JP2015139434A - Microorganism detection method, and microorganism detection kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting living cells of a microorganism in a specimen conveniently in a short time by discriminating the living cells from dead cells or damaged cells.SOLUTION: A living cell of a microorganism in a specimen can be detected by discriminating the living cell from a dead cell or a damaged cell through following steps of: a) adding, to the specimen, an agent which inhibits an amplification by nucleic acid amplifying method of the nucleic acid of the microorganism selectively for a dead cell; b) performing a treatment which improves permeability of the cell of the microorganism; c) amplifying a target region of a nucleic acid of the microorganism in the specimen by isothermal nucleic acid amplification method using a chain-substituted type nucleic acid elongation enzyme without extracting the nucleic acid from the cell; and d) analyzing an amplification product.

Description

  The present invention relates to a method for detecting microorganisms contained in foods and biological samples, microorganisms contained in environments such as industrial water and city water, and a microorganism detection kit. More specifically, the present invention relates to a detection method and a microorganism detection kit that can selectively detect living cells of microorganisms contained in an environment such as foods, biological samples, wiped samples, industrial water, and city water.

  Conventionally, a plate culture method has been used to measure the number of general live bacteria in foods, biological samples, wiped samples, or the environment. However, the plate culture method has a problem that it takes about 2 days to 1 month to obtain the result, and it is difficult to identify bacteria.

  In recent years, a test sample is treated with a crosslinking agent that crosslinks DNA such as ethidium monoazide (EMA), a topoisomerase inhibitor and / or a DNA gyrase inhibitor, and then a chromosome in a microorganism in the sample. A technique for detecting viable bacteria in a sample by selectively amplifying DNA by a nucleic acid amplification reaction has been proposed and has been achieved (Patent Documents 1 to 4).

  When the above crosslinking agent, topoisomerase inhibitor and DNA gyrase inhibitor enter the cell, they bind to or intercalate with DNA to inhibit the action of topoisomerase or DNA gyrase (enzyme), Alternatively, the DNA is cross-linked, and as a result, the chromosomal DNA is destroyed (fragmentation / cutting). Since these drugs are more permeable to the cell walls of dead and damaged bacteria than the cell walls of live bacteria, the chromosomal DNA of the damaged or dead bacteria is preferentially fragmented over the live bacteria. Therefore, viable bacteria can be selectively detected compared with damaged or dead bacteria by PCR targeting a specific region of chromosomal DNA.

  Conventionally, nucleic acids extracted from microbial cells contained in a test sample have been used as the PCR template. However, a drug that suppresses the action of a nucleic acid amplification inhibitor without extracting nucleic acids from cells is used. A method of rapidly detecting viable bacteria by carrying out PCR in the presence has been disclosed (Patent Document 4). Hereinafter, such a method of performing nucleic acid amplification without extracting nucleic acid from cells may be referred to as “direct method”. In this method, nucleic acid amplification is preferably performed by adding a drug that suppresses the function of a nucleic acid amplification inhibitor, a magnesium salt, and an organic acid salt or phosphate to the test sample.

As a method for amplifying a nucleic acid, a method (LAMP (Loop-mediated Isothermal Amplification) method) in which a gene amplification reaction proceeds isothermally using a DNA polymerase having strand displacement activity has been developed (Patent Document 5). This method uses four types of primers, and two of them recognize two different regions in the target nucleic acid sequence on the 3 ′ and 5 ′ sides, and the sequence on the 5 ′ side is the 3 It is designed to anneal in the complementary strand region synthesized by the extension reaction from the side. The amplification reaction is a dumbbell type with a stem-loop structure that is generated by these inner primers and two types of outer primers that are used to remove the DNA strand synthesized from the inner primer as a single strand from the template DNA. It proceeds by repeating the self-elongation reaction and the strand displacement synthesis reaction starting from the structure of In this method, the amplification reaction is performed isothermally (60-65 ° C).
In addition, by using two types of loop primers having sequences complementary to the single-stranded portion of the loop on the 5 ′ end side of the dumbbell structure, it becomes possible to increase the starting point of DNA synthesis and shorten the reaction time. (Patent Document 6).

The LAMP method has the advantage of high amplification efficiency and does not require a special device. Detection of the amplification reaction in the LAMP method is performed using white turbidity due to magnesium pyrophosphate generated in association with the amplification reaction or calcein which is a fluorescent dye. Calcein is quenched by binding to manganese ions, but as the nucleic acid amplification reaction proceeds, pyrophosphate ions generated along with the amplification reaction deprive calcein of manganese ions, causing calcein to become free and fluoresce. become. The amplification reaction is detected in real time by measuring the white turbidity of magnesium pyrophosphate using a turbidity measuring device, which requires a dedicated device. When using calcein, the reagent kit used in the LAMP method (Eiken Chemical)
Loopamp fluorescence / visual detection reagent. According to the instructions of Loopamp (trademark of the company), fluorescence is observed after completion of the amplification reaction. That is, real-time detection of an amplification reaction using calcein is not usually performed.

As a nucleic acid amplification method using a nucleic acid elongation enzyme having strand displacement activity (also called isothermal nucleic acid amplification method), in addition to the LAMP method, ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) (Patent Document 7) ), SDA (Strand Displacement Amplification) method (Patent Document 8), LCR (Ligase Chain Reaction) method (Non-Patent Document 1), TMA (Transcription Mediated Amplification) method (Patent Document 9), SMAP (SMart Amplification Process) method ( Non-Patent Document 2) and TRC (Transcription-Reverse Transcription-Concerted)
) Method (Non-Patent Document 3) and the like are known.

  As described above, various isothermal amplification methods using a nucleic acid elongation enzyme having a strand displacement activity, such as the LAMP method, have been developed. However, it is not known that the isothermal amplification method is applied to a method of performing nucleic acid amplification without extracting nucleic acid from cells (direct method).

International Publication No. 2001/077379 International Publication No. 2007/094077 International Publication No. 2009/022558 International Publication No. 2011/010740 International Publication No. 00/28082 International Publication No. 2002/024902 International Publication No. 00/56877 U.S. Pat.No. 5,455,166 International Publication No. 91/01384

Barany, F., Proc. Natl. Acad. Sci. USA, 88: 189-193, 1991 Mitani, Y., Seibutsu Butsuri Kagaku, 52 (4): 183-187, 2008 Nakaguchi, Y. et al., J. Clin. Microbiol., 42 (9): 4284-4292, 2004

  An object of the present invention is to provide a technique for performing nucleic acid amplification without directly extracting nucleic acids from cells (direct method) in a short time.

The present inventor has conceived that nucleic acid amplification in the direct method is performed by an isothermal amplification method such as the LAMP method. However, the present inventors have found that nucleic acid amplification is not performed simply by combining the direct method and the LAMP method, and there are conditions necessary for performing nucleic acid amplification by combining both.

In addition, the present inventor examined a method capable of detecting nucleic acid amplification in real time. Nucleic acid detection reagents such as SYBR Green and acridine used in real-time PCR are not used in the LAMP method. These reagents are DNA intercalating agents. However, in an isothermal amplification method that does not involve high-temperature treatment such as PCR, these intercalating agents are inserted and fixed in the double strand of the template nucleic acid. Then, it was thought that when the nucleic acid was extended by the strand displacement type nucleic acid extender, the intercalating agent interfered with it and gene amplification could not be performed. Then, the present inventor examined real-time detection of nucleic acid amplification using calcein.
As a result of the above studies, the present invention has been completed.

That is, the present invention is a method for detecting a living cell of a microorganism in a test sample by distinguishing it from a dead cell or a damaged cell, which comprises the following steps:
a) a step of adding, to the test sample, an agent that selectively inhibits the dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
b) performing a treatment for increasing the permeability of cells of microorganisms;
c) a step of amplifying a target region of a nucleic acid of a microorganism in a test sample by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme without extracting the nucleic acid from the cell, and d) an amplification product Process to analyze,
And the process of increasing the permeability of the microorganism cell is thereby a process that allows selective nucleic acid amplification in living cells without the extraction of nucleic acids from the cells.

In the method, the drug is selected from a drug that is covalently bonded to a nucleic acid by light irradiation with a wavelength of 350 nm to 700 nm and a platinum group element complex, and the drug is covalently bonded to a nucleic acid by light irradiation with a wavelength of 350 nm to 700 nm. In the case of a drug, a preferred embodiment includes a step of performing a light irradiation treatment with a wavelength of 350 nm to 700 nm on a test sample to which the drug is added.
The method is a drug in which the drug is covalently bonded to a nucleic acid by irradiation with light having a wavelength of 350 nm to 700 nm, and is ethidium monoazide, ethidium diazide, propidium monoazide, psoralen, 4,5 ′, 8− A preferred embodiment is selected from trimethylpsoralen and 8-methoxypsoralen.

Moreover, the said method makes it a preferable aspect that the said chemical | medical agent is a complex of a platinum group element, and is chosen from a platinum complex, a palladium complex, and an iridium complex.
In the method, the complex of the platinum group element is a platinum complex, and NH ₃ , RNH ₂ , a halogen element, a carboxylate group, a pyridine group, H ₂ O, CO ₃ ²⁻ , OH ⁻ , NO ₃ ⁻ , ROH _{, N 2 H 4, PO 4} 3-, R 2 O, RO -, ROPO 3 2-, (RO) 2 PO 2 -, R 2 S, R 3 P, RS -, CN -, RSH, RNC, ( RS) ₂ PO ₂ ⁻ , (RO) ₂ P (O) S ⁻ , SCN ⁻ , CO, H ⁻ , and R ⁻ (wherein “R” represents a saturated or unsaturated organic group). It is a preferable aspect to include a ligand selected from:
In the method, the platinum group complex is a palladium complex, NH ₃ , RNH ₂ , halogen element, carboxylate group, H ₂ O, CO ₃ ²⁻ , OH ⁻ , NO ₃ ⁻ , ROH, N _{2 ^{H 4, PO 4 3-, R}} 2 O, RO -, ROPO 3 2-, (RO) 2 PO 2 -, R 2 S, R 3 P, RS -, CN -, RSH, RNC, (RS) 2 PO ₂ ⁻ , (RO) ₂ P (O) S ⁻ , SCN ⁻ , CO, H ⁻ , R ⁻ (where “R” represents a saturated or unsaturated organic group), NO ₂ ⁻ , Ar— A preferred embodiment includes a ligand selected from NH ₂ , Ar—CN (Ar is an unsaturated organic group), N ₂ , SO ₃ ²⁻ , an imidazole ring, an unsaturated cyclic organic group, and N ₃ ⁻ . .
In the method, the platinum group element complex is an iridium complex, and NH ₃ , RNH ₂ , a halogen element (Cl, F, Br, I, At), a carboxylate (—CO—O—) group, a pyridine group ^{_{, H 2 O, CO 3 2-}} , OH -, NO 3 -, ROH, N 2 H 4, PO 4 3-, R 2 O, RO -, ROPO 3 2-, (RO) 2 PO 2-, NO ^{_{2 -, N 2, N 3}} -, R 2 S
^{_{, R 2 P -, R 3}} P, RS -, CN -, RSH, RNC, (RS) 2 PO 2 -, (RO) 2 P (O) S -, SCN -, CO, H -, and R ^-
(However, “R” represents a saturated or unsaturated organic group.) A preferred embodiment includes a ligand selected from the group consisting of.

Moreover, the said method makes it the preferable aspect that the process which improves the permeability | transmittance of the cell of the said microorganism is chosen from heat processing, electron beam irradiation, voltage application, enzyme treatment, and an osmotic shock.
Moreover, the said method makes it the preferable aspect that the said process is heat processing and process conditions are 65-100 degreeC and 0.5 to 30 minutes.

  In the above method, the isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme is preferably selected from the LAMP method, ICAN method, SDA method, LCR method, TMA method, SMAP method, and TRC method. .

Moreover, the said method makes it the preferable aspect to perform the said process c) in the solution of the following composition.
Tris-HCl (pH 7 ~ 9) 10mM ~ 25mM
KCl 5mM ~ 15mM
MgSO ₄ 5mM~40mM
Surfactant 0.1% -0.4%
Betaine 0.5M-1M
dNTPs 1mM to 1.5mM each
Strand displacement type nucleic acid elongation enzyme 0.2-0.6 U / μl

In the method, the surfactant is preferably polyethylene glycol sorbitan monolaurate.
Moreover, the said method makes it a preferable aspect that strand displacement type | mold nucleic acid elongase is Bst polymerase and / or Csa polymerase.

Moreover, the said method makes it a preferable aspect that the said target region is a 50-5000 base target region.
In the method, the target region is a 5S rRNA gene of a nucleic acid of a test sample, 1
A preferred embodiment is a target region corresponding to a gene selected from 6S rRNA gene, 23S rRNA gene, and tRNA gene.
Further, the method is preferably configured such that amplification of the target region is performed in the presence of calcein, and the amplification product is detected in real time by fluorescence of calcein.

  The method also includes isothermal nucleic acid amplification, a set of primers having the sequences of SEQ ID NOs: 1, 2, 3, 4, 9, and 10, and sequences of SEQ ID NOs: 10, 11, 12, 13, 14, and 17. It is a preferred embodiment to use a set of primers and a set of primers selected from the set of primers having the sequences of SEQ ID NOs: 18, 19, 20, and 21.

Further, the present invention is a kit for detecting a living cell of a microorganism in a test sample by distinguishing it from a dead cell or a damaged cell by an isothermal nucleic acid amplification method using a strand displacement nucleic acid elongation enzyme, Provide a kit containing the following elements:
1) a drug that selectively inhibits dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
2) a reagent for preparing a reaction solution having the following composition;
Tris-HCl (pH 7 ~ 9) 10mM ~ 25mM
KCl 5mM ~ 15mM
MgSO ₄ 5mM~40mM
Surfactant 0.1% -0.4%
Betaine 0.5M-1M
dNTPs 1mM to 1.5mM each
3) A primer for amplifying the target region of the nucleic acid of the microorganism to be detected by an isothermal nucleic acid amplification method.

In a preferred embodiment, the kit further includes a strand displacement type nucleic acid elongation enzyme.
In addition, the kit preferably includes calcein.

  In the kit, the agent, surfactant, and primer that selectively inhibit the dead cell from amplifying the strand displacement nucleic acid elongase, the nucleic acid of the microorganism by the nucleic acid amplification method are the same as those described for the method. .

Further, the present invention is a method for detecting a living cell of a microorganism in a test sample by distinguishing it from a dead cell or a damaged cell, which comprises the following steps:
a) a step of adding, to the test sample, an agent that selectively inhibits the dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
c) a step of amplifying a target region of a nucleic acid of a microorganism in a test sample by a nucleic acid amplification method, and d) a step of analyzing an amplification product,
A process comprising performing a) in a cell suspension containing 0.5 to 10% by mass of a component selected from the group consisting of proteins, sugars, lipids, and yeast extracts To do.

  In the method, the microorganism to be detected is Legionella bacteria, Campylobacter bacteria, Vibrio bacteria, Listeria bacteria, Clostridium bacteria, Helicobacter bacteria, Mycobacterium bacteria, Chlamydiaceae bacteria, Rickettsia bacteria, and A preferred embodiment is a bacterium belonging to the genus Neisseria.

  According to the method of the present invention, living cells of microorganisms can be detected by distinguishing them from dead cells or damaged cells. According to the present invention, it is possible to discriminate between living cells, damaged cells, and dead cells of microorganisms in an environment such as food and biological samples, wiped samples, industrial water, environmental water, wastewater, and the like by a nucleic acid amplification method. The method and kit of the present invention can be applied to self-inspection and are excellent in economy.

Standard curve of living microorganisms by direct-LAMP method (platinum complex treatment or untreated). The horizontal axis represents the number of living cells, and the vertical axis represents the Ct value (the same applies to FIG. 2). “Direct-LAMP” indicates untreated, and “Pt-Direct-LAMP” indicates platinum complex treatment. Standard curve of living microorganisms by direct-quantitative PCR method (platinum complex treatment or untreated). “Direct-qPCR” indicates untreated, and “Pt-Direct-qPCR” indicates platinum complex treatment.

Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be freely changed within the scope of the present invention. In the present specification, percentages are expressed by mass unless otherwise specified.
In the method of the present invention, any kind of nucleic acid may be used as a detection target as long as it can be amplified by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme. The template of the isothermal nucleic acid amplification method is usually double-stranded DNA, but when the detection target is RNA, it can be used as a template by forming a double strand from RNA by reverse transcription and DNA polymerase reaction.

<1> Method of the Present Invention The method of the present invention is a method for detecting living cells of microorganisms in a test sample by distinguishing them from dead cells or damaged cells, and the following steps:
a) a step of adding, to the test sample, an agent that selectively inhibits the dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
b) performing a treatment for increasing the permeability of cells of microorganisms;
c) a step of amplifying a target region of a nucleic acid of a microorganism in a test sample by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme without extracting the nucleic acid from the cell, and d) an amplification product Process to analyze,
including. The treatment for increasing the permeability of the cells of the microorganism is a treatment that enables nucleic acid amplification selective to living cells without extracting nucleic acids from the cells.

In the present specification, the “test sample” is a target for detecting living cells of microorganisms present therein, and the presence is detected by amplification of a specific region of chromosomal DNA or RNA by a nucleic acid amplification method. Although it will not restrict | limit if it is possible, A foodstuff, biological sample, drinking water, industrial water, environmental water, drainage, soil, or a wipe sample etc. are mentioned.
In particular, as food, beverages such as soft drinks, carbonated drinks, nutrient drinks, fruit juice drinks, lactic acid bacteria drinks (including concentrated concentrates and powders for preparation of these drinks); ice confectionery such as ice cream, ice sherbet, shaved ice; Dairy products such as milk, milk drinks, fermented milk, butter; enteral nutrition foods, liquid foods, milk for childcare, sports drinks; functional foods such as foods for specified health use and health supplements are preferred.

Biological samples include blood samples, urine samples, spinal fluid samples, synovial fluid samples, pleural effusion samples, sputum samples, stool samples, nasal mucus samples, laryngeal mucus samples, gastric lavage fluid samples, pus juice samples, skin mucosa samples, oral cavity Examples include mucus samples, respiratory mucosa samples, digestive mucosa samples, eye conjunctiva samples, placenta samples, germ cell samples, birth canal samples, breast milk samples, saliva samples, vomiting, or blister contents.
Furthermore, examples of the environmental water include city water, ground water, river water, and rain water.

  In the present invention, the test sample may be a food, biological sample, drinking water, industrial water, environmental water, waste water, soil, or a wipe sample itself as described above, or a diluted or concentrated product thereof. Alternatively, pretreatment other than the treatment according to the method of the present invention may be performed. Examples of the pretreatment include heat treatment, filtration, and centrifugation.

  In addition, cells other than microorganisms, protein colloid particles, fats and carbohydrates, etc. present in the test sample may be removed or reduced by treatment with an enzyme having the activity of decomposing them. Examples of cells other than microorganisms present in the test sample include bovine leukocytes and mammary epithelial cells when the test sample is milk, dairy products, milk or foods made from dairy products. When the test sample is a biological sample such as a blood sample, urine sample, spinal fluid sample, synovial fluid sample or pleural effusion sample, red blood cells, white blood cells (granulocytes, neutrophils, basophils, monocytes, lymphoid cells) Spheres), and platelets.

The enzyme is not particularly limited as long as it can decompose the contaminants and does not damage the living cells of the microorganism to be detected. For example, a lipolytic enzyme, a proteolytic enzyme, and a carbohydrase Enzymes. As the enzyme, one kind of enzyme may be used alone, or two or more kinds of enzymes may be used in combination, but both lipolytic enzyme and proteolytic enzyme, or lipolytic enzyme, proteolytic enzyme It is preferable to use all of saccharide-degrading enzymes.
Examples of the lipolytic enzyme include lipase and phosphatase, examples of the proteolytic enzyme include serine protease, cysteine protease, proteinase K, and pronase, and examples of the carbohydrase include amylase and cellulase.

  A “microorganism” is an object to be detected by the method of the present invention, and can be detected by a nucleic acid amplification method, and the action of a drug that inhibits the amplification of microbial DNA by the nucleic acid amplification method is a living cell. Although it will not restrict | limit especially if it is different with a dead cell and a damaged cell, Preferably bacteria, a filamentous fungus, yeast, or a virus is mentioned. Bacteria include both gram-positive bacteria and gram-negative bacteria.

Gram-positive bacteria include Staphylococcus, such as Staphylococcus aureus and Staphylococcus epidermidis; Micrococcus, Streptococcus pyogenes, Streptococcus pneumoniae, and other Streptococcus; Enterococcus faecalis, Enterococcus; Erococcus, Bacillus cereus Bacillus subtilis and Bacillus licheniformis (preferably vegetative cells); Clostridium such as Clostridium botulinum and Clostridium perfringens; Mycobacterium tuberculosis and bovine tuberculosis Mycobacterium genus (mycobacteria and atypical mycobacteria group) such as Mycobacterium intracellulare, Mycobacterium abium; Rae bacteria; Actinomyces; Nocardia; Nocardiopsis; Actinomadura; Streptomyces Genus; Dell Tofirusu genus; Eubacterium; Corynebacterium; Propionibacterium genus, and the like.

  Gram-negative bacteria include Legionella spp .; Salmonella spp .; Enterohemorrhagic Escherichia coli including O157, O26, O11, O145; Campylobacter spp .; Alcobacter spp .; Helicobacter spp .; Pseudomonas spp. Deria; Acinetobacter; Alkagenes; Chryseobacterium; Moraxella; Coxella; Brucella; Francisella, such as Francisella tularensis; Balticella; Bordeterra; Haemophilus influenzae, Genus; Pasteurella genus; Chromobacterium genus; Streptobacillus genus; Shigella genus such as Shigella, Yersinia, Escherichia coli, etc. Enterobacteriaceae such as Genus, Proteus, Providencia, Morganella, Serratia, etc .; Vibrio; Eromonas; Bacteroides; Prevoterra; Porphyroonas; Fusobacterium; Leptotricia; Examples include Treponema; swine dysentery spirochete; Borrelia; Mycoplasma; Rickettsia; Chlamydia and the like.

  Viruses include Poxviridae, Herpesviridae, Adenoviridae, Papillomaviridae, Polyomaviridae, Parvoviridae, Picornaviridae, Caliciviridae, Astroviridae, Coronaviridae, Togaviridae, Examples include Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Bornaviridae, Arenaviridae, Bunyaviridae, Reoviridae, Retroviridae, and Hepatitis Virus.

Regarding viruses, a method of measuring only activated viruses by RT-PCR is known in the method of measuring activation / deactivation of viruses in water by allowing photoreactive nucleic acid cross-linking agent (EMA) to act. (Http://www.recwet.tu-tokyo.ac.jp/furumailab/j/sotsuron/H21/H21sotsuron.html, Development of ethidium monoazide (EMA) -RT-PCR for selective detection of enteric viruses.15th International Symposium on Health-Related Water Microbiology. (May 31-Jun 05, 2009, Ursulines Conference Centre, Naxos, Greece)).
In other words, EMA does not permeate activated viruses, only permeates only inactivated viruses with nucleocapsids that are severely physically damaged, and EMA can distinguish between activated viruses (Live) and inactivated viruses (Dead). It has been suggested that it is possible. Therefore, it is considered that the present invention can be applied not only to bacteria, filamentous fungi and yeasts but also to viruses.

  Further, as shown in Examples below, it was shown that the method of the present invention can distinguish between live and dead cells of Gram negative bacteria and Gram positive bacteria. Therefore, it is considered that a virus having the outermost envelope having the same component as the cell wall outer membrane of Gram-negative bacteria can be used for distinguishing between live cells and dead cells. In addition, since a virus having only a so-called nucleocapsid (protein membrane) without an envelope does not have an outer membrane and is relatively similar to a Gram-positive bacterium in which the peptidoglycan layer directly contacts the outside world, the living cell according to the present invention It is considered possible to identify dead cells.

In the present invention, a “live cell” is a state that can grow when cultured under suitable culture conditions and exhibits the metabolic activity of the microorganism (Viable-and-Culturable state). A microorganism that has almost no damage to the cell wall. The metabolic activity mentioned here can be exemplified by ATP activity and esterase activity. In the present invention, virus particles are also referred to as “cells” for convenience. “Live cell” refers to a state in which a mammalian cell can be infected and propagated with respect to a virus.

“Dead cells” are microorganisms that cannot grow even when cultured under suitable culture conditions and do not exhibit metabolic activity (Dead). In addition, although the structure of the cell wall is maintained, the cell wall itself is highly damaged, and a weakly permeable nuclear stain such as propidium iodide penetrates the cell wall. Regarding virus, it means a state in which mammalian cells cannot be infected.

“Injured cells” (Viable-but-Non Culturable cells) are damaged by human or environmental stress, and are generally proliferated even when cultured under suitable culture conditions. Although it is difficult, the microorganism has a metabolic activity that is lower than that of living cells, but is significantly more active than that of dead cells. Regarding virus, it means a state in which, even if a mammalian cell is infected, it cannot grow in the cell.
In the present specification, unless otherwise specified, “live cells”, “dead cells” and “damaged cells” mean live cells, dead cells and damaged cells of microorganisms.

  In particular, in food hygiene inspections and clinical tests, attention has been focused on the detection of bacteria exhibiting damaged cells by mild heat treatment and antibiotic administration. In the present invention, not only the detection of living cells but also the detection of living cells. It is an object of the present invention to provide a method for detecting a microorganism that can distinguish cells from dead cells or damaged cells.

The unit of cell number of living cells, damaged cells and dead cells is usually the number of cells / cells / cell.
Expressed in ml.
The number of living cells can be approximated by the number of colonies formed (cfu / ml (colony forming units / ml)) when cultured on a suitable plate medium under suitable conditions. In addition, a standard sample of damaged cells can be prepared, for example, by subjecting a living cell suspension to heat treatment, for example, heat treatment in boiling water. In this case, the number of damaged cells is heat treated. It can be approximated by cfu / ml of the previous live cell suspension. In addition, although the heating time in the boiling water for preparing damaged cells changes with kinds of microorganisms, in the case of bacteria described in the examples, damaged cells can be prepared in about 50 seconds. In addition, standard samples of damaged cells can also be prepared by antibiotic treatment, in which case the number of damaged cells is determined by treating the live cell suspension with antibiotics and then removing the antibiotics. By measuring the transmittance of visible light (wavelength 600 nm), that is, turbidity, and comparing it with the turbidity of a live cell suspension whose live cell number concentration is known in advance, the preferable conditions on a suitable plate medium Can be approximated by the number of colonies formed (cfu / ml).
In viruses, the cell number unit is plaque-forming unit (pfu or PFU (plaque-forming).
units)).
In this specification, the number of cells is expressed in logarithm, and “a log cfu / ml” and “a log cells / ml” represent 10 ^a cfu / ml and 10 ^a cells / ml, respectively.

  The method of the present invention is intended for detection of live cells, and the microorganisms distinguished from live cells may be damaged cells or dead cells.

  In the present invention, “detection of living cells” includes both determination of presence / absence of living cells in the test sample and determination of the amount of living cells. Further, the amount of living cells is not limited to an absolute amount, and may be an amount relative to a control sample. Further, “detecting a living cell by discriminating it from a dead cell or a damaged cell” means selectively detecting a dead cell or a damaged cell. Note that “discrimination between live cells and dead cells or damaged cells” includes discrimination between live cells and both dead cells and damaged cells.

  Hereinafter, the method of the present invention will be described step by step. As described above, prior to the following steps, as an optional step, the test sample may have an activity of degrading cells other than microorganisms, protein colloid particles, fat, or carbohydrates present in the test sample. The process of processing with the enzyme which has may be included.

(1) Step a)
To the test sample, an agent that selectively inhibits dead cells from amplifying microorganism nucleic acid (DNA or RNA) by the nucleic acid amplification method is added. That is, the microorganism in the test sample is treated with the drug. Examples of the drug include a drug covalently bonded to a nucleic acid by irradiation with light having a wavelength of 350 nm to 700 nm, and a platinum group element complex.

  A drug that is covalently bonded to a nucleic acid by irradiation with light having a wavelength of 350 nm to 700 nm intercalates into double-stranded DNA or RNA, and is covalently bonded by irradiation with light to crosslink between the molecules. In addition, it is estimated that the drug is covalently bonded to single-stranded DNA or RNA by light irradiation to inhibit the nucleic acid amplification reaction. Hereinafter, the drug may be simply referred to as “crosslinking agent”.

It is preferable that the cross-linking agent has a different action on living cells, damaged cells or somatic cells such as dead cells and bovine leukocytes, leukocytes, platelets, etc., more specifically, damage more than the cell walls of live cells. It is preferable that it is highly permeable to cell walls of cells or dead cells, or somatic cells such as bovine leukocytes, and cell membranes such as leukocytes and platelets.
Examples of the cross-linking agent include ethidium monoazide, ethidium diazide, psolaren, 4,5 ′, 8-trimethyl psoralen (4,5 ′, 8-trimethyl psolaren), And 8-methoxypsoralen (8-methoxy
psolaren), propidium monoazide and the like. A crosslinking agent may be used individually by 1 type, and may use 2 or more types together.
The conditions for the treatment with the crosslinking agent can be set as appropriate, and for example, the conditions disclosed in International Publication No. 2011/010740 can be adopted.

When a cross-linking agent is used as the drug, a test sample to which the cross-linking agent is added is subjected to light irradiation treatment with a wavelength of 350 nm to 700 nm.
The light having a wavelength of 350 nm to 700 nm may be light having a wavelength of at least 350 nm to 700 nm, and may be single wavelength light or composite light. Further, all components may be in the range of 350 nm to 700 nm, and may include light having a shorter wavelength than 350 nm and / or light having a longer wavelength of 700 nm or more, but the peak in the intensity distribution is 350 nm. It is preferable to be within a range of ˜700 nm. It should be noted that it is preferable not to include a component having a short wavelength enough to cleave the chromosomal DNA of a microorganism only by light irradiation.
When a cross-linking agent is used, it is preferable to keep it under light shielding in a dark room, etc., except for light irradiation to the sample, in order to prevent chemical modification due to exposure.

  Examples of the platinum group element complex include platinum complexes, palladium complexes, and iridium complexes (PCT / JP2013 / 070662, PCT / JP2013 / 070663, Japanese Patent Application 2014-010257).

Platinum complexes include NH ₃ , RNH ₂ , halogen elements, carboxylate groups, pyridine groups, H ₂ O, CO ₃ ²⁻ , OH ⁻ , NO ₃ ⁻ , ROH, N ₂ H ₄ , PO ₄ ³⁻ , R ^{_{2 O, RO -, ROPO 3}} 2-, (RO) 2 PO 2 -, R 2 S, R 3 P, RS -, CN -, RSH, RNC, (RS) 2 PO 2 -, (RO) 2 P (O) A complex containing a ligand selected from the group consisting of S ⁻ , SCN ⁻ , CO, H ⁻ , and R ⁻ (wherein “R” represents a saturated or unsaturated organic group). .
Specific examples of platinum complexes include cisplatin, carboplatin, cis-diammine (pyridine) chloroplatinum (II) chloride, dichloro (ethylenediamine) platinum (II), cis-bis (benzonitrile) dichloroplatinum (II), tetrakis (tri Phenylphosphine) platinum (II), dinitrate (ethylenediamine) platinum iodide (II) dimer, oxaliplatin, nedaplatin, and transplatin.

  Further, the platinum complex may be a platinum complex formed by dissolving a platinum compound in an organic solvent capable of binding to platinum as a ligand or a solution containing a substance capable of binding to platinum as a ligand. . Examples of the platinum compound include platinum chloride, platinum bromide, platinum fluoride, platinum iodide, platinum hydroxide, platinum nitrate, platinum carbonate, platinum acetate, dimethoxyplatinum, platinum methoxyphosphate, platinum phosphate, chloroplatinic acid, Examples include disulfumethylplatinum, dicyanoplatinum, dithiocyanateplatinum, platinum dihydride, and dimethylplatinum. Examples of the solvent include dimethyl sulfoxide.

Examples of the palladium complex include NH ₃ , RNH ₂ , halogen element, carboxylate group, H ₂ O, CO ₃ ²⁻ , OH ⁻ , NO ₃ ⁻ , ROH, N ₂ H ₄ , PO ₄ ³⁻ , R ₂ O, ^{_{RO -, ROPO 3 2-, (}} RO) 2 PO 2 -, R 2 S, R 3 P, RS -, CN -, RSH, RNC, (RS) 2 PO 2 -, (RO) 2 P (O) S ⁻ , SCN ⁻ , CO, H ⁻ , R ⁻ (where “R” represents a saturated or unsaturated organic group), NO ₂ ⁻ , Ar—NH ₂ , Ar—CN (Ar is an unsaturated organic group) Group), N ₂ , SO ₃ ²⁻ , an imidazole ring, an unsaturated cyclic organic group, and a complex containing a ligand selected from N ₃ ^— .
Specific examples of palladium complexes include dichloro (η-cycloocta-1,5-diene) palladium (II), bis (benzonitrile) dichloropalladium (II), diamminedichloropalladium (II), dichloro (ethylenediamine) palladium (II ) And bis (triphenylphosphine) palladium (II) diacetate.

  The palladium complex may be a palladium complex formed by dissolving a palladium compound in an organic solvent capable of binding to palladium as a ligand or a solution containing a substance capable of binding to palladium as a ligand. . Examples of the palladium compound include palladium chloride, palladium fluoride, palladium bromide, palladium iodide, palladium hydroxide, palladium (II) dinitrate, palladium (IV) tetranitrate, palladium acetate, palladium phosphate, dimethoxypalladium, methoxy Examples include palladium phosphate, palladium sulfite, dinitropalladium, and palladium diazide. Examples of the solvent include dimethyl sulfoxide.

Examples of iridium complexes include NH ₃ , RNH ₂ , halogen elements (Cl, F, Br, I, At), carboxylate (—CO—O—) groups, pyridine groups, H ₂ O, CO ₃ ²⁻ , ^{_{OH -, NO 3 -, ROH}} , N 2 H 4, PO 4 3-
^{_{, R 2 O, RO -,}} ROPO 3 2-, (RO) 2 PO 2-, NO 2 -, N 2, N 3 -, R 2 S, R 2 P -, R 3 P, RS -, CN - , RSH, RNC, (RS) 2 PO 2 -, (RO) 2 P (O) S -, SCN -, CO, H -, and R ^- (wherein "R" are both saturated or unsaturated organic group And a complex containing a ligand selected from the group consisting of:
Specific examples of iridium complexes include di-μ-chlorobis [(η-cycloocta-1,5-diene).
Iridium (I)] and 2-hydroxy-N-pyridine (pentamethylcyclopentadienyl)
Examples include iridium (III) dichloride.

  Further, the iridium complex may be an iridium complex produced by dissolving an iridium compound in an organic solvent capable of binding to iridium as a ligand or a solution containing a substance capable of binding to iridium as a ligand. . Examples of the iridium compound include iridium chloride, iridium bromide, iridium fluoride, iridium iodide, iridium hydroxide, iridium nitrate, iridium carbonate, iridium acetate, dimethoxyiridium, iridium methoxyphosphate, iridium phosphate, iridium chloride, Examples thereof include disulfmethyl iridium, dicyano iridium, dithiocyanate iridium, iridium dihydride, methyl iridium, iridium oxide, iridium pentachloride (IV) diammonium (hexachloroiridium (IV) diammonium acid), and the like. Examples of the solvent include dimethyl sulfoxide.

  Conditions for treatment with a drug can be set as appropriate. For example, various concentrations of a drug are added to a suspension of living and dead cells or damaged cells of a microorganism to be detected and left for various times. After that, the cells are separated by centrifugation or the like, and analyzed by the nucleic acid amplification method, whereby the conditions for easily distinguishing live cells from dead cells or damaged cells can be determined. Furthermore, after adding various concentrations of drugs to living cells of microorganisms to be detected and somatic cells such as bovine leukocytes or suspensions of platelets, and leaving them to stand for a predetermined time, the cells and the above-mentioned various kinds are obtained by centrifugation or the like. By separating the cells and analyzing them by the nucleic acid amplification method, it is possible to determine conditions that make it easy to distinguish between living cells and various cells.

In the case of a platinum complex, the final concentration of cisplatin is 10 to 3000 μM, preferably 25 to 3000 μM, 4 to 43 ° C., 5 minutes to 2 hours. With transplatin, a final concentration of 10 to 3000 μM, 4 to 43 ° C., 5 minutes to 2 hours are exemplified.
In carboplatin, the final concentration is 10 to 3000 μM, preferably 250 to 3000 μM, 4 to 43 ° C., 5 minutes to 2 hours. For oxaliplatin or nedaplatin, the final concentration is 10 to 3000 μM, 4 to 43 ° C., 5 minutes to 2 hours.

In cis-diammine (pyridine) chloroplatinum (II) chloride, the final concentration is 10 to 3000 μm.
M, preferably 25 to 3000 μM, 4 to 43 ° C., 5 minutes to 2 hours. For dichloro (ethylenediamine) platinum (II), a final concentration of 10 to 3000 μM, 4 to 43 ° C., and 5 minutes to 2 hours are exemplified. For cis-bis (benzonitrile) dichloroplatinum (II), a final concentration of 10
Examples thereof include 3000 μM, preferably 100 to 3000 μM, 4 to 43 ° C., and 5 minutes to 2 hours. For tetrakis (triphenylphosphine) platinum (II), the final concentration is 10 to 3000 μM, preferably 25 to 3000 μM, 4 to 43 ° C., 5 minutes to 2 hours. In dinitric acid (ethylenediamine) platinum iodide (II) dimer, the final concentration is 10 to 3000 μM, preferably 400 to 3000 μM, 4 to 43 ° C., 5 minutes to 2 hours.

In complexes obtained by dissolving platinum chloride (II), platinum chloride (IV), or chloroplatinic acid (or chloroplatinic acid hexahydrate) in DMSO, platinum (II) chloride, platinum (IV) The final concentration of chloroplatinic acid is 10 to 3000 μM, preferably 10 to 100 μM, 4 to 43 ° C., 5 minutes to 2 hours.

In the case of palladium complex, dichloro (η-cycloocta-1,5-diene) palladium (II)
Then, the final concentration is 10 to 3000 μM, preferably 10 to 100 μM, 4 to 43 ° C., and 5 minutes to 2 hours. Bis (benzonitrile) dichloropalladium (II) has a final concentration of 10-3.
000 μM, preferably 10 to 100 μM, 4 to 43 ° C., 5 minutes to 2 hours are exemplified. In diamminedichloropalladium (II), the final concentration is 10 to 3000 μM, preferably 10 to 1
Examples are 00 μM, 4 to 43 ° C., and 5 minutes to 2 hours. In dichloro (ethylenediamine) palladium (II), the final concentration is 10 to 3000 μM, preferably 10 to 250 μM, 4 to 43 ° C.
Examples are 5 minutes to 2 hours. In the case of bis (triphenylphosphine) palladium (II) diacetate, the final concentration is 10 to 3000 μM, preferably 10 to 100 μM, 4 to 43 ° C., and 5 minutes to 2 hours.

  Further, in the complex obtained by dissolving palladium (II) chloride in DMSO, the final concentration is 10 to 3000 μM, preferably 10 to 100 μM, 4 to 43 ° C., 5 minutes to 2 hours as the amount of palladium (II). Is done. In the complex obtained by dissolving palladium (II) acetate in DMSO, the final concentration is 1 to 300 μM, preferably 1 to 10 μM, 4 to 43 minutes, 5 minutes to 2 hours as the amount of palladium (II). .

In the case of an iridium complex, in Di-μ-chlorobis [(η-cycloocta-1,5-diene) iridium (I)], the final concentration is 20 to 3000 μM, preferably 25 to 300 μM, 4 to 43 ° C., 5 minutes to 2 Time is illustrated. In 2-Hydroxy-N-pyridine (pentamethylcyclopentadienyl) iridium (III) dichloride, the final concentration is 20 to 3000 μM, preferably 50 to 300 μM, 4 to 43 ° C., and 5 minutes to 2 hours.

  The addition of the drug to the test sample may be performed by adding the drug to the suspension of the test sample as described above, or may be performed by adding the test sample to the drug solution. .

  In addition, when a platinum group metal complex is used, light irradiation and light shielding are not necessary.

  One of these drugs may be used alone, or two or more may be used in combination.

  Such drugs are more permeable to the cell walls of dead and damaged cells than the cell walls of living cells. Accordingly, it is considered that the cell wall / cell membrane of living cells of microorganisms does not substantially permeate within an appropriate action time, and the membrane of damaged cells, dead cells, or somatic cells that are dead cells permeate. As a result, the drug enters the dead cells of somatic cells and dead cells of microorganisms and cells of damaged cells and subsequently binds directly or indirectly to nucleic acids (chromosomal DNA or RNA), resulting in drugs It is presumed that the nucleic acid bound with is no longer a template for the nucleic acid amplification reaction.

  When the drug permeates preferentially to damaged or dead cells over live cells, the target region of nucleic acid (chromosomal DNA or RNA) is amplified by the nucleic acid amplification method in live cells, whereas in damaged or dead cells, nucleic acid is amplified. Since the drug binds directly or indirectly to (chromosomal DNA or RNA) and the nucleic acid amplification reaction is inhibited, live cells can be selectively detected compared to damaged cells or dead cells.

The step a) and the light irradiation treatment performed as necessary may be performed by repeating two cycles or more. In that case, the concentration of the drug is preferably higher in the first step a) than in the second and subsequent steps, and lower in the second and subsequent steps a) than in the first.
In the first drug treatment, it is preferable to shorten the treatment time compared to the second and subsequent drug treatments.

  A step of removing the unreacted drug may be added between the previous drug processing and the subsequent drugs. Examples of the method for removing the drug include a method of centrifuging a test sample, separating a precipitate containing a microorganism and a supernatant containing a drug, and removing the supernatant. In this case, it is possible to add a step of washing the microorganism with a cleaning agent as appropriate after removing the drug.

  In addition, it is preferable to perform the process of removing a chemical | medical agent between the process a) and the following process b). This is to prevent the remaining drug from entering the living cells whose cell wall and / or cell membrane permeability has been increased by step b).

(2) Step b)
In this step, a treatment for increasing the permeability of the cells of the microorganism is performed. This treatment is for enabling nucleic acid amplification selective to living cells without extracting nucleic acids from the cells. As will be described later, in step c), the target region of the nucleic acid of the microorganism in the test sample is amplified by isothermal nucleic acid amplification using a strand displacement type nucleic acid elongation enzyme without extracting the nucleic acid from the cell. The

In the detection method of living cells using a drug that selectively inhibits nucleic acid amplification of dead cells (life-determining agent), a nucleic acid amplification reaction is conventionally performed using a nucleic acid extracted from the cells as a template. A microbial cell suspension or a suspension of microbial cells treated with a proteolytic enzyme, lipolytic enzyme, glycolytic enzyme or the like is used as a template without nucleic acid extraction.
When the PCR method is used as the nucleic acid amplification method in the direct method (hereinafter sometimes referred to as “direct PCR”), nucleic acid amplification occurs using such a suspension of microbial cells as a template. As shown in Example 1, no nucleic acid amplification was observed when an isothermal nucleic acid amplification reaction was performed by the LAMP method using a suspension of microbial cells as a template.

  Therefore, the present inventor paid attention to the difference between the PCR method and isothermal nucleic acid amplification, and in the direct PCR method, the permeability of the cells is increased by repeated high-temperature treatment performed for the thermal denaturation of the nucleic acid, and the PCR reagent becomes intracellular. The target region was amplified in the cell. It was found that when the cells were heat-treated, a nucleic acid amplification reaction by the LAMP method occurred using a suspension of microbial cells as a template.

  From the above, step b) can be rephrased as a step for permeating the reagent necessary for the isothermal nucleic acid amplification reaction into the cells of the microorganism, preferably while maintaining the cell morphology. That is, “transparency of cell” means a property that allows a reagent necessary for isothermal nucleic acid amplification reaction to permeate cells of an organism. The treatment for increasing the permeability of the cells of the microorganism may be a treatment for perforating the cell wall and / or cell membrane of the microorganism or a treatment for loosening the structure of the cell wall and / or cell membrane.

  Examples of the treatment for increasing the cell permeability of microorganisms include heat treatment, electron beam irradiation, voltage application, enzyme treatment, and osmotic pressure shock. For such treatment, a transformation method according to the type of microorganism can be referred to.

  The conditions suitable for the treatment for increasing the permeability are the microbial cells subjected to the treatment in the step a) under various conditions, the treatment for increasing the permeability of the microbial cells under various conditions, followed by isothermal nucleic acid amplification. It can be set by observing the amplification reaction. For example, in the case of heat treatment, the heating temperature is usually 65 to 100 ° C, preferably 70 to 96 ° C, more preferably 90 to 96 ° C. As processing time, it is 0.5-30 minutes normally, Preferably it is 1-10 minutes, More preferably, 1-3 minutes are mentioned.

  The step b) is preferably performed after the step a). However, the reagent necessary for the isothermal nucleic acid amplification reaction can permeate into the cells of the microorganism and selectively inhibits the nucleic acid amplification of dead cells. Step b) may be performed before step a) if the drug to be entered is less likely to enter the cells of living cells than dead cells.

Step c)
Subsequently, the target region of the nucleic acid of the microorganism in the test sample is amplified by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme without extracting the nucleic acid from the cell.

  Specifically, an amplification reaction is performed by mixing a test sample subjected to the process of step b) and a reagent necessary for an isothermal nucleic acid amplification reaction. Of the reagents necessary for the isothermal nucleic acid amplification reaction, those that are not inactivated or denatured by heat treatment or the like may be contained in advance in the cell suspension in step b).

  The isothermal nucleic acid amplification method is not particularly limited as long as it does not require dissociation of double-stranded DNA by heat denaturation and can amplify a template nucleic acid by performing an extension reaction at a constant temperature using a strand displacement type DNA polymerase. For example, LAMP method (International Publication No. 00/28082, International Publication No. 2002/024902), ICAN Method (International Publication No. 00/56877), SDA Method (US Pat. No. 5,455,166), LCR Method (Barany, F., Proc. Natl. Acad. Sci. USA, 88: 189-193, 1991), TMA method (WO 91/01384), and SMAP method (Mitani, Y., Seibutsu Butsuri Kagaku, 52 (4) : 183-187, 2008). In these methods, the amplification product is DNA. The target is usually DNA, but even RNA can be used as a template by synthesizing complementary DNA (cDNA) by reverse transcription.

  In the isothermal nucleic acid amplification method, RNA is targeted by synthesizing complementary DNA (cDNA) by reverse transcriptase, and further by synthesizing double-stranded DNA by the DNA polymerase activity of reverse transcriptase. The TRC method (Nakaguchi, Y. et al., J. Clin. Microbiol., 42 (9): 4284-4292, 2004) in which only a specific region of RNA is increased by RNA polymerase is exemplified. In the TRC method, the amplification product is RNA.

Examples of reagents necessary for the isothermal nucleic acid amplification reaction include a strand displacement type nucleic acid elongation enzyme, dNTP (dATP, dCTP, dGTP, dTTP), and primers. As the strand displacement type nucleic acid elongation enzyme, Bst polymerase (Geobacillus stearothermophilus-derived DNA polymerase or a large fragment thereof, distributor: Wako Pure Chemical Industries, Ltd., manufacturer: Nippon Gene, code No .: 311-07481), And Csa polymerase (for example, distributor: Wako Pure Chemical Industries, Ltd., manufacturer: Nippon Gene, code No .: 319-07281).

One of the strand displacement type nucleic acid elongation enzymes may be used alone, or two or more thereof may be used in combination. The concentration of the strand displacement type nucleic acid elongation enzyme in the reaction solution is preferably 0.2 to 0.6 U / μl, more preferably 0.35.
~ 0.45 U / μl. The concentration of each dNTP is preferably 1 mM to 1.5 mM, more preferably 1.1 to 1.4 mM. The primer will be described later. Strand displacement type nucleic acid elongation enzymes are commercially available and can be used.

  The reaction solution for performing the isothermal nucleic acid amplification method preferably contains a buffer, a salt, a surfactant, and betaine.

Examples of the buffer include Tris-HCl, K ₂ HPO _{4 and the} like. The pH of the reaction solution is usually 7 to 9, preferably 7 to 8.9, more preferably 7.6 to 8.8. When Tris-HCl is used as a buffer, the concentration is preferably 10 mM to 25 mM, more preferably 16 to 22 mM.

Examples of the salt include KCl, MgSO ₄ , MgCl ₂ , (NH ₄ ) ₂ SO ₄ , and the reaction solution preferably contains both KCl and MgSO ₄ . The concentration of KCl is preferably 5 mM to 15 m, and more preferably 8 to 12 mM. The concentration of MgSO ₄ is preferably 10 mM to 40 mM, more preferably 15 mM to 25 mM. The concentration of MgCl ₂ is preferably 0.1 mM to ₁ mM, more preferably 0.3 mM to 0.8 mM. The concentration of (NH ₄ ) ₂ SO ₄ is preferably 10 mM to 30 mM, more preferably 15 mM to 25 mM.

Surfactants include nonionic surfactants such as Triton (registered trademark of Union Carbide), Nonidet (shell), Tween (registered trademark of ICI), Brij (registered trademark of ICI), SDS ( Anionic surfactants such as sodium dodecyl sulfate) and cationic surfactants such as stearyldimethylbenzylammonium chloride.
Triton X-100 (polyethylene glycol tert-octylphenyl ether) as Triton, Nonidet P-40 (octylphenyl-polyethylene glycol) etc. as Nonidet, Tween 20 (polyethylene glycol sorbitan monolaurate) as Tween, Tween 40 (polyethylene glycol sorbitan monopalmitate), Tween 60 (polyethylene glycol sorbitan monostearate), Tween 80 (polyethylene glycol sorbitan monooleate), etc. Brij is Brij 56 (polyoxyethylene (10) cetyl ether) , Brij58 (polyoxyethylene (20) cetyl ether) and the like. When Tween 20 (polyethylene glycol sorbitan monolaurate) is used, the concentration is preferably 0.1% to 0.4%, more preferably 0.15% to 0.25%. Other surfactants may be set at a concentration that does not inhibit the activity of the strand displacement type nucleic acid elongation enzyme according to the above concentration.

The type and concentration of the surfactant in the nucleic acid amplification reaction solution are not particularly limited as long as the reaction of the strand displacement type nucleic acid elongation enzyme is not inhibited. For example, when an anionic surfactant is used, a range of 0.0005 to 0.01% is preferable, and when a cationic surfactant is used, a range of 0.0005 to 0.01% is preferable.
Specifically, the concentration in the case of SDS is usually 0.0005 to 0.01%, preferably 0.0.
It is 01 to 0.01%, more preferably 0.001 to 0.005%, and more preferably 0.001 to 0.002%.

In the case of other surfactants, for example, the concentration when a nonionic surfactant is used is preferably in the range of 0.001 to 1.5%.
Specifically, the concentration in the case of Nonidet P-40 is usually in the range of 0.001 to 1.5%, preferably 0.002 to 1.2%, and 0.9 to 1.1. % Is more preferable.
The concentration in the case of Tween 20, Tween 40, Tween 60, or Tween 80 is preferably 0.1 to 0.4%, more preferably 0.15 to 0.25%.
The concentration in the case of Brij56 or Brij58 is usually in the range of 0.1 to 1.5%, preferably 0.4 to 1.2%, more preferably 0.7 to 1.1%.
When the enzyme solution used for the nucleic acid amplification reaction contains a surfactant, only the surfactant derived from the enzyme solution may be used, or the same or different surfactant may be added.

  Examples of betaine include trimethylglycine and carnitine. When using trimethylglycine, the concentration is usually 0.5M to 1M, preferably 0.6M to 0.8M.

A typical reaction solution composition is as follows.
Tris-HCl (pH7 ~ 9) 10mM ~ 25mM
KCl 5mM ~ 15mM
MgSO ₄ 5mM~40mM
Surfactant 0.1% -0.4%
Betaine 0.5M-1M
dNTPs 1mM to 1.5mM each
Strand displacement type nucleic acid elongation enzyme 0.2-0.6 U / μl
In addition, this reaction liquid composition does not prevent that other arbitrary components are included unless a nucleic acid amplification reaction is inhibited.

In the present invention, the “target region” is a region that can be amplified by isothermal nucleic acid amplification method among chromosomal DNA or RNA, and is not particularly limited as long as it can detect a microorganism to be detected. It can be set appropriately depending on the situation. For example, when the test sample includes cells of a different type from the microorganism to be detected, the target region preferably has a sequence specific to the microorganism to be detected. Further, depending on the purpose, it may have a sequence common to a plurality of types of microorganisms.
Furthermore, the target area may be single or plural. Using a primer set corresponding to the target region specific to the detection target microorganism and a primer set corresponding to the nucleic acid of a wide range of microorganisms, the amount of living cells of the detection target microorganism and the number of living cells of many types of microorganisms can be calculated. Can be measured simultaneously. The length of the target region usually includes 50 to 5000 bases.

  Primers used for nucleic acid amplification can be appropriately set based on the principles of various nucleic acid amplification methods, and are not particularly limited as long as they can specifically amplify the target region.

Examples of preferred 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, and a region spanning two or more genes may be targeted. Isothermal nucleic acid amplification method kits corresponding to target regions specific to microbial species are commercially available, and primers contained therein may be used.

  When primers common to a plurality of types of microorganisms are used, viable cells of a plurality of types of microorganisms in a test sample can be detected. In addition, when a primer specific to a specific bacterium is used, a living cell of a specific bacterial species in a test sample can be detected. For example, when the primers described in Example 4 below (F3, B3, FIP, BIP, LoopF, LoopB in Tables 15 to 17) are used, living cells of Gram-positive bacteria and Gram-negative bacteria can be detected simultaneously. it can. Specifically, a set of primers having the sequences of SEQ ID NOs: 1, 2, 3, 4, 9, and 10, a set of primers having the sequences of SEQ ID NOs: 10, 11, 12, 13, 14, and 17, and Examples include a set of primers having the sequences of SEQ ID NOs: 18, 19, 20, and 21.

  In the case of an influenza virus having an envelope, hemagglutinin (H protein) gene, neuraminidase (N protein) gene, RNA polymerase gene of caliciviridae virus represented by norovirus, gene regions encoding various capsid proteins, etc. Can be mentioned. In addition to noroviruses, rotaviruses and adenoviruses are available as food poisoning viruses. The target genes are gene regions encoding RNA polymerase genes and capsid proteins as in the case of noroviruses.

  As described above, when the detection target is RNA, it can be used as a template by forming a double strand from RNA by reverse transcription and DNA polymerase reaction. In that case, before step c), reverse transcriptase, a primer and, if necessary, a nucleic acid elongation enzyme are added to the test sample to generate double-stranded DNA from RNA.

  The concentration of the primer is not particularly limited, but is preferably 0.01 μM to 3 μM, more preferably 0.02 μM to 1.8 μM.

If calcein is added to the nucleic acid amplification reaction solution, real-time nucleic acid amplification can be detected. In that case, the reaction solution preferably does not contain a chelating agent. In the preferred form of the direct PCR method, an organic acid salt is contained in the reaction solution. However, since organic acid works as a chelating agent, when calcein is used in the LAMP method, manganese is deprived from calcein that has been quenched by binding to manganese. As a result, it emits fluorescence regardless of the presence or absence of amplification.

  In the present invention, the conditions for the isothermal nucleic acid amplification reaction are not particularly limited, and the normal conditions for the isothermal nucleic acid amplification reaction can be employed.

(4) Step d)
Analyze amplification products amplified by isothermal nucleic acid amplification. Analysis of the amplification product is performed following step c) or simultaneously with step c). For example, when detecting amplification reaction in real time, step d) can be performed simultaneously with step c).

The analysis method is not particularly limited as long as the amplification product can be detected or quantified, and examples thereof include electrophoresis. The presence or absence of an amplification product can also be determined by analyzing the melting temperature (TM) pattern of the amplification product. In addition, the amplification product can be indirectly analyzed by the progress of the amplification reaction. For example, since the amplification efficiency is high in the LAMP method, the amplification reaction can be detected by white turbidity due to magnesium pyrophosphate generated in association with the amplification reaction. This cloudiness can be measured with a turbidimeter, but can also be observed visually. Further, as described above, the amplification reaction can be detected using calcein.
The optimum excitation wavelength for calcein to emit fluorescence is 495 nm, and the maximum fluorescence wavelength is 515 nm. Usually, ultraviolet rays of 240 to 260 nm and 350 to 370 nm are used as excitation light, but visible light around 488 nm can also be used as excitation light.
The concentration of calcein is not particularly limited, but is usually 2 to 5%, preferably 3 to 4%.

  The above detection method can also be used for optimization of various conditions in the method of the present invention.

  When detecting live cells by the method of the present invention, the analysis of the amplification product is performed by using a standard curve indicating the relationship between the amount of microorganisms prepared using the standard sample of the identified microorganism and the amplification product. Presence or absence or the accuracy of quantification can be increased. A standard curve prepared in advance can be used, but it is preferable to use a standard curve prepared by performing each step of the present invention on the standard sample simultaneously with the test sample. If the correlation between the amount of microorganism and the amount of DNA or RNA is examined in advance, DNA or RNA isolated from the microorganism can also be used as a standard sample.

<2> Kit of the Present Invention The kit of the present invention detects the living cells of microorganisms in a test sample by distinguishing them from dead cells or damaged cells by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme. A kit for performing the following steps.
1) a drug that selectively inhibits dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
2) a reagent for preparing a reaction solution having the following composition;
Tris-HCl (pH 7 ~ 9) 10mM ~ 25mM
KCl 5mM ~ 15mM
MgSO ₄ 5mM~40mM
Surfactant 0.1% -0.4%
Betaine 0.5M-1M
dNTPs 1mM to 1.5mM each
Strand displacement type nucleic acid elongation enzyme 0.2-0.6 U / μl
3) A primer for amplifying the target region of the nucleic acid of the microorganism to be detected by an isothermal nucleic acid amplification method.

  Preferred surfactants, betaines, and strand displacement nucleic acid extenders are as described above.

  Each reagent is not particularly limited as long as it can prepare a reaction solution having the above composition. For example, each reagent is contained in a separate container as a concentrated solution, and is appropriately mixed and diluted so as to have the above concentration at the time of use. Alternatively, two or more reagents may be mixed and accommodated in the same container. It is preferable that the strand displacement type nucleic acid extending enzyme is accommodated in a container separate from other reagents.

  The kit of the present invention may contain calcein.

  In addition, an enzyme having an activity of degrading cells other than microorganisms, protein colloid particles, fat, or carbohydrates present in a test sample can be added to the kit of the present invention.

The enzyme should be capable of degrading cells other than microorganisms, protein colloid particles, fats and carbohydrates, etc. present in the test sample and not damaging the living cells of the microorganism to be detected. Examples thereof include, but are not limited to, lipolytic enzymes, proteolytic enzymes, and carbohydrases. As the enzyme, one kind of enzyme may be used alone, or two or more kinds of enzymes may be used in combination, but both lipolytic enzyme and proteolytic enzyme, or lipolytic enzyme, proteolytic enzyme It is preferable to use all of saccharide-degrading enzymes.
Examples of the lipolytic enzyme include lipase and phosphatase, examples of the proteolytic enzyme include serine protease, cysteine protease, proteinase K, and pronase, and examples of the carbohydrase include amylase and cellulase.

  The kit of the present invention may further contain a diluent, an enzyme for isothermal nucleic acid amplification, instructions describing the method of the present invention, and the like.

<3> Life / death judgment of microorganisms that are easily killed Microorganisms that are easily killed when suspended in physiological saline or sterilized water, microorganisms that do not have catalase, etc., have a life / death judgment agent that penetrates into living cells. It may be difficult to make a life / death determination. Even for such microorganisms, treatment with a life-and-death determination agent is performed using D-MEM (Dullbecco's Modified Eagle Media), RPMI 1640, Ham's F-12, D-MEM / F-12 (1: 1), Eagle MEM (Eagle's Minimum Essential Media), Alpha MEM (Alpha modification of Eagle's MEM), Eagle Basal Medium Eagle, McCoy's 5A Modified Medium, M-199 medium, Iscove's Modified DMEM, etc. By performing in a cell suspension containing at least one nutritional component excluding salts, antibiotics, pigments, etc. from the basal medium, it is possible to make life / death determination easier. As such a nutrient component, any one or more selected from the group consisting of proteins, saccharides and lipids are preferable, and yeast extract is particularly preferable.
The concentration of the nutrient component is preferably 0.5% to 10%, more preferably 1% to 5%. This density | concentration is a density | concentration in a total amount, when using multiple types of nutrient components.

That is, the present invention is a method for detecting a living cell of a microorganism in a test sample by distinguishing it from a dead cell or a damaged cell, which comprises the following steps:
a) a step of adding, to the test sample, an agent that selectively inhibits the dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
c) a step of amplifying a target region of a nucleic acid of a microorganism in a test sample by a nucleic acid amplification method, and d) a step of analyzing an amplification product,
A method is provided wherein step a) is performed in a cell suspension containing 1% to 5% yeast extract.

  The above microorganisms include Legionella genus bacteria that are likely to be killed in physiological saline, Campylobacter bacteria such as Campylobacter jejuni and Campylobacter coli, which are microaerobic bacteria, and natural saline because the salt concentration is too low. Vibrio bacteria such as Vibrio parahaemolyticus that are likely to die, psychrophilic bacteria (and Listeria spp.) That are easily damaged at laboratory temperatures, obligate anaerobes (Clostridial bacteria such as Clostridium botulinum and Clostridium perfringens, Helicobacter Helicobacter bacteria such as Helicobacter pylori), bacteria that are difficult to cultivate artificially (obligate intracellular parasitic bacteria such as Mycobacterium, Chlamydiaceae, Rickettsia, etc.) And Neiseria bacteria.

  In addition, microorganisms such as those mentioned above include obligate intracellular parasitic microorganisms that can only grow in cells of another organism and cannot grow on their own, such as viruses having only nucleocapsids (for example, Norovirus) and viruses with an envelope (influenza virus) and the like in general. Specifically, Poxviridae, Herpesviridae, Adenoviridae, Papillomaviridae / Polyomaviridae, Parvoviridae, Picornaviridae, Caliciviridae / Astroviridae / Coronaviridae, Togaviridae -Viruses such as Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae / Firoviridae / Bornaviviridae, Arenaviridae / Bunijaviridae, Reoviridae, Retroviridae, Hepatitis Virus, etc. .

  Examples of the life / death determining agent in the above-described method include the aforementioned agent that is covalently bonded to a nucleic acid upon irradiation with light having a wavelength of 350 nm to 700 nm, and a platinum group element complex. In the case where a drug that is covalently bonded to a nucleic acid by irradiation with light having a wavelength of 350 nm to 700 nm is used as a life / death determination agent, a light irradiation treatment with a wavelength of 350 nm to 700 nm is performed on a test sample to which the drug is added.

  The nucleic acid amplification reaction may be a method of repeating a cycle consisting of denaturation, annealing, and extension reaction, such as PCR, or may be an isothermal nucleic acid amplification method. The analysis of the amplification product can be appropriately selected according to the nucleic acid amplification method.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Example 1] Examination of master mix for direct LAMP method (1)
The composition of the reaction solution (master mix for direct LAMP method) for performing nucleic acid amplification by LAMP method without extracting nucleic acid from cells was examined.

1-1) Test method Legionella pneumophila ATCC33153 strain was cultured in BCYEα medium at 37 ° C for 2 days, colonies were picked, suspended in physiological saline, and 2.1 ± 0.32 log cfu / ml A live cell suspension was prepared. The ATCC strain can be obtained from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America).

Centrifuge the live cell suspension above (4 ° C, 10 minutes, 3,000 x G), remove the supernatant completely, and add 1 ml
After washing with sterile water, the supernatant was completely removed by the same cooling centrifugation treatment. The precipitate (pellet) was suspended by adding 30 μl of sterile water, and the suspension was quantitatively transferred to a new PCR tube.
The PCR tube was treated at 96 ° C. for 3 minutes and then rapidly cooled to 4 ° C. and an unheated sample were prepared.

Further, as a master mix used for gene amplification, a LAMP master mix 1 having the composition shown in Table 1 and a LAMP master mix 2 shown in Table 2 were prepared.
Master mix 1 for the LAMP method is obtained by adding a fluorescent dye calcein (Calcein, using Eiken Fluorescent Detection Reagent) to the basic composition of Eiken Chemical Loopamp Legionella detection reagent kit E.
The master mix 2 for LAMP method is the same as the master mix 1 for LAMP method, but it is the concentration of a mixture of drugs that suppress the action of nucleic acid amplification inhibitors necessary for efficient PCR without extracting nucleic acids from cells. (Table 3. This concentrated solution is described as a concentrated direct buffer component, “cDBC”. See International Publication No. 2011/010740).
The “RM leg” (reaction mixture for Legionella) in Tables 1 and 2 includes primers (FIP, BIP, Loop-F, Loop-B, F3, and B3) for amplifying Legionella 16S rRNA gene. ing.

cDBC is bovine serum albumin (BSA; Sigma A7906), trisodium citrate dihydrate (TSC: Tri-Sodium Citrate Dihydrate; Kanto Chemical, Tokyo), magnesium chloride hexahydrate (31404-15 Nacalai Tesque, Kyoto) ), Egg white lysozyme (126-02671 Lysozyme from egg white;
Each stock solution of Wako Pure Chemical (Osaka) and Brij58 (P5884-100G; Sigma) was mixed to the concentration shown in Table 3.
According to the basic composition manufacturer manual of Eiken Chemical Legionella Detection Reagent Reagent Kit E, Master Mix 2 is presumed to contain MgSO ₄ equivalent to 8 mM as the final concentration, so the total Mg ²⁺ is It was estimated to be equivalent to 11 mM. Brij 58, MgCl ₂ , and TSC were dissolved in sterilized water, autoclaved (121 ° C., 20 minutes), cooled to room temperature, and used as a stock solution. For BSA and Lysozyme, a stock solution was prepared with sterilized water, and sterilized by filtration through a 0.22 μm filter to obtain a stock solution.

The heated and unheated Legionella live cell suspension obtained above was cooled and centrifuged (4 ° C., 10 minutes, 3,000 × G), and the supernatant was almost removed. Each master mix shown in Table 2 was added, and LAMP amplification (65 ° C., 100 minutes; 80 ° C., 2 minutes; 4 ° C., 2 minutes) (twice) was performed. In LAMP amplification, one minute in the gene amplification step at 65 ° C. was defined as one cycle. The degree of target gene amplification was grasped as the total amount of fluorescence of calcein, the fluorescence boundary value was set to 20,000, and the gene amplification time (minute or number of cycles) exceeding the first was set as the Ct value.

1-2) Test results and discussion Table 4 shows the test results.

As shown in Table 4, amplification of the target gene was detected only when heat treatment (96 ° C., 3 minutes) was performed before nucleic acid amplification by the LAMP method and Master Mix 1 was used.
The reason why amplification was not detected in Master Mix 2 may be that components effective for PCR such as BSA and lysozyme contained in cDBC inhibited the strand displacement polymerase reaction. In addition, TSC (trisodium citrate dihydrate) in cDBC is a metal chelator, and when it reacts with manganese that has quenched calcein, no increase in fluorescence associated with the amplification reaction was observed. Probability is high.

Even when Master Mix 1 was used, amplification was not observed unless the cell suspension was previously heat-treated. In PCR, a long denaturation treatment (heat treatment) is usually performed before the reaction cycle.
Is done. In direct PCR, it is presumed that the nucleic acid amplification reaction mainly occurs in cells. Specifically, the cell morphology is maintained by high-temperature treatment of the cell in the nucleic acid amplification reaction, and chromosomal DNA is left in the cell, but pinholes or voids are formed in the cell membrane or cell wall of the microorganism, the primer and It is considered that an enzyme necessary for nucleic acid amplification flows into the cell and an amplification reaction occurs in the cell. It is estimated that a part of the amplified product remains in the cell or flows out of the cell depending on the gene length of the amplified product (International Publication No. 2011/010740). In the direct LAMP method, the amplification reaction was observed only when the cell suspension was previously heat-treated, suggesting that the above estimation was correct.

[Example 2] Study of master mix composition for direct LAMP method (2)
2-1) Test method Legionella pneumophila ATCC33153 strain was cultured in BCYEα medium at 37 ° C for 2 days, colonies were picked, suspended in physiological saline, and 2.5 ± 0.21 log cfu / ml A live cell suspension was prepared.

Next, 1 ml of the above live cell suspension is used as a specimen, and a multi-stage EMA treatment is performed 3 times in a final concentration of 5 μg / ml EMA (the first EMA treatment: 5 μg / ml for 5 minutes on ice, protected from light) The third EMA treatment: 5 μg / ml for 5 minutes, the third EMA treatment: 5 μg / ml for 5 minutes). Visible light was irradiated for 5 minutes after each EMA treatment. In addition, the cleaning between each EMA treatment was not performed (the same applies to other examples unless otherwise specified). An untreated group was also prepared for each specimen.

Cool and centrifuge the multi-stage EMA treatment group and untreated group (4 ° C, 10 minutes, 3,000 x G), completely remove the supernatant, wash with 1 ml of sterilized water, and then perform the same cooling centrifuge treatment The supernatant was completely removed, 30 μl of sterile water was added to the pellet to suspend it, and the suspension was quantitatively transferred to a new PCR tube. The PCR tube was treated at 96 ° C. for 3 minutes and then rapidly cooled to 4 ° C. so that components such as enzymes in the master mix for direct LAMP could permeate bacterial cells effectively.

Next, cool and centrifuge (4 ° C, 10 minutes, 3,000 x G) to remove the supernatant. Add 11-17 µl of direct LAMP master mix to the pellet (equivalent to 2.5 µl) and add 65 ° C. 100 minutes; 80 ° C., 2 minutes; LAMP amplification for 2 minutes at 4 ° C. The master mix for direct LAMP is added to the basic composition of Eiken Chemical Loopamp Legionella Detection Reagent Kit E with a fluorescent dye calcein (Calcein, using Eiken Fluorescent Detection Reagent) for detection of amplified genes, and isothermal DNA extension. The enzyme Csa polymerase (Nippon Gene) is added. In the gene amplification process at 65 ° C., a 1 minute reaction was set as one cycle, and the degree of target gene amplification by the LAMP method was grasped as the total fluorescence of calcein. At that time, the fluorescence boundary value was set to 20,000, and the gene amplification time (minute (min) or cycle number) exceeding the first was defined as the Ct value. Details are shown below.

First, the basic master mix composition used as a control group (control) is shown in Table 5. This basic master mix is the basic composition of Eiken Chemical Legionella Detection Reagent Kit E with calcein added according to the package insert of this kit. In the reaction system, a DNA solution equivalent to 2.5 μl was added as a template.

Next, add 1 μl of Csa polymerase (8 U / μl) to the basic master mix for control.
And a master mix to which 1 μl of the additives shown in Table 6 was added was prepared. In Table 6, “+”
Indicates that Csa polymerase or an additive was added. Additives include (NH ₄ ) ₂ SO ₄ (Kanto Chemical, Tokyo), Tween20 (Bio-Rad, Richmond, CA, USA), KCl (Kanto Chemical, Tokyo), betaine (trimethylglycine, Acros, New Jersey, USA) and dNTPs (Takara Bio, Shiga) were used, and except for dNTPs, they were dissolved in milli-Q water at the concentrations shown in Table 6 and then autoclaved (121 ° C., 15 minutes).

2-2) Test results and discussion Table 7 shows the test results.

As shown in Table 7, with respect to the untreated group of Legionella live cells, except for Compositions 2 and 6, there was no significant difference from the Ct value of Composition 1, which is the basic master mix. On the other hand, the Ct value for living cells obtained by EMA treatment of living Legionella cells in physiological saline is the highest, with the Ct value of composition 1 being the highest at 35.3, and the most prominent target due to EMA that may have partially penetrated living cells Gene amplification suppression was observed.
As a suitable master mix performance, a master mix composition that exhibits a low Ct value of living cells compared to composition 1 is the best regardless of whether it is untreated or EMA treated. From the above, it is considered that composition 4 is appropriate in the present example for the direct LAMP method.

[Example 3] Examination of direct LAMP method master mix composition (2)
As a master mix composition for the direct LAMP method in Example 2, a composition in which isothermal DNA elongation enzyme Csa polymerase, (NH ₄ ) ₂ SO ₄ , and Tween 20 are reinforced is suitable for the conventional typical basic master mix. I understood. In this example, suitable concentrations of each additive were examined.

3-1) Test method
A colony obtained by culturing Legionella pneumophila ATCC33153 strain in BCYEα medium at 37 ° C. for 2 days was picked and suspended in physiological saline to prepare a 2.7 ± 0.18 log cfu / ml live cell suspension. This live cell suspension was subjected to multistage EMA treatment and LAMP amplification in the same manner as in Example 2, and the Ct value was measured. However, a master mix (14 μl) having the composition shown in Table 8 was used for the direct LAMP master mix. The basic master mix is shown in Table 5. In Table 8, “+” indicates that Csa polymerase or an additive was added.

The target gene amplification time (65 ° C, 100 minutes) greatly exceeds the recommended time of 60 minutes for the kit, but this indicates that the direct LAMP method master mix includes enzymes and reagents other than the kit. This is because the possibility of non-specific amplification reaction due to chemical contamination of DNA and the like was also considered (the same applies to Example 2).

3-2) Test results and discussion Table 9 shows the test results.

According to Table 9, composition 6 was judged to have a lower Ct value than composition 1 regardless of the EMA untreated group or the treated group. However, as a result of LAMP amplification (75 to 100 minutes) of the negative control with composition 6, gene amplification presumed to be a non-specific reaction was observed, so the amount of Csa polymerase (8 U / μl) added in composition 6 1 μl (8U) is reduced to 0.4 μl (3.2U), and 0.6 μl of sterilized water is added instead. (NH ₄ ) ₂ SO ₄ and Tween 20 are added in the same composition as in composition 6. Master mix. The composition is shown in Table 10.

  Table 11 shows the above composition converted to concentration.

[Example 4] Detection of Gram-negative bacteria and Gram-positive bacteria by direct LAMP method using platinum complex As shown in Table 7 of Example 2 and Table 9 of Example 3, the master mix composition is optimized. By doing so, it was found that live cells of Legionella can be detected in real time by the direct LAMP method.
In this example, a direct LAMP method using tetrakis (triphenylphosphine) platinum (II) (etrakis (triphenylphosphine) platinum (II)) as a reagent capable of distinguishing living cells of microorganisms from dead cells or damaged cells. Based on the above, live / dead cells of Gram-negative bacteria and Gram-positive bacteria other than Legionella were determined.

4-1) Test method Citrobacter freundii NBRC12681
E. coli JM109, Klebsiella pneumoniae NBRC3321, Cronobacter sakazakii (former name: Enterobacter sakazakii) ATCC51329, Enterobacter
Enterobacter cloacae IFO13535, E. coli O157 VT1 IS001, and E. coli DH5α were cultured in BHI broth at 37 ° C. for 18 hours, and the cells were washed with sterilized water, suspended in sterilized water, A live cell suspension was obtained.

Gram-negative bacterium Vibrio vulnificus L-1 opacity is cultured in tryptosoy broth with 3% salt solution for 18 hours, and then the cells are washed with physiological saline, suspended in sterile water, and living cells A suspension was obtained. Although this strain is likely to die in sterilized water, the platinum complex becomes difficult to coordinate with nucleic acids of dead cells in a physiological saline environment, and therefore, sterilized water was used and exposure to the platinum complex was promptly performed. A part of this live cell suspension was immersed in boiling water for 3 minutes to prepare a dead cell suspension. The dead cell suspension contains damaged cells and dead cells, and these are collectively referred to as “dead cells” hereinafter.

Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA), Micrococcus luteus ATCC9341, and Bacillus cereus JCM2152 are cultured in BHI broth at 37 ° C for 2 days. Was washed with sterile water and suspended in sterile water. Bacillus cereus mainly used vegetative cells for testing. A part of these live cell suspensions was immersed in boiling water for 3 minutes to prepare a dead cell suspension.
For the gram-negative bacteria, 90 μl of sterilized water was added to 10 μl of the live cell suspension to prepare a live cell suspension of about 8 log cfu / ml. In the same manner, a suspension of Gram-negative bacteria and dead cells in the order of 8 log cells / ml was prepared. 90 μl of these live cell suspensions and dead cell suspensions were subjected to the platinum complex exposure described below.
For Gram-positive bacteria, 90 μl (approximately 8 log cfu / ml order) of a live cell suspension or a dead cell suspension was directly exposed to the following platinum complex.

Tetrakis (triphenylphosphine) platinum (II) (Sigma) 7.21 mg (5.11 μmol) was accurately weighed and dissolved in 1275.9 μl of DMSO to prepare a 4 mM platinum complex solution. Thereafter, it was diluted 40-fold with physiological saline to prepare a 100 μM platinum complex aqueous solution.

Add 10 μl of 100 μM platinum complex aqueous solution to 90 μl of the above-mentioned gram-negative and positive bacteria live cell suspension (8 log cfu / ml order level) or dead cell suspension (8 log cells / ml order level). The platinum complex action concentration was adjusted to 10 μM, which was similar to that of EMA in the above Example, and maintained at 37 ° C. for 15 minutes in a constant temperature water bath (PERSONAL-11, TAITEC, Tokyo, Japan). Thereafter, the supernatant is removed by cooling centrifugation (4 ° C., 10,000 × G, 5 minutes), the pellet is suspended in 150 μl of sterilized water, stirred well, and then the same cooling centrifugation is performed. The pellet was suspended by adding 30 μl of sterilized water, and the suspension was quantitatively transferred to a new LAMP amplification reaction tube (quantitative PCR tube).

These tubes are treated at 96 ° C for 3 minutes (96 ° C, 10 minutes for detecting Gram-positive bacteria), then rapidly cooled to 4 ° C, and then the components such as enzymes in the master mix for direct LAMP. Effectively penetrated the bacterial cells.
Next, cool and centrifuge (4 ° C, 10 minutes, 3,000 x G) to remove the supernatant. Add 10.5 µl of the master mix for direct LAMP to the pellet (equivalent to 2.5 µl), and add 63 ° C, 90 ° C. Minutes (
Only when GN_GP_ID_4 was used as a primer, a non-specific reaction occurred in 90 minutes, so it was set to 50 minutes); 80 ° C., 2 minutes; 4 ° C., 2 minutes of LAMP amplification. In addition, the composition of the master mix for direct LAMP of LAMP method is shown in Tables 12-14. In Tables 12 to 14, 2 × RM attached to the Loopamp DNA amplification reagent kit corresponds to the RM leg in Table 10, but this “2 × RM” does not include the LAMP primer for detecting Legionella. However, according to these compositions, there is no significant difference from the composition of Table 10 except for the primer.

Primers for LAMP were designed as follows. 16S rRNA gene information on the following microorganisms was obtained from the GenBank database (http://www.ebi.ac.uk/genbank/). The accession number and sequence length are shown in parentheses.
Bacillus cereus Se07 (JN700112; 1,438_bp)
Citrobacter koseri NBRC 105690 (AB682264; 1,467_bp)
Citrobacter freundii 5N09 (JQ271810; 1,430_bp)
Enterococcus faecium JCM8905 (AB690254; 1,449_bp) Klebsiella pneumoniae (X80684; 1,459_bp)
Listeria monocytogenes isolate 44 (AJ535697; 1,404_bp),
Mycobacterium avium subsp. Paratuberculosis ATCC19698 (EF521896; 1,442_bp)
Salmonella enteritidis strain E1 (EU118100; 1,546_bp)
Lactobacillus acidophilus ATCC4356 (AB008203; 1,553_bp)
Staphylococcus aureus ATCC12600 (X68417; 1,555_bp)

  All the gene information is arranged in a row, each gene region is analyzed by ClustalW, the bases that match in all the genera, and the bases that did not maintain even the perfect match, are identified, 16S of each microorganism The conserved region and variant region in the rRNA gene region were analyzed.

The specific method is shown below, using the 16S rRNA gene base sequence of Staphylococcus aureus ATCC12600 as a representative example. Manually register the 16S rRNA gene base sequence of Staphylococcus aureus ATCC12600, and the regions and variant regions stored for all the genera in the analysis software (PrimerExplorer Ver.3, Eiken Chemical; Fujitsu) did. The following restrictions were placed on the F2 and F1c parts constituting the FIP primer described later, the B2 and B1c parts constituting the BIP primer, and the F3 and B3 primers. As for the F2, F3, B2, and B3 primers, the 5 ′ end of the oligonucleotide and the inner (intermediate) part may not be kept complementary to the template DNA. The terminal side was set so as to be completely complementary to the template DNA. In addition, regarding the F1c part and B1c part, the 3 ′ end of the oligonucleotide does not have to be kept complementary to the template DNA, and the 5 ′ end is set to be completely complementary to the template DNA. .

In addition, the distance between F2 and B2 (the number of bases sandwiched between FIP and BIP) is 120 to 300_bp, the distance between F1c and F2 is 40 to 60_bp, the distance between F2 and F3 is 0 to 450_bp, and the distance between F1c and B1c is The setting was changed within the range that is scientifically and theoretically acceptable from 0 to 260_bp. The other parameters have default settings.

Loop primers (LoopF and LoopB primers described later) were prepared according to the primer primer creation manual of PrimerExplorer Ver.3 after the FIP, F3, BIP, and B3 primers were determined. According to these settings, two types of LAMP amplified fragments (minimum unit: a base of Flc portion added to one end of the fragment and a base of Blc portion added to the other end) are obtained from all the microorganisms. It was theoretically possible.
This minimum unit means the minimum size of a LAMP amplified fragment having a dumbbell structure at both ends by FIP and BIP described later, specifically, the 5 ′ end of the S. aureus 16S rRNA gene registered in the above Genbank. When the smallest unit containing the base sequence corresponding to the 690th to 889th base sequence from the first and the smallest unit containing the 1050th to 1203rd base sequence from the 5 ′ end of the S. aureus 16S rRNA gene are mainly produced Conceivable.

In the case of the former minimum unit, it is inferred that the nucleotide sequence corresponding to positions 709 to 870 from the 5 ′ end of the S. aureus 16S rRNA gene is necessarily included.
It is presumed that the base sequence corresponding to the 1068th to 1185th positions from the 5 ′ end of the aureus 16S rRNA gene is necessarily included. LAMP amplified fragments 2 ⁿ times longer than those smallest units are
Obtained as the process progresses.
That is, in order to detect all the microorganisms (especially in the case of bacteria) all at once by the LAMP method, it was found that only the above-mentioned two types of minimum units could be obtained. Decided to consider what would go on.

  Tables 15 to 17 show the base sequences (detailed information) of the primers of the LAMP primer set (GN_GM_ID_3, GN_GM_ID_4, GN_GM_ID_9) for simultaneous detection of Gram negative bacteria and Gram positive bacteria, respectively.

The items described in Tables 15 to 17 are as follows.
a) For each primer dimer of four types FIP, BIP, F3, and B3, when the amount of change in the gypsum free energy (dG) of the primer dimer most likely to be generated is less than -4 The possibility of dimer generation increases and the possibility of inhibiting LAMP amplification increases. In this system, since dG = -2.27, which is higher than -4, the generation of primer dimer is negligible.
b) FIP means a primer linked by the sequence 5'-F1c-F2-3 '. BIP is 5'-B1c-B2-3
It means a primer linked by 'sequence. Refer to Sequence in j) for FIP and BIP sequences. LoopF and LoopB are primers that assist FIP and BIP to anneal to self-extending oligonucleotides, respectively, and to form a loop, and are also defined as loop primers. c) A position based on the 5 ′ end of the 16S rRNA gene base sequence (1555_bp) of the accession number S. aureus ATCC12600 (X68417) of the GenBank database (http://www.ebi.ac.uk/genbank/). The position of the 5 ′ end of each primer that anneals in the 16S rRNA gene is shown.
d) A position based on the 5 'end of the 16S rRNA gene base sequence of the accession number S. aureus ATCC12600 (X68417) of the GenBank database (http://www.ebi.ac.uk/genbank/). The position of the 3 ′ end of each primer that anneals (adheres) in the 16S rRNA gene is shown.
e) The length of each primer.
f) Tm (melting temperature) of each primer.
g) Indicates the degree of annealing of the 5 ′ end of each primer to the template DNA, and indicates the amount of change in the free energy of the cast during the annealing reaction. If the value is less than -4, a good annealing reaction occurs.
h) Indicates the degree of annealing of the 3 ′ end of each primer to the template DNA, and indicates the amount of change in the free energy of the cast during the annealing reaction. If the value is less than -4, a good annealing reaction occurs.
i) GC content of each primer.
j) Base sequence information of each primer. Bold italics are primer regions that do not necessarily maintain complementarity when the primer anneals to the 16S rRNA gene region in various gram-negative and gram-positive bacteria to detect total bacteria This is a region where the base sequence of the primer is varied. The region other than the bold italic is the region where the complementarity of the primer is completely maintained for all total bacteria.

In addition, although Gram-positive bacteria do not have an outer membrane, there is a peptidoglycan layer that is significantly thicker than Gram-negative bacteria, so when performing the direct LAMP method, direct enzymes such as Bst polymerase and Csa polymerase are used. It was unclear whether each reagent of the LAMP master mix penetrated into microbial cells. Especially in the case of Gram-positive bacteria, when the final direct LAMP method was negative, it was unclear whether the penetration of each reagent of the LAMP method into living cells was insufficient or the primer design was inappropriate Therefore, LAMP amplification using DNA extracted from bacteria as a template was also performed.
Specifically, from 1 ml of each Gram-negative bacteria and Gram-positive bacteria culture solution, DNA was prepared according to the operation manual using NucleoSpin Tissue XS (manufactured by MACHEREY-NAGEL GmbH & Co. KG, Duren, Germany; TaKaRa-Bio). The DNA was extracted and finally dissolved in sterilized water. The optical concentration of the extracted and purified DNA aqueous solution was measured, and it was confirmed that only DNA was extracted with high efficiency.

Next, adjust the concentration of each purified DNA aqueous solution to 5 ng / μl with sterilized water, add 2 μl to the direct LAMP master mix composition 1, 2, and 3, and perform the LAMP amplification reaction in the same way as for direct LAMP. Carried out. That is, 10 ng of DNA was contained per LAMP amplification tube, which corresponded to 2 × 10 ⁶ cells in bacterial cells.

4-2) Test results and discussion The results are shown in Tables 18-21. In the table, “ND” indicates that target gene amplification is not detected, and “ND × 2” indicates that the same result was obtained in two operations. The same applies hereinafter.

According to Tables 18 to 20, Gram-negative bacteria and Gram-positive bacteria could be detected simultaneously by the direct LAMP master mix optimized in Example 4 (see Tables 12 to 14). Further, as shown in Table 21, it was possible to amplify target genes from all bacterial species by the direct LAMP method using direct DNA as a template.
In the experiments shown in Tables 18 to 20, assuming that the recovery rate of each bacterial cell is 100%, the LAMP amplification reaction is performed in approximately 9 × 10 ⁶ cfu / tube, and the recovery rate is increased by centrifugation or the like. Even though it is partially reduced, 6 log cfu / tube level is available for the reaction.
On the other hand, in the experiment of Table 21, since 10 ng of DNA usually corresponds to the number of bacterial cells of 2 × 10 ⁶ cfu, a significant difference from the number of cells in Tables 18 to 20 cannot be considered.

  Even if the Ct value of each living cell in Tables 18 to 20 is compared with the corresponding Ct value in Table 21, no significant delay in the Ct value is basically observed. It was thought that there was no particular element.

When distinguishing live and dead cells by the direct LAMP method, 10 μM Tetrakis (triphenyl
In phosphine) platinum (II), LAMP amplification of various gram-negative and gram-positive bacterial dead cells was completely suppressed by a single treatment. In the examples shown below, EMA did not completely suppress LAMP amplification of 6.2 log cells / ml Legionella dead cells even after three consecutive treatments of 11.9 μM (5 μg / ml). Compared to, platinum complexes may be preferred for the direct LAMP method.

  From the above, it is considered that a suitable direct LAMP master mix is capable of simultaneous amplification of a wide range of gram-negative and gram-positive bacteria.

[Example 5] Evaluation of the degree of DNA damage (inactivation) when exposed to a reagent for determining living cells / dead cells. Live cells of E. coli by direct LAMP method and direct real-time PCR method using platinum complex The identification of dead cells was compared.

5-1) Test method
E. coli JCM109 strain was cultured in BHI broth at 37 ° C for 18 hours, washed with sterilized water, suspended in sterilized water, and live cell suspension (1.1 x 10 ⁶ cfu / ml; 6.0 log cfu / ml) was prepared. A part of this live cell suspension was immersed in boiling water for 3 minutes to prepare a dead cell suspension (1.1 × 10 ⁶ cells / ml). Each 1 ml of these was subjected to the following platinum complex exposure.

tetrakis (triphenylphosphine) platinum (II) (Sigma, molecular weight 1244.22) 6.04 mg (4.85
μmols) was accurately weighed and dissolved in 1213.6 μl DMSO to prepare a 4 mM solution. This solution was diluted 2-fold with DMSO to prepare a 2000 μM platinum complex solution.

Add 5 μl of the above 2000 μM platinum complex solution to 1 ml of the above live cell suspension or 1 ml of dead cell suspension.
After the addition, the lid was carefully closed and the test sample was stirred all at once, and then held at 37 ° C. for 12.5 minutes. Thereafter, the mixture was cooled and centrifuged (4 ° C., 8,000 × G, 5 minutes), the supernatant was removed, and the pellet was washed with 1 ml of sterile water.
Add 60 μl of sterilized water to the washed pellet, stir well, transfer 30 μl to a new LAMP amplification reaction tube (quantitative PCR tube), treat the tube at 96 ° C for 3 minutes, and then rapidly cool to 4 ° C. Each component such as an enzyme in the direct LAMP master mix effectively permeated bacterial cells.

Next, the supernatant was almost removed by cooling and centrifuging (4 ° C, 10 minutes, 3,000 x G), and the master mix for direct LAMP of Table 13: Composition 2 (GN_GM_ID_4) was applied to the pellet (equivalent to 2.0 µl). In addition, the total amount was adjusted to 12.5 μl, and LAMP amplification was performed at 65 ° C., 100 minutes; 80 ° C., 2 minutes; 4 ° C., 2 minutes).

On the other hand, real-time PCR was performed as follows. Transfer 30 μl of the live cell suspension and dead cell suspension after exposure to the platinum complex to a quantitative PCR tube, cool and centrifuge (4 ° C, 10 minutes, 3,000 x G), and remove the supernatant. The direct real-time PCR master mix shown in Table 22 below was added to prepare a total volume of 25 μl. Taq DNA Polymerase with Standard Taq Buffer (New England Biolabs Japan Inc .; M0273S) (described as “NEB 10 × buffer”) was used as a qPCR buffer (quantitative PCR buffer).

  In addition, as shown in Table 22, direct real-time PCR (DqPCR) master mix prepared by adding Taq polymerase x4 times the usual amount and further adding a predetermined amount of cDBC (10 x DBC) (see Table 3). DqPCR amplification (45 cycles) was performed twice using (Japanese Patent Application 2013-542301). “NEB” refers to the New England Biolabs product.

For PCR amplification, Primer ENT-16S forward: Enterobacteriaceae-specific 16S rRNA gene detection forward primer (5'-GTTGTAAAGCACTTTCAGTGGTGAGGAAGG-3 ': SEQ ID NO: 26) and Primer ENT-16S reverse : Enterobacteriaceae specific 16S rRNA gene detection reverse primer (5′-GCCTCAAGGGCACAACCTCCAAG-3 ′: SEQ ID NO: 27) was used as a PCR primer (both primers were outsourced to Nippon Gene). The fragment length of the amplified rRNA gene is 424 bp.
Enterobacteriaceae ENT-16S TaqMan probe uses an oligonucleotide having a sequence of (5 '-/ 56-FAM / AACTGCATC / ZEN / TGATACTGGCAGGCT / 3lABkFQ / -3': SEQ ID NO: 28) It was. This probe has a specification in which a fluorescent substance 56-FAM is placed at the 5 'end of the oligonucleotide, a quencher dye called ZEN at the center and 3ABkFQ is placed at the 3' end, and is commissioned by Integrated DNA Technologies. . In addition, the base sequence information regarding the primer for detecting Enterobacteriaceae group is obtained from Nakano, S. et al., J. Food Prot. 66: 1798-1804, 2003, and the base sequence information regarding ENA-16S TaqMan probe. Was obtained by selecting the complementary region of the 16S rRNA gene in the Enterobacteriaceae family from the GenBank database (http://www.ebi.ac.uk/genbank/).

Real-time PCR was performed twice using the real-time PCR apparatus (StepOnePlus Real-Time PCR System; Applied Biosystems) under the following PCR thermal cycle conditions.
1) 95 ℃, 20 seconds (1 cycle)
2) 95 ℃, 5 seconds; 60 ℃, 1 minute (45 cycles)
As a negative control, 5 μl of sterilized water was used as a template.

5-2) Test results and discussion Table 23 shows the results of the direct LAMP method and the direct PCR method. Moreover, the standard curve created from this result is shown in FIG. 1 (direct LAMP method) and FIG. 2 (direct PCR method).

According to the results of this test, when the platinum complex was allowed to act on 6 log cfu / ml of E. coli live cells, the direct LAMP method already increased by about 3.3 compared to the corresponding untreated live Ct. (
However, in direct / real-time PCR, Ct of this platinum-exposed live cell remained at an increase (delay) of about 1 compared to the corresponding untreated live / dead cell Ct.
Similarly, comparing the direct LAMP method and the direct real-time PCR method for live cells of 5.0 to 1.0 log cfu / ml, the direct LAMP method detected the platinum complex that slightly penetrated the live cells more sensitively. I found out.

In comparison between the Ct value of the direct LAMP method and the Ct value of the direct real-time PCR method, the standard curve of the untreated group in each method (Figs. 1 and 2) and E. exposed to the platinum complex.
By comparing the Ct value of living cells in Escherichia coli and using the degree of reduction in the number of living cells with intact chromosomes as a result of exposure to the platinum complex as an index, either the direct LAMP method or the direct real-time PCR method can be used. It can be seen whether exposed live cells are detected sensitively.

In the direct LAMP method, for example, when 5.0 log cfu / ml of live cells are exposed to the platinum complex, the Ct value is about 25.3, which is 1.0 log cfu / ml at the standard Ct value of untreated live cells. It is considered that the viable cell number of 5.0 log cfu / ml was lowered to less than 1.0 log cfu / ml by the platinum complex.
On the other hand, in direct real-time PCR, Ct value 28.9 when 5.0 log cfu / ml live cells are exposed to this platinum complex is the number of live cells that retain intact DNA using standard Ct values of untreated live cells. When the degree of reduction is evaluated, it is close to the Ct value of 4 log cfu / ml of untreated living cells. Therefore, even if exposed to this platinum complex, the viable cell count of 5.0 log cfu / ml is reduced to 4 log cfu / ml viable cell count, but the reduction is only 1 log cells unit order level. .

That is, the direct LAMP method is more sensitive to exposure of the present platinum complex to living cells, and may detect a small amount of DNA damage to living cells with high sensitivity. On the other hand, even when the degree of gene amplification suppression from dead cells was evaluated using the same platinum complex, the direct LAMP method is considered to suppress the amplification more intensely than the direct real-time PCR method.

From the above results, gene amplification from living cells was significantly suppressed when the direct LAMP method was used compared to the direct real-time PCR method. Similarly, regarding the dead cells, it was revealed that the direct LAMP method can suppress the gene amplification derived from the dead cells.

In the direct real-time PCR method, the temperature change at 95 ° C. and 60 ° C. is performed continuously and alternately, so that the dissociation process from the double strand to the single strand of the template DNA is repeated 40 to 50 times. Therefore, platinum elements that are incompletely coordinated to the target gene region of a living cell / chromosome are detached from the chromosome, but platinum elements that have not coordinated to the chromosome at all through the living cell are It is considered that there is no coordinate bond to the target gene of the cell chromosome. Therefore, it is considered that there is a certain proportion of intact (unmodified) target gene regions derived from living cells, which are thought to contribute to amplification of target genes derived from living cells.
However, in the direct LAMP method, heating is performed only once at 96 ° C for about 3 minutes, so the dissociation step from the double strand to the single strand of the living cell template DNA is performed only once, and thereafter The gene is extended at 60 to 65 ° C. by a strand displacement type DNA polymerase. Therefore, platinum elements that are incompletely coordinated to the target gene region of living cells / chromosomes are significantly less likely to detach from the chromosome than direct real-time PCR, and therefore, intact ( It is speculated that the unmodified target gene region is significantly reduced.
Furthermore, while the template DNA double strand is dissociated at 60 to 65 ° C. with a strand displacement type DNA polymerase, gene elongation will be performed based on each dissociated single-stranded DNA,
Since the platinum element that has been cross-linked to the template DNA duplex in advance by coordination bond inhibits dissociation of the template DNA duplex into a single strand by the enzyme, subsequent gene extension does not proceed. It is guessed. Therefore, it is considered that amplification of target genes derived from living cells is significantly suppressed.
As described above, it is considered that gene amplification from living cells is significantly suppressed when the LAMP method is used. Similarly, with respect to dead cells, the direct LAMP method is more likely to suppress gene amplification derived from dead cells.

Furthermore, as shown in FIG. 1, the direct LAMP method is considered to be quantitative in the concentration range of 1 to 6 log cfu / ml, and the platinum complex treatment-direct LAMP method is 3 to 6 log cfu / ml. It is considered to be quantitative in the concentration range.
Similarly, direct real-time PCR is considered to be quantitative in the concentration range of 1-6 log cfu / ml, and platinum complex treatment-direct real-time PCR method in the concentration range of 2-6 log cfu / ml. It is considered to be quantitative.

Based on the above, the LAMP method can be used to detect sensitively the effects of permeated nucleic acid inactivating agents (coordinating bonds or photoreactive nucleic acid covalent bonds) regardless of whether the cells are live or dead. Is considered to be superior to the PCR method in principle and in test.

[Example 6] Identification of live and dead cells of Legionella by direct LAMP method Legionella is a representative example of microorganisms that are likely to die in a normal test environment and typically used in physiological saline. In this example, it was verified whether the Legionella live cells or dead cells could be identified by the direct LAMP method.

6-1) Test method The component which suppresses the transmission | permeation of EMA to a Legionella live cell and assists the permeation | transmission to a dead cell was examined.

As a suitable artificial medium for Legionella, GVPC selective medium is known. The composition of the medium is Legionella CYE agar basal medium (CM0655; Kanto Chemical, Tokyo; including activated carbon, yeast extract, agar), Legionella BCYEα growth supplement (Regionella essential nutrients ACES / potassium hydroxide buffer, ferric pyrophosphate , L-cysteine hydrochloride, α-ketoglutaric acid), Legionella GVPC selective supplement (a group of various antibiotics that kills bacteria other than Legionella, including glycine (ammonia free), vancomycin hydrochloride, polymyxin B sulfate, Cycloheximide).
Since Legionella does not have catalase, it cannot decompose hydrogen peroxide produced by this bacterium, and as a result, it is killed by DNA damage caused by hydroxyl radicals. However, EMA is also adsorbed on activated carbon. Moreover, the various antibiotic groups of Legionella GVPC selection supplement are added to a culture medium in order to suppress the proliferation of miscellaneous bacteria other than Legionella, and are not essential for Legionella viable cells.

Therefore, yeast extract and Legionella BCYEα growth supplement are candidates for adding EMA-treated suspension to suppress EMA penetration into living cells and help maintain the penetration of dead cells. And evaluated their effects.

Legionella pneumophila ATCC33153 strain was cultivated in BCYEα medium at 37 ° C for 2 days. The colony was picked and sterilized with 1% Bacto Yeast Extract aqueous solution (hereinafter referred to as "Y-SW"), Y-SW Furthermore, it was made to suspend in the aqueous solution (henceforth "YS-SW") which added Legionella BCYE alpha growth supplement, or physiological saline, and each 9.4 +/- 0.12 log cfu / ml live cell suspension was prepared.
This 9.4 log cfu / ml live cell suspension was diluted 10 ² to 10 ⁷ times with the above various aqueous solutions to prepare live cell suspensions of various concentrations. Further, the above 9.4 log cfu / ml live cell suspension was boiled for 10 minutes, and then diluted 10 ² times with various aqueous solutions to prepare dead cell suspensions. A variety of the above-mentioned live cell suspensions (10 ² to 10 ⁷ times diluted solution) and dead cell suspension (10 ² times diluted solution) 1 ml were used as test specimens.

Details of the method for preparing Y-SW and YS-SW are shown below.
For Y-SW, 1 g of Bacto ^™ Yeast Extract (BD, Sparks, MD, USA) was dissolved in 99 ml of MilliQ water and then autoclaved. YS-SW is a Bacto ^TM Yeast Extract 1 g MilliQ water 89
After dissolving in ml, autoclaved, cooled to 55 ° C, Legionella BCYEα growth supplement (SR110; Kanto Chemical, Tokyo; ACES / potassium hydroxide buffer 1.0 g, ferric pyrophosphate 0.025 g, L-cysteine hydrochloride 0.04 g and 0.1 g of α-ketoglutaric acid in 1 vial (for preparation of 100 ml) were prepared by adding an aqueous solution in which 1 vial was dissolved in 10 ml of warm sterilized water.

Next, the concentration of EMA is increased from the above example, and a total of three multi-stage EMA treatments at a final concentration of EMA 25-35 μg / ml (first EMA treatment, 35 μg / ml on dark ice for 16 minutes, second time EMA treatment 30 μg / ml 16 minutes, 3rd EMA treatment 25 μg / ml 16 minutes). Visible light irradiation after each EMA treatment was 10 minutes. An untreated group was also prepared for each specimen.

Cool and centrifuge the multi-stage EMA treatment group and the untreated group (4 ° C, 10 minutes, 13,000 x G), completely remove the supernatant, add 30 μl of sterile water to the pellet, suspend and quantitate The suspension
Transfer to tube. The PCR tube was treated at 96 ° C. for 3 minutes and then rapidly cooled to 4 ° C. so that components such as enzymes in the master mix for direct LAMP could permeate bacterial cells effectively.
Thereafter, the supernatant was almost removed by cooling and centrifugation (4 ° C., 10 minutes, 3,000 × G), and 14 μl of the master mix for direct LAMP shown in Table 10 was added to the pellet (equivalent to 2.5 μl).
, 100 minutes; 80 ° C., 2 minutes; 4 ° C., 2 minutes).

6-2) Results and discussion Table 24 shows the results.

In Table 24, the symbols are as follows.
a) Bacto Yeast Extract (1%)
b) Bacto Yeast Extract (1%) + SR110
c) Test sample in which Legionella live or dead cells are suspended in physiological saline instead of Y-SW or YS-SW
d) L. pneumophila ATCC33153 (9.4 ± 0.12 log cells / ml) in 10 ^double dead cells-suspension liquid dilution and live cells Ken Nigoeki (Ken Nigoeki is, Y-SW, YS-SW , or saline )
e) LAMP amplification was performed twice and the Ct value was displayed in Mean ± SD
f) Two results indicate LAMP unamplified (negative).

According to Table 24, the live Legionella cells suspended in the physiological saline corresponding to the subject group had cell concentrations of 2.4 log cfu / ml (10 ⁷ dilution) and 3.4 log cfu / ml (10 ⁶ dilution). The result was determined to be negative, but it was determined to be positive even after EMA treatment at 7.4 log cfu / ml (10 ² fold dilution). Therefore, it was shown that if the cell concentration is high, it is possible to distinguish between live and dead cells even when EMA treatment is performed with a physiological saline suspension.

For Legionella live and dead cells suspended in YS-SW, 7.4 log cfu / ml
Ct value of dead cells treated with EMA was delayed by about 12 (cycles or minutes) compared to untreated Ct, and gene amplification of dead cells was confirmed despite multi-step EMA treatment Therefore, it was considered unsuitable as a suspension for EMA treatment.

On the other hand, in the case of Y-SW, dead cells treated with EMA at 7.4 log cfu / ml were determined to be negative, the Ct value of the untreated group was 9.9, and good gene amplification by the LAMP method was confirmed. Furthermore, when compared with the untreated group in the same cell viable cell concentration range of 2.4 to 7.4 log cfu / ml, the target gene amplification was confirmed by the gene amplification time within 36.4 minutes although the Ct value was large.
Comprehensive evaluation showed that the suspension of Legionella in 1% Bacto ^TM Yeast Extract aqueous solution and multistage EMA treatment enables clearer determination of live and dead cells. .

By comparing the results of Y-SW and YS-SW dead cells, SR110 ACES / potassium hydroxide buffer, ferric pyrophosphate, L-cysteine hydrochloride, α-ketoglutaric acid originally produced Legionella It is suggested that components essential for cell growth may inhibit the penetration of EMA into Legionella dead cells or bind these components to positively charged EMA and inactivate EMA .

[Example 7] Effect of yeast extract in distinguishing live and dead Legionella cells by multi-stage EMA treatment-direct LAMP method and multi-stage EMA treatment-alkali DNA extraction LAMP method As shown in Table 24 of Example 6 above, It was shown that by performing EMA treatment of Legionella with a cell suspension containing yeast extract, it is possible to clearly distinguish between live and dead cells even at high dead cell concentrations. In this example, the effect of the yeast extract in the alkaline DNA extraction-LAMP method was further verified.

7-1) Test method
Legionella pneumophila ATCC33153 strain was cultured in BCYEα medium at 37 ° C for 2 days, colonies were picked, suspended in sterile 1% Bacto Yeast Extract aqueous solution (Y-SW), and 8.9 log cfu / ml live cell suspension A liquid was prepared. Thereafter, a suspension diluted 10 ² times with Y-SW was prepared and boiled for 10 minutes to prepare a dead cell suspension. In addition, a 10 ² to 10 ⁸ times diluted suspension of the 8.9 log cfu / ml live cell suspension was prepared by Y-SW.
Using 1 ml of each of the above live and dead cell suspensions as a test sample, a total of 3 multi-stage EMA treatments (final concentration of EMA 25-35 μg / ml) (first EMA treatment 35 μg / ml on ice protected from light) 15 minutes, second EMA treatment 30 μg / ml 15 minutes, third EMA treatment 25 μg / ml 10 minutes). Visible light irradiation after each EMA treatment was 10 minutes. An untreated group was also prepared for each specimen.

Cool and centrifuge the multi-stage EMA treatment group and the untreated group (4 ° C, 10 minutes, 13,000 x G), completely remove the supernatant, add 30 μl of sterile water to the pellet, suspend and quantitate The suspension
Transfer to tube. The PCR tube was heated at 96 ° C. for 3 minutes so that each component such as an enzyme in the direct LAMP master mix effectively permeated the bacterial cells.
Thereafter, the supernatant was almost removed by cooling and centrifugation (4 ° C., 10 minutes, 3,000 × G), and 14 μl of the master mix for direct LAMP shown in Table 10 was added to the pellet (equivalent to 2.5 μl).
, 100 minutes; 80 ° C., 2 minutes; 4 ° C., 2 minutes).

On the other hand, the multistage EMA treatment-alkali DNA extraction-LAMP method was performed as follows.
Each sample after multi-stage EMA treatment was concentrated to 20 μl by cooling centrifugation (4 ° C., 10 minutes, 13,000 × G), then 25 μl Extraction Solution for Legionella (EX Leg), and 4 μl of 1M Tris-HCl ( pH 7.0) was added, and 2.5 μl of the centrifugal supernatant was added to the LAMP method master mix (see Table 5). That is, alkaline DNA extraction followed the manual attached to Legionella detection reagent kit E.

Further, as a control group, living cells and dead cells were suspended in physiological saline in the same manner as described above, and multistage EMA treatment-direct LAMP method and multistage EMA treatment-alkaline DNA extraction-LAMP method were performed.

7-2) Results and Discussion Tables 25 and 26 show the test results.

The symbols in Table 25 and Table 26 are as follows.
a) Y.SW of L. pneumophila ATCC33153 or saline suspension (8.9 ± 0.31 log cells / ml)
b) ± dead cell suspension of L. pneumophila ATCC33153 (6.9 ± 0.31 log cells / ml) or 8.9 log cells / ml of live cell suspension 10 ² times with Y-SW or physiological saline Dilute live cell suspension.
c) Perform LAMP amplification twice and describe the Ct value (number of cycles or min) as Mean ± SD (n = 2).
d) Two results indicate LAMP unamplified (negative).

  As shown in Table 25, when an aqueous yeast extract solution is used as an aqueous solution for suspending Legionella live cells and dead cells, discrimination between live cells and dead cells can be achieved even at a lower cell concentration than when using physiological saline according to Table 26. Was shown to be possible.

In addition, the test method using the multi-step EMA treatment-direct LAMP method is negative for high-concentration Legionella dead cells (6.9 log cells / ml), and low-concentration Legionella live cells of 1.9 log cfu / ml can be detected. Therefore, it is considered to be a more appropriate method for inspection.

[Example 8] Simultaneous detection of live Legionella cells by multi-stage EMA treatment-direct LAMP method In this example, the detection limit of live cells of various Legionella bacteria by the direct LAMP method was examined.

8-1) Test method
Legionella pneumophila ATCC33153, ATCC33154, ATCC33215, JLP1008, and JLP1024 strains were cultured in BCYEα medium at 37 ° C. for 2 days. A 9.0 log cfu / ml live cell suspension was prepared. The JLP strain can be obtained from the Department of Bacteriology, Kyushu University Graduate School of Medicine (3-1-1 Ude, Higashi-ku, Fukuoka, 812-8582, Japan).

Thereafter, a suspension diluted 10 ² times with Y-SW was prepared and boiled for 10 minutes to prepare a dead cell suspension. In addition, the suspension obtained by diluting 10 ⁶ to 10 ⁸ times the 8.2 to 9.0 log cfu / ml live cell suspension
It was prepared by.
Using 1 ml of each of the above live and dead cell suspensions as a test sample, a total of 3 multi-stage EMA treatments (final EMA 27.5-30 μg / ml) (first EMA treatment 30 μg / ml on light protected from light) 16.5 minutes, second EMA
Treatment 27.5 μg / ml for 15 minutes, 3rd EMA treatment 27.5 μg / ml for 15 minutes). Visible light irradiation after each EMA treatment was 10 minutes.

After multi-stage EMA treatment, cool and centrifuge (4 ° C, 10 minutes, 13,000 x G), completely remove the supernatant, add 30 μl of sterile water to the pellet, suspend, and quantitatively suspend the suspension. The solution was transferred to a PCR tube. The PCR tube was heated at 96 ° C. for 3 minutes so that each component such as an enzyme in the direct LAMP master mix effectively permeated the bacterial cells.
Thereafter, the supernatant was almost removed by cooling and centrifugation (4 ° C., 10 minutes, 3,000 × G), and 14 μl of the master mix for direct LAMP shown in Table 10 was added to the pellet (equivalent to 2.5 μl).
75 minutes; 80 ° C., 2 minutes; 4 ° C., 2 minutes).

8-2) Results and discussion Table 27 shows the test results.

The symbols in Table 27 are as follows.
a) Concentration of L. pneumophila ATCC33153 suspension at the start of the study.
b) Dead cell suspension: L. pneumophila suspension (8.8 ± 0.29 log cells / ml) was diluted 10 ² times (6.8 ± 0.29 log cells / ml) and then boiled for 10 minutes. Live cell suspension:. L pneumophila suspension 10 ⁶ fold dilution of (8.8 ± 0.29 log cells / ml ) (2.8 ± 0.29 log cells / ml) were prepared.
c) Indicates that the number of unamplified (undetected) is 5 or 2.
d) When measuring conditions (65 ° C, 75 minutes, 80 ° C, 2 minutes, and 4 ° C, 2 minutes) 2 or 5 times, reaction time exceeding the boundary value at the beginning: Ct value (minutes): mean ± SD (n = 5 or 2) is shown.
e) 4 times indicate unamplified (not detected), 1 time indicate Ct value 66.2.
f) Shows the measured Ct values for each of the two measurements.
g) Detection limit
h) 2 measurements each (67.2, unamplified)
i) Two measurements (32.3, unamplified)

According to Table 27, in the multi-stage EMA-direct LAMP method, Legionella dead cells were determined to be negative because LAMP amplification was not observed at a concentration of 6.2 to 7.0 log cells / ml. Moreover, LAMP amplification was confirmed by the density | concentration of 0.2-2.8 log cfu / ml, and all the live Legionella cells were determined to be positive.

  It is said that the Legionella dead cell concentration in the hot spring water is 6-7 log cells / 100 ml by the Legionella test by PCR, etc. From the above results, the LAMP amplification derived from Legionella dead cells according to the present invention is 6 log cells / ml. Therefore, it is speculated that the multistage EMA-direct LAMP method according to the present invention can completely suppress LAMP amplification of Legionella dead cells in hot springs.

In addition, the JLP1008 strain can be detected at a concentration of 0.2 log cfu / ml or more, the ATCC33153 strain can be detected at a concentration of 2.8 log cfu / ml or more, and the remaining 3 strains can also be detected at 2.0 log cfu / ml. Since detection was possible at a concentration of ml or more, it was confirmed that any Legionella viable cell could be detected by the present invention.
As described above, when the multi-stage EMA treatment-LAMP method of the present invention is carried out, it is possible to specifically detect only living Legionella cells in hot spring water directly from the specimen without requiring pre-culture of the specimen.
In the case of ATCC33153, considering that the generation time of living cells is 1 h, the Legionella viable cells in the test water are cultured for about 3 hours in Legionella's preferred medium, and then the multistage EMA treatment-LAMP method is performed. For example, only the live Legionella cells can be specifically detected for the ATCC33153 strain.

[Reference Example 1] Identification of live and dead cells of Esherichia coli by Di-μ-chlorobis [(η-cycloocta-1,5-diene) iridium (I)] Live of E. coli using iridium complexes As an iridium complex, Di-μ-chlorobis [(η-cycloocta-1,5-) is an iridium complex dimer (a dimer having two iridium elements in one complex). diene) iridium (I)] was used.

1. Test Materials and Methods 1-1) Preparation of cell suspension A sterile cell suspension (1.2 × 10 ⁷ CFU / ml) of E. coli JCM1649 was prepared. A portion of this live cell suspension is immersed in boiling water for 3 minutes to give a damaged / dead cell suspension (1.2 × 10 ⁷ cells / ml. Hereinafter, the damaged and dead cells are collectively referred to as dead cells). Prepared). 90 μl of each of these live cell suspensions and dead cell suspensions was subjected to the following test.

1-2) Preparation of iridium complex solution
Di-μ-chlorobis [(η-cycloocta-1,5-diene) iridium (I)] (Wako) 3.92 mg (5.84 μmols) was accurately weighed and 116.7 μl of dimethyl sulfoxide (DMSO, D8418-50ML , Sigma)
To prepare a 50 mM solution. This solution was diluted with physiological saline to prepare 100 μM, 250 μM and 1000 μM iridium complex solutions.

1-3) Treatment of test sample with iridium complex 10 μl of each of the above iridium complex solutions was added to 90 μl of the live cell suspension or 90 μl of the dead cell suspension and kept at 37 ° C. for 30 minutes in a constant temperature water bath. . Thereafter, the mixture was cooled and centrifuged (4 ° C., 15,000 × G, 5 minutes), and the supernatant was removed. The precipitate (pellet) was washed with 1 ml of sterilized water. The washed pellet (corresponding to 5 μl of cell suspension) was used as a PCR amplification sample.

1-4) PCR amplification Next, using cDBC (10 × DBC) shown in Table 3 above, real-time PCR (without extraction of nucleic acids from cells) is performed without extraction of nucleic acids from cells. A master mix for performing real-time PCR (hereinafter referred to as “direct real-time PCR”) (master mix for direct real-time PCR) was prepared.
Specifically, using the Taq DNA Polymerase with Standard Taq Buffer as a qPCR buffer, add 4 times the amount of Taq normally used to this, and add a predetermined amount of cDBC (10 × DBC, see Table 3) to the same buffer. A direct real-time PCR (DqPCR) master mix was prepared.
The master mix for direct real-time PCR was added to the previously prepared PCR amplification sample, and real-time PCR amplification (40 cycles) was performed twice. Hereinafter, the New England Biolabs product is referred to as NEB.

  As the primer, the primer of SEQ ID NO: 26 to 28 described in Example 5 was used.

Real-time PCR was performed twice using the real-time PCR apparatus (StepOnePlus Real-Time PCR System; Applied Biosystems) under the following PCR thermal cycle conditions.
1) 95 ℃, 20 seconds (1 cycle)
2) 95 ℃, 5 seconds; 60 ℃, 1 minute (40 cycles)
As a negative control, 5 μl of sterilized water was used as a template.

2. Results and Discussion Table 29 shows the results of real-time PCR. “No Agent” indicates that no iridium complex is added.

According to Table 29, when Di-μ-chlorobis [(η-cycloocta-1,5-diene) iridium (I)] was allowed to act on E. coli live cells and dead cells, the Ct value of dead cells was PCR amplification derived from dead cells was completely suppressed at a concentration of 100 μM of the iridium complex.
On the other hand, with respect to living cells, a slight drug permeation phenomenon is observed as the concentration increases. However, the drug concentration at which the PCR from dead cells is completely suppressed is 3.6% compared with the Ct value of untreated living cells. Only an increase (delay in amplification) was observed, and it was possible to clearly distinguish between live cells and dead cells with the 100 μM iridium complex.

Claims (31)

A method for detecting living cells of microorganisms in a test sample by distinguishing them from dead cells or damaged cells, comprising the following steps:
a) a step of adding, to the test sample, an agent that selectively inhibits the dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
b) performing a treatment for increasing the permeability of cells of microorganisms;
c) a step of amplifying a target region of a nucleic acid of a microorganism in a test sample by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme without extracting the nucleic acid from the cell, and d) an amplification product Process to analyze,
Wherein the treatment of increasing the permeability of the cells of the microorganism is a treatment thereby enabling nucleic acid amplification selective to living cells without extraction of nucleic acids from the cells.   When the drug is selected from a drug that is covalently bonded to a nucleic acid by light irradiation with a wavelength of 350 nm to 700 nm and a platinum group element complex, and the drug is a drug that is covalently bonded to a nucleic acid by light irradiation with a wavelength of 350 nm to 700 nm. The method according to claim 1, further comprising a step of performing a light irradiation treatment with a wavelength of 350 nm to 700 nm on the test sample to which the drug is added. The drug is a drug that is covalently bonded to a nucleic acid by irradiation with light having a wavelength of 350 nm to 700 nm, ethidium monoazide, ethidium diazide, propidium monoazide, psoralen, 4,5 ′, 8-trimethylpsoralen, and 3. A process according to claim 2 selected from 8-methoxypsoralen.   The method according to claim 2, wherein the agent is a complex of a platinum group element and is selected from a platinum complex, a palladium complex, and an iridium complex. The platinum group element complex is a platinum complex, NH ₃ , RNH ₂ , halogen element, carboxylate group, pyridine group, H ₂ O, CO ₃ ²⁻ , OH ⁻ , NO ₃ ⁻ , ROH, N ₂ H _{4 ^{, PO 4 3-, R 2 O}} , RO -, ROPO 3 2-, (RO) 2 PO 2 -, R 2 S, R 3 P, RS -, CN -, RSH, RNC, (RS) 2 PO 2 _{^{-, (RO) 2 P (}} O) S -, SCN -, CO, H -, and R ^- (where "R" is either represents a saturated or unsaturated organic group) configuration selected from the group consisting of The method of claim 4, comprising a child. The platinum group element complex is a palladium complex, NH ₃ , RNH ₂ , halogen element, carboxylate group, H ₂ O, CO ₃ ²⁻ , OH ⁻ , NO ₃ ⁻ , ROH, N ₂ H ₄ , PO _{4 ^{3-, R 2 O, RO -}} , ROPO 3 2-, (RO) 2 PO 2 -, R 2 S, R 3 P, RS -, CN -, RSH, RNC, (RS) 2 PO 2 -, ( RO) ₂ P (O) S ⁻ , SCN ⁻ , CO, H ⁻ , R ⁻ (where “R” represents a saturated or unsaturated organic group), NO ₂ ⁻ , Ar—NH ₂ , Ar— The method according to claim 4, comprising a ligand selected from CN (Ar is an unsaturated organic group), N ₂ , SO ₃ ²⁻ , an imidazole ring, an unsaturated cyclic organic group, and N ₃ ⁻ . The complex of the platinum group element is an iridium complex, NH ₃ , RNH ₂ , halogen element (Cl, F, Br, I, At), carboxylate (—CO—O—) group, pyridine group, H ₂ O, ^{_{CO 3 2-, OH -, NO}} 3 -, ROH
_{, N 2 H 4, PO 4} 3-, R 2 O, RO -, ROPO 3 2-, (RO) 2 PO 2-, NO 2 -, N 2, N 3 -, R 2 S, R 2 P - , R ₃ P, RS ^{-
, CN -, RSH, RNC,} (RS) 2 PO 2 -, (RO) 2 P (O) S -, SCN -, CO, H -, and R ^- (provided that both "R" is a saturated or unsaturated The method according to claim 4, comprising a ligand selected from the group consisting of: a saturated organic group.   The method according to any one of claims 1 to 7, wherein the treatment for increasing cell permeability of the microorganism is selected from heat treatment, electron beam irradiation, voltage application, enzyme treatment, and osmotic shock. The method according to claim 8, wherein the treatment is a heat treatment, and the treatment conditions are 65 to 100 ° C. and 0.5 to 30 minutes.   The isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme is selected from the LAMP method, the ICAN method, the SDA method, the LCR method, the TMA method, the SMAP method, and the TRC method. The method described in 1. The method according to claim 1, wherein the step c) is performed in a solution having the following composition:
Tris-HCl (pH 7 ~ 9) 10mM ~ 25mM
KCl 5mM ~ 15mM
MgSO ₄ 5mM~40mM
Surfactant 0.1% -0.4%
Betaine 0.5M-1M
dNTPs 1mM to 1.5mM each
Strand displacement type nucleic acid elongation enzyme 0.2-0.6 U / μl   12. The method of claim 11, wherein the surfactant is polyethylene glycol sorbitan monolaurate.   The method according to claim 11 or 12, wherein the strand displacement type nucleic acid elongation enzyme is Bst polymerase and / or Csa polymerase.   The method according to claim 1, wherein the target region is a target region of 50 to 5000 bases. The method according to claim 14, wherein the target region is a target region corresponding to a gene selected from a 5S rRNA gene, a 16S rRNA gene, a 23S rRNA gene, and a tRNA gene of a nucleic acid of a test sample.   The method according to any one of claims 1 to 15, wherein the amplification of the target region is performed in the presence of calcein, and the amplification product is detected in real time by fluorescence of calcein.   Isothermal nucleic acid amplification, a set of primers having the sequences of SEQ ID NO: 1, 2, 3, 4, 9, and 10; a set of primers having the sequences of SEQ ID NOs: 10, 11, 12, 13, 14, and 17; and The method according to any one of claims 1 to 16, which is carried out using a set of primers selected from a set of primers having the sequences of SEQ ID NOs: 18, 19, 20, and 21. A kit for detecting a living cell of a microorganism in a test sample by distinguishing it from a dead cell or a damaged cell by an isothermal nucleic acid amplification method using a strand displacement type nucleic acid elongation enzyme, comprising the following elements: :
1) a drug that selectively inhibits dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
2) a reagent for preparing a reaction solution having the following composition;
Tris-HCl (pH 7 ~ 9) 10mM ~ 25mM
KCl 5mM ~ 15mM
MgSO ₄ 5mM~40mM
Surfactant 0.1% -0.4%
Betaine 0.5M-1M
dNTPs 1mM to 1.5mM each
3) A primer for amplifying the target region of the nucleic acid of the microorganism to be detected by an isothermal nucleic acid amplification method.   Furthermore, the kit of Claim 18 containing a strand displacement type | mold nucleic acid extension enzyme.   The kit according to claim 19, wherein the strand displacement type nucleic acid elongation enzyme is Bst polymerase and / or Csa polymerase.   The agent that selectively inhibits dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method is selected from agents that covalently bind to nucleic acids by irradiation with light having a wavelength of 350 nm to 700 nm, and platinum group element complexes. The kit according to any one of 18 to 20. The drug is a drug that is covalently bonded to a nucleic acid by irradiation with light having a wavelength of 350 nm to 700 nm, ethidium monoazide, ethidium diazide, propidium monoazide, psoralen, 4,5 ′, 8-trimethylpsoralen, and The kit according to claim 21, wherein the kit is selected from 8-methoxypsoralen.   The kit according to claim 21, wherein the agent is a complex of a platinum group element and is selected from a platinum complex, a palladium complex, and an iridium complex. The platinum group element complex is a platinum complex, NH ₃ , RNH ₂ , halogen element, carboxylate group, pyridine group, H ₂ O, CO ₃ ²⁻ , OH ⁻ , NO ₃ ⁻ , ROH, N ₂ H _{4 ^{, PO 4 3-, R 2 O}} , RO -, ROPO 3 2-, (RO) 2 PO 2 -, R 2 S, R 3 P, RS -, CN -, RSH, RNC, (RS) 2 PO 2 _{^{-, (RO) 2 P (}} O) S -, SCN -, CO, H -, and R ^- (where "R" is either represents a saturated or unsaturated organic group) configuration selected from the group consisting of 24. The kit according to claim 23, comprising a child. The platinum group element complex is a palladium complex, NH ₃ , RNH ₂ , halogen element, carboxylate group, H ₂ O, CO ₃ ²⁻ , OH ⁻ , NO ₃ ⁻ , ROH, N ₂ H ₄ , PO _{4 ^{3-, R 2 O, RO -}} , ROPO 3 2-, (RO) 2 PO 2 -, R 2 S, R 3 P, RS -, CN -, RSH, RNC, (RS) 2 PO 2 -, ( RO) ₂ P (O) S ⁻ , SCN ⁻ , CO, H ⁻ , R ⁻ (where “R” represents a saturated or unsaturated organic group), NO ₂ ⁻ , Ar—NH ₂ , Ar— The kit according to claim 23, comprising a ligand selected from CN (Ar is an unsaturated organic group), N ₂ , SO ₃ ²⁻ , an imidazole ring, an unsaturated cyclic organic group, and N ₃ ⁻ . The platinum group element complex is an iridium complex, NH ₃ , RNH ₂ , halogen element, carboxylate group, pyridine group, H ₂ O, CO ₃ ²⁻ , OH ⁻ , NO ₃ ⁻ , ROH, N ₂ H _{4 ^{, PO 4 3-, R 2 O}} , RO -, ROPO 3 2-, (RO) 2 PO 2-, NO 2 -, N 2, N 3 -, R 2 S, R 2 P -, R 3 P, ^{RS -, CN -, RSH,} RNC, (RS) 2 PO 2 -, (RO) 2 P (O) S -, SCN -, CO, H -, and R ^- (provided that both "R" saturated 24. The kit according to claim 23, comprising a ligand selected from the group consisting of: or an unsaturated organic group.   The kit according to any one of claims 18 to 26, wherein the surfactant is polyethylene glycol sorbitan monolaurate.   The kit according to any one of claims 18 to 27, further comprising calcein.   A set of primers having the sequences of SEQ ID NOs: 1, 2, 3, 4, 9, and 10; a set of primers having the sequences of SEQ ID NOs: 10, 11, 12, 13, 14, and 17; and 27. A kit according to any one of claims 18 to 26, selected from a set of primers having the sequences 18, 19, 20, and 21. A method for detecting living cells of microorganisms in a test sample by distinguishing them from dead cells or damaged cells, comprising the following steps:
a) a step of adding, to the test sample, an agent that selectively inhibits the dead cells from amplifying microorganism nucleic acids by a nucleic acid amplification method;
c) a step of amplifying a target region of a nucleic acid of a microorganism in a test sample by a nucleic acid amplification method, and d) a step of analyzing an amplification product,
A step comprising performing step a) in a cell suspension containing 0.5 to 10% of a component selected from the group consisting of proteins, sugars, lipids, and yeast extracts.   The microorganisms to be detected are Legionella, Campylobacter, Vibrio, Listeria, Clostridium, Helicobacter, Mycobacterium, Chlamydiaceae, Rickettsia, and Niseria. 32. The method of claim 30, wherein:

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