JP7350716B2 - Method for measuring microbial cells and/or viruses - Google Patents

Description

The present invention relates to a method for measuring microbial cells and/or viruses in a test sample.

Currently, foods to which microorganisms such as lactic acid bacteria are added, including dairy products, are widely accepted by consumers (Non-Patent Documents 1 to 4). In order to provide food containing microorganisms such as lactic acid bacteria, it is necessary to measure the microbial content in the food through shipping inspection of the food, acceptance inspection at the recipient, and the like.

As a technique for detecting and quantifying microorganisms, for example, a method is known in which a test sample is filtered with a membrane filter, cultured in an appropriate medium, and grown colonies are observed (Patent Document 1).

Furthermore, 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. Furthermore, there is a method that utilizes ATP in the bacterial body and uses chemiluminescence due to the luciferin-luciferase reaction (Non-patent Document 7, Non-patent Document 8), and a method that uses changes in the electrical conductivity of the medium to control bacterial growth in the medium. A capturing method (Non-Patent Document 9) is also known.

In addition, virus detection and quantification are widely performed for the purpose of detecting contamination and virus infection.

As a technique for detecting and quantifying a virus, for example, a method is known that utilizes an antigen-antibody reaction between an antibody against a virus and a nuclear protein of the virus for the purpose of detecting and quantifying an influenza virus (Patent Document 2).

Japanese Unexamined Patent Publication No. 6-311894 Japanese Patent Application Publication No. 2005-164496

Yoshikawa, T. et al. 2009. Biosci. Biotechnol. Biochem. 73: 1439-1442. Inoue, R. et al. 2010. Immunol Med Microbiol. 61: 94-102. Iwabuchi, N. et al. 2012. Immunol Med Microbiol. 66: 230-239. Nishibayashi, R. et al. 2015. Plos One | DOI:10.1371/journal.pone.0129806. Back, W. 1980. Brauwelt. 120: 1562. Taguchi, H. et al. 1990. J. Am. Soc. Brew. Chem. 48: 72. Hysert, D. W. et al. 1976. J. Am. Soc. Brew. Chem. 34: 145. Soejima, T. et al. 2009. FEMS Microbiol. Lett. 294: 74-81. Vogel, H. & Bohak, I. 1990. Brauwelt. 130: 414.

In view of the above-mentioned background, there has been a need for a technology 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 that can quickly and accurately measure microbial cells and viruses in a test sample.

The present invention for solving the above problems includes:
a measurement sample preparation step of preparing a measurement sample from a test sample containing microbial cells and/or viruses;
an amplification product measurement step of amplifying the target region of microbial cell and/or virus-specific DNA or RNA in the measurement sample by digital PCR and measuring the amplification product;
a measuring step of measuring the number of cells and/or viruses of the microorganism based on the measurement results of the amplification product measuring step;
has
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 is characterized by including a dispersion operation in which ultrasonic treatment and intermittent treatment are continuously repeated on the microorganism cells and/or viruses contained in the test sample.

According to the present invention, microbial cells and/or viruses in a test sample can be measured with high accuracy. Furthermore, since no operation is performed to extract DNA from microbial cells, the microbial cells in the test sample can be rapidly measured.

In a preferred form of the invention, said cells are living cells.

In a preferred embodiment of the present invention, the number of repetitions of the dispersion operation is 20 to 100 times.
By repeating the dispersion operation 20 to 100 times, microbial cells and/or viruses in the test sample can be measured with higher accuracy.

In a preferred embodiment of the present invention, the ultrasonic treatment is performed under conditions of an output of 10 W to 100 W and a treatment time of 0.1 seconds to 1 second.
By setting the ultrasonic treatment conditions to an output of 10 W to 100 W and a processing time of 0.1 seconds to 1 second, microbial cells and/or viruses in the test sample can be measured with higher accuracy.

According to the present invention, microbial cells and/or viruses in a test sample can be measured with high accuracy.

Next, preferred embodiments 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> Method for measuring microbial cells and/or viruses The method of the present invention is a method for measuring microbial cells and/or viruses in a test sample by digital PCR.

The method of the present invention is not limited to methods for determining the amount of microbial cells and/or viruses, but also includes methods for detecting the presence of microbial cells and/or viruses together with a measurement of the amount.

The microorganism cells to be measured are not particularly limited as long as the DNA or RNA of the microorganism can be amplified by digital PCR, and examples thereof include cells such as bacteria, filamentous fungi, and yeast.

Among these, the method of the present invention preferably targets Gram-positive bacterial cells. Examples of Gram-positive bacteria include Lactobacillus bacteria, Olsenella bacteria, Carnobacterium bacteria, Weissella bacteria, Enterococcus bacteria, or Bifidobacterium ( Examples include bacteria of the genus Bifidobacterium.

In particular, some bacteria of the genus Lactobacillus and Bifidobacterium exhibit physiological activities that are advantageous to the human body. It is preferable to apply this method to the measurement of cells of bacteria belonging to the genus Bifidobacterium.

Lactobacillus bacteria include Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus gasseri, and Lactobacillus saliba. Lactobacillus salivarius and the like can be mentioned.

Bifidobacterium bacteria include Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, and Bifidobacterium ad. Resentis ( Bifidobacterium adolescentis) and Bifidobacterium infantis (reclassified as Bifidobacterium longum subsp. infantis).

Viruses are not particularly limited as long as the DNA or RNA of the virus can be amplified by digital PCR, but examples include poxviridae, herpesviridae, adenoviridae, papillomaviridae, polyomaviridae, and parvoviruses. Family, Picornaviridae, Caliciviridae, Astroviridae, Coronaviridae, Togaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Bornaviridae, Alena Viridae, Bunyaviridae, Reoviridae, Retroviridae, hepatitis virus, etc. can be mentioned. Furthermore, the presence or absence of the outermost envelope is not limited.

Furthermore, there are no particular limitations on the test sample that can be used in the method of the present invention, and examples include food, biological samples, vaccine preparations, drinking water, industrial water, environmental water, wastewater, soil, and wiped samples. Can be done.

In particular, from the viewpoint of the usefulness of measuring cells of microorganisms that have physiological activities advantageous to living organisms, it is preferable to use food as the test sample. Foods include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit juice drinks, and lactic acid bacteria drinks (including concentrated stock solutions and powders for preparation of these drinks); frozen desserts such as ice cream, ice sorbet, and shaved ice; chocolate, caramel, Confectionery such as candies, cakes, biscuits, and cookies; Dairy products such as milk, processed milk, milk drinks, fermented milk, and butter; Highly nutritious liquid foods such as enteral nutritional foods, milk for infants, and sports drinks; Foods for specified health uses , functional foods such as health supplements.

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, etc., or a diluted or concentrated sample thereof. , or may be subjected to any other pretreatment. Preferable examples of pretreatment include heat treatment, filtration, centrifugation, and the like.

In addition, contaminants such as cells of the microorganism to be measured and/or cells other than viruses, protein colloid particles, fats, and carbohydrates present in the test sample are removed by treatment with enzymes that have the activity to decompose them. Or you may reduce it. If the test sample is milk, dairy products, or food made from milk or dairy products, bovine leukocytes, mammary gland epithelial cells, etc. may be used as cells other than microbial cells and/or viruses present in the test sample. can be mentioned.

The enzyme is not particularly limited as long as it can decompose impurities, and examples include lipolytic enzymes, proteolytic enzymes, and carbohydrate degrading enzymes.
One type of enzyme may be used alone, or two or more types of enzymes may be used in combination. Among these, it is preferable to use both a lipolytic enzyme and a protease, or all of a lipolytic enzyme, a proteolytic enzyme, and a carbohydrate degrading enzyme.

Examples of lipolytic enzymes include lipase and phosphatase.
Furthermore, examples of proteases include serine protease, cysteine protease, proteinase K, and pronase (registered trademark).
Furthermore, examples of carbohydrate degrading enzymes include amylase, cellulase, and N-acetylmuramidase.

Further, in the present invention, it is more preferable to dilute the test sample. By diluting the test sample, microbial cells and/or viruses contained in the test sample can be more efficiently dispersed by the dispersion operation described below.

The dilution ratio of the test sample is preferably in the range of 3 to 35 times, more preferably 5 to 30 times.
When the dilution ratio of the test sample is within the above range, microbial cells and/or viruses contained in the test sample can be more efficiently dispersed in the dispersion operation described below.

<2> Regarding each step in the method of the present invention Each step in the method of the present invention will be described in detail below.

(1) Measurement sample preparation process The measurement sample preparation process involves adding necessary reagents and processing to the test sample, and preparing the measurement sample (nucleic acid amplification reaction This is the process of preparing liquid).

The measurement sample preparation step includes a dispersion operation in which microbial cells and/or viruses contained in the test sample are continuously subjected to ultrasonic treatment and intermittent treatment.
By including a dispersion operation that continuously repeats ultrasonic treatment and intermittent treatment, it prevents aggregates containing multiple cells from being dispensed into one well of a digital PCR chip and improves quantitative performance. can be done.

More preferable forms of dispersion conditions will be described below.

In the present invention, the time per ultrasonic treatment is preferably 0.1 seconds to 1 second, more preferably 0.3 seconds to 0.9 seconds, and still more preferably 0.4 seconds to 0.8 seconds. , particularly preferably from 0.45 seconds to 0.75 seconds.

Further, in the present invention, the time for each intermittent treatment is preferably 0.1 seconds to 0.7 seconds, more preferably 0.25 seconds to 0.55 seconds.

Further, in the present invention, the output of the ultrasonic treatment is preferably 10W to 100W, more preferably 25W to 45W, and still more preferably 30W to 40W.

Further, in the present invention, the number of times the dispersion operation is repeated is preferably 20 to 100 times, more preferably 35 to 80 times.

Here, when the time per ultrasonic treatment is 0.3 seconds 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.

Further, when the time per ultrasonic treatment is 0.45 seconds to 0.75 seconds, the number of repetitions of the dispersion operation is preferably 25 to 85 times, more preferably 30 to 50 times. be.

Further, when the time per ultrasonic treatment is 0.75 seconds to 0.9 seconds, the number of repetitions of the dispersion operation is preferably 20 to 80 times, more preferably 25 to 45 times. be.

Further, the total processing time of the ultrasonic treatment (time per ultrasonic treatment x number of repetitions) is preferably 2 seconds to 100 seconds, more preferably 12 seconds to 41 seconds.

With the above conditions, microbial cells and viruses in the test sample can be measured more quickly and with higher accuracy.

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 device used.

Incidentally, the dispersion operation can be performed at any stage before or after the addition of various reagents and medicines, which will be described later.

Furthermore, in the measurement sample preparation step, DNA or RNA of microorganism cells and/or viruses contained in the test sample is not extracted. By not extracting the DNA or RNA of microbial cells and/or viruses contained in the test sample, the microbial cells and/or viruses contained in the test sample can be measured more quickly.

In the present invention, "extracting DNA or RNA" means an operation of actively destroying or lysing cells to collect or purify nucleic acids. That is, allowing components necessary for nucleic acid amplification, such as primers, to flow into cells without substantially destroying or lysing the cells, and allowing a portion of the amplified product to remain within the cells or to flow out of the cells. Not included in "extracting DNA or RNA".

In the measurement sample preparation step, reagents commonly used in digital PCR are added. Specifically, in addition to primers for amplifying the target region and probes for measuring the amplified product, which will be described later, reagents used in normal digital PCR such as a dNTP mixture and DNA polymerase are added. As reagents other than primers and probes, for example, when using a digital PCR device described below, QuantStudio (registered trademark) 3D digital PCR Master Mix v2 (x2) (Thermo Fisher Scientific) can be used.

Furthermore, in the measurement sample preparation step, it is preferable to add a drug that suppresses the action of the nucleic acid amplification inhibitor to the test sample from the viewpoint of improving quantitative performance.
In particular, in the present invention, in which DNA or RNA of microorganism cells and/or viruses in the test sample is not extracted 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 quantitative performance.

Here, the term "nucleic acid amplification inhibitor" refers to a substance that inhibits a nucleic acid amplification reaction or a nucleic acid elongation reaction, such as a positively charged inhibitor that adsorbs to a DNA template or a substance that adsorbs to a nucleic acid synthase (such as DNA polymerase). Examples include negative charge inhibiting substances. Examples of the positive charge inhibiting substance include calcium ions, polyamines, heme, and the like. Further, examples of negative charge inhibiting substances include phenol, phenolic compounds, heparin, and the outer membrane of cell walls of Gram-negative bacteria. Foods and clinical samples are said to contain many substances that inhibit such nucleic acid amplification reactions.

Examples of drugs that suppress the effects of nucleic acid amplification inhibitors include albumin, dextran, T4 Gene 32 protein, acetamide, betaine, dimethyl sulfoxide, formamide, glycerol, polyethylene glycol, soybean trypsin inhibitor, α2-macroglobulin, and tetra Examples include hydrophilic drugs such as methylammonium chloride, lysozyme, phosphorylase, and lactate dehydrogenase. These hydrophilic drugs may be used alone or in combination.

Among the above-mentioned hydrophilic drugs, polyethylene glycol 400 or polyethylene glycol 4000 can be preferably exemplified as the polyethylene glycol. Examples of betaine include trimethylglycine and its derivatives. Examples of phosphorylase and lactate dehydrogenase include glycogen phosphorylase and lactate dehydrogenase derived from rabbit muscle. In addition, as glycogen phosphorylase, glycogen phosphorylase b is preferable.
In particular, it is preferable to use albumin, dextran, T4 gene 32 protein, and lysozyme.

It has been suggested that albumin, represented by BSA (bovine serum albumin), may reduce inhibition of nucleic acid amplification by binding to nucleic acid amplification inhibitors such as heme (Abu Al-Soud). and others).

In addition, T4 Gene 32 protein is a single-stranded DNA-binding protein that prevents the template from being degraded by nucleolytic enzymes by binding in advance to the single-stranded DNA that serves as a template during the nucleic acid amplification process. Alternatively, 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)).

Furthermore, BSA, T4 Gene 32 protein, and proteinase inhibitors reduce proteolytic activity by binding to proteinases, making it possible to maximize the function of nucleic acid synthases. sex is suggested. In fact, proteolytic enzymes may remain in milk and blood, and in such cases, the addition of BSA or protease inhibitors (soybean trypsin inhibitor and α2-macroglobulin) can prevent the nucleic acid synthase from being degraded. A case in which the nucleic acid amplification reaction progressed favorably has also been introduced (Abu Al-Soud et al.).

Furthermore, dextran is a polysaccharide that is generally synthesized by lactic acid bacteria using glucose as a raw material. It has also 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), and that dextran negatively It is surmised that there is a good possibility of binding to charge inhibiting substances (adsorbed to nucleic acid synthase) or positive charge inhibiting substances (adsorbed to nucleic acids) by adsorbing these inhibitors in advance.

Furthermore, lysozyme adsorbs nucleic acid amplification inhibitors, which are contained in large numbers in milk (Abu Al-Soud et al., supra).

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.

Examples of albumin include bovine serum albumin, ovalbumin, milk albumin, and human serum albumin. Among these, bovine serum albumin (BSA) is a preferred example. Albumin may be a purified product, and may be used in combination with other components such as globulin as long as the effects of the present invention are not impaired. Moreover, albumin may be a fraction.

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 0.0% by mass. It is 6% by mass.
In the measurement sample preparation step, albumin is preferably added to the test sample so that the concentration of albumin in the measurement sample falls within the above range.

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%, and more preferably 1 to 4%.
In the measurement sample preparation step, dextran is preferably added to the test sample so that the concentration of dextran in the measurement sample falls within the above range.

As the T4 Gene 32 protein, a commercially available product (for example, manufactured by Roche, also called gp32) may be used.
The concentration of 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. It is .02%.
In the measurement sample preparation step, T4 Gene 32 protein is preferably added to the test sample so that the concentration of T4 Gene 32 protein in the measurement sample falls within the above range.

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.

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 protein-derived nucleic acid amplification inhibitors contained in foods, adding these to a test sample can improve the quantification of microbial cells and/or viruses.

Furthermore, in the measurement sample preparation step, it is particularly preferable to use lysozyme and polyethylene glycol in combination as a drug that suppresses the action of the nucleic acid amplification inhibitor.
Lysozyme and polyethylene glycol act cooperatively on nucleic acid amplification inhibitors and alter the surface structure of the nucleic acid amplification inhibitor, so if they are used in combination, they can more efficiently remove nucleic acid amplification inhibitors derived from proteins contained in the test sample. can be inhibited.

Further, in the measurement sample preparation step, it is preferable to add a magnesium salt, an organic acid salt, a phosphate, etc. to the test sample or a solution of DNA or RNA extracted from the test sample.

Examples of magnesium salts include magnesium chloride, magnesium sulfate, and magnesium carbonate.
The test sample or extracted from the test sample such that the concentration of 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. Preferably, magnesium salts are added to the DNA or RNA solution.

Examples of organic acid salts include salts of citric acid, tartaric acid, propionic acid, butyric acid, and the like. Examples of the salt include sodium salt, potassium salt, and the like. Moreover, pyrophosphoric acid etc. can be mentioned as a phosphate. These may be used alone or in a mixture of two or more.
So that 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 a phosphate to a test sample or a solution of DNA or RNA extracted from the test sample.

In addition, in the measurement sample preparation process, nucleic acid amplification using the digital PCR method including the aforementioned primers and probes, reagents for measuring amplified products, drugs that suppress the action of nucleic acid amplification inhibitors, magnesium salts, and organic The order of addition of acid salts or phosphates does not matter, and they may be added at the same time (including in the form of pre-mixing).

(2) Amplification product measurement step The amplification product measurement step is a step of amplifying the target region of the cell DNA or RNA in the measurement sample prepared in the measurement sample preparation step by digital PCR method and measuring the amplification product. be.

In the digital PCR method, a sample containing DNA or RNA to be measured is distributed to a chip with many wells, nucleic acid amplification is performed individually in each well, and the presence or absence of amplification in each well is detected, and a signal is detected. This method directly calculates the number of copies of the target as the number of wells containing the target.

For digital PCR, a commercially available digital PCR device can be used. As the digital PCR, it is preferable to use, for example, QuantStudio (registered trademark) 3D digital PCR (Thermo Fisher Scientific), which performs analysis using a hydrophobic chip with 20,000 wells.

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, etc.

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. If a probe labeled with a fluorescent molecule is used, the fluorescence emitted from the well can be detected using a device such as a chemiluminometer.

In the present invention, the "target region of DNA or RNA" is a region of DNA or RNA of a microorganism and/or virus to be measured that is targeted for amplification by digital PCR. For example, if the test sample contains cells of a different type from the microorganism and/or virus to be measured, the target region of DNA or RNA should contain a sequence specific to the microorganism and/or virus to be measured. It is preferable to set it to . Furthermore, depending on the purpose, it may have a sequence common to multiple types of microorganisms and/or viruses. Furthermore, the target region of DNA or RNA may be single or multiple.

The length of the target region of DNA or RNA is usually 50 to 5000 bases or 50 to 3000 bases. Primers used for nucleic acid amplification can be appropriately set based on the principles of nucleic acid amplification methods, and are not particularly limited as long as they can specifically amplify the target region of the DNA or RNA.

Examples of preferred DNA or RNA target regions are various specific genes such as 5S rDNA genes, 16S rDNA genes, 23S rDNA genes, tDNA genes, and pathogenic genes. One or a portion of these genes may be targeted, or a region spanning two or more genes may be targeted.

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 portion of the 16S rDNA gene of Lactobacillus paracasei.

In particular, when measuring Bifidobacterium breve cells, the primer set 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 portion of the 16S rDNA gene of Bifidobacterium breve.

Furthermore, by using primers common to multiple types of microorganisms and/or viruses, cells and/or viruses of multiple types of microorganisms in the test sample can be measured. Further, by using primers specific to a specific microorganism and/or virus, cells of a specific microorganism and/or virus in a test sample can be measured.

Moreover, when the measurement target is live bacteria, it is preferable to include a heating step.
Here, in the heating step in the present invention, the thermal cycle of PCR during amplification product measurement may also serve as the heating step, or it may be a step of separately applying heat to the test sample or measurement sample.

By including the heating process, the cell membrane is damaged without the DNA of living bacteria leaking out, and components such as primers, DNA polymerase, and probes enter the cells, so that the specific microorganism cells and/or Alternatively, the virus can be measured more accurately.

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, even more preferably 95°C or higher.
By using this form, the cell membrane is damaged without the DNA of living bacteria leaking out, and components such as primers, DNA polymerase, probes, etc. enter the cells, so that the cells of specific microorganisms in the test sample are and/or viruses can be measured with higher accuracy.

Further, the heating time in the heating step is preferably 5 minutes or more, more preferably 7 minutes or more, and still more preferably 9 minutes or more.
This form damages the cell membrane of living bacteria and allows components such as primers, DNA polymerase, and probes to enter the cells, making it more difficult to detect specific microorganism cells and/or viruses in the test sample. Can be measured with high precision.

Further, the heating time in the heating step is preferably 20 minutes or less, more preferably 15 minutes or less, and still more preferably 12 minutes or less.
By using this form, the cell membrane is damaged without the DNA of living bacteria leaking out, and components such as primers, DNA polymerase, probes, etc. enter the cells, so that the cells of specific microorganisms in the test sample are and/or viruses can be measured with higher accuracy.

Note that various conditions in the heating step can be adjusted by changing the settings of the digital PCR device used.

(3) Step of measuring the number of biological cells and/or viruses The measuring step is a step of measuring the number of microbial cells and/or viruses based on the measurement results of the amplification product measuring step.

In the amplification product measurement step described above, information regarding the number or percentage of wells in which the nucleic acid amplification reaction is positive or negative with respect to all wells included in the digital PCR chip is obtained. The method of calculating the quantitative value of microbial cells and/or viruses in the test sample based on this information is not particularly limited, and includes, for example, a method of analysis by adapting to a Poisson distribution model. Note that calculations for calculating quantitative values of microbial cells and/or viruses from information regarding wells in which the nucleic acid amplification reaction is positive or negative can also be performed using dedicated cloud-type analysis software.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

[Test Example 1] Regarding the influence of dispersion conditions on measurement results In Test Example 1, the influence of dispersion conditions on measurement results was investigated.

(1) Preparation of test sample Killed Lactobacillus paracasei cells were added to 10 ml of clinical food so that the cell concentration was 2.0 x 10 ⁸ cells/ml. Thereafter, it was diluted 20 times using the dilution solvent shown in Table 5 to prepare a test sample.

(2) Preparation of sample for measurement (2-1) Dispersion operation 3 ml of the test sample obtained in (1) above was collected. The collected test samples were 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 Kagaku Trading Co., Ltd.).

(2-2) Preparation of sample for measurement A sample for measurement was prepared by adding 2 μl of the test sample after the dispersion operation to the PCR master mix shown in Table 1.

Here, cDBC (abbreviation for concentrated direct component) in Table 1 is a drug that suppresses the action of nucleic acid amplification inhibitors. cDBC is bovine serum albumin (Sigma, hereinafter referred to as BSA), trisodium citrate dihydrate (Kanto Kagaku, hereinafter referred to as TSC), and magnesium chloride hexahydrate (Nacalai Tesque, hereinafter referred to as TSC). It was prepared by mixing egg white lysozyme (hereinafter referred to as MgCl ₂ ), egg white lysozyme (Wako Pure Chemical Industries, hereinafter simply referred to as lysozyme), and Brij58 (registered trademark: Sigma) to the concentrations shown in Table 2.

Further, Table 3 shows the sequences of the forward primer, reverse primer, and TaqMan (registered trademark) probe used.

(3) Measurement of amplification products and quantification of cells The prepared measurement sample was placed in each well (approximately 18,000 wells) in the chip of QuantStudio (registered trademark) 3D Digital PCR 20K Chip Kit v2 (Thermo Fisher Scientific). ) was dispensed in 865 pL portions using a dedicated loader. After dispensing, digital PCR was performed using a digital PCR device (QuantStudio (registered trademark) 3D Digital PCR System, Thermo Fisher Scientific) under the PCR thermal cycle conditions shown in Table 4.

Using a chip reader dedicated to the kit, the number of wells emitting green fluorescence and the fluorescence intensity were measured, and the amplified products were measured.
Based on the measurement results of the amplified products, the copy number of the Lactobacillus paracasei-specific gene contained in the test sample subjected to digital PCR was calculated according to Poisson distribution using dedicated cloud-based analysis software.

(4) Results Table 5 shows the results of digital PCR.

(5) Discussion From the results shown in Table 5, it was found that microbial cells in the test sample can be measured with high accuracy by performing a dispersion operation that continuously repeats ultrasonic treatment and intermittent treatment.

Here, from a comparison of the results of Comparative Example 1 and each Example, by repeating the dispersion operation 20 times or more, the aggregation of microorganism cells in the test sample can be suppressed, and the number of microorganisms in the test sample can be reduced. It was found that cells could be measured with high accuracy.

In addition, from a comparison of the results of Examples 1 to 3 and Examples 4 to 5, it was found that by repeating the dispersion operation 100 times or less, microbial cells in the test sample could be accurately measured. It turns out that it can be done. It is considered that when the dispersion operation is repeated many times, the cell membrane collapses and the DNA leaks out, resulting in a decrease in the accuracy of quantifying the microorganism cells in the test sample.

[Test Example 2] Regarding the influence of selection of bacterial species and the viability of bacterial bodies on the measurement results In Test Example 2, the selection of bacterial species and the influence of the viability of bacterial bodies on the measurement results were investigated.

(1) Preparation of test sample (1-1) Preparation of live bacterial 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 bacterial suspension. When the concentration of the prepared viable cell suspension was measured using a bacteria counting board, it was found to be 1.6×10 ¹⁰ cells/ml.

(1-2) Preparation of killed bacteria suspension Bifidobacterium breve (Bifidobacterium breve) MCC1274 (B-3 strain) was anaerobically cultured in MRS broth containing L-Cystein for 16 hours. After culturing, heat treatment was performed at 70° C. for 2 minutes using an autoclave machine. After the heat treatment, the cells were washed and suspended in the same volume of 0.1% Tween 80-PBS to prepare a killed bacteria suspension. Thereafter, the dead bacteria suspension was cultured on a TOS agar medium, and it was confirmed that no colonies were formed. Further, when the concentration of the prepared killed bacteria suspension was measured using a bacteria counting board, it was found to be 5.3×10 ⁸ cells/ml.

(1-3) Preparation of test sample The suspension containing each bacterial cell prepared in (1-1) and (1-2) was prepared so that the concentration of each bacterial cell was 2.0×10 ⁸ cells/ml. It was added to commercially available milk (manufactured by Morinaga Milk Industry Co., Ltd.). Thereafter, a test sample was prepared by diluting the milk containing bacterial cells 20 times using 0.1% Tween 80-PBS.

(2) Preparation of sample for measurement (2-1) Dispersion operation 3 ml of the test sample obtained in (1) above was collected. The collected test samples were 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 Kagaku Trading Co., Ltd.).

(2-2) Preparation of sample for measurement A sample for measurement was prepared by adding 2 μl of the test sample after the dispersion operation to the PCR master mix shown in Table 6.

Here, cDBC in Table 6 was the same as in Test Example 1.

Furthermore, Table 7 shows the sequences of the forward primer, reverse primer, and TaqMan (registered trademark) probe used.

(3) Measurement of amplification products and cell measurements Digital PCR was performed in the same manner as in Test Example 1.

(4) Results The results of digital PCR are shown in Table 8.

(5) Discussion From the results in Table 8, a comparative example in which no dispersion operation is performed by performing a dispersion operation that continuously repeats ultrasonic treatment and intermittent treatment, regardless of the selection of bacterial species and whether the bacterial cells are alive or dead. 2. Compared to Comparative Example 3, it was found that microbial cells in the test sample could be measured with high accuracy.

[Test Example 3] Regarding the influence of the selection of the test sample and the dilution rate of the test sample on the measurement results In Test Example 3, we will discuss the influence of the selection of the test sample and the dilution rate of the test sample on the measurement results. Study was carried out.

(1) Preparation of test sample As shown in Table 9, bacterial cells were added to food. Thereafter, test samples were prepared by diluting the bacterial cell-containing food using 0.1% Tween 80-PBS to the dilution ratio shown in Table 9.

(2) Preparation of sample for measurement (2-1) Dispersion operation 3 ml of the test sample obtained in (1) above was collected. The collected test samples were 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 Kagaku Trading Co., Ltd.).

(2-2) Preparation of sample for measurement 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 removing insoluble matter to the PCR master mix shown in Table 1.

(3) Measurement of amplification products and cell measurements Digital PCR was performed in the same manner as in Test Example 1.

(4) Results The results of digital PCR are shown in Table 9.

(5) Discussion From the results in Table 9, it is clear that regardless of the dilution ratio of the test sample, microbial cells in the test sample can be accurately dispersed by performing a dispersion operation that continuously repeats ultrasonic treatment and intermittent treatment. It turned out that it can be measured well.
Additionally, from the results in Table 9, regardless of the type of test sample, microbial cells in the test sample can be measured accurately by performing a dispersion operation that continuously repeats ultrasonic treatment and intermittent treatment. I found out that it is possible.

[Test Example 4] Effect of the presence of microbial cells other than the measurement target on the measurement results In Test Example 4, the influence of the presence of microbial cells other than the measurement target on the measurement results was investigated.

(1) Preparation of test sample Lactobacillus paracasei was added to 10 g of food containing bacterial cells other than Lactobacillus paracasei at the bacterial cell concentration shown in Table 10. Thereafter, test samples were prepared by diluting them into foods using 0.1% Tween 80-PBS at the dilution ratio shown in Table 10.

(2) Preparation of sample for measurement (2-1) Dispersion operation 3 ml of the test sample obtained in (1) above was collected. The collected test samples were 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 Kagaku Trading Co., Ltd.).

(2-2) Preparation of measurement sample After the dispersion operation, measurement samples were prepared in the same manner as Test Examples 1 and 2.

(3) Measurement of amplification products and cell measurements Digital PCR was performed in the same manner as in Test Example 1.

(4) Results The results of digital PCR are shown in Table 10.

(5) Discussion From the results in Table 10, we found that by performing a dispersion operation that continuously repeats ultrasonic treatment and intermittent treatment, regardless of the presence of microbial cells other than those to be measured, the microbial cells in the test sample It was found that it was possible to measure with high accuracy.

The present invention can be applied to the measurement of bacterial cell content and the quantification of viruses in shipping inspections of foodstuffs, etc. and acceptance inspections at receiving sites.

Claims (5)

A method for measuring the amount of microbial cells in a test sample, the method comprising:
a measurement sample preparation step of preparing a measurement sample from a test sample containing microbial cells ;
an amplification product measurement step of amplifying a target region of cell-specific DNA or RNA of the microorganism in the measurement sample by a digital PCR method and measuring the amplification product;
a measuring step of measuring the number of cells of the microorganism based on the measurement results of the amplification product measuring step;
has
The measurement sample preparation step does not include an operation of extracting DNA or RNA from microbial cells ,
The measurement sample preparation step includes a dispersion operation in which ultrasonic treatment and intermittent treatment are continuously repeated on the cells of the microorganism contained in the test sample ,
A method characterized in that the number of times the dispersion operation is repeated is 20 to 100 times . The method according to claim 1, characterized in that the cells are living cells. The method according to claim 1 or 2, wherein the total processing time of the ultrasonic treatment (time per ultrasonic treatment x number of repetitions) is 2 seconds to 100 seconds . The method according to any one of claims 1 to 3, wherein the ultrasonic treatment is performed under conditions of an output of 10 W to 100 W and a treatment time of 0.1 seconds to 1 second. The test sample is a food, biological sample, vaccine preparation, drinking water, industrial water, environmental water, wastewater, soil, or a wipe sample,
comprising diluting the test sample,
The method according to any one of claims 1 to 4, wherein the dilution ratio of the test sample is within the range of 5 times to 30 times.