JP6979269B2 - Method for measuring dead cells and / or inactivated viruses of microorganisms - Google Patents

Description

The present invention relates to a measuring method for quantifying dead cells and / or inactivated viruses of microorganisms. The present invention particularly relates to a measuring method for quantifying dead cells of Gram-positive bacteria added to foods and the like.

Conventionally, the detection and quantification of microorganisms or viruses has been carried out for those having a proliferative ability, as typified by the detection of contamination by microorganisms and the detection of infections by viruses.
As a technique for detecting and quantifying microorganisms, for example, for the purpose of detecting and quantifying contamination by live lactic acid bacteria cells in a beverage in which lactic acid bacteria do not exist, the test sample is filtered with a membrane filter and then cultured in an appropriate medium. A method of observing a grown colony is known (Patent Document 1). Further, a method of using a modified NBB medium as a lactic acid bacterium detection medium (Non-Patent Document 1) and a method of using a KOT medium (Non-Patent Document 2) are also known. Furthermore, a method using ATP in the cells and using chemiluminescence by the luciferin-luciferase reaction (Non-Patent Documents 3 and 4), and a method of capturing the growth of the bacteria in the medium by the change in the electrical conductivity of the medium (Non-Patent Documents 3 and 4). Non-Patent Document 5) is also known.
As a technique for detecting / quantifying a virus, for example, a method using an antigen-antibody reaction between an antibody against a virus and a nuclear protein of the virus is known for the purpose of detecting / quantifying an influenza virus (Patent Document 2).
All of these methods target microorganisms or viruses having proliferative ability for detection and quantification, and do not have a viewpoint of quantification of dead cells or inactivated viruses of microorganisms.
In addition, quantification of microorganisms and viruses using the PCR method has also been proposed. For example, quantification of lactic acid bacteria or bifidobacteria by reverse transcription PCR method (Non-Patent Document 6), and lactic acid bacteria by adding propidium iodide (PMA). Alternatively, selective quantification of living cells of bifidobacteria (Non-Patent Document 7), quantification of virus by real-time PCR method (Patent Document 3), and the like are known.

By the way, lactic acid bacteria in foods are called good bacteria, and various foods (fermented milk, cheese, etc.) to which they are added are widely accepted by consumers (Non-Patent Documents 8 to 11). In recent years, lactic acid bacteria have also been sterilized by heat treatment for the purpose of suppressing changes in flavor and physical properties during storage of these foods.
Then, lactic acid bacteria (Patent Documents 4 to 6) having the same activity as live cells even if they are dead cells have been reported.

As can be said from the above report, in recent years, research on microorganisms having no proliferative ability has been actively conducted.

Further, as a titer test of an inactivated vaccine, a method of inoculating an animal with a vaccine and measuring the antibody titer in blood after a certain period of time, a method of measuring a virus in an inactivated vaccine by ELISA, and the like are known. (Non-Patent Document 12).

Japanese Unexamined Patent Publication No. 6-31189 Japanese Unexamined Patent Publication No. 2005-164496 Japanese Unexamined Patent Publication No. 2004-305092 International Publication No. 2015/004949 Japanese Unexamined Patent Publication No. 2010-6801 Japanese Unexamined Patent Publication No. 2008-09623

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. Matsuda, K. et al. 2009. Appl. Environ. Microbiol. 75: 1961-1969. Fujimoto, J. et al. 2013. Appl. Environ. Microbiol. 79: 2182-2188. 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. Dynamic Drug Inspection News, No. 267, Published by National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, October 2005, p. 16

In the background described above, it is an object of the present invention to provide a novel technique for quantifying dead cells or inactivated viruses of microorganisms in a test sample. In particular, it is an object to measure dead cells of microorganisms in foods easily and with good quantitativeness.

As a result of diligent research to solve the above problems, the present inventors have found that the digital PCR method is suitable for quantifying dead cells or inactivated viruses of microorganisms, and have completed the present invention.

That is, the present invention that solves the above problems
A measurement method for quantifying dead cells and / or inactivated viruses of microorganisms in a test sample using a digital PCR method.
The measurement sample preparation process for preparing a measurement sample from the test sample,
An amplification product measurement step of amplifying a dead cell of the microorganism and / or a target region of DNA or RNA of the inactivating virus in the measurement sample by a digital PCR method and measuring the amplification product.
It is characterized by having a quantification step for quantifying dead cells and / or the inactivated virus of the microorganism based on the measurement result.
According to the present invention, dead cells and / or inactivated viruses of microorganisms in a test sample can be quantitatively measured.

Among the microorganisms, Gram-positive bacteria are known to have a thick peptidoglycan layer. Therefore, among microorganisms, there is a problem in measuring Gram-positive bacteria with good quantitativeness. Under these circumstances, the present invention is suitable for quantitative measurement of Gram-positive bacteria.

In recent years, it has been reported that some bacteria belonging to the genus Lactobacillus show favorable physiological activity to the human body even if they are dead cells (dead bacteria) (Patent Documents 4 and 6). There is a demand for quantitative measurement of dead cells of Lactobacillus spp., Which can be an active ingredient of medicine. Under these circumstances, the present invention is suitable for quantitative measurement of dead cells of Lactobacillus spp.

Among the bacteria of the genus Lactobacillus, some species of Lactobacillus paracasei show particularly high physiological activity in the state of dead cells (dead bacteria) (Patent Document 5). Therefore, the measuring method of the present invention is particularly suitable for quantitative measurement of dead cells (dead bacteria) of Lactobacillus paracasei.

The measuring method of the present invention can be applied to various test samples. Above all, it is suitable when the test sample is a sample containing oils and fats and the test sample contains dead cells of Gram-positive bacteria.
This is because dead cells of Gram-positive bacteria are hydrophobic, so it is difficult to form aggregates in a sample containing oils and fats, and the amplification product can be measured with high quantitativeness by the digital PCR method. be.

In a preferred embodiment of the present invention, the measurement sample preparation step comprises dispersing dead cells of microorganisms and / or the inactivated virus in the test sample.
By performing an operation of dispersing dead cells and / or the inactivated virus of the microorganism in the test sample in the sample preparation step for measurement, a plurality of dead cells and / or the above-mentioned microorganism of the microorganism are placed in one well of the digital PCR chip. It is possible to prevent the agglomerates containing the inactivating virus from being dispensed, and to measure the amplified product by the digital PCR method with high quantitativeness.

In a preferred embodiment of the present invention, the measurement sample preparation step comprises adding a drug that suppresses the action of a nucleic acid amplification inhibitor to the test sample, and the dead cells of the microorganism and / or the inactivating virus. Does not include extracting the DNA or RNA of the virus.
By adding a drug that suppresses the action of nucleic acid amplification inhibitor without extracting DNA or RNA of dead cells, it is possible to easily measure dead cells and / or inactivated viruses of microorganisms with good quantification. can.

In a preferred embodiment of the invention, the agent that suppresses the action of the nucleic acid amplification inhibitor comprises at least lysozyme and / or polyethylene glycol.
By using a drug containing lysoteam or polyethylene glycol as a drug that suppresses the action of the nucleic acid amplification inhibitor, the nucleic acid amplification inhibitor contained in the test sample such as food can be more efficiently inhibited, and the dead cells of the microorganism and the dead cells of the microorganism and / Or the inactivated virus can be measured with good quantitativeness.

Further, the present invention comprises a Lacticaseibacillus paracasei in a test sample using a digital PCR method, which comprises an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 1 and an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 2. (Lactobacillus paracasei) A primer set for quantitative measurement of dead cells.
According to the primer set of the present invention, the dead cells of Lactobacillus paracasei in the test sample can be easily measured with good quantification by using the digital PCR method.

According to the present invention, dead cells and / or inactivated viruses of microorganisms can be quantitatively measured. Further, according to the preferred embodiment of the present invention, the dead cells of Gram-positive bacteria, particularly Lactobacillus paracasei, in the test sample can be easily measured with good quantification.

3 is a standard curve showing the relationship between the concentration of dead cells of Lactobacillus paracasei in the test sample prepared from the results of Examples 3 and 7 and the quantitative value by the digital PCR method. 3 is a standard curve showing the relationship between the concentration of dead cells of Lactobacillus paracasei in the test sample prepared from the results of Examples 3 and 8 and the quantitative value by the digital PCR method. 3 is a standard curve showing the relationship between the concentration of dead cells of Lactobacillus paracasei in the test sample prepared from the results of Examples 3 and 9 and the quantitative value by the digital PCR method. 4 is a standard curve showing the relationship between the concentration of dead cells of Lactobacillus paracasei in the test sample prepared from the results of Example 4 and Table 10 and the quantified value by the digital PCR method. 4 is a standard curve showing the relationship between the concentration of dead cells of Lactobacillus paracasei in the test sample prepared from the results of Example 4 and Table 11 and the quantitative value by the digital PCR method.

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 changed within the scope of the present invention.

<1> Measurement method for dead cells and / or inactivated virus of microorganisms The measurement method of the present invention is a measurement method for quantifying dead cells and / or inactivated viruses of microorganisms in a test sample by a digital PCR method.

The measuring method of the present invention is not limited to a method for determining the amount of dead cells and / or inactivated virus of a microorganism, and is a method for detecting the presence of dead cells and / or inactivated virus of a microorganism together with a measured value for the amount. Also includes.
That is, the present invention is a method for detecting dead cells and / or inactivated viruses of microorganisms in a test sample by using a digital PCR method, and prepares a measurement sample from the test sample. The step, the amplification product measurement step of amplifying the dead cell of the microorganism and / or the target region of the DNA or RNA of the inactivated virus in the measurement sample by a digital PCR method, and measuring the amplification product, and the measurement result. There is also a method having a quantification step of quantifying dead cells and / or the inactivated virus of the microorganism based on the above.

The "dead cells of microorganisms" to be measured in the present invention are cells of microorganisms in a state (Dade) in which proliferation is impossible even when cultured under suitable culture conditions and the cells do not show metabolic activity. Is. Further, although the structure of the cell wall is maintained, the cell wall itself is highly damaged, and it is a microbial cell in a state where a weakly permeable nuclear stain such as propidium iodide permeates the cell wall.

The dead cells of a microorganism 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 dead cells such as bacteria, filamentous fungi, and yeast.

Above all, the measuring method of the present invention preferably targets dead cells of Gram-positive bacteria. Gram-positive bacteria include Lactobacillus, Olsenella, Carnobacterium, Weissella, Enterococcus, or Bifidobacteria. Bacteria genus Bacteria and the like can be mentioned.

In particular, since some Lactobacillus bacteria show favorable physiological activity to the human body even if they are dead cells, the measurement method of the present invention is a quantification of dead cells of Lactobacillus bacteria among Gram-positive bacteria. It is preferable to apply it to the measurement.

Examples of Lactobacillus bacteria include Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus gasseri, and Lactobacillus salivarius.

Among them, some Lactobacillus paracasei show particularly high physiological activity in the state of dead cells. Therefore, the measuring method of the present invention is preferably used for quantitative measurement of dead cells of Lactobacillus paracasei. More specifically, the measuring method of the present invention is particularly preferably used for quantitative measurement of dead cells of Lactobacillus paracasei MCC1849 (NITE BP-01633).

Further, the "inactivated virus" to be measured in the present invention means a virus that has lost the infectivity to cells and, for example, a virus that does not have the ability to propagate in the body of a mammal.
The inactivated virus can also be artificially prepared by chemical treatment, heating treatment, ultraviolet irradiation, or the like.

The inactivated virus is not particularly limited as long as it can amplify the DNA or RNA of the virus by the digital PCR method, and is, for example, Poxvirus family, Herpesvirus family, Adenovirus family, Papillomavirus family, Polyomavirus family, and the like. Parvovirus, Picornavirus, Calicivirus, Astrovirus, Coronavirus, Togavirus, Flavivirus, Orthomixovirus, Paramyxovirus, Rabdovirus, Philovirus, Bornavirus , Arenavirus family, Bunyavirus family, Leovirus family, Retrovirus family, Hepatitis virus and the like. Also, the presence or absence of the outermost envelope is not limited.

Among them, the virus having only the non-enveloped nucleocapsid (protein membrane) has no outer membrane, and its structure is relatively similar to that of Gram-positive bacteria in which the peptidoglycan layer is in direct contact with the outside world. The method is preferably applicable.

Further, the test sample that can be used in the measurement method of the present invention is not particularly limited, and examples thereof include foods, biological samples, vaccine preparations, drinking water, industrial water, environmental water, wastewater, soil, and wiped samples. Be done.

In particular, from the viewpoint of the usefulness of quantitatively measuring dead cells of microorganisms having physiological activity favorable to the living body, it is preferable to use food as a test sample. Foods include soft drinks, carbonated drinks, nutritional drinks, fruit juice drinks, lactic acid bacteria drinks and other beverages (including concentrated stock solutions and preparation powders for these beverages); ice cream, ice sherbets, shaved ice and other cold confectionery; chocolate, caramel. , Candy, cakes, biscuits, cookies and other confectionery; sterilized milk, processed milk, milk drinks, fermented milk, butter and other dairy products; Examples include functional foods such as foods for food and health supplements.

Further, the measuring method of the present invention is particularly suitable for measuring a sample containing oils and fats containing dead cells of Gram-positive bacteria from the viewpoint of enhancing the quantitativeness by using the digital PCR method.
This is because dead cells of Gram-positive bacteria are hydrophobic and difficult to form aggregates in a sample containing oils and fats. Therefore, when performing digital PCR, it is possible to prevent the aggregate of dead cells from being dispensed into one well of the digital PCR chip.

The fats and oils are not particularly limited, and include both natural fats and oils and synthetic fats and oils. Examples of fats and oils contained in foods include animal fats and oils such as milk fat and vegetable fats and oils. Examples of the sample containing fats and oils include foods containing fats and oils, such as chocolate, caramel, cakes, sweets such as cookies, cold confectionery such as ice cream, sterilized milk, processed milk, milk drinks, and fermentation. Examples thereof include dairy products such as milk and butter, milk for raising children, and highly nutritious liquid foods such as enteric nutritional foods.

In the present invention, the test sample may be the above-mentioned food, biological sample, vaccine preparation, drinking water, industrial water, environmental water, wastewater, soil, wiped sample or the like itself, and these are diluted or concentrated. It may be a product or any other pretreated product. Examples of the pretreatment include heat treatment, filtration, centrifugation and the like.

In addition, dead cells and / or cells other than inactivated virus, protein colloidal particles, fats, sugars and other contaminants of the microorganism to be measured present in the test sample are treated with an enzyme having an activity to decompose them. It may be removed or reduced by such means. Examples of cells other than dead cells and / or inactivated viruses of microorganisms present in the test sample include bovine leukocytes and / or bovine leukocytes when the test sample is milk, dairy products, or foods made from milk or dairy products. Examples include mammary epithelial cells.

The enzyme is not particularly limited as long as it can decompose impurities, and examples thereof include lipid-degrading enzymes, proteolytic enzymes, and glycolytic enzymes. As the enzyme, one type of enzyme may be used alone, or two or more types of enzymes may be used in combination, but both a lipid-degrading enzyme and a proteolytic enzyme, or a lipid-degrading enzyme, a proteolytic enzyme, And it is preferable to use all of the glycolytic enzymes.
Lipase, phosphatase, etc. as lipid-degrading enzymes, serine protease, cysteine protease, proteinase K, pronase (registered trademark), etc. as proteolytic enzymes, amylases, cellulase, N-acetylmuramidase, etc. as glycolytic enzymes, etc. Can be mentioned.

Hereinafter, each step in the measurement method of the present invention will be described in detail.

(1) Measurement sample preparation step In the measurement sample preparation step, a measurement sample (nucleic acid amplification) for adding or treating a test sample with necessary reagents and using it for measurement of an amplified product using a digital PCR method. This is a step of preparing a reaction solution).

In the measurement sample preparation step, it is not necessary to extract the DNA or RNA of the dead cells and / or the inactivated virus of the microorganism contained in the test sample. This is because the measurement method of the present invention targets dead cells and / or inactivated viruses of microorganisms. That is, the cell wall of the microorganism to be measured and the capsid of the virus are not a little damaged, and in the measurement of nucleic acid amplification and amplification products by the digital PCR method described later, components such as primers, DNA polymerase, and probe are used as dead cells of the microorganism. And / or because it can act on the DNA or RNA of the inactivated virus, the measurement method of the present invention does not require extraction of the dead cells of the microorganism and / or the DNA or RNA of the inactivated virus contained in the test sample. ..
In the present invention, "extracting DNA or RNA" means an operation of actively destroying or lysing cells to collect or purify nucleic acid. That is, to allow components necessary for nucleic acid amplification such as primers to flow into the cell without substantially destroying or dissolving the cell, to allow a part of the amplification product to remain inside the cell, or to allow a part of the amplification product to flow out of the cell. Leaving chromosomal DNA out of the cell is not included in "extracting DNA or RNA".

Of course, in the measurement sample preparation step, DNA or RNA can also be extracted from dead cells and / or inactivated viruses of microorganisms.
In such a form, it is preferable to perform a cooling centrifugation treatment and a glass bead crushing operation on the test sample before lysis with a peptidoglycan cleaving enzyme or other chemicals. By adding such an operation, the DNA or RNA extraction efficiency of the dead cells and / or the inactivated virus of the microorganism can be improved, and the quantitativeness of the dead cells and / or the inactivated virus of the microorganism in the test sample can be improved. Can be improved.
The method for extracting DNA or RNA of dead cells and / or inactivated viruses of microorganisms is not particularly limited and can be carried out by a conventional method, and a commercially available DNA extraction kit may be used.

In the measurement sample preparation step, reagents usually used for the digital PCR method are added. Specifically, in addition to a primer for amplifying a target region described later and a probe for measuring an amplification product, a reagent used for ordinary digital PCR such as a dNTP mixture and a DNA polymerase is added. As the reagent other than the primer and the probe, for example, when a digital PCR device described later is used, QuantStudio (registered trademark) 3D digital PCR Master Mix v2 (x2) (Thermo Fisher Scientific) can be used.

In the measurement sample preparation step, it is preferable to dilute the test sample as necessary and then disperse the dead cells and / or inactivated viruses of the microorganisms contained in the test sample from the viewpoint of improving the quantification. This makes it possible to prevent the aggregate containing a plurality of dead cells from being dispensed into one well of the digital PCR chip in the digital PCR method described later, and to improve the quantification.
In particular, when the DNA or RNA of the dead cells and / or the inactivated virus of the microorganism in the test sample is not extracted in the sample preparation step for measurement, the dead cells of the microorganism and / or the aggregate of the inactivated virus are collected. Dispersion is effective for improving quantitativeness.

The means for dispersing the agglomerates is not particularly limited, and examples thereof include a method using an ultrasonic or pressure type disperser or a homogenizer.

Further, 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 the quantitativeness.
In particular, when the DNA or RNA of dead cells of microorganisms and / or inactivated virus in the test sample is not extracted in the sample preparation step for measurement, a drug that suppresses the action of a nucleic acid amplification inhibitor in the test sample. Is effective for improving the quantitativeness.

Here, the "nucleic acid amplification inhibitor" is a substance that inhibits a nucleic acid amplification reaction or a nucleic acid extension reaction, and is, for example, a positive charge inhibitor that is adsorbed on a DNA template or a nucleic acid synthase (DNA polymerase, etc.). Nucleic acid inhibitors and the like. Examples of the positive charge inhibitor include calcium ions, polyamines, heme and the like. Examples of the negative charge inhibitor include phenol, phenolic compounds, heparin, cell wall outer membrane of Gram-negative bacteria, and the like. It is said that foods and clinical specimens contain many substances that inhibit such nucleic acid amplification reactions.

Drugs that suppress the action of nucleic acid amplification inhibitors as described above include albumin, dextran, T4 gene 32 protein, acetamide, betaine, dimethylsulfoxide, formamide, glycerol, polyethylene glycol, soybean trypsin inhibitor, α2-macroglobulin, tetra. Hydrophilic agents such as methylammonium chloride, lysoteam, phosphorylase, and lactate dehydrogenase can be exemplified. Only one kind of these hydrophilic agents may be used, or a plurality of kinds may be used in combination.

Among the above-mentioned hydrophilic agents, polyethylene glycol 400 or polyethylene glycol 4000 is 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. As the glycogen phosphorylase, glycogen phosphorylase b is preferable.
In particular, it is preferred to use albumin, dextran, T4 gene 32 protein, and lysozyme.

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

Further, the T4 gene 32 protein is a single-stranded DNA-binding protein, and by pre-binding to the single-stranded DNA used as a template in the nucleic acid amplification process, does it prevent the template from being degraded by the nucleic acid-degrading enzyme? , Or it is believed that the 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 inhibitor can reduce proteolytic activity by binding to proteolytic enzyme (proteinase) and maximize the function of nucleic acid synthase. Sex is suggested. In fact, proteolytic enzymes may remain in milk and blood, and the addition of BSA or proteolytic enzyme inhibitors (soy trypsin inhibitor or α2-macroglobulin) prevents the nucleic acid synthase from being degraded. Cases in which the nucleic acid amplification reaction proceeded well have also been introduced (Abu Al-Soud et al.).

Dextran is a polysaccharide generally synthesized by lactic acid bacteria using glucose as a raw material. A similar polysaccharide-peptide complex called mucin has also been reported to adhere to the intestinal mucosa (Ruas-Madiedo, P., Applied and Environmental Microbiology, 74: 1936-1940, 2008), with dextran being a negative charge inhibitor. It is presumed that there is a sufficient possibility of binding to these inhibitors by (adsorbing to nucleic acid synthase) or pre-adsorbing to positive charge inhibitor (adsorbing to nucleic acid).

In addition, it is presumed that lysozyme is adsorbed to a nucleic acid amplification inhibitor that is considered to be contained in a large amount in milk (Abu Al-Soud et al.).

From the above, it can be said that the hydrophilic agents represented by albumin, T4 gene 32 protein, dextran, and lysozyme are agents that suppress the action of nucleic acid amplification inhibitors.

Examples of albumin include bovine serum albumin, egg white albumin, lactic albumin, human serum albumin and the like. Among these, bovine serum albumin (BSA) can be preferably exemplified. Albumin may be a refined product and may be used in combination with other components such as globulin as long as the effect of the present invention is not impaired. Albumin may also 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%. It is 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 is within the above range.

Examples of the dextran include dextran 40 and dextran 500, and dextran 40 is particularly preferable.
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, it is preferable to add dextran to the test sample so that the concentration of dextran in the measurement sample is within the above range.

As the T4 gene 32 protein, a commercially available product (for example, manufactured by Roche: also referred to as 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%, and more preferably 0.01 to 0. It is 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 is within the above range.

As the lysozyme, lysozyme derived from egg white is 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, it is preferable to add lysozyme to the test sample so that the concentration of lysozyme in the measurement sample is 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 nucleic acid amplification inhibitors derived from proteins contained in foods, adding them to a test sample can improve the quantification of dead cells and / or inactivated viruses of microorganisms. can.

Further, 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.
Since lysoteam and polyethylene glycol act synergistically with the nucleic acid amplification inhibitor and alter the surface structure of the nucleic acid amplification inhibitor, when these are used in combination, the nucleic acid amplification inhibitor derived from the protein contained in the test sample is more efficient. Can be inhibited.

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

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 the magnesium salt to the solution of DNA or RNA.

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, potassium salt and the like. Further, examples of the phosphate include pyrophosphoric acid and the like. These may be one kind or a mixture of two kinds or three kinds 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, preferably 1 to 10 mM, more preferably 1 to 5 mM in total amount. It is preferable to add an organic acid salt or a phosphate to the test sample or a solution of DNA or RNA extracted from the test sample.

In the measurement sample preparation step, nucleic acid amplification using the digital PCR method including the above-mentioned primers and probes, reagents for measuring amplification products, drugs that suppress the action of nucleic acid amplification inhibitors, magnesium salts, and organic substances. The order of addition of the acid salts or phosphates is not limited, and the acid salts or phosphates may be added simultaneously (including premixed forms).

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

In the digital PCR method, a sample containing DNA or RNA to be measured is distributed to many wells so that one copy is made per well, nucleic acid amplification is performed individually for each well, and each well is subjected to nucleic acid amplification. This is a method of directly calculating the number of wells with a signal as the number of copies of the target by detecting the "presence or absence of amplification" of.

Digital PCR can be performed by a commercially available digital PCR device. For example, it is preferred to use Quant Studio® 3D digital PCR (Thermo Fisher Scientific) for analysis with a hydrophobic chip with 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. 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 chemilimiphotometer.

In the present invention, the "target region of DNA or RNA" is a region of the DNA or RNA of the microorganism and / or the inactivated virus to be measured, which is the target region for amplification by digital PCR. For example, if the test sample contains cells of a different type than the microorganism and / or inactivated virus to be measured, the target region of DNA or RNA is specific to the microorganism and / or inactivated virus to be measured. It is preferable to set it to include an sequence. Further, depending on the purpose, it may have a sequence common to a plurality of types of microorganisms and / or inactivated viruses. Further, the target region of DNA or RNA may be single or plural.

The length of the target region of DNA or RNA usually includes 50 to 5000 bases, or 50 to 3000 bases. The primer used for nucleic acid amplification can be appropriately set based on the principle of the nucleic acid amplification method, and is not particularly limited as long as it 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 rRNA gene, 16S rRNA gene, 23S rRNA gene, tRNA gene, and pathogenic gene. One or a part of these genes may be targeted, or a region spanning two or more genes may be targeted.

In particular, when dead cells of Lactobacillus paracasei are to be measured, the primer set shown in SEQ ID NOs: 1 and 2 and the probe shown in SEQ ID NO: 3 can be used (see Table 3). The primer set can specifically amplify a portion of the 16S rRNA gene of Lactobacillus paracasei.

In addition, by using a primer common to a plurality of types of microorganisms and / or an inactivated virus, it is possible to quantitatively measure the dead cells and / or the inactivated virus of the plurality of types of microorganisms in the test sample. In addition, by using a primer specific to a specific microorganism and / or an inactivated virus, the dead cells and / or the inactivated virus of the specific microorganism in the test sample can be quantitatively measured.

(3) Quantitative step The quantification step is a step of quantifying dead cells and / or inactivated viruses of microorganisms contained in the test sample based on the result of the amplification product measurement step.

In the amplification product measurement step described above, information on the number or proportion of wells in which the nucleic acid amplification reaction is positive or negative is obtained with respect to all the wells provided in the digital PCR chip. The method for calculating the quantitative value of the dead cells and / or the inactivated virus of the microorganism in the test sample based on this information is not particularly limited, and examples thereof include a method of fitting to a Poisson distribution model for analysis. The calculation for calculating the quantitative value of dead cells of microorganisms and / or inactivated virus from the information on wells in which the nucleic acid amplification reaction is positive or negative can also be performed using a dedicated cloud-type analysis software.

<2> Primer set for measuring dead cells of lactobacillus paracasei The primer set of the present invention is for quantitatively measuring dead cells of lactobacillus paracasei in a test sample using a digital PCR method. A primer set containing an oligonucleotide containing the base sequence shown in SEQ ID NO: 1 and an oligonucleotide containing the base sequence shown in SEQ ID NO: 2 (see Table 3 below).
In addition, the primer set of the present invention quantifies the dead cells of Lactobacillus paracasei in the test sample by the digital PCR method by combining with the reagents such as DNA polymerase and dNTP mixture used in the above-mentioned digital PCR method. It can also be a kit for measuring the target.

The kit preferably contains, in addition to the primer set, a probe containing the nucleotide sequence shown in SEQ ID NO: 3 (Table 3 below).
Further, it is preferable that the kit further contains a drug that suppresses the action of the nucleic acid amplification inhibitor. As the preferred form of the drug that suppresses the action of the nucleic acid amplification inhibitor, the matters described in the above item <1> can be applied.

In addition, the kit may be an embodiment containing a magnesium salt and a reagent such as an organic acid salt or a phosphate commonly used in the digital PCR method. Further, a diluted solution, a buffer solution and the like may be included as elements of the kit.
The matters described in the above item <1> can also be applied to these reagents.

Steps necessary for measuring dead cells and / or inactivated viruses of microorganisms in a test sample using the primer set of the present invention and the kit, that is, a sample preparation step for measurement, an amplification product measurement step, and quantification. The matters described in the above-mentioned item <1> can also be applied to the process.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Example 1]
In a quantitative test of Gram-positive bacteria using digital PCR, the effect of a drug that suppresses the action of a nucleic acid amplification inhibitor on the quantitativeness was investigated.

(1) Preparation of test sample Two types of samples (test sample) of the following Lactobacillus paracasei dead cell DNA aqueous solution and Lactobacillus paracasei dead cell suspension were prepared by the following method.

(1-1) Preparation of Lactobacillus paracasei dead cell DNA aqueous solution (0.4 ng / μL) Lactobacillus paracasei MCC1849 (NITE BP-01633) (hereinafter, simply referred to as Lactobacillus paracasei) was cultured in MRS medium. The cells were collected and washed with phosphate buffered saline (PBS, pH 7.4). The washed cells were heat-treated (96 ° C., 15 min) to prepare Lactobacillus paracasei dead cells.

Suspension (1 mL) of Lactobacillus paracasei dead cells is subjected to cold centrifugation (8000 × G, 10 min, 4 ° C), and after removing the supernatant, 300 mg of glass beads having a diameter of 1 mm is added and a vortex mixer is used. After vigorous vibration for 2 minutes, the cells were spun down using a tabletop centrifuge. Then, DNA was extracted and purified from Lactobacillus paracasei dead cells using a DNA extraction kit (QuickGene SP Kit DNA tissue (SP-DT), Kurabo Industries Ltd.).

The purified DNA is dissolved in sterile distilled water, and the absorbance ratio (OD260 nm / OD280 nm) is measured using an ultra-trace spectrophotometer (NanoDrop 2000c Spectrophotometer, Thermo Fisher Scientific) to obtain RNA into a DNA solution. It was confirmed that there was no contamination.
This DNA solution was diluted with sterile distilled water to obtain a Lactobacillus paracasei dead cell DNA aqueous solution (0.4 ng / μL) sample (test sample). The DNA concentration was determined by absorbance measurement (OD 260 nm) using the same ultra-trace spectrophotometer.

(1-2) Preparation of Lactobacillus / Paracasei Dead Cell Suspension (2.8 × 10 ⁹ cells / mL) Specimen (Test Sample) Lactobacillus prepared by the same procedure as the above method (1-1). -Dead paracasei cells were diluted with sterile distilled water to prepare a lactobacillus paracasei dead cell suspension (2.8 × 10 ⁹ cells / mL) sample (test sample).
The number of dead cells was counted with a stereomicroscope (× 400 times) using a bacterial counter hemocytometer (Sunlead Glass Co., Ltd.).

(2) Preparation of measurement sample (2-1) Preparation of drug that suppresses the action of nucleic acid amplification inhibitor Next, a drug that suppresses the action of nucleic acid amplification inhibitor (hereinafter, cDBC (abbreviation of concentrated direct component)) Notated) was prepared.
Specifically, bovine serum albumin (Sigma, hereinafter referred to as BSA), trisodium citrate dihydrate (Kanto Kagaku, hereinafter referred to as TSC), magnesium chloride hexahydrate (Nakalitesk, Inc., Hereinafter, MgCl ₂ ), egg white lysoteam (Wako Junyaku, hereinafter simply referred to as lysodium), and Brij58 (registered trademark: Sigma) were mixed so as to have the concentrations shown in Table 1 to prepare cDBC.

(2-2) Preparation of cDBC-containing digital PCR master mix and cDBC-free digital PCR master mix A cDBC-containing digital PCR master mix was prepared with the composition shown in Table 2. In addition, in the composition shown in Table 2, distilled water was added instead of cDBC to prepare a cDBC-free digital PCR master mix.
The sequences of the forward and reverse primers used, as well as the TaqMan® probe, are shown in Table 3.

(2-3) Preparation of Nucleic Acid Amplification Reaction Solution (Sample for Measurement) In the cDBC-containing digital PCR master mix (16 μL) prepared in (2-2), 2 μL of Lactobacillus paracasei dead cell DNA aqueous solution as a test sample was added. Alternatively, a lactobacillus paracasei dead cell suspension was added to prepare a total of 18 μL of nucleic acid amplification reaction solution (measurement sample).
Similarly, 2 μL of Lactobacillus paracasei dead cell DNA aqueous solution or Lactobacillus paracasei dead cell suspension was added to the cdBC-free digital PCR master mix (16 μL), and a total of 18 μL of nucleic acid amplification reaction solution ( A sample for measurement) was prepared.

(3) Measurement of Amplification Product and Quantification of Dead Cells This nucleic acid amplification reaction solution (measurement sample) is applied to each well of the chip of QuantStudio (registered trademark) 3D Digital PCR 20K Chip Kit v2 (Thermo Fisher Scientific). Using a dedicated loader, 865 pL was dispensed at a time. After dispensing, digital PCR was performed using a digital PCR device (QuantStudio® 3D Digital PCR System, Thermo Fisher Scientific) under the PCR thermal cycle conditions shown in Table 4.

The number of wells emitting green fluorescence and the fluorescence intensity thereof were measured using a chip reader dedicated to the kit, and the amplification product was measured.
Based on the measurement results 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) Results Table 5 shows the results of digital PCR.

(5) Discussion Digital PCR was performed on Lactobacillus paracasei dead cell DNA aqueous solution (0.4 ng / μL) using a cDBC-free digital PCR master mix. As a result, the target gene of Lactobacillus paracasei per 1 μL of the reaction solution was performed. The number of copies remained at 129.42 copies.
On the other hand, when digital PCR was performed using a digital PCR master mix containing cDBC, the number of target gene copies of lactobacillus paracasei per 1 μL of the reaction solution was 711.72 copies, which was digital using the digital PCR master mix containing no cdBC. The quantitative value was 5.5 times that of the case where PCR was performed.

Similarly, for Lactobacillus paracasei dead cell suspension ^{(2.8 × 10 9 cells / mL} ), the case of performing digital PCR also found the following when using the CDBC containing digital PCR Master Mix, CDBC non The quantitative value was about 3 times higher than that when the contained digital PCR master mix was used.

The above results show that when quantitative measurement of dead cells of Gram-positive bacteria is performed by the digital PCR method, the quantitativeness is improved by adding a drug that suppresses the action of a nucleic acid amplification inhibitor to the test sample. Shows that it can be done.

[Example 2]
Next, when quantitative measurement of dead cells by digital PCR method using chocolate containing dead cells of Lactobacillus paracasei or liquid food (dairy product) as a test sample, a drug that suppresses the action of nucleic acid amplification inhibitor The effect on quantification was investigated.

(1) Preparation of test sample (1-1) Preparation of Lactobacillus paracasei dead cell-containing chocolate sample (test sample) Lactobacillus paracasei dead cell-containing chocolate (Lactobacillus paracasei dead cell 2 × 10 ⁹ cells / g) 5 g was added to 45 mL of sterile distilled water and heated at 40 ° C. for 30 minutes to obtain a uniform solution. This solution was further diluted 10-fold with sterile distilled water, and diluted 100-fold in total. 1 mL of this 100-fold diluted solution of chocolate was collected, subjected to cooling centrifugation (8000 × G, 10 minutes, 4 ° C), the supernatant was removed, and then 1 mL of sterile distilled water was added to add Lactobacillus paracasei dead cells. A chocolate-containing (100-fold diluted) sample (test sample) was used.

(1-2) Preparation of Lactobacillus paracasei dead cell-containing liquid food (dairy product) sample (test sample) Lactobacillus paracasei dead cell-containing liquid food (dairy product) (1.25 × 10 ⁸ cells / mL) After adding 5 mL to 45 mL of sterile distilled water to dilute 10-fold, 1 mL is collected and subjected to cooling centrifugation (8000 × G, 10 minutes, 4 ° C.), and after removing the supernatant, 1 mL of sterile distilled water is added. In addition, a liquid diet (dairy product) sample (test sample) containing Lactobacillus paracasei dead cells was used.

(2) Preparation of measurement sample For each test sample, a measurement sample was prepared using the cDBC-containing digital PCR master mix and the cDBC-free digital PCR master mix in the same manner as in Example 1.
(3) Measurement of amplification product and quantitative measurement of dead cells Digital PCR was performed by the same method as in Example 1.

(4) Results The results of digital PCR are shown in Table 6.

(5) Discussion As shown in Table 6, when digital PCR was performed using the cDBC-containing digital PCR master mix for the quantitative measurement of dead cells of the same bacterium contained in Lactobacillus paracasei dead cell-containing chocolate, cDBC. Compared with the case of using the non-containing digital PCR master mix, the quantitative value was 4.5 times higher.

Similarly, for the quantitative measurement of dead cells of the same bacterium contained in the liquid diet containing Lactobacillus paracasei dead cells, when digital PCR is performed using the cDBC-containing digital PCR master mix, the cDBC-free digital PCR is also performed. The quantitative value was 2.8 times higher than that when the master mix was used.

The results of Example 2 show that even when the test sample is food, quantitative measurement of dead cells of Gram-positive bacteria can be performed by digital PCR without extracting DNA. Further, as in the result of Example 1, the result of Example 2 also shows that the quantitativeness can be improved by adding a drug that suppresses the action of the nucleic acid amplification inhibitor to the test sample.

[Example 3]
In Example 3, a quantitative comparison was made with and without the step of dispersing the aggregates of dead cells in the food.

(1) Preparation of test sample Highly nutritious liquid food containing oils and fats (product name: A (Ace) 1.5, manufactured by Clinico Co., Ltd.) is diluted 10-fold to be highly nutritious without lactobacillus paracasei dead cells. A liquid food diluent was prepared. To this, lactobacillus paracasei dead cells (4 × 10 ¹⁰ cells / mL) were added stepwise, 1.0 × 10 ⁷ cells / mL, 2.0 × 10 ⁷ cells / mL, 4.0 × 10 ⁷ A highly nutritious liquid food diluent containing cells / mL Lactobacillus paracasei dead cells was prepared.

The Lactobacillus paracasei dead cell-free high-nutrient liquid food diluent prepared in this way and the three types of Lactobacillus paracasei dead cell-containing high-nutrient liquid food diluted solution were designated as Group A.
Further, the sample group obtained by ultrasonically treating the samples of the A group using an ultrasonic homogenizer was designated as the B group.

In addition, Lactobacillus paracasei dead cells (4 × 10 ¹⁰ cells / mL) were added stepwise to ^{sterile distilled water, and 1.0 × 10 7} cells / mL, 2.0 × 10 ⁷ cells / mL, 4. Sterile distilled water containing 0 × 10 ⁷ cells / mL Lactobacillus paracasei dead cells was prepared.
Then, the sterilized distilled water containing lactobacillus paracasei dead cells and the sterilized distilled water containing no dead cells were ultrasonically treated using an ultrasonic homogenizer. These sample groups after ultrasonic treatment are referred to as group C.

(2) Preparation of sample for measurement Using the samples of groups A to C prepared in (1) above as test samples, dead cells contained therein were quantified by digital PCR. The addition amount (μl) of each reagent in Table 2 was scaled up to 1.125 times, and 2 μl of the test sample was added to the cDBC-containing digital PCR master mix having a total amount of 18 μl to prepare a sample for measurement.

(3) Measurement of amplification product and quantitative measurement of dead cells The measurement of amplification product and quantitative measurement of dead cells were performed under the same PCR thermal cycle conditions as in Example 1. For the same test, parallel measurements were performed 6 times.

(4) Results The results are shown in Tables 7-9. In addition, the results of Tables 7 to 9 were plotted on a graph to create a standard curve (FIGS. 1 to 3).

(5) Consideration From Tables 7, 8 and 1 and 2, the quantitative value of dead cells of Lactobacillus paracasei was improved by performing ultrasonic treatment on the test sample, and R ^{2 of the standard curve.} It can be seen that the value approaches 1 more.
The result is that by applying an operation such as sonication to disperse dead cell aggregates in the test sample, aggregates containing multiple dead cells are dispensed into one well of the digital PCR chip. It is suggested that this can be prevented and the quantitativeness can be improved.

In Group A of Table 7, the recovery rate of dead cells of Lactobacillus paracasei represented by "number of cells" is 10.6% on average, and this value is the recovery rate calculated in Table 6. It is presumed that the half of the value of about 20% was due to the difference in the effect of the sample matrix on digital PCR due to the difference in the target products.

Comparing Tables 8 and 9, and FIGS. 2 and 3, the death of Lactobacillus paracasei was higher when a highly nutritious liquid food containing fats and oils was used than when sterile distilled water was used. It can be seen that the quantitative value of cells is improved. This result suggests that the measuring method of the present invention is suitable for quantitative measurement of dead cells of Gram-positive bacteria contained in oil and fat foods.

This result shows that the dead cells have a structure in which the hydrophobic part is exposed on the outer surface, so that the degree of hydrophobicity is high as a whole, and it is easy to disperse in a sample containing oils and fats that are also highly hydrophobic, and aggregates. It is considered that this is due to the fact that it is difficult to form.

Further, the results of Tables 7 to 9 and FIGS. 1 to 3 clearly show that the measurement method of the present invention clearly shows the concentration even if the concentration difference of the dead cells of Gram-positive bacteria in the test sample is about twice. The difference can be measured separately, indicating that high quantification is exhibited.

[Example 4]
Next, a method for measuring dead cells of Gram-positive bacteria including a DNA extraction step was examined.

(1) Preparation of test sample First, 10 g of tofu containing no dead cells of Lactobacillus paracasei was diluted 2.5 times with distilled sterilized water. Separately, dead cells of lactobacillus paracasei previously ultrasonically treated with an ultrasonic homogenizer were added to this diluted solution, and 0, 2 × 10 ⁷ cells / g, 4 × 10 ⁷ cells / g, 8 × 10 A standard solution containing dead cells was prepared at a concentration of ^{7 cells / g.}

At the same time, Lactobacillus paracasei dead cell-containing tofu (theoretical ^{amount: 6.67 × 10 7 cells / g} ) was diluted to 2.5 times with distilled sterile water, bean curd 2.5 fold dilution (theoretical amount 2 .67 × 10 ⁷ cells / g) was prepared.

(2) Preparation of measurement sample (2-1) Tofu pellet DNA extraction sample Measurement sample 200 μl was separated from the 2.5-fold diluted solution of tofu prepared in (1) above, and cooled and centrifuged (8000 × G). 10 minutes, 4 ° C.) to prepare pellets. Then, 300 mg of glass beads having a diameter of 1 mm was added to the obtained pellets, and the mixture was vigorously stirred for 1 minute to disrupt dead cells. DNA was extracted from this crushed dead cell using KURABO DNA extraction Kit (Kurabo Industries Ltd.) to obtain 200 μl of a DNA aqueous solution. Of these, 2 μl of a DNA aqueous solution was mixed with 18 μl of a commercially available cDBC-free digital PCR master mix (manufactured by Thermo Fisher Scientific Life Technologies Japan) to prepare a sample for measuring a tofu pellet DNA extraction sample.
Further, the same operation was applied to the standard solution prepared in (1) above to prepare a sample for measuring a standard pellet DNA extraction sample.

(2-2) Tofu Suspension DNA Extraction Sample Measurement Sample 200 μl is separated from the 2.5-fold diluted solution of tofu prepared in (1) above, and DNA is obtained using KURABO DNA extraction Kit (Kurabo Industries Ltd.). Extraction was performed to obtain 200 μl of an aqueous DNA solution. A 2 μl DNA aqueous solution was mixed with 18 μl of a commercially available cdBC-free digital PCR master mix (manufactured by Thermo Fisher Scientific Life Technologies Japan) to prepare a sample for measuring a tofu suspension DNA extraction sample.
Further, the same operation was applied to the standard solution prepared in (1) above to prepare a sample for measuring a standard suspension DNA extract sample.

(3) Measurement of amplification product and quantitative measurement of dead cells Digital PCR was performed on each measurement sample prepared in (2-2) of (2-1) above by the same method as in Example 1 and amplified. The product was measured.
Based on the measurement results, the dead cells of Lactobacillus paracasei contained in each measurement sample were quantitatively measured.

(4) Results The results are shown in Tables 10 and 11. In addition, standard curves were created from the results of Tables 10 and 11, and are shown in FIGS. 4 and 5, respectively.

Furthermore, using the standard curves of FIGS. 4 and 5, the dead cells contained in the tofu containing Lactobacillus paracasei dead cells before the 2.5-fold dilution were quantitatively measured. The measurement results are shown in Table 12.

(5) Consideration As shown in Tables 10, 11 and 4 and 5, even when the method of Example 4, that is, the DNA extraction operation is performed, a small concentration difference of about 2 times is distinguished and Lactobacillus is distinguished. -It is possible to measure dead cells of Paracasei with good quantitativeness.

Further, as shown in the results of Table 12, 85.9% of the theoretical addition amount of Lactobacillus paracasei dead cell-containing tofu was obtained when DNA extraction was performed by the method (2-1), (2-). When DNA extraction was performed by the method of 2), the quantitative value was 77.5%. This result shows that the quantitativeness is improved by adding an operation of vigorously stirring the dead cells and the glass beads together and crushing the dead cells before performing the DNA extraction operation.

The present invention can be applied to quality control of health foods to which dead cells of microorganisms exhibiting effective physiological activity on the human body are added, and quantification of viruses of inactivated vaccines.

NITE BP-01633

Claims (6)

The ShiHoso cells of Gram-positive bacteria in a test sample, a method for measuring by using the digital PCR method,
The measurement sample preparation process for preparing a measurement sample from the test sample,
The gram-positive bacterium ShiHoso cells DNA or RNA of the target area of the measurement sample was amplified by digital PCR method, the amplified product measuring step of measuring the amplification products,
Anda quantitative quantifying the ShiHoso cells of the gram-positive bacteria on the basis of the measurement result,
The test sample is a sample containing oils and fats, and is
Said assay sample preparation process, the include adding inhibit hydrophilic drugs act of nucleic acid amplification inhibitors in the test sample, and extracting the ShiHoso cells of DNA or RNA of the Gram-positive bacterium Not included, method. The method according to claim 1, wherein the Gram-positive bacterium is a bacterium belonging to the genus Lactobacillus. The method according to claim 2, wherein the bacterium belonging to the genus Lactobacillus is Lactobacillus paracasei. The method according to any one of claims 1 to 3, wherein the test sample is a food containing animal fat and / or vegetable fat. The assay sample preparation step comprises causing said Gram positive bacteria ShiHoso cells of dispersed using a dispersing machine ultrasonic or pressure type, the method according to any one of claims 1-4. The method according to any one of claims 1 to 5, wherein the hydrophilic agent that suppresses the action of the nucleic acid amplification inhibitor contains at least lysozyme and polyethylene glycol.

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