JP2018068211A (ja) * 2016-10-28 2018-05-10 森永乳業株式会社 微生物の死細胞及び／又は不活化ウイルスの測定方法

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JP2013255455A (ja) 2012-06-13 2013-12-26 Ihi Corp 細胞壁穿孔方法、および微生物検出方法
JP5565455B2 (ja) 2012-12-27 2014-08-06 株式会社Ｉｈｉ 微生物の検出方法及びその装置
JP2016195575A (ja) 2015-04-06 2016-11-24 アサヒ飲料株式会社 微生物液の調製方法

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JPH02203799A (ja) * 1988-11-17 1990-08-13 Becton Dickinson & Co エステラーゼの色原体基質及びそれを用いるイムノアッセイ
WO2011010740A1 (fr) * 2009-07-24 2011-01-27 森永乳業株式会社 Procédé et trousse pour la détection de microorganismes
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