WO2019188552A1 - Method for measuring cells of microorganisms and/or viruses - Google Patents

Method for measuring cells of microorganisms and/or viruses 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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PCT/JP2019/011355
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French (fr)
Japanese (ja)
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隆志 副島
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森永乳業株式会社
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Priority to JP2020510728A priority Critical patent/JP7350716B2/en
Publication of WO2019188552A1 publication Critical patent/WO2019188552A1/en

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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

The present invention addresses the problem of providing a method for precisely quantifying cells of microorganisms and/or viruses in a test sample. The means for solving the problem of the present invention is a method for measuring cells of microorganisms and/or viruses in a test sample, the method being characterized by having: a measurement sample preparation step for preparing a measurement sample from a test sample including cells of microorganisms and/or viruses; an amplified product measurement step for amplifying, by using a digital PCR method, a target region of DNA or RNA which is intrinsic to cells of microorganisms and/or viruses in the measurement sample, and for measuring the amplified product; and a measurement step for measuring the number of cells of the microorganisms and/or viruses on the basis of the measurement result of the amplified product measurement step, wherein the measurement sample preparation step does not include an operation for extracting DNA or RNA from cells of microorganisms and/or viruses, and the measurement sample preparation step includes a dispersion operation which continuously repeats ultrasonic treatment and intermittent treatment on cells of the microorganisms and/or viruses included in the test sample.

Description

微生物の細胞及び／又はウイルスの測定方法Method for measuring microorganism cell and / or virus
　本発明は、被検試料中の微生物の細胞及び／又はウイルスの測定方法に関する。 The present invention relates to a method for measuring microbial cells and / or viruses in a test sample.
　現在、乳製品をはじめとして、乳酸菌などの微生物を添加した食品が広く消費者に受け入れられている（非特許文献１～４）。ここで、乳酸菌などの微生物を含む食品を提供するにあたり、食品等の出荷検査や受け入れ先の受け入れ検査等で食品中の微生物含有量を測定する必要がある。 Currently, 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). Here, in providing a food containing microorganisms such as lactic acid bacteria, 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.
　微生物の検出・定量の技術として、例えば、被検試料をメンブランフィルターでろ過後、適当な培地中で培養し、生育したコロニーを観察する方法が知られている（特許文献１）。 As a technique for detecting and quantifying microorganisms, for example, 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 (Patent Document 1).
　また、乳酸菌検出培地として改変ＮＢＢ培地を使用する方法（非特許文献５）、ＫＯＴ培地を使用する方法（非特許文献６）も知られている。さらに、菌体中のＡＴＰを利用し、ルシフェリン－ルシフェラーゼ反応による化学発光を用いる方法（非特許文献７、非特許文献８）、また、培地中の菌の生育を培地の電気伝導度の変化で捕らえる方法（非特許文献９）も知られている。 Further, a method using a modified NBB medium as a lactic acid bacteria detection medium (Non-patent Document 5) and a method using a KOT medium (Non-patent Document 6) are also known. In addition, a method using chemiluminescence by luciferin-luciferase reaction using ATP in bacterial cells (Non-patent Document 7, Non-patent Document 8), and the growth of bacteria in the medium can be determined by changing the electrical conductivity of the medium. A capturing method (Non-Patent Document 9) is also known.
　また、汚染の検出や、ウイルスによる感染の検出を目的として、ウイルスの検出・定量が広く行われている。 In addition, detection and quantification of viruses are widely performed for the purpose of detection of contamination and detection of infection by viruses.
　ウイルスの検出・定量の技術としては、例えば、インフルエンザウイルスの検出・定量を目的として、ウイルスに対する抗体とウイルスの核蛋白質との抗原抗体反応を利用する方法が知られている（特許文献２）。 As a technique for detecting and quantifying viruses, for example, 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 (Patent Document 2).
特開平６－３１１８９４号公報JP-A-6-31894 特開２００５－１６４４９６号公報JP 2005-16496 A
　前述した背景において、被検試料中の微生物の細胞やウイルスを迅速かつ精度よく測定することのできる技術が求められていた。すなわち、本発明は、被検試料中の微生物の細胞やウイルスを迅速かつ精度よく測定することのできる技術を提供することを課題とする。 In the background described above, there has been a demand for a technique that can quickly and accurately measure microbial cells and viruses in a test sample. That is, 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.
　前記課題を解決する本発明は、
　微生物の細胞及び／又はウイルスを含む被検試料から測定用試料を調製する測定用試料調製工程と、
　前記測定用試料中の微生物の細胞及び／又はウイルス固有のＤＮＡ又はＲＮＡのターゲット領域をデジタルＰＣＲ法により増幅し、増幅産物を測定する増幅産物測定工程と、
　増幅産物測定工程の測定結果に基づき前記微生物の細胞及び／又はウイルスの数を測定する測定工程と、
　を有し、
　前記測定用試料調製工程は、微生物の細胞及び／又はウイルスからＤＮＡ又はＲＮＡを抽出する操作を含まず、
　前記測定用試料調製工程は、前記被検試料に含まれる前記微生物の細胞及び／又はウイルスに対し、超音波処理と間欠処理とを連続して繰り返す分散操作を含むことを特徴とする。 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,
A measurement step of measuring the number of cells and / or viruses of the microorganism based on the measurement result of the amplification product measurement step;
Have
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.
　本発明によれば、被検試料中の微生物の細胞及び／又はウイルスを精度良く測定することができる。また、微生物の細胞からＤＮＡを抽出する操作を行わないため、被検試料中の微生物の細胞を迅速に測定することができる。 According to 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.
　本発明の好ましい形態では、前記細胞は生細胞である。 In a preferred embodiment of the present invention, the cell is a living cell.
　本発明の好ましい形態では、前記分散操作の繰り返し回数は２０回～１００回である。
　分散操作の繰り返し回数を２０回～１００回とすることで、被検試料中の微生物の細胞及び／又はウイルスをより精度良く測定することができる。 In a preferred embodiment of the present invention, the dispersion operation is repeated 20 to 100 times.
By setting the number of repetitions of the dispersion operation to 20 to 100 times, the cells and / or viruses of the microorganisms in the test sample can be measured with higher accuracy.
　本発明の好ましい形態では、前記超音波処理が、出力１０Ｗ～１００Ｗ、処理時間０．１秒～１秒の条件の超音波処理である。
　超音波処理の条件を出力１０Ｗ～１００Ｗ、処理時間０．１秒～１秒の条件とすることで、被検試料中の微生物の細胞及び／又はウイルスをより精度良く測定することができる。 In a preferred embodiment of the present invention, 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.
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.
　本発明によれば、被検試料中の微生物の細胞及び／又はウイルスを精度よく測定することができる。 According to the present invention, it is possible to accurately measure microorganism cells and / or viruses in a test sample.
　次に、本発明の好ましい実施形態について詳細に説明する。但し、本発明は以下の実施形態に限定されず、本発明の範囲内で自由に変更することができる。 Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be freely modified within the scope of the present invention.
＜１＞微生物の細胞及び／又はウイルスの測定方法
　本発明の方法は、被検試料中の微生物の細胞及び／又はウイルスをデジタルＰＣＲ法により測定する方法である。 <1> Method for Measuring Microbial Cells and / or Viruses 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.
　本発明の方法は、微生物の細胞及び／又はウイルスの量を決定する方法に限定されず、微生物の細胞及び／又はウイルスの存在を量についての測定値と共に検出する方法も含む。 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.
　測定対象となる微生物の細胞としては、デジタルＰＣＲ法により、該微生物のＤＮＡ又はＲＮＡを増幅し得る限り特に制限されないが、例えば、細菌、糸状菌、酵母等の細胞を挙げることができる。 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.
　中でも、本発明の方法は、グラム陽性細菌の細胞を対象とすることが好ましい。グラム陽性細菌としては、ラクトバチルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ）属細菌、オルセネラ（Ｏｌｓｅｎｅｌｌａ）属細菌、カルノバクテリウム（Ｃａｒｎｏｂａｃｔｅｒｉｕｍ）属細菌、ウェイセラ（Ｗｅｉｓｓｅｌｌａ）属細菌、エンテロコッカス（Ｅｎｔｅｒｏｃｏｃｃｕｓ）属細菌、又はビフィドバクテリウム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ）属細菌等を挙げることができる。 Of these, the method of the present invention is preferably directed to cells of Gram-positive bacteria. 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.
　特にラクトバチルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ）属細菌やビフィドバクテリウム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ）属細菌には人体に有利な生理活性を示すものがあることから、本発明の方法は、ラクトバチルス属細菌及び／又はビフィドバクテリウム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ）属細菌の細胞の測定に適用することが好ましい。 In particular, there are bacteria belonging to the genus Lactobacillus and Bifidobacterium that exhibit physiological activity advantageous to the human body. Therefore, 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.
　ラクトバチルス属細菌としては、ラクトバチルス・パラカゼイ（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐａｒａｃａｓｅｉ）、ラクトバチルス・プランタラム（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍ）、ラクトバチルス・ガセリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｇａｓｓｅｒｉ）、ラクトバチルス・サリバリウス（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｓａｌｉｖａｒｉｕｓ）等を挙げることができる。 Examples of bacteria belonging to the genus Lactobacillus include Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus gasseri, and Lactobacillus L.
　ビフィドバクテリウム属細菌としては、ビフィドバクテリウム・ロンガム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｌｏｎｇｕｍ）、ビフィドバクテリウム・ビフィダム（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｉｆｉｄｕｍ）、ビフィドバクテリウム・ブレーベ（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｒｅｖｅ）、ビフィドバクテリウム・アドレセンティス（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ａｄｏｌｅｓｃｅｎｔｉｓ）、及び、ビフィドバクテリウム・インファンティス（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｉｎｆａｎｔｉｓ、ビフィドバクテリウム・ロンガム・サブスピーシーズ・インファンティスに再分類されている）を挙げることができる。 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).
　ウイルスとしては、デジタルＰＣＲ法により、該ウイルスのＤＮＡ又はＲＮＡを増幅し得る限り特に制限されないが、例えば、ポックスウイルス科、ヘルペスウイルス科、アデノウイルス科、パピローマウイルス科、ポリオーマウイルス科、パルボウイルス科、ピコルナウイルス科、カリシウイルス科、アストロウイルス科、コロナウイルス科、トガウイルス科、フラビウイルス科、オルトミクソウイルス科、パラミクソウイルス科、ラブドウイルス科、フィロウイルス科、ボルナウイルス科、アレナウイルス科、ブニヤウイルス科、レオウイルス科、レトロウイルス科、肝炎ウイルス等を挙げることができる。また、最外殻エンベロープの有無も制限されない。 The virus is not particularly limited as long as the DNA or RNA of the virus can be amplified by a digital PCR method. For example, 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.
　また、本発明の方法に用いることのできる被検試料に特に制限はなく、例えば、食品、生体試料、ワクチン製剤、飲料水、工業用水、環境用水、排水、土壌、又は拭き取り試料等を挙げることができる。 Moreover, there is no restriction | limiting in particular in the test sample which can be used for the method of this invention, For example, food, a biological sample, a vaccine formulation, drinking water, industrial water, environmental water, drainage, soil, or a wipe sample etc. are mentioned. Can do.
　特に、生体に有利な生理活性を有する微生物の細胞を測定することの有用性の観点から、食品を被検試料とすることが好ましい。食品として、清涼飲料、炭酸飲料、栄養飲料、果汁飲料、乳酸菌飲料等の飲料（これらの飲料の濃縮原液及び調製用粉末を含む）；アイスクリーム、アイスシャーベット、かき氷等の冷菓；チョコレート、キャラメル、キャンディ、ケーキ、ビスケット、クッキー等の菓子；牛乳、加工乳、乳飲料、発酵乳、バター等の乳製品；経腸栄養食品等の高栄養流動食品、育児用ミルク、スポーツ飲料；特定保健用食品、健康補助食品等の機能性食品を挙げることができる。 In particular, it is preferable to use 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.
　本発明において、被検試料は、前述の食品、生体試料、ワクチン製剤、飲料水、工業用水、環境用水、排水、土壌、又は拭き取り試料等そのものであってもよく、これらを希釈もしくは濃縮したもの、又はその他任意の前処理をしたものであってもよい。前処理としては、加熱処理、濾過、遠心分離等を好ましく挙げることができる。 In the present invention, 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. Or any other pretreatment. Preferred examples of the pretreatment include heat treatment, filtration, and centrifugation.
　また、被検試料中に存在する測定対象の微生物の細胞及び／又はウイルス以外の細胞、タンパク質コロイド粒子、脂肪及び糖質等の夾雑物は、これらを分解する活性を有する酵素による処理等によって除去又は低減させてもよい。被検試料が乳、乳製品、乳又は乳製品を原料とする食品である場合には、被検試料中に存在する微生物の細胞及び／又はウイルス以外の細胞としてウシ白血球及び乳腺上皮細胞等を挙げることができる。 In addition, 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. When 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.
　前記酵素としては、夾雑物を分解することができるものであれば特に制限されないが、例えば、脂質分解酵素、タンパク質分解酵素、及び糖質分解酵素を挙げることができる。
酵素は、１種類の酵素を単独で用いてもよいし、２種又はそれ以上の酵素を併用してもよい。中でも、脂質分解酵素及びタンパク質分解酵素の両方、又は脂質分解酵素、タンパク質分解酵素、及び糖質分解酵素の全てを用いることが好ましい。 The enzyme is not particularly limited as long as it can decompose impurities, and examples thereof include lipolytic enzymes, proteolytic enzymes, and carbohydrases.
As 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.
　脂質分解酵素としては、リパーゼ、フォスファターゼ等を挙げることができる。
　また、タンパク質分解酵素としてはセリンプロテアーゼ、システインプロテアーゼ、プロテイナーゼＫ、プロナーゼ（登録商標）等を挙げることができる。
　また、糖質分解酵素としてはアミラーゼ、セルラーゼ、Ｎ－アセチルムラミダーゼ等を挙げることができる。 Examples of the lipolytic enzyme include lipase and phosphatase.
Examples of proteolytic enzymes include serine protease, cysteine protease, proteinase K, and pronase (registered trademark).
Examples of the saccharide-degrading enzyme include amylase, cellulase, N-acetylmuramidase and the like.
　また、本発明においては、被検試料を希釈することがより好ましい。被検試料を希釈することで、後述する分散操作で被検試料に含まれる微生物の細胞及び／又はウイルスをより効率よく分散させることができる。 In the present invention, it is more preferable to dilute the test sample. By diluting 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.
　被検試料の希釈倍率は、好ましくは３倍～３５倍、より好ましくは５倍～３０倍の範囲内とすることがより好ましい。
　被検試料の希釈倍率が上記範囲内にあることで、後述する分散操作で被検試料に含まれる微生物の細胞及び／又はウイルスをより効率よく分散させることができる。 The dilution rate of the test sample is preferably 3 to 35 times, more preferably 5 to 30 times.
When 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.
＜２＞　本発明の方法における各工程について
　以下、本発明の方法における各工程について、詳細に説明する。 <2> About each process in the method of this invention Hereinafter, each process in the method of this invention is demonstrated in detail.
（１）測定用試料調製工程
　測定用試料調製工程は、被検試料に必要な試薬の添加や処理を行い、デジタルＰＣＲ法を用いた増幅産物の測定に供するための測定用試料（核酸増幅反応液）を調製する工程である。 (1) Measurement sample preparation process The 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).
　そして、測定用試料調製工程は、被検試料に含まれる微生物の細胞及び／又はウイルスを、超音波処理と間欠処理とを連続して繰り返す分散操作を含む。
　超音波処理と間欠処理とを連続して繰り返す分散操作を含むことで、デジタルＰＣＲチップの一つのウェルに複数の細胞が含まれた凝集体が分注されてしまうことを防ぎ、定量性を向上させることができる。 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.
By including 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.
　以下、分散条件のより好ましい形態について説明する。 Hereinafter, a more preferable form of the dispersion condition will be described.
　本発明における、超音波処理の一回当たりの時間は、好ましくは０．１秒～１秒、より好ましくは０．３秒～０．９秒、さらに好ましくは０．４秒～０．８秒、特に好ましくは、０．４５秒～０．７５秒である。 In the present invention, 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.
　また、本発明における、間欠処理の一回当たりの時間は、好ましくは０．１秒～０．７秒、より好ましくは０．２５秒～０．５５秒である。 In the present invention, the time per intermittent treatment is preferably 0.1 seconds to 0.7 seconds, more preferably 0.25 seconds to 0.55 seconds.
　また、本発明における、超音波処理の出力は、好ましくは１０Ｗ～１００Ｗ、より好ましくは２５Ｗ～４５Ｗ、さらに好ましくは３０Ｗ～４０Ｗである。 In the present invention, 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.
　また、本発明における、分散操作の繰り返し回数は、好ましくは２０回～１００回、より好ましくは３５回～８０回である。 In the present invention, the number of repetitions of the dispersion operation is preferably 20 to 100 times, more preferably 35 to 80 times.
　ここで、超音波処理の一回当たりの時間が０．３秒～０．４５秒である場合の、分散操作の繰り返し回数は、好ましくは３０回～９０回、より好ましくは４０回～６０回である。 Here, when the time per ultrasonic treatment is 0.3 to 0.45 seconds, the number of repetitions of the dispersion operation is preferably 30 to 90 times, more preferably 40 to 60 times. It is.
　また、超音波処理の一回当たりの時間が０．４５秒～０．７５秒である場合の、分散操作の繰り返し回数は、好ましくは２５回～８５回、より好ましくは３０回～５０回である。 Further, when the time per ultrasonic treatment is 0.45 to 0.75 seconds, the number of repetitions of the dispersion operation is preferably 25 to 85 times, more preferably 30 to 50 times. is there.
　また、超音波処理の一回当たりの時間が０．７５秒～０．９秒である場合の、分散操作の繰り返し回数は、好ましくは２０回～８０回、より好ましくは２５回～４５回である。 Further, when the time per ultrasonic treatment is 0.75 to 0.9 seconds, the number of repetitions of the dispersion operation is preferably 20 to 80 times, more preferably 25 to 45 times. is there.
　また、超音波処理の総処理時間（超音波処理の一回あたりの時間×繰り返し回数）は、好ましくは２秒～１００秒、より好ましくは１２秒～４１秒である。 Also, 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.
　以上の条件とすることで、被検試料中の微生物の細胞やウイルスをより迅速かつより精度よく測定することができる。 By setting it as the above conditions, the microbial cell and virus in a test sample can be measured more rapidly and more accurately.
　分散操作には、超音波装置を用いることができる。そして、前述の各種条件は、用いる超音波装置の設定を変更することにより調整することができる。 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.
　なお、分散操作は後述する各種試薬や薬剤の添加前、各種試薬や薬剤の添加後の何れの段階であっても行うことができる。 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.
　また、測定用試料調製工程では、被検試料に含まれる微生物の細胞及び／又はウイルスのＤＮＡ又はＲＮＡの抽出を行わない。被検試料に含まれる微生物の細胞及び／又はウイルスのＤＮＡ又はＲＮＡの抽出を行わないことで、より迅速に被検試料に含まれる微生物の細胞及び／又はウイルスを測定することができる。 Also, in the measurement sample preparation step, the extraction of microbial cells and / or viral DNA or RNA contained in the test sample is not performed. By not extracting the microorganism cells and / or virus DNA or RNA contained in the test sample, the microorganism cells and / or viruses contained in the test sample can be measured more rapidly.
　なお、本発明において、「ＤＮＡ又はＲＮＡの抽出を行う」とは、積極的に細胞を破壊又は溶解して核酸を採取又は精製する操作を意味する。すなわち、細胞を実質的に破壊又は溶解せずにプライマー等の核酸増幅に必要な成分を細胞内に流入させること、増幅産物の一部分を細胞内に留まらせること若しくは細胞外に流出させることは、「ＤＮＡ又はＲＮＡを抽出する」に含まない。 In the present invention, “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”.
　測定用試料調製工程では、デジタルＰＣＲ法に通常用いられる試薬を添加する。具体的には、後述するターゲット領域を増幅するためのプライマー、増幅産物を測定するためのプローブのほか、ｄＮＴＰ混合液、ＤＮＡポリメラーゼ等通常のデジタルＰＣＲに用いられる試薬を添加する。プライマー及びプローブ以外の試薬としては、例えば後述するデジタルＰＣＲ装置を用いる場合には、ＱｕａｎｔＳｔｕｄｉｏ（登録商標）　３Ｄ　ｄｉｇｉｔａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ　ｖ２（×２）（サーモフィッシャーサイエンティフィック社）を用いることができる。 In the measurement sample preparation process, 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. As 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.
　また、測定用試料調製工程では、被検試料に核酸増幅阻害物質の働きを抑制する薬剤を添加することが定量性の向上の観点から好ましい。
　特に、測定用試料調製工程で被検試料中の微生物の細胞及び／又はウイルスのＤＮＡ又はＲＮＡの抽出を行わない本発明においては、被検試料に核酸増幅阻害物質の働きを抑制する薬剤を添加することが、定量性の向上のために有効である。 In 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.
In particular, in the present invention in which extraction of microbial cells and / or viral DNA or RNA in a test sample is not performed in the measurement sample preparation step, 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.
　ここで「核酸増幅阻害物質」とは、核酸増幅反応又は核酸伸張反応を阻害する物質であって、例えば、ＤＮＡの鋳型に吸着する正電荷阻害物質、又は核酸合成酵素（ＤＮＡポリメラーゼなど）に吸着する負電荷阻害物質等を挙げることができる。前記正電荷阻害物質としては、カルシウムイオン、ポリアミン、ヘム（ｈｅｍｅ）等を挙げることができる。また、負電荷阻害物質としては、フェノール、フェノール系化合物、ヘパリン、グラム陰性菌の細胞壁外膜等を挙げることができる。食品や臨床検体中には、このような核酸増幅反応を阻害する物質が多く含まれているといわれている。 Here, the “nucleic acid amplification inhibitor” is a substance that inhibits a nucleic acid amplification reaction or a nucleic acid extension reaction. For example, a positive charge inhibitor that adsorbs to a DNA template, or a nucleic acid synthase (DNA polymerase, etc.). And negative charge inhibitory substances. Examples of the positive charge inhibitor include calcium ions, polyamines, and heme. In addition, 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.
　前述したような核酸増幅阻害物質の働きを抑制する薬剤としては、アルブミン、デキストラン、Ｔ４ジーン３２プロテイン、アセトアミド、ベタイン、ジメチルスルホキシド、ホルムアミド、グリセロール、ポリエチレングリコール、大豆トリプシンインヒビター、α２－マクログロブリン、テトラメチルアンモニウムクロライド、リゾチームから、ホスホリラーゼ、及び乳酸脱水素酵素等の親水性薬剤が例示できる。これら親水性薬剤は１種のみを用いてもよいし、複数種を組み合わせて用いてもよい。 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, α2-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.
　前述した親水性薬剤のうち、ポリエチレングリコールとしては、ポリエチレングリコール４００又はポリエチレングリコール４０００が好ましく例示できる。ベタインとしては、トリメチルグリシンやその誘導体等を挙げることができる。また、ホスホリラーゼ及び乳酸脱水素酵素としては、ウサギ筋肉由来のグリコーゲンホスホリラーゼ及び乳酸脱水素酵素を挙げることができる。なお、グリコーゲンホスホリラーゼとしては、グリコーゲンホスホリラーゼｂが好ましい。
　特に、アルブミン、デキストラン、Ｔ４ジーン３２プロテイン、及びリゾチームを使用することが好ましい。 Among the hydrophilic drugs mentioned above, polyethylene glycol 400 or polyethylene glycol 4000 can be preferably exemplified as polyethylene glycol. Examples of betaine include trimethylglycine and its derivatives. Examples of phosphorylase and lactate dehydrogenase include rabbit muscle-derived glycogen phosphorylase and lactate dehydrogenase. As glycogen phosphorylase, glycogen phosphorylase b is preferable.
In particular, it is preferable to use albumin, dextran, T4 gene 32 protein, and lysozyme.
　ＢＳＡ（ウシ血清アルブミン）に代表されるアルブミンは、ヘム（ｈｅｍｅ）のような核酸増幅阻害物質に結合することにより、核酸増幅阻害を低減させている可能性が示唆されている（Abu Al-Soudら）。 It has been suggested that albumin typified by BSA (bovine serum albumin) may reduce nucleic acid amplification inhibition by binding to a nucleic acid amplification inhibitor such as heme (Abu Al-Soud). Et.)
　また、Ｔ４ジーン３２プロテインは１本鎖ＤＮＡ結合性タンパク質であり、核酸増幅過程で鋳型となっている１本鎖ＤＮＡに予め結合することにより鋳型が核酸分解酵素によって分解されることを防いでいるか、または、ＢＳＡと同様の核酸増幅阻害物質に結合することにより核酸増幅阻害を低減しているのであろうと考えられている（Abu Al-Soud, W. et al, Journal of Clinical Microbiology, 38:4463-4470, 2000））。 In addition, 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)).
　さらに、ＢＳＡ、Ｔ４ジーン３２プロテイン、及びタンパク質分解酵素阻害剤（proteinase inhibitor）は、タンパク質分解酵素（proteinase）に結合することによりタンパク質分解活性を低減させ、核酸合成酵素の働きを最大限に引き出す可能性が示唆されている。事実、牛乳や血液にはタンパク質分解酵素が残存していることもあり、その際ＢＳＡ又はタンパク質分解酵素阻害剤（大豆トリプシンインヒビターやα２－マクログロブリン）の添加により核酸合成酵素が分解を受けずに核酸増幅反応が良好に進行したケースも紹介されている（Abu Al-Soudら）。 In addition, BSA, T4 Gene 32 protein, and proteinase inhibitor (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 α2-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.).
　また、デキストランは、一般にグルコースを原料として乳酸菌が合成する、多糖類である。ここで、ムチンという同様の多糖類－ペプチド複合体が腸管粘膜に接着することも報告されており（Ruas-Madiedo, P., Applied and Environmental Microbiology, 74:1936-1940, 2008）、デキストランが負電荷阻害物質（核酸合成酵素に吸着）、又は正電荷阻害物質（核酸に吸着）に予め吸着することにより、それら阻害物質に結合する可能性は十分あるものと推察される。 Dextran is a polysaccharide generally synthesized by lactic acid bacteria using glucose as a raw material. Here, it has been reported that a similar polysaccharide-peptide complex called 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.
　また、リゾチームは、牛乳中に多数含まれている核酸増幅阻害物質と吸着する（前記Abu Al-Soudら）。 In addition, lysozyme adsorbs with nucleic acid amplification inhibitors contained in a large amount in milk (AbuSoAl-Soud et al.).
　以上のことから、アルブミン、Ｔ４ジーン３２プロテイン、デキストラン、及びリゾチームに代表される親水性薬剤は、核酸増幅阻害物質の働きを抑制する薬剤であるといえる。 From the above, it can be said that hydrophilic drugs represented by albumin, T4 gene 32 protein, dextran, and lysozyme are drugs that suppress the action of nucleic acid amplification inhibitors.
　アルブミンとしては、ウシ血清アルブミン、卵白アルブミン、乳アルブミン、ヒト血清アルブミン等を挙げることができる。これらの中ではウシ血清アルブミン（ＢＳＡ）が好ましく例示できる。アルブミンは精製品でもよく、本発明の効果を損わない限りグロブリン等の他の成分と組み合わせて用いてもよい。また、アルブミンは分画物であってもよい。 Examples of albumin 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.
　測定用試料（核酸増幅反応液）中のアルブミンの濃度は、例えば、通常０．０００１～１質量％であり、好ましくは０．０１～１質量％であり、より好ましくは０．２～０．６質量％である。
　測定用試料調製工程においては、測定用試料におけるアルブミンの濃度が前記範囲となるように、被検試料にアルブミンを添加することが好ましい。 The concentration of albumin in the measurement sample (nucleic acid amplification reaction solution) 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.
In the measurement sample preparation step, it is preferable to add albumin to the test sample so that the concentration of albumin in the measurement sample falls within the above range.
　デキストランとしては、デキストラン４０やデキストラン５００等が挙げられ、特にデキストラン４０を好ましく挙げることができる。
　測定用試料（核酸増幅反応液）中のデキストランの濃度は、例えば、通常１～８％であり、好ましくは１～６％であり、より好ましくは１～４％である。
　測定用試料調製工程においては、測定用試料におけるデキストランの濃度が前記範囲となるように、被検試料にデキストランを添加することが好ましい。 Examples of dextran include dextran 40 and dextran 500, with dextran 40 being particularly preferred.
The concentration of dextran in the measurement sample (nucleic acid amplification reaction solution) is, for example, usually 1 to 8%, preferably 1 to 6%, more preferably 1 to 4%.
In the measurement sample preparation step, it is preferable to add dextran to the test sample so that the concentration of dextran in the measurement sample falls within the above range.
　Ｔ４ジーン３２プロテインとしては、市販品（例えば、ロシュ社製：ｇｐ３２とも呼ばれる）を用いてもよい。
　Ｔ４ジーン３２プロテインの測定用試料（核酸増幅反応液）中の濃度は、通常０．０１～１％であり、好ましくは０．０１～０．１％であり、より好ましくは０．０１～０．０２％である。
　測定用試料調製工程においては、測定用試料におけるＴ４ジーン３２プロテインの濃度が前記範囲となるように、被検試料にＴ４ジーン３２プロテインを添加することが好ましい。 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 (nucleic acid amplification reaction solution) 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.
　リゾチームとしては、卵白由来のリゾチームを好ましく挙げることができる。
　測定用試料（核酸増幅反応液）中のリゾチームの濃度は、例えば、通常１～２０μｇ／ｍＬであり、好ましくは６～１５μｇ／ｍＬであり、より好ましくは９～１３μｇ／ｍＬである。
　測定用試料調製工程においては、測定用試料におけるリゾチームの濃度が前記範囲となるように、被検試料にリゾチームを添加することが好ましい。 As the lysozyme, lysozyme derived from egg white can be preferably mentioned.
The concentration of lysozyme in the measurement sample (nucleic acid amplification reaction solution) is, for example, usually 1 to 20 μg / mL, preferably 6 to 15 μg / mL, and more preferably 9 to 13 μg / mL.
In the measurement sample preparation step, lysozyme is preferably added to the test sample so that the concentration of lysozyme in the measurement sample falls within the above range.
　測定用試料調製工程においては、核酸増幅阻害物質の働きを抑制する薬剤として、リゾチーム及びポリエチレングリコールの少なくとも一方を用いることが好ましい。
　リゾチームとポリエチレングリコールは食品に含まれる蛋白質由来の核酸増幅阻害物質を阻害するため、これらを被検試料に添加することにより、微生物の細胞及び／又はウイルスの定量性を向上させることができる。 In the measurement sample preparation step, it is preferable to use at least one of lysozyme and polyethylene glycol 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.
　また測定用試料調製工程において、核酸増幅阻害物質の働きを抑制する薬剤として、リゾチーム及びポリエチレングリコールを組み合わせて用いることが特に好ましい。
　リゾチームとポリエチレングリコールが核酸増幅阻害物質に協奏的に作用し核酸増幅阻害物質の表面構造を変質させるため、これらを組み合わせて用いれば、被検試料に含まれる蛋白質由来の核酸増幅阻害物質をより効率的に阻害することができる。 In the measurement sample preparation step, it is particularly preferable to use a combination of 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.
　また、測定用試料調製工程においては、被検試料又は被検試料より抽出したＤＮＡ又はＲＮＡの溶液にマグネシウム塩、及び有機酸塩又はリン酸塩等を添加することが好ましい。 In the measurement sample preparation step, 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.
　マグネシウム塩としては、塩化マグネシウム、硫酸マグネシウム、炭酸マグネシウム等を挙げることができる。
　測定用試料（核酸増幅反応液）中のマグネシウム塩の濃度が、例えば、１～１０ｍＭ、好ましくは２～６ｍＭ、より好ましくは２～５ｍＭとなるように、被検試料又は被検試料より抽出したＤＮＡ又はＲＮＡの溶液にマグネシウム塩を添加することが好ましい。 Examples of the magnesium salt 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.
　有機酸塩としては、クエン酸、酒石酸、プロピオン酸、酪酸等の塩を挙げることができる。塩の種類としては、ナトリウム塩、カリウム塩等を挙げることができる。また、リン酸塩として、ピロリン酸等を挙げることができる。これらは１種でもよく、２種又は３種以上の混合物であってもよい。
　測定用試料（核酸増幅反応液）中の有機酸塩又はリン酸塩の濃度が、例えば、合計量で０．１～２０ｍＭ、好ましくは１～１０ｍＭ、より好ましくは１～５ｍＭとなるように、被検試料又は被検試料より抽出したＤＮＡ又はＲＮＡの溶液に有機酸塩又はリン酸塩を添加することが好ましい。 Examples of the organic acid salt include salts of citric acid, tartaric acid, propionic acid, butyric acid and the like. Examples of the salt include sodium salt and potassium salt. Moreover, 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 (nucleic acid amplification reaction solution) 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.
　なお、測定用試料調製工程において、前述したプライマー、プローブを含むデジタルＰＣＲ法を用いた核酸増幅、増幅産物の測定のための試薬、核酸増幅阻害物質の働きを抑制する薬剤、マグネシウム塩、及び有機酸塩又はリン酸塩の添加の順序は問わず、また、同時に（あらかじめ混合する形態を含む）添加してもよい。 In addition, in the sample preparation process for measurement, 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).
（２）増幅産物測定工程
　増幅産物測定工程は、測定用試料調製工程で調製した測定用試料中の前記細胞のＤＮＡ又はＲＮＡのターゲット領域をデジタルＰＣＲ法により増幅し、増幅産物を測定する工程である。 (2) Amplification product measurement step 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.
　デジタルＰＣＲ法は、測定対象となるＤＮＡ又はＲＮＡを含む試料を多数のウェルを備えるチップに分配し、ウェルごとに個別に核酸増幅を行い、各ウェルでの「増幅の有無」を検出し、シグナルのあるウェルの数をターゲットのコピー数として直接的に算出する方法である。 In the digital PCR method, 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.
　デジタルＰＣＲは、市販のデジタルＰＣＲ装置を用いることができる。デジタルＰＣＲとして、例えば、２００００ウェルを備える疎水性のチップにより解析を行うＱｕａｎｔＳｔｕｄｉｏ（登録商標）　３Ｄ　ｄｉｇｉｔａｌ　ＰＣＲ（サーモフィッシャーサイエンティフィック社）を用いることが好ましい。 Digital PCR can use a commercially available digital PCR device. As 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.
　核酸増幅反応の条件は特に限定されず、ＤＮＡ又はＲＮＡのターゲット領域の長さ、プライマーのＴＭ値などを考慮して適宜設定することができる。 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.
　各ウェルにおける核酸増幅の有無は、蛍光分子などで標識されたプローブを増幅産物にハイブリダイズさせることにより判別することができる。すなわち、核酸増幅反応が起こったウェルではプローブに由来するシグナルが観察される。蛍光分子で標識されたプローブを用いる場合には、ケミルミフォトメータなどの装置によってウェルから発する蛍光を検出することができる。 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. When 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.
　本発明において「ＤＮＡ又はＲＮＡのターゲット領域」とは、測定対象である微生物及び／又はウイルスのＤＮＡ又はＲＮＡのうち、デジタルＰＣＲによる増幅の目的とする領域である。例えば、被検試料に測定対象の微生物及び／又はウイルスと異なる種類の細胞が含まれる場合には、ＤＮＡ又はＲＮＡのターゲット領域は、測定対象の微生物及び／又はウイルスに特異的な配列を含むように設定することが好ましい。また、目的によっては複数種の微生物及び／又はウイルスに共通する配列を有するものであってもよい。さらに、ＤＮＡ又はＲＮＡのターゲット領域は単一であっても、複数であってもよい。 In the present invention, 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. For example, when the test sample contains a different type of cell from 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. Furthermore, the target region of DNA or RNA may be single or plural.
　ＤＮＡ又はＲＮＡのターゲット領域の長さとしては、通常５０～５０００塩基、又は５０～３０００塩基を挙げることができる。核酸の増幅に用いるプライマーは核酸増幅法の原理に基づいて適宜設定することが可能であって、上記ＤＮＡ又はＲＮＡのターゲット領域を特異的に増幅することができるものであれば特に制限されない。 As the length of the target region of DNA or RNA, usually 50 to 5000 bases or 50 to 3000 bases can be mentioned. 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.
　好ましいＤＮＡ又はＲＮＡのターゲット領域の例は、５Ｓ　ｒＤＮＡ遺伝子、１６Ｓ　ｒＤＮＡ遺伝子、２３Ｓ　ｒＤＮＡ遺伝子、ｔＤＮＡ遺伝子、及び病原遺伝子等の各種特異遺伝子である。これらの遺伝子の一つ又はその一部をターゲットとしてもよく、２又はそれ以上の遺伝子にまたがる領域をターゲットとしてもよい。 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.
　特に、ラクトバチルス・パラカゼイの細胞を測定対象とする場合には、配列番号１及び配列番号２に示すプライマーセット、配列番号３に示すプローブを用いることができる（表３参照）。同プライマーセットは、ラクトバチルス・パラカゼイの１６Ｓ　ｒＤＮＡ遺伝子の一部を特異的に増幅することができる。 In particular, when Lactobacillus paracasei cells are to be measured, 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.
　特に、ビフィドバクテリウム・ブレーベの細胞を測定対象とする場合には、配列番号４及び配列番号５に示すプライマーセット、配列番号６に示すプローブを用いることができる（表７参照）。同プライマーセットは、ビフィドバクテリウム・ブレーベの１６Ｓ　ｒＤＮＡ遺伝子の一部を特異的に増幅することができる。 In particular, when Bifidobacterium breve cells are to be measured, 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.
　また、複数種の微生物及び／又はウイルスに共通するプライマーを用いると、被検試料中の複数種の微生物の細胞及び／又はウイルスを測定することができる。また、特定の微生物及び／又はウイルスに特異的なプライマーを用いると、被検試料中の特定の微生物の細胞及び／又はウイルスを測定することができる。 In addition, when primers common to a plurality of types of microorganisms and / or viruses are used, the cells and / or viruses of the plurality of types of microorganisms in the test sample can be measured. In addition, when a primer specific to a specific microorganism and / or virus is used, cells and / or viruses of the specific microorganism in the test sample can be measured.
　また、測定対象が生菌である場合には、加熱工程を含むことが好ましい。
　ここで、本発明における加熱工程は、増幅産物測定時のＰＣＲのサーマルサイクルが加熱工程を兼ねていてもよく、また、別途被検試料若しくは測定用試料に熱を加える工程であってもよい。 Moreover, when a measuring object is a living microbe, it is preferable to include a heating process.
Here, 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.
　加熱工程を含むことで、生菌のＤＮＡが流出することなく細胞膜が損傷し、プライマー、ＤＮＡポリメラーゼ、プローブ等の成分が細胞内に侵入するため、被検試料中の特定の微生物の細胞及び／又はウイルスをより精度よく測定することができる。 By including a heating step, 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.
　加熱工程における加熱温度は、好ましくは８０℃以上、より好ましくは８５℃以上、より好ましくは９０℃以上、さらに好ましくは９５℃以上である。
　このような形態とすることで、生菌のＤＮＡが流出することなく細胞膜が損傷し、プライマー、ＤＮＡポリメラーゼ、プローブ等の成分が細胞内に侵入するため、被検試料中の特定の微生物の細胞及び／又はウイルスをより精度よく測定することができる。 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.
By adopting such a form, the cell membrane is damaged without the outflow of viable DNA, and components such as primers, DNA polymerase, and probes enter the cell. And / or virus can be measured more accurately.
　また、加熱工程における加熱時間は、好ましくは５分以上、より好ましくは７分以上、さらに好ましくは９分以上である。
　このような形態とすることで、生菌の細胞膜が損傷し、プライマー、ＤＮＡポリメラーゼ、プローブ等の成分が細胞内に侵入するため、被検試料中の特定の微生物の細胞及び／又はウイルスをより精度よく測定することができる。 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.
By adopting such a form, the cell membrane of live bacteria is damaged, and components such as primers, DNA polymerase, and probes enter the cell. Therefore, more specific cells and / or viruses in the test sample can be used. It can be measured with high accuracy.
　また、加熱工程における加熱時間は、好ましくは２０分以下、より好ましくは１５分以下、さらに好ましくは１２分以下である。
　このような形態とすることで、生菌のＤＮＡが流出することなく細胞膜が損傷し、プライマー、ＤＮＡポリメラーゼ、プローブ等の成分が細胞内に侵入するため、被検試料中の特定の微生物の細胞及び／又はウイルスをより精度よく測定することができる。 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.
By adopting such a form, the cell membrane is damaged without the outflow of viable DNA, and components such as primers, DNA polymerase, and probes enter the cell. And / or virus can be measured more accurately.
　なお、加熱工程における各種条件は、用いるデジタルＰＣＲ装置の設定を変更することにより、調整することができる。 Various conditions in the heating process can be adjusted by changing the settings of the digital PCR device to be used.
（３）生物の細胞及び／又はウイルスの数の測定工程
　測定工程は、増幅産物測定工程の測定結果に基づき微生物の細胞及び／又はウイルスの数を測定する工程である。 (3) Step of measuring the number of living cells and / or viruses 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.
　前述した増幅産物測定工程では、デジタルＰＣＲチップに備えられた全ウェルに対する、核酸増幅反応が陽性又は陰性であるウェルの数又は割合に関する情報が得られる。この情報に基づき被検試料中の微生物の細胞及び／又はウイルスの定量値を算出する方法は、特に制限されず、例えば、ポアソン分布モデルに適合させて解析する方法を挙げることができる。なお、核酸増幅反応が陽性又は陰性のウェルに関する情報から微生物の細胞及び／又はウイルスの定量値を算出する計算は、専用のクラウド型解析ソフトを用いて行うこともできる。 In the amplification product measurement step described above, information on the number or ratio of wells that are positive or negative for the nucleic acid amplification reaction is obtained for all the wells provided in the digital PCR chip. 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.
　以下、実施例を示して本発明をさらに具体的に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
［試験例１］分散条件が測定結果に与える影響について
　試験例１では、分散条件が測定結果に与える影響について検討を行った。 [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.
（１）被検試料の調製
　クリニカル食品１０ｍｌに、ラクトバチルス・パラカゼイ（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐａｒａｃａｓｅｉ）死菌体をその菌体濃度が２．０ × １０８ｃｅｌｌｓ／ｍｌとなるよう添加した。その後、表５に示す希釈溶媒を用いて２０倍希釈し、被検試料とした。 (1) Preparation of 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.
（２）測定用試料の調製
（２－１）分散操作
　前記（１）で得られた被検試料を３ｍｌ採取した。採取した被検試料に対し、超音波機器（ＢＲＡＮＳＯＮ　ＡＤＶＡＮＣＥＤ　ＳＯＮＩＦＩＥＲ　ＭＯＤＥＬ４５０ＡＡ　株式会社セントラル科学貿易　社製）を用いて、氷水中で表５に示す条件の分散操作を行った。 (2) Preparation of measurement sample (2-1) Dispersion operation 3 ml of the test sample obtained in (1) was collected. The collected test sample was subjected to a dispersion operation under the conditions shown in Table 5 in ice water using an ultrasonic device (BRANSON ADVANCED SONIFIER MODEL 450AA, manufactured by Central Scientific Trading Co., Ltd.).
（２－２）測定用試料の調製
　分散操作後の被検試料２μｌを表１に示すＰＣＲマスターミックスに加えることで、測定用試料を調製した。 (2-2) Preparation of Measurement Sample A measurement sample was prepared by adding 2 μl of the test sample after the dispersion operation to the PCR master mix shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　ここで、表１中のｃＤＢＣ（濃縮ダイレクトコンポーネントの略）は、核酸増幅阻害物質の働きを抑制する薬剤である。そして、ｃＤＢＣは、ウシ血清アルブミン（シグマ社、以下、ＢＳＡと表記）、クエン酸三ナトリウム２水和物（関東化学社、以下、ＴＳＣと表記）、塩化マグネシウム６水和物（ナカライテスク社、以下、ＭｇＣｌ２と表記）、卵白リゾチーム（和光純薬、以下、単にリゾチームと表記）、Ｂｒｉｊ５８（登録商標：シグマ社）を、表２に示す濃度となるよう混合することにより、調製した。 Here, 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.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　また、使用したフォワードプライマー及びリバースプライマー、並びにＴａｑＭａｎ（登録商標）プローブの配列を表３に示す。 In addition, Table 3 shows the sequences of the forward primer, reverse primer, and TaqMan (registered trademark) probe used.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（３）増幅産物の測定及び細胞の定量
　調製した測定用試料を、ＱｕａｎｔＳｔｕｄｉｏ（登録商標）３Ｄ　Ｄｉｇｉｔａｌ　ＰＣＲ　２０Ｋ　Ｃｈｉｐ　Ｋｉｔ　ｖ２（サーモフィッシャーサイエンティフィック社）のチップ中の各ウェル（およそ１８，０００ウェル）に、専用のローダーを用いて８６５ｐＬずつ分注した。分注後、デジタルＰＣＲ装置（ＱｕａｎｔＳｔｕｄｉｏ（登録商標）　３Ｄ　Ｄｉｇｉｔａｌ　ＰＣＲ　Ｓｙｓｔｅｍ、サーモフィッシャーサイエンティフィック社）を用いて、表４に示すＰＣＲサーマルサイクル条件により、デジタルＰＣＲを実施した。 (3) Measurement of amplification product and quantification of cells The prepared measurement sample was used for each well (approximately 18,000 wells) in the chip of QuantStudio (registered trademark) 3D Digital PCR 20K Chip Kit v2 (Thermo Fisher Scientific). 865 pL at a time using a dedicated loader. After dispensing, digital PCR was performed under the PCR thermal cycle conditions shown in Table 4 using a digital PCR apparatus (Quant Studio (registered trademark) 3D Digital PCR System, Thermo Fisher Scientific).
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　キット専用のチップリーダーを用いて緑色蛍光を発するウェル数及びその蛍光強度を計測し、増幅産物の測定を行った。
　その増幅産物の測定結果に基づき、ポアソン分布に従ってデジタルＰＣＲに供した被検試料に含まれるラクトバチルス・パラカゼイ特異遺伝子のコピー数を専用のクラウド型解析ソフトを用いて算出した。 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.
（４）結果
　表５にデジタルＰＣＲの結果を示す。 (4) Results Table 5 shows the results of digital PCR.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
（５）考察
　表５に示す結果より、超音波処理と間欠処理とを連続して繰り返す分散操作を行うことにより、被検試料中の微生物の細胞を精度よく測定することができることがわかった。 (5) Consideration From the results shown in Table 5, it was found that the cells of the microorganisms in the test sample can be accurately measured by performing a dispersion operation in which ultrasonic treatment and intermittent treatment are repeated continuously.
　ここで、比較例１と各実施例の結果の比較から、分散操作の繰り返し回数を２０回以上とすることで、被検試料中の微生物の細胞の凝集を抑え、被検試料中の微生物の細胞を精度よく測定することができることがわかった。 Here, from the comparison of the results of Comparative Example 1 and each example, by making the number of repetitions of the dispersion operation 20 times or more, the aggregation of the cells of the microorganisms in the test sample is suppressed, and the microorganisms in the test sample It was found that cells can be measured with high accuracy.
　また、実施例１～実施例３と、実施例４～実施例５の結果の比較から、分散操作の繰り返し回数を１００回以下とすることで、被検試料中の微生物の細胞を精度よく測定することができることがわかった。なお、分散操作の繰り返し回数が多い場合は、細胞膜が崩壊し、ＤＮＡが流出してしまうことにより、被検試料中の微生物の細胞の定量精度が低下したものと考えられる。 In addition, by comparing the results of Examples 1 to 3 and Examples 4 to 5, 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. In addition, when 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.
［試験例２］菌種の選択及び、菌体の生死が測定結果に与える影響について
　試験例２では、菌種の選択及び、菌体の生死が測定結果に与える影響について検討を行った。 [Test Example 2] Effect of selection of bacterial species and viability of bacterial cells on measurement results In Test Example 2, the effect of selection of bacterial species and viability of bacterial cells on measurement results was examined.
（１）被検試料の調製
（１－１）生菌懸濁液の調製
　ビフィドバクテリウム・ブレーべ（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｒｅｖｅ）ＭＣＣ１２７４（Ｂ－３株）をＬ－Ｃｙｓｔｅｉｎ含有ＭＲＳブロスで１６時間嫌気培養した。
　培養後洗浄し、同容量の０．１％Ｔｗｅｅｎ８０－ＰＢＳと混合し、生菌懸濁液を調製した。調製した生菌懸濁液の濃度をバクテリア計算盤を用いて測定をしたところ、１．６×１０１０ｃｅｌｌｓ／ｍｌであった。 (1) Preparation of 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.
（１－２）死菌懸濁液の調製
　ビフィドバクテリウム・ブレーべ（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ　ｂｒｅｖｅ）ＭＣＣ１２７４（Ｂ－３株）をＬ－Ｃｙｓｔｅｉｎ含有ＭＲＳブロスで１６時間嫌気培養した。培養後に、オートクレーブ機を用い７０℃、２分加熱処理した。加熱処理後洗浄し、同容量の０．１％Ｔｗｅｅｎ８０－ＰＢＳに懸濁させ、死菌懸濁液を調製した。その後、死菌懸濁液をＴＯＳ寒天培地で培養し、コロニーを形成しないことを確認した。また、調製した死菌懸濁液の濃度をバクテリア計算盤を用いて測定をしたところ、５．３×１０８ｃｅｌｌｓ／ｍｌであった。 (1-2) Preparation of dead bacterial suspension Bifidobacterium breve MCC1274 (B-3 strain) was anaerobically cultured in MRS broth containing L-Cystein for 16 hours. After the culture, heat treatment was performed at 70 ° C. for 2 minutes using an autoclave. After the heat treatment, it was washed and suspended in the same volume of 0.1% Tween 80-PBS to prepare a dead cell suspension. Thereafter, the dead bacterial suspension was cultured on a TOS agar medium, and it was confirmed that no colonies were formed. Moreover, it was 5.3 * 10 < 8 > cells / ml when the density | concentration of the prepared dead suspension was measured using the bacteria calculation board.
（１－３）被検試料の調製
　（１－１）、（１－２）で調製した各菌体を含む懸濁液を、各菌体の濃度が２．０×１０８ｃｅｌｌｓ／ｍｌとなるよう、市販の牛乳（森永乳業株式会社製）に添加した。その後、菌体を含む牛乳を０．１％　Ｔｗｅｅｎ８０－ＰＢＳを用いて２０倍に希釈することにより、被検試料を調製した。 (1-3) Preparation of 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.
（２）測定用試料の調製
（２－１）分散操作
　前記（１）で得られた被検試料を３ｍｌ採取した。採取した被検試料に対し、超音波機器（ＢＲＡＮＳＯＮ　ＡＤＶＡＮＣＥＤ　ＳＯＮＩＦＩＥＲ　ＭＯＤＥＬ４５０ＡＡ　株式会社セントラル科学貿易　社製）を用いて、氷水中で表８に示す条件の分散操作を行った。 (2) Preparation of measurement sample (2-1) Dispersion operation 3 ml of the test sample obtained in (1) was collected. The collected test sample was subjected to a dispersion operation under the conditions shown in Table 8 in ice water using an ultrasonic device (BRANSON ADVANCED SONIFIER MODEL 450AA, manufactured by Central Scientific Trading Co., Ltd.).
（２－２）測定用試料の調製
　分散操作後の被検試料２μｌを表６に示すＰＣＲマスターミックスに加えることで、測定用試料を調製した。 (2-2) Preparation of Measurement Sample A measurement sample was prepared by adding 2 μl of the test sample after the dispersion operation to the PCR master mix shown in Table 6.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　ここで、表６中のｃＤＢＣは、試験例１と同様のものを用いた。 Here, the cDBC in Table 6 was the same as in Test Example 1.
　また、使用したフォワードプライマー及びリバースプライマー、並びにＴａｑＭａｎ（登録商標）プローブの配列を表７に示す。 In addition, Table 7 shows the sequences of the forward primer, reverse primer, and TaqMan (registered trademark) probe used.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
（３）増幅産物の測定及び細胞の測定
　試験例１と同様の方法で、デジタルＰＣＲを実施した。 (3) Measurement of amplification product and measurement of cells Digital PCR was carried out in the same manner as in Test Example 1.
（４）結果
　デジタルＰＣＲの結果を表８に示す。 (4) Results Table 8 shows the results of digital PCR.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
（５）考察
　表８の結果より、菌種の選択及び菌体の生死を問わず、超音波処理と、間欠処理とを連続して繰り返す分散操作を行うことにより、分散操作を行わない比較例２、比較例３に比して、被検試料中の微生物の細胞を精度よく測定することができることがわかった。 (5) Discussion From the results in Table 8, a comparative example in which a dispersion operation is not performed by performing a dispersion operation that continuously repeats ultrasonic treatment and intermittent treatment regardless of the selection of the bacterial species and the viability of the cells. 2. Compared to Comparative Example 3, it was found that the cells of microorganisms in the test sample can be measured with high accuracy.
［試験例３］被検試料の選択、及び被検試料の希釈倍率が測定結果に与える影響について
　試験例３では、被検試料の選択、及び被検試料の希釈倍率が測定結果に与える影響について検討を行った。 [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.
（１）被検試料の調製
　表９に示すとおり、菌体を食品に添加した。その後、０．１％　Ｔｗｅｅｎ８０－ＰＢＳを用いて表９に示す希釈倍率となるよう菌体含有食品を希釈することにより、被検試料を調製した。 (1) Preparation of 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.
（２）測定用試料の調製
（２－１）分散操作
　前記（１）で得られた被検試料を３ｍｌ採取した。採取した被検試料に対し、超音波機器（ＢＲＡＮＳＯＮ　ＡＤＶＡＮＣＥＤ　ＳＯＮＩＦＩＥＲ　ＭＯＤＥＬ４５０ＡＡ　株式会社セントラル科学貿易　社製）を用いて、氷水中で表９に示す条件の分散操作を行った。 (2) Preparation of measurement sample (2-1) Dispersion operation 3 ml of the test sample obtained in (1) was collected. The collected test sample was subjected to a dispersion operation under the conditions shown in Table 9 in ice water using an ultrasonic device (BRANSON ADVANCED SONIFIER MODEL 450AA, manufactured by Central Scientific Trading Co., Ltd.).
（２－２）測定用試料の調製
　分散操作後の被検試料を、フィルターバッグを用いて濾過し、不溶成分を除去した。
　不溶性分除去後の被検試料２μｌを表１に示すＰＣＲマスターミックスに加えることで、測定用試料を調製した。 (2-2) Preparation of measurement sample The test sample after the dispersion operation was filtered using a filter bag to remove insoluble components.
A sample for measurement was prepared by adding 2 μl of the test sample after removal of insoluble matter to the PCR master mix shown in Table 1.
（３）増幅産物の測定及び細胞の測定
　試験例１と同様の方法で、デジタルＰＣＲを実施した。 (3) Measurement of amplification product and measurement of cells Digital PCR was carried out in the same manner as in Test Example 1.
（４）結果
　デジタルＰＣＲの結果を表９に示す。 (4) Results Table 9 shows the results of digital PCR.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
（５）考察
　表９の結果より、被検試料の希釈倍率を問わず、超音波処理と、間欠処理とを連続して繰り返す分散操作を施すことにより、被検試料中の微生物の細胞を精度よく測定することができることがわかった。
　また、表９の結果より、被検試料の種類に関係なく、超音波処理と、間欠処理とを連続して繰り返す分散操作を施すことにより、被検試料中の微生物の細胞を精度よく測定することができることがわかった。 (5) Consideration From the results in Table 9, 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.
［試験例４］測定対象以外の菌体の存在が測定結果に与える影響について
　試験例４では、測定対象以外の菌体の存在が測定結果に与える影響について検討を行った。 [Test Example 4] Effect of the presence of cells other than the measurement target on the measurement result In Test Example 4, the effect of the presence of the cell other than the measurement target on the measurement result was examined.
（１）被検試料の調製
　ラクトバチルス・パラカゼイ（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐａｒａｃａｓｅｉ）以外の菌体を含む食品１０ｇに、ラクトバチルス・パラカゼイ（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐａｒａｃａｓｅｉ）を表１０に示す菌体濃度となるよう添加した。その後、０．１％　Ｔｗｅｅｎ８０－ＰＢＳを用いて表１０に示す希釈倍率となるよう食品に希釈することにより、被検試料を調製した。 (1) Preparation of 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.
（２）測定用試料の調製
（２－１）分散操作
　前記（１）で得られた被検試料を３ｍｌ採取した。採取した被検試料に対し、超音波機器（ＢＲＡＮＳＯＮ　ＡＤＶＡＮＣＥＤ　ＳＯＮＩＦＩＥＲ　ＭＯＤＥＬ４５０ＡＡ　株式会社セントラル科学貿易　社製）を用いて、氷水中で表１０に示す条件の分散操作を行った。 (2) Preparation of measurement sample (2-1) Dispersion operation 3 ml of the test sample obtained in (1) was collected. The collected test sample was subjected to a dispersion operation under the conditions shown in Table 10 in ice water using an ultrasonic device (BRANSON ADVANCED SONIFIER MODEL 450AA, manufactured by Central Scientific Trading Co., Ltd.).
（２－２）測定用試料の調製
　分散操作後に、試験例１、試験例２と同様の方法で、測定用試料を調製した。 (2-2) Preparation of measurement sample After the dispersion operation, a measurement sample was prepared in the same manner as in Test Example 1 and Test Example 2.
（３）増幅産物の測定及び細胞の測定
　試験例１と同様の方法で、デジタルＰＣＲを実施した。 (3) Measurement of amplification product and measurement of cells Digital PCR was carried out in the same manner as in Test Example 1.
（４）結果
　デジタルＰＣＲの結果を表１０に示す。 (4) Results Table 10 shows the results of digital PCR.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
（５）考察
　表１０の結果より、測定対象以外の菌体の存在に関係なく、超音波処理と、間欠処理とを連続して繰り返す分散操作を施すことにより、被検試料中の微生物の細胞を精度よく測定することができることがわかった。 (5) Discussion From the results of Table 10, regardless of the presence of cells other than the measurement target, 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.
　本発明は食品等の出荷検査や受け入れ先の受け入れ検査における菌体含有量の測定及びウイルスの定量に応用することができる。 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.

Claims (4)

  1. 　被検試料中の微生物の細胞及び／又はウイルスを測定する方法であって、
    　微生物の細胞及び／又はウイルスを含む被検試料から測定用試料を調製する測定用試料調製工程と、
    　前記測定用試料中の微生物の細胞及び／又はウイルス固有のＤＮＡ又はＲＮＡのターゲット領域をデジタルＰＣＲ法により増幅し、増幅産物を測定する増幅産物測定工程と、
    　増幅産物測定工程の測定結果に基づき前記微生物の細胞及び／又はウイルスの数を測定する測定工程と、
    　を有し、
    　前記測定用試料調製工程は、微生物の細胞及び／又はウイルスからＤＮＡ又はＲＮＡを抽出する操作を含まず、
    　前記測定用試料調製工程は、前記被検試料に含まれる前記微生物の細胞及び／又はウイルスに対し、超音波処理と間欠処理とを連続して繰り返す分散操作を含むことを特徴とする、方法。     A method for measuring microbial cells and / or viruses in a test sample, comprising:
    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,
    A measurement step of measuring the number of cells and / or viruses of the microorganism based on the measurement result of the amplification product measurement step;
    Have
    The measurement sample preparation step does not include an operation of extracting DNA or RNA from microbial cells and / or viruses,
    The method for preparing a sample for measurement includes a dispersion operation in which ultrasonic treatment and intermittent treatment are continuously repeated on the cells and / or viruses of the microorganisms contained in the test sample.
  2. 　前記細胞が生細胞であることを特徴とする、請求項１に記載の方法。 The method according to claim 1, wherein the cell is a living cell.
  3. 　前記分散操作の繰り返し回数が、２０回～１００回である、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the number of repetitions of the dispersing operation is 20 to 100 times.
  4. 　前記超音波処理が、出力１０Ｗ～１００Ｗ、処理時間０．１秒～１秒の条件の超音波処理である、請求項１～３の何れか１項に記載の方法。

          The method according to any one of claims 1 to 3, wherein the ultrasonic treatment is an ultrasonic treatment under conditions of an output of 10W to 100W and a treatment time of 0.1 second to 1 second.

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