WO2014021351A1 - Procédé de détection de micro-organismes et trousse de détection de micro-organismes - Google Patents

Procédé de détection de micro-organismes et trousse de détection de micro-organismes Download PDF

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WO2014021351A1
WO2014021351A1 PCT/JP2013/070662 JP2013070662W WO2014021351A1 WO 2014021351 A1 WO2014021351 A1 WO 2014021351A1 JP 2013070662 W JP2013070662 W JP 2013070662W WO 2014021351 A1 WO2014021351 A1 WO 2014021351A1
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platinum
cells
nucleic acid
amplification
chloride
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PCT/JP2013/070662
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Japanese (ja)
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隆志 副島
淳一 南
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森永乳業株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • 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.
  • a plate culture method has been used to measure the number of general viable bacteria in foods, biological samples, wiped samples, or the environment.
  • 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.
  • 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.
  • EMA ethidium monoazide
  • a technique for detecting viable bacteria in a sample by selectively amplifying DNA by a nucleic acid amplification reaction has been proposed, and results have been achieved (Patent Documents 1 to 4).
  • 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.
  • the EMA crosslinks between DNA molecules by hydrogen bonding to DNA and then irradiation with light having a wavelength of 350 to 700 nm. Therefore, although light irradiation to a sample is essential, in order to prevent the sample from being heated by a light source, light irradiation is usually performed by immersing the sample in ice water, and the process is complicated. In addition, a method using an LED as a light source has been proposed, but there are problems such as insufficient light intensity and a decrease in the crosslinking ability of the crosslinking agent over time. Furthermore, a drug such as EMA or a sample containing the drug needs to be shielded from light such as in a dark room except for light irradiation to the sample in order to prevent the drug from being denatured.
  • a PCR target region is a region having a certain length or more, for example, a region having a length of 900 bases (bp) or more. It is common.
  • bp bases
  • Patent Document 3 EMA and a topoisomerase inhibitor or DNA gyrase inhibitor are added. It is known that by using together (Patent Document 2), it is possible to distinguish between live bacteria and dead or damaged bacteria even in a target region of about 100 bp. However, these methods involve complicated processes or drug preparation.
  • platinum complexes such as cisplatin (cisplatin, cis-diammineplatinum (II) dichloride, cis-diammineplatinum (II) dichloride), carboplatin (Carboplatin) are known as antineoplastic agents (Non-Patent Documents 1 and 2).
  • Their mechanism of action is inhibition of tumor cell DNA synthesis, and the mode of action of cell killing is said to be concentration-dependent fast-acting.
  • cisplatin and carboplatin are linked via a covalent bond (covalent bond) to nucleic acids (adenine (A) or guanine (G), which are defined as intermediate Lewis bases in bioinorganic chemistry). In recent years, it has been used clinically as an anticancer agent.
  • Non-patent Document 3 Usually, antibiotics are said to exert the most power in the logarithmic growth phase of bacteria, but cisplatin exerts an antitumor effect at any stage of tumor cells.
  • Non-patent Documents 4 and 5 suggested that cationic platinum complexes intercalate with DNA or bind to the phosphodiester site of DNA as a search for the mechanism of action as an anticancer agent. ing.
  • platinum complexes can be used to determine whether microorganisms are alive or dead.
  • An object of the present invention is to provide a method capable of detecting a living cell of a microorganism in a simple process and, in a preferred embodiment, even if a target region is relatively short.
  • the present inventors treat a test sample with a drug and selectively amplify chromosomal DNA in a microorganism in the sample by a nucleic acid amplification reaction to use a drug used in a technique for detecting a living cell in the sample.
  • a drug used in a technique for detecting a living cell in the sample was examined.
  • platinum complex it is possible to detect living cells of microorganisms without requiring light irradiation, cooling, and light shielding environment, and when a relatively short chain length region is set as the target region
  • the present inventors have found that living cells of microorganisms can be detected with high accuracy and have completed the present invention.
  • 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, and includes the following steps: a) adding a platinum complex to the test sample; b) a step of amplifying a target region of DNA or RNA of a microorganism contained in a test sample by a nucleic acid amplification method, and c) a step of analyzing an amplification product, I will provide a.
  • the amplification of the target region is preferably performed without extracting nucleic acid from cells.
  • the said method makes it a preferable aspect that amplification of the said target area
  • the said method makes it the preferable aspect that amplification of the said target area
  • the said method makes it a preferable aspect that the said test sample is any one of a foodstuff, a biological sample, drinking water, industrial water, environmental water, drainage, soil, or a wiping sample. Moreover, the said method makes it a preferable aspect that the said microorganisms are bacteria or a virus. Moreover, the said method makes it a preferable aspect that the said bacteria are Gram negative bacteria or Gram positive bacteria. Further, the method is preferably such that the target region is a target region of 50 to 5000 bases.
  • the method has a preferable aspect in which the target region is a target region corresponding to a gene selected from 5S rRNA gene, 16S rRNA gene, 23S rRNA gene, and tRNA gene of DNA of the test sample.
  • the nucleic acid amplification method is preferably a PCR method, an RT-PCR method, a LAMP method, an SDA method, an LCR method, a TMA method, a TRC method, an HC method, an SMAP method, or a microarray method. It is said.
  • the method is preferably such that the PCR method is performed by a real-time PCR method, and PCR and amplification product analysis are performed simultaneously.
  • the said method makes it a preferable aspect that the analysis of the said amplification product is performed using the standard curve which shows the relationship between the amount of microorganisms produced using the standard sample of microorganisms, and an amplification product.
  • 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 a nucleic acid amplification method, and includes the following elements: 1) Platinum complex or A platinum compound that forms a platinum complex when dissolved 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; 2) a primer for amplifying a target region of DNA or RNA of a microorganism to be detected by a nucleic acid amplification method; I will provide a.
  • the kit of the present invention preferably further includes an organic solvent capable of binding to platinum as a ligand.
  • the kit of the present invention preferably further includes a drug that suppresses the action of the nucleic acid amplification inhibitor, a magnesium salt, and an organic acid salt or phosphate.
  • the kit preferably includes a surfactant.
  • the platinum complex is NH 3 , RNH 2 , halogen element, carboxylate group, pyridine group, H 2 O, CO 3 2 ⁇ , OH ⁇ , NO 3 ⁇ , ROH, N 2 H.
  • the ligand is preferably selected from the group consisting of NH 3 , RNH 2 , a halogen element, a carboxylate group, a pyridine group, R 3 P, and RNC.
  • the platinum complex may be cisplatin, carboplatin, cis-diammine (pyridine) chloroplatinum (II) chloride, dichloro (ethylenediamine) platinum (II), cis-bis (benzonitrile) dichloroplatinum (II). ), Tetrakis (triphenylphosphine) platinum (II), dinitric acid (ethylenediamine) platinum iodide (II) dimer, oxaliplatin, nedaplatin, and transplatin.
  • the method and kit are generated by dissolving the platinum complex in a solution containing a platinum compound as an organic solvent capable of binding to platinum as a ligand or a substance capable of binding to platinum as a ligand.
  • a platinum compound as an organic solvent capable of binding to platinum as a ligand or a substance capable of binding to platinum as a ligand.
  • a preferred form is a platinum complex.
  • the platinum compound is platinum chloride, platinum bromide, platinum fluoride, platinum iodide, platinum hydroxide, platinum nitrate, platinum carbonate, platinum acetate, dimethoxyplatinum, platinum methoxyphosphate, phosphorus
  • a preferred embodiment is selected from the group consisting of platinum acid chloride, chloroplatinic acid, disulfurmethyl platinum, dicyano platinum, dithiocyanate platinum, platinum dihydride, and dimethyl platinum.
  • the platinum compound is preferably platinum (II) chloride, platinum (IV) chloride, or chloroplatinic acid.
  • the organic solvent is preferably dimethyl sulfoxide.
  • 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 includes the following steps. a) adding a platinum complex to the test sample; b) a step of amplifying a target region of DNA or RNA of a microorganism contained in a test sample by a nucleic acid amplification method; and c) a step of analyzing an amplification product.
  • the target of amplification may be any nucleic acid in general, and specific examples include single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA. it can.
  • 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.
  • a nucleic acid amplification method A foodstuff, biological sample, drinking water, industrial water, environmental water, drainage, soil, or a wipe sample etc. are mentioned.
  • foods include soft drinks, carbonated drinks, nutrition drinks, fruit juice drinks, lactic acid bacteria drinks and other drinks (including concentrated concentrates and powders for preparation of these drinks); ice cream such as ice cream, ice sherbet and shaved ice; Dairy products such as milk, processed milk, milk drinks, fermented milk, butter; enteral nutritional 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.
  • examples of the environmental water include city water, ground water, river water, and rain water.
  • 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.
  • 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.
  • 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 an 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.
  • 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.
  • a lipolytic enzyme a proteolytic enzyme, and a carbohydrase Enzymes.
  • 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.
  • lipolytic enzyme examples include lipase and phosphatase
  • examples of the proteolytic enzyme include serine protease, cysteine protease, proteinase K, and pronase
  • examples of the saccharide-degrading enzyme include amylase, cellulase, and N-acetylmuramidase.
  • a “microorganism” is an object to be detected by the method of the present invention, can be detected by a nucleic acid amplification method, and the action of a platinum complex binding to DNA or RNA on a microorganism is a living cell and a dead cell.
  • preferred examples include bacteria, filamentous fungi, yeasts, viruses, and the like.
  • Bacteria include both gram-positive bacteria and gram-negative bacteria.
  • Gram-positive bacteria include Staphylococcus bacteria such as Staphylococcus epidermidis, Streptococcus pneumoniae, Streptcoccus pyogenes Streptcoccus sp. Bacteria, Listeria bacteria such as Listeria monocytogenes, Bacillus ⁇ cereus, Bacillus anthracis, Bacillus anthracis, Mycobacterium tuberculosis ( Mycoba such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium intracellulare Examples include bacteria belonging to the genus Cterium, Clostridium bacteria such as Clostridium botulinum (Clostridium botulinum) and Clostridium perfringens.
  • Gram-negative bacteria include Escherichia bacteria such as Escherichia coli, Enterobacter bacteria such as Cronobacter sakazakii (formerly Enterobacter sakazakii), Citrobacter ⁇ Citrobacter koseri and other Citrobacter bacteria, Klebsiella oxytoca and other intestinal bacteria such as Klebsiella oxytoca, Salmonella bacteria, Vibrio bacteria, Pseudomonas bacteria, Legionella bacteria, Legionella bacteria, etc. Is mentioned.
  • Viruses include viruses such as an influenza virus having an envelope, and noroviruses, rotaviruses, adenoviruses and the like that do not have an envelope and have only a nucleocapsid.
  • cisplatin cis-diammineplatinum (II) dichloride
  • cis-diammine (pyridine) chloroplatinum (II) chloride which are platinum complexes.
  • Active virus (live virus) platinum complexes such as cisplatin having an Envelope is not transmitted, inactive virus (dead virus) with damaged envelope can be inferred easily be transmitted through the platinum complex. Furthermore, as shown in Example 5, it was shown that cisplatin can distinguish between live and dead cells of Staphylococcus aureus. From these results, it is considered that the platinum complex can be used to distinguish between living cells and dead cells for all microorganisms.
  • a “live cell” is a state (Viable-and-Culturable cell state) that can proliferate when cultured under suitable culture conditions and exhibits the metabolic activity of the microorganism.
  • the metabolic activity mentioned here can be exemplified by ATP activity and esterase activity.
  • 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).
  • 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.
  • 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.
  • live cells”, “dead cells” and “damaged cells” mean live cells, dead cells and damaged cells of microorganisms.
  • the unit of the number of living cells, damaged cells, and dead cells is usually expressed by the number of cells (cells) / ml.
  • the number of viable cells can be approximated by the number of colonies formed (cfu / ml (colony forming units / ml)) when cultured under suitable conditions on a suitable plate medium.
  • a standard sample of damaged cells can be prepared by, for example, subjecting a living cell suspension to a heat treatment, for example, a heat treatment in boiling water. In that case, the number of damaged cells is heat-treated. It can be approximated by the cfu / ml of the previous live cell suspension.
  • damaged cells can be prepared in about 50 seconds.
  • a standard sample 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. Measure the transmittance of visible light (wavelength 600nm), that is, turbidity, and compare it with the turbidity of the live cell suspension whose live cell number concentration is known in advance, on a suitable plate medium Can be approximated by the number of colonies formed (cfu / ml).
  • the cell number unit is represented by plaque-forming units (pfu or PFU (plaque-forming units)).
  • 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.
  • detection of living cells includes both determination of the presence or 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.
  • 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.
  • Step a) A platinum complex (hereinafter also referred to as “agent of the present invention” or simply “agent”) is added to the test sample. That is, the microorganism in the test sample is treated with the drug. As will be described later, it is presumed that the drug directly binds to nucleic acid (DNA or RNA) and inhibits the PCR reaction of the target region.
  • the agent of the present invention can be an agent that binds to nucleic acids.
  • the bond between the agent of the present invention and DNA or RNA may be a coordinate bond (covalent bond).
  • the drug has a different action on living cells and damaged cells or dead cells and bovine leukocytes and other somatic cells, leukocytes, platelets, and the like, more specifically, damaged cells rather than living cell walls. Or it is preferable that it is highly permeable to cell walls of dead cells, or somatic cells such as bovine leukocytes, and cell membranes such as leukocytes and platelets.
  • the platinum complex is not particularly limited as long as it has different permeability to the cell wall of living cells, damaged cells, or dead cells, and can bind to nucleic acid in the cells to inhibit the PCR reaction of the target region.
  • a ligand at least 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 2-, 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 -, or R - platinum complex comprising one selected from the like (provided that The above-mentioned “R” represents any saturated or unsaturated organic group).
  • saturated organic group examples include alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, octyl, and cyclooctyl.
  • unsaturated organic group examples include a benzyl group (benzene ring), a naphthyl group (naphthalene ring), an allyl group, and a cyclooctadienyl group. These saturated organic groups and unsaturated organic groups may have a substituent.
  • Examples of the ligand include NH 3 , RNH 2 , halogen element (Cl, F, Br, I, At), carboxylate group, pyridine group, H 2 O, CO 3 2- , OH ⁇ , NO 3 2- , ROH, N 2 H 4 , PO 4 3 ⁇ , R 2 O, RO ⁇ , ROPO 3 2 ⁇ , (RO) 2 PO 2 ⁇ , R 3 P, and RNC are preferable (provided that the expression “R” is used) Are all saturated or unsaturated organic groups).
  • NH 3 , RNH 2 , halogen element, carboxylate group, pyridine group, R 3 P, and RNC are particularly preferable.
  • As the halogen element Cl (chlorine) is preferable.
  • Tables 2 and 1 include metals (soft, , Hard Lewis acids) and ligands (soft, intermediate, hard Lewis bases), although with individual exceptions, soft Lewis acids tend to bind soft Lewis bases and soft Such Lewis acids are said to be easier to bind to soft Lewis bases than hard Lewis bases (see pages 21-22 of the same book).
  • the agent of the present invention is preferably a compound in which a hard or soft Lewis base and platinum are coordinated and more preferably a compound in which a hard Lewis base and platinum are coordinated.
  • Intermediate Lewis base e.g., NO 2 -, Ar-NH 2 [Ar unsaturated organic group including an aromatic ring], N 2, SO 3 2- , N 3 -, or imidazole ring
  • platinum complex contained as can be used in the present invention a platinum complex in which platinum is coordinated with a hard or soft Lewis base and platinum is preferable from the viewpoint of sensitivity or accuracy in determining the viability of microorganisms.
  • dead cells are generally more morphologically damaged than living cells (damaged cells are mildly damaged), and complexes containing hard or soft Lewis bases as ligands are generally more active than live cells. Dead cells (including damaged cells) are considered to be more permeable.
  • the complex is less likely to penetrate into living cells when the positive charge or the negative charge as a whole than when it is nonpolar. Therefore, the drug of the present invention is preferably positively charged or negatively charged as a whole.
  • the compound as a whole is nonpolar, such as cisplatin, permeation into living cells can be significantly suppressed by shortening the action time to some extent (see Examples). Good.
  • platinum complex examples include the following compounds. Cisplatin (cisplatin, cis-diammineplatinum (II) dichloride, cis-diammineplatinum (II) dichloride, or cis-dichlorodiammine platinum (II); cis-DDP), Carboplatin (Carboplatin, cis-diammine (1,1-cyclobutanedicarboxylate) platinum), cis-diammine (pyridine) chloroplatium (II) chloride; cis-DPCP; Dichloro (ethylenediamine) platinum (II), Transplatin (transplatin, trans-diamminedichloroplatinum (II)), Chloro (2,2 ': 6', 2 ''-terpyridine) platinum (II) chloride dihydrate, Bis (acetylacetonato) platinum (II), cis-dichlorobis (pyridine) platinum (II)
  • Preferred examples of the complex include the following compounds. Cisplatin (chemical formula 1, molecular weight 300.04), Carboplatin (chemical formula 2, molecular weight 371.25), cis-diammine (pyridine) chloroplatinum (II) chloride (chemical formula 3, molecular weight 379.14), Dichloro (ethylenediamine) platinum (II) (chemical formula 4, molecular weight 326.10), cis-bis (benzonitrile) dichloroplatinum (II) (chemical formula 5, molecular weight 472.23), Tetrakis (triphenylphosphine) platinum (II) (chemical formula 6, molecular weight 1244.22), Chloroplatinic acid hexahydrate (molecular weight 517.90) Dinitric acid (ethylenediamine) platinum iodide (II) dimer (ethylenediamine) iodoplatinum (II) dimer dinitrate (chemical formula 7, molecular weight 888.17).
  • examples of the platinum complex include 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 such a platinum compound include a platinum compound that forms a macromolecule by a covalent bond between platinum and another element or group.
  • the element or group include halogen elements (Cl, F, Br, I, At), OH ⁇ , NO 3 ⁇ , CH 3 COO ⁇ , PO 4 3 ⁇ , RO ⁇ , CO 3 2 ⁇ , ROPO 3 2 ⁇ .
  • the notation represents a saturated or unsaturated organic group).
  • 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, and chloride. Examples include platinum acid, disulfomethylplatinum, dicyanoplatinum, dithiocyanate platinum, platinum dihydride, and methylplatinum.
  • preferable compounds include platinum chloride, platinum bromide, platinum fluoride, and platinum iodide, and particularly preferable compounds include platinum chloride.
  • platinum chloride include platinum (II) chloride (monomer molecular weight 265.99) and platinum chloride (IV) (monomer molecular weight 336.89).
  • the organic solvent include dimethyl sulfoxide (DMSO), benzonitrile and the like. Examples of the complex obtained by dissolving platinum chloride in DMSO include dichlorobis (dimethylsulfoxide) platinum (II) and tetrakis (dimethylsulfoxide) platinum (II).
  • Examples of the solution containing a substance capable of binding to platinum as a ligand include a harmaline solution, such as an aqueous solution of harmaline hydrochloride, and a diferrocenyl-phosphine solution, such as DMSO in diferrocenyl phosphine. And the like.
  • a harmaline solution such as an aqueous solution of harmaline hydrochloride
  • a diferrocenyl-phosphine solution such as DMSO in diferrocenyl phosphine.
  • platinum-containing macromolecules When such platinum-containing macromolecules are dissolved in these solutions, platinum is rebound to harmaline or diferrocenyl phosphine as a ligand, and the molecular weight is reduced as a platinum complex.
  • the platinum complex thus produced can also be used in the present invention.
  • the platinum complex may be a multimer such as a dimer. Examples of the dimer include dinitric acid (ethylenediamine) platinum iod
  • platinum complexes and platinum compounds that form macromolecules by covalent bonding of platinum and other elements or groups are commercially available (for example, Wako Pure Chemical Industries, Sigma), and can be used.
  • one type of drug may be used alone, or two or more types may be used in combination.
  • 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 cells of a microorganism to be detected and dead cells or damaged cells, and the mixture is left for various periods of time. Thereafter, the bacterial cells are separated by centrifugation or the like, and analyzed by a nucleic acid amplification method, whereby 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.
  • Such conditions include a final concentration of cisplatin of 10 to 3000 ⁇ M, preferably 25 to 3000 ⁇ M, 4 to 43 ° C., and 5 minutes to 2 hours.
  • the final concentration is 10 to 3000 ⁇ M, 4 to 43 ° C., 5 minutes to 2 hours.
  • carboplatin the final concentration is 10 to 3000 ⁇ M, preferably 250 to 3000 ⁇ M, 4 to 43 ° C., 5 minutes to 2 hours.
  • the final concentration is 10 to 3000 ⁇ M, 4 to 43 ° C., 5 minutes to 2 hours.
  • the final concentration is 10 to 3000 ⁇ M, preferably 25 to 3000 ⁇ M, 4 to 43 ° C., 5 minutes to 2 hours.
  • Examples of dichloro (ethylenediamine) platinum (II) include final concentrations of 10 to 3000 ⁇ M, 4 to 43 ° C., and 5 minutes to 2 hours.
  • the final concentration is 10 to 3000 ⁇ M, preferably 100 to 3000 ⁇ M, 4 to 43 ° C., 5 minutes to 2 hours.
  • the final concentration is 10 to 3000 ⁇ M, preferably 25 to 3000 ⁇ M, 4 to 43 ° C., 5 minutes to 2 hours.
  • the final concentration is 10 to 3000 ⁇ M, preferably 400 to 3000 ⁇ M, 4 to 43 ° C., 5 minutes to 2 hours.
  • platinum (II), platinum chloride (IV), or chloroplatinic acid or chloroplatinic acid hexahydrate
  • platinum (II) chloride platinum (IV)
  • the amount of chloroplatinic acid is 10 to 3000 ⁇ M, preferably 10 to 100 ⁇ M, preferably 4 to 43 ° C., 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. .
  • the agent of the present invention is more permeable to the cell walls of dead cells and damaged cells than the cell walls of living cells. Therefore, it is considered that the cell wall and cell membrane of living cells of microorganisms do not substantially permeate within the action time shown above, and the membranes of somatic cells that are damaged or dead cells of microorganisms or somatic cells are permeated. .
  • the drug enters the dead cells of somatic cells and dead cells of microorganisms and cells of damaged cells, and subsequently binds to chromosomal DNA or RNA, so that the DNA or RNA to which the drug is bound is It is presumed that it will no longer be a template for nucleic acid amplification reaction.
  • the target region of chromosomal DNA or RNA is amplified by the nucleic acid amplification method in live cells, whereas in damaged or dead cells, chromosomal DNA or RNA Since the drug binds to the nucleic acid and the nucleic acid amplification reaction is inhibited, live cells can be selectively detected compared to damaged cells and dead cells.
  • the treatment with the agent in step a) may be performed once or may be repeated a number of times.
  • the concentration of the drug is preferably higher in the first drug treatment than in the second time and lower, and lower in the second and subsequent drug processes than in the first time. 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.
  • 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.
  • the platinum element in this drug binds and binds favorably to the nucleic acid adenine (A) and guanine (G), so after the action of this drug, a new nucleic acid or oligonucleotide not containing the target gene should be added.
  • this unreacted drug can be removed (inactivated).
  • Step b) the target region of the DNA or RNA of the microorganism contained in the test sample after the drug treatment is amplified by a nucleic acid amplification method.
  • the DNA or RNA used as a template for nucleic acid amplification may be extracted from a microbial cell, or a sample treated with a drug without extracting nucleic acid from the cell may be used as it is. It is preferable not to extract the nucleic acid.
  • a nucleic acid amplification reaction may be performed by adding an agent that suppresses the action of a nucleic acid amplification inhibitor to a nucleic acid amplification reaction solution containing a test sample. Preferred (see Japanese Patent No. 4825313, WO2011 / 010740).
  • a magnesium salt and an organic acid salt or phosphate it is more preferable to add a magnesium salt and an organic acid salt or phosphate to the nucleic acid amplification reaction solution containing the test sample. Further, it is particularly preferable to add a surfactant to the nucleic acid amplification reaction solution containing the test sample.
  • a surfactant e.g., sodium EDTA
  • a magnesium salt e.g., sodium bicarbonate
  • an organic acid salt or a phosphate e.g., sodium bicarbonate
  • a phosphate e.g., sodium bicarbonate
  • a nucleic acid elongation enzyme may be added at a concentration 2 to 10 times the concentration used in the normal PCR method.
  • a nucleic acid amplification inhibitor is a substance that inhibits a nucleic acid amplification reaction or a nucleic acid extension reaction.
  • the positive charge inhibitor include calcium ions, polyamines, and heme.
  • Examples of the negative charge inhibitor include phenol, phenolic compounds, heparin, and Gram-negative bacterial cell wall outer membrane. Foods and clinical specimens are said to contain many substances that inhibit such nucleic acid amplification reactions.
  • drugs that suppress the action of the nucleic acid amplification inhibitor as described above include albumin, dextran, T4 gene 32 protein, acetamide, betaine, dimethyl sulfoxide, formamide, glycerol, polyethylene glycol, soybean trypsin inhibitor, ⁇ 2-macroglobulin, tetra
  • examples thereof include one or more selected from phosphorylase and lactate dehydrogenase from methylammonium chloride and lysozyme.
  • polyethylene glycol examples include polyethylene glycol 400 and polyethylene glycol 4000.
  • betaine examples include trimethylglycine and its derivatives.
  • phosphorylase and lactate dehydrogenase examples include glycogen phosphorylase and lactate dehydrogenase derived from rabbit muscle.
  • glycogen phosphorylase glycogen phosphorylase b is preferable. In particular, it is preferable to use albumin, dextran, T4 gene 32 protein, or lysozyme.
  • albumin typified by BSA may reduce nucleic acid amplification inhibition by binding to a nucleic acid amplification inhibitor such as heme (the Abu Al- Soud et al.)
  • T4 Gene 32 protein is a single-stranded DNA-binding protein that binds in advance to the single-stranded DNA that is the template in the nucleic acid amplification process and the template is free from degradation by nucleolytic enzymes, thus inhibiting the nucleic acid amplification reaction.
  • Two possibilities are considered that nucleic acid amplification proceeds without being inhibited by binding to a nucleic acid amplification inhibitor similar to BSA (Abu Al-Soud et al.) .
  • BSA, T4 Gene 32 protein, and proteinase inhibitor can reduce proteolytic activity by binding to proteinase and maximize the function of nucleic acid synthase.
  • proteolytic enzymes may remain in milk and blood, and at that time, nucleic acid synthase is degraded by the addition of BSA or proteolytic enzyme inhibitors (soybean trypsin inhibitor or ⁇ 2-macroglyblin).
  • BSA proteolytic enzyme inhibitors
  • Dextran is a polysaccharide generally synthesized by lactic acid bacteria using glucose as a raw material. It has been reported that a similar polysaccharide-peptide complex called mucin adheres to the intestinal mucosa (Ruas-Madiedo, P., Applied and Environmental Microbiology, 74: 1936-1940, 2008), and dextran is a negative charge inhibitor. It is presumed that there is a possibility of binding to these inhibitory substances by adsorbing in advance (adsorbed on nucleic acid synthase) or positive charge inhibitory substance (adsorbed on nucleic acid). In addition, it is inferred that lysozyme is adsorbed to a nucleic acid amplification inhibitor thought to be contained in a large amount in milk (Abu Al-Soud et al.).
  • albumin T4 gene 32 protein
  • dextran a substance represented by albumin
  • lysozyme drugs that suppress the action of nucleic acid amplification inhibitors.
  • Albumin includes bovine serum albumin, ovalbumin, milk albumin, human serum albumin and the like. Of these, bovine serum albumin (BSA) is preferred. Albumin may be a purified product and may contain other components such as globulin as long as the effects of the present invention are not impaired. Moreover, a fraction may be sufficient.
  • the concentration of albumin in the test sample (nucleic acid amplification reaction solution) is, for example, usually 0.0001 to 1% by mass, preferably 0.01 to 1% by mass, more preferably 0.2 to 0.6% by mass. is there.
  • dextran examples include dextran 40 and dextran 500. Of these, dextran 40 is preferred.
  • concentration of dextran in the test sample (nucleic acid amplification reaction solution) is, for example, usually 1 to 8%, preferably 1 to 6%, more preferably 1 to 4%.
  • the concentration of T4 gene 32 protein (for example, Roche: also called gp32) in the test sample (nucleic acid amplification reaction solution) is usually 0.01 to 1%, preferably 0.01 to 0.1%. Preferably, the content is 0.01 to 0.02%.
  • Lysozyme is lysozyme derived from egg white.
  • concentration of lysozyme in the test sample (nucleic acid amplification reaction solution is, for example, usually 1 to 20 ⁇ g / ml, preferably 6 to 15 ⁇ g / ml, more preferably 9 to 13 ⁇ g / ml.
  • 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 ( And anionic surfactants such as sodium dodecyl sulfate) and cationic surfactants such as stearyldimethylbenzylammonium chloride.
  • Triton X-100 polyethylene glycol tert-octylphenyl ether
  • Triton P-40 octylphenyl-polyethylene glycol
  • Tween 20 polyethylene glycol sorbitan monolaurate
  • Tween 40 polyethylene glycol sorbitan monopalmitate
  • Tween 60 polyethylene glycol sorbitan monostearate
  • Tween 80 polyethylene glycol sorbitan monooleate
  • Brij as Brij56 (polyoxyethylene (10) cetyl ether), Brij58 (polyoxyethylene (20) cetyl ether) and the like.
  • the type and concentration of the surfactant in the sample to be tested is not particularly limited as long as it promotes the permeation of the PCR reagent into the cells of the microorganism and does not substantially inhibit the nucleic acid amplification reaction.
  • the nucleic acid amplification reaction solution is usually 0.0005 to 0.01%, preferably 0.001 to 0.01%, more preferably 0.001 to 0.005%, and more preferably 0.001 to 0%. 0.002%.
  • Nonidet P-40 is usually 0.001 to 1.5%, preferably 0.002 to 1.2%, more preferably 0.9 to 1.1. %
  • Tween 20 is usually 0.001 to 1.5%, preferably 0.002 to 1.2%, more preferably 0.9 to 1.1%
  • Brij56 and Brij58 are usually It is 0.1 to 1.5%, preferably 0.4 to 1.2%, more preferably 0.7 to 1.1%.
  • 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.
  • magnesium salts include magnesium chloride, magnesium sulfate, magnesium carbonate and the like.
  • concentration of the magnesium salt in the test sample (nucleic acid amplification reaction solution) is, for example, usually 1 to 10 mM, preferably 2 to 6 mM, more preferably 2 to 5 mM.
  • organic acid salts include salts of citric acid, tartaric acid, propionic acid, butyric acid, and the like.
  • the salt include sodium salt and potassium salt.
  • pyrophosphate etc. are mentioned as a phosphate. These may be one kind, or a mixture of two or more kinds.
  • the concentration of the organic acid salt or phosphate in the test sample (nucleic acid amplification reaction solution) is, for example, usually 0.1 to 20 mM, preferably 1 to 10 mM, more preferably 1 to 5 mM in total amount (patent) No. 4127847, see WO2007 / 094077).
  • the extraction method is not particularly limited as long as the extracted DNA can function as a template in nucleic acid amplification, and is performed according to a commonly used method for extracting microbial DNA. be able to.
  • nucleic acid When nucleic acid is not extracted from the test sample, DNA or RNA present in the cell in the presence of a drug that suppresses the function of the nucleic acid amplification inhibitor and, if necessary, other components Is amplified by a nucleic acid amplification method.
  • a microbial cell suspension or a suspension of microbial cells treated with proteolytic enzyme, lipolytic enzyme, glycolytic enzyme, etc. is used, and nucleic acid is not extracted for template preparation. It is preferable.
  • the target region is amplified by a nucleic acid amplification method by an ordinary method using the extracted DNA or RNA as a template.
  • the nucleic acid amplification method preferably includes a step of heat denaturation of nucleic acid at a high temperature, for example, 90 to 95 ° C., preferably 93 to 95 ° C., more preferably 94 to 95 ° C.
  • Nucleic acid amplification methods include PCR methods (White, TJ et al., Trends Genet., 5, 185 (1989)), LAMP method (Loop-Mediated Isothermal Amplification: Principle and application of novel gene amplification method (LAMP method), Nobutomi, Nobuyoshi, Hase Satoshi, BIO INDUSTRY, Vol.18, No.2, 15-23, 2001), SDA method (Strand Displacement Amplification: Edward L. Chan, et al., Arch. Pathol. Lab. Med., 124: 1649-1652, 2000), LCR method (Ligase Chain Reaction: Barany, F., Proc. Natl. Acad.cadSci.
  • TMA method Transcription-Mediated -Amplification: Sarrazin C. et al., J. Clin. Microbiol., Vol.39: p.2850-2855 (2001)
  • TRC method Transcription-Reverse Transcription-Concerted method : Nakaguchi Y. et al., J Clin. Microbiol., Vol.42: p.4248-4292 (2004)
  • HC method Hybrid Capture: Nazarenko I., Kobayashi L. et al., J. Virol.
  • PCR method is particularly preferably used, but is not limited thereto.
  • the “target region” refers to a region of chromosomal DNA or RNA that can be amplified by a nucleic acid amplification method using a primer used in the present invention, and can detect a microorganism to be detected. If it does not restrict
  • 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.
  • 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 is usually 50 to 5000 bases or 50 to 3000 bases. In the method of the present invention, even if the target region is shorter than the conventional method, for example, about 400 bases in length, it is possible to distinguish between live cells, dead cells, and damaged cells.
  • 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.
  • 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.
  • a part of the 16S rRNA gene specific to Cronobacter / Sakazaki can be amplified.
  • a commercially available primer for 16S rRNA gene amplification may also be used.
  • the target region includes a pathogenic gene.
  • pathogenic genes include Listeria ricin O (hlyA) gene of Listeria, enterotoxin (enterotoxin) gene and invasion (invA) gene of Salmonella, pathogenic E. coli O-157, O-26, O-111, etc.
  • Verotoxin gene Enterobacter or Cronobacter bacterium outer-membrane-proteinA (ompA) gene (Cronobacter sakazaki) and macromolecular synthesis (MMS) operon (Cronobacter sakazaki), Legionella bacterium invasion protein (mip) gene, heat-resistant hemolytic toxin gene of Vibrio parahaemolyticus, heat-resistant hemolytic toxin-like toxin gene, Shiga and intestinal invasive Escherichia coli ipa gene (invasion plasmid antigen gene), invE gene (invasion gene), Staphylococcus aureus enterotoxin gene, Bacillus cerevisiae Mouse bacteria cereulide (emetic toxin) gene and enterotoxin gene, various toxin genes such as Clostridium botulinum and the like.
  • ompA Cronobacter sakazaki
  • MMS macromolecular synthesis
  • hemagglutinin (H protein) gene 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.
  • 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.
  • a primer common to multiple types of microorganisms living cells of multiple types of microorganisms in a test sample can be detected.
  • a primer specific to a specific bacterium used, a living cell of a specific bacterial species in a test sample can be detected.
  • the conditions for the nucleic acid amplification reaction are specific amplification in accordance with the principle of each nucleic acid amplification method (PCR method, LAMP method, SDA method, LCR method, TMA method, TRC method, HC method, SMAP method, microarray method, etc.) As long as this occurs, it is not particularly limited and can be set as appropriate.
  • Step c) Analyze amplification products amplified by the nucleic acid amplification method.
  • the analysis of the amplification product is performed following step b) or simultaneously with step b), depending on the nucleic acid amplification method employed in step b).
  • step c) can be performed simultaneously with step b).
  • the analysis method is not particularly limited as long as the nucleic acid amplification product can be detected or quantified, and examples thereof include electrophoresis.
  • real-time PCR Nogva et al., Appl. Environ. Microbiol., Vol. 66, 2000, pp. 4266-4271, Nogva et al., Appl. Environ Microbiol., Vol. 66, 2000, pp. 4029-4036
  • the amount and size of the nucleic acid amplification product can be evaluated. Further, according to the real-time PCR method, the PCR amplification product can be rapidly quantified.
  • the change in fluorescence intensity is generally a noise level and is equal to zero until the number of amplification cycles is 1 to 10. Therefore, these are regarded as sample blanks with zero amplification products, and their standard deviation SD is calculated.
  • a value obtained by multiplying the SD value by 10 is referred to as a threshold value, and the number of PCR cycles that first exceeds the threshold value is referred to as a cycle threshold value (Ct value).
  • the presence or absence of an amplification product can also be determined by analyzing the melting temperature (TM) pattern of the amplification product.
  • TM melting temperature
  • analysis of nucleic acid amplification products can be performed using a standard curve that shows the relationship between the amount of microorganisms prepared using a standard sample of the identified microorganism and the amplification product.
  • 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.
  • kit of the present invention is a kit for distinguishing and detecting a living cell of a microorganism in a test sample from a dead cell or a damaged cell by a nucleic acid amplification method. Including.
  • the kit of the present invention may further include a primer for amplifying a target region of DNA or RNA of a microorganism to be detected by a nucleic acid amplification method.
  • the kit of the present invention may further contain a drug that suppresses the action of a nucleic acid amplification inhibitor, a magnesium salt, and an organic acid salt or phosphate, or two or more of these. In a more preferred embodiment, it may contain all of an agent that suppresses the action of a nucleic acid amplification inhibitor, a magnesium salt, and an organic acid salt or phosphate. Furthermore, it is more preferable that the nucleic acid elongation enzyme contains a concentration 2 to 10 times the concentration used in normal PCR or normal real-time PCR. Moreover, the kit of the present invention may further contain a surfactant.
  • the kit of the present invention can be used for carrying out the method of the present invention.
  • 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 nucleic acid amplification reaction is preferably PCR method, LAMP method, SDA method, LCR method, TMA method, TRC method, HC method, SMAP method, or microarray method.
  • the crosslinking agent and the medium are the same as those described in the method of the present invention.
  • Preferred examples of the platinum complex contained in the kit of the present invention are the same as the compounds described for the method of the present invention.
  • drugs that suppress the action of nucleic acid amplification inhibitors include albumin, dextran, and T4 gene 32 protein, acetamide, betaine, dimethyl sulfoxide, formamide, glycerol, polyethylene glycol, soybean trypsin inhibitor, ⁇ 2-macroglobulin, tetramethyl Any one or plural kinds selected from ammonium chloride, lysozyme, phosphorylase, and lactate dehydrogenase can be exemplified.
  • examples of the magnesium salt include magnesium chloride, magnesium sulfate, magnesium carbonate and the like.
  • organic acid salt examples include salts of citric acid, tartaric acid, propionic acid, butyric acid and the like.
  • examples of the salt include sodium salt and potassium salt.
  • pyrophosphate etc. are mentioned as a phosphate. These may be one kind, or a mixture of two or more kinds.
  • the enzyme can decompose non-microorganism cells, protein colloid particles, fats and carbohydrates, etc. present in the test sample, and does not damage the living cells of the target microorganism. If it is, it will not restrict
  • 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.
  • lipolytic enzyme examples include lipase and phosphatase
  • examples of the proteolytic enzyme include serine protease, cysteine protease, proteinase K, and pronase
  • examples of the saccharide-degrading enzyme include amylase, cellulase, and N-acetylmuramidase.
  • the kit of the present invention may further contain a diluent, a reaction solution for reaction with a platinum complex, an enzyme and reaction solution for nucleic acid amplification, instructions describing the method of the present invention, and the like.
  • Example 1 Examination of PCR amplification suppression effect by cisplatin and carboplatin Cisplatin for Cronobacter sakazakii (former name: Enterobacter sakazakii), which is representative of coliform bacteria And the inhibitory effect on PCR amplification by carboplatin was examined.
  • the strain can be obtained from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America).
  • Purified DNA was obtained from 1 ml of the culture solution using a DNA extraction kit (QuickGene SP kit DNA tissue SP-DT; Fujifilm Corp., Tokyo, Japan). Then, it adjusted to 50 ng / 100 microliters with the sterilized water in the microtube. Assuming that the chromosomal DNA is 5 ⁇ 10 ⁇ 15 g (5 fg) per cell, the DNA in 100 ⁇ l of the DNA solution corresponds to 10 7 cells of Cronobacter Sakazaki, and the bacterial cell concentration is 10 8 cells / cell. It can be regarded as equivalent to ml.
  • the purity of the purified DNA was evaluated by OD 260 / OD 280 .
  • Carboplatin (Sigma, molecular weight 371.25) 3.60 mg (9.7 ⁇ mol) was accurately weighed and dissolved in 970 ⁇ l of sterilized water (carboplatin is insoluble in DMSO) to prepare a 10 mM aqueous solution. This solution was diluted with sterilized water to prepare 5 ⁇ M, 50 ⁇ M, 200 ⁇ M, 500 ⁇ M, 1000 ⁇ M, 2 mM, and 5 mM carboplatin aqueous solutions.
  • each sample was placed under light shielding during this period, but it may be placed in a normal laboratory environment (the same applies to the following examples). Further, as a non-treated sample, the same treatment was performed using 100 ⁇ l of physiological saline or sterilized water instead of each platinum complex physiological saline solution as a positive control.
  • PCR amplification a master mix for real-time PCR shown in Table 1 below was prepared.
  • Primer 16S forward C. sakazakii 16S rRNA gene detection forward primer (5'-TAACAGGGAGCAGCTTGCTGCTCTG-3 ': SEQ ID NO: 1)
  • Primer 16S reverse C. sakazakii 16S rRNA gene detection reverse primer (5' -CGGGTAACGTCAATTGCTGCGGT-3 ': SEQ ID NO: 2) was used as a PCR primer.
  • the fragment length of the amplified rRNA gene was 426 bp.
  • 16S TaqMan probe an oligonucleotide having the sequence of SEQ ID NO: 3 (5′-CCGCATAACGTCTACGGACCAAA-3 ′) was used.
  • the nucleotide sequence information of each primer and 16S TaqMan probe was obtained from Kang, Eun Sil et al., J. Microbiol. Biotechnol. 17: 516-519, 2007. Real-time PCR amplification was performed twice.
  • Real-time PCR was carried out under the following PCR thermal cycle conditions using a real-time PCR apparatus (StepOnePlus Real-Time PCR System; Applied Biosystems, the same applies to other examples). 1) 95 °C, 20 seconds (1 cycle) 2) 95 °C, 5 seconds; 55 °C, 10 seconds; 72 °C, 30 seconds (45 cycles) As a negative control, 5 ⁇ l of sterilized water was used as a template.
  • Example 2 Identification of live and dead cells (Lactose broth suspension) of Cronobacter sakazaki by cisplatin Clarification of live and dead bacteria using cisplatin in Cronobacter sakazaki We examined the conditions for this.
  • Test Material and Culture Method 1-1) Cronobacter sakazaki ATCC29544 was cultured with BHI broth at 37 ° C. for 16 hours. 1 ml of the above culture solution is cooled and centrifuged (4 ° C., 3,000 ⁇ G, 10 minutes), and after removing the supernatant, 1 ml of physiological saline is added to the pellet. A live cell suspension (1.2 ⁇ 10 8 CFU / ml) was prepared by doubling dilution.
  • the mixture is cooled and centrifuged (4 ° C., 10,000 ⁇ G, 5 minutes), the supernatant is removed, the pellet is suspended in 150 ⁇ l of sterilized water, stirred well, and subjected to the same cooling and centrifugation. Was removed.
  • the obtained pellet (corresponding to 5 ⁇ l of cell suspension) was used as a sample for PCR amplification.
  • PCR amplification A concentrated solution of a mixture of drugs that suppresses the action of a nucleic acid amplification inhibitor necessary for efficient PCR without extracting nucleic acids from cells (this concentrated solution is used as a concentrated direct buffer component). , Described as cDBC).
  • 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; Wako Pure Chemicals, Osaka), Brij58 (P5884-100G; Sigma) stock solutions were mixed to the concentrations shown in Table 3, and cDBC was mixed. Prepared.
  • each stock solution of 16.6% Brij58, 4.8% BSA, 333 mM TSC, 1 M MgCl 2 , 2.5 mg / ml lysozyme is in a volume of 250 ⁇ l, 200 ⁇ l, 15 ⁇ l, 15 ⁇ l, 20 ⁇ l, respectively.
  • 500 ⁇ l of cDBC (10 ⁇ DBC) shown in Table 3 can be prepared.
  • qPCR quantitative polymerase
  • MgCl 2 equivalent to 2 mM was estimated to be equivalent to 5 mM.
  • Brij 56, MgCl 2 and TSC were dissolved in sterilized water, autoclaved (121 ° C., 20 minutes), cooled to room temperature, and used as a stock solution.
  • a stock solution was prepared with sterilized water, and sterilized by filtration through a 0.22 ⁇ m filter to obtain a stock solution.
  • real-time PCR performed without extraction of nucleic acids from cells is referred to as “direct real-time PCR”.
  • a master mix master mix for direct real-time PCR
  • PCR amplification was performed using the same primers as in Example 1 and using the PCR amplification sample as a template.
  • Real-time PCR amplification was performed twice.
  • real-time PCR was performed under the following PCR thermal cycle conditions. 3) 95 °C, 2 minutes (1 cycle) 4) 95 °C, 5 seconds; 55 °C, 10 seconds; 72 °C, 20 seconds (45 cycles)
  • FIG. 1 shows the result of electrophoresis of the PCR final amplification product (cis-DDP concentration 2000 ⁇ M).
  • Example 3 Discrimination of Cronobacter Sakazaki Live and Dead Cells (Saline Suspension) with Cisplatin
  • Cl ions were used to efficiently bind cisplatin to dead cell chromosomes.
  • Lactose broth which is a suitable environment for living cells, was examined (however, the cell suspension diluted with lactose broth is suspended in physiological saline, so compared to physiological saline. 1/10 concentration of Cl ions). Even if cisplatin once permeates the cell wall / cell membrane of living cells, it was intended to activate the ATP-dependent efflux pump and to discharge this drug out of the living cells from various transporters. Therefore, in this Example 3, Lactose broth was replaced with physiological saline for examination.
  • Test Material and Culture Method 1-1) Live cell suspension (6.4 ⁇ 10 7 CFU / ml) and damaged cells / death of Cronobacter sakazaki ATCC 29544 using physiological saline in the same manner as in Example 2 above. A cell suspension (6.4 ⁇ 10 7 cells / ml, hereinafter referred to as “dead cell suspension”) was prepared. The above-mentioned Chronobacter / Sakazaki live cell suspension or dead cell suspension was diluted 10-fold with physiological saline (6.4 ⁇ 10 6 cells / ml), and 90 ⁇ l of each was subjected to the following test.
  • each cisplatin saline solution was added to 90 ⁇ l of the above live cell suspension or dead cell suspension, and kept at 37 ° C. for 30 minutes in a constant temperature water bath. Then, cool and centrifuge (4 ° C, 10,000 x G, 5 minutes), remove the supernatant, suspend the pellet in 150 ⁇ l of sterilized water, stir well, and then perform the same cooling centrifuge. Qing was removed. The obtained pellet (corresponding to 5 ⁇ l of cell suspension) was used as a sample for PCR amplification.
  • Example 4 Discrimination of Cronobacter / Sakazaki Live Cells / Dead Cells (Sterilized Water Suspension) Using Cisplatin
  • a suspension of Cronobacter / Sakazaki live cells and dead cells prepared in sterile water was used. To identify each.
  • Test Material and Culture Method 1-1) Live cell suspension (1.2 ⁇ 10 8 cfu / ml) and dead cell suspension of Cronobacter sakazaki ATCC 29544 using physiological saline in the same manner as in Example 2 above. A liquid (1.2 ⁇ 10 8 cells / ml) was prepared. The above-mentioned Chronobacter / Sakazaki live cell suspension or dead cell suspension was diluted 10-fold with sterilized water (1.2 ⁇ 10 7 cells / ml), and 90 ⁇ l of each was subjected to the following test.
  • Live cells could be distinguished from dead cells at a cisplatin concentration of 25 ⁇ M. From the results of Examples 2 to 4, although the method of the present invention can be carried out with or without Cl ions, the lower the Cl ion concentration in the cisplatin treatment, the lower the concentration of cisplatin. It was shown that it is possible to distinguish between live cells and dead cells.
  • Example 5 Identification of Staphylococcus aureus live cells and dead cells by cisplatin
  • Staphylococcus aureus a gram-positive bacterium having no outer membrane in the outermost shell and having peptidoglycan was used.
  • Staphylococcus aureus Staphylococcus aureus
  • live cells and dead cells were identified by cisplatin.
  • Test Material and Culture Method 1-1) Using Staphylococcus aureus ATCC 6538P strain, 1 ml of live cell suspension (9.4 ⁇ 10 7 cfu / ml) and dead cell suspension (9.4 ⁇ 10 7 cells / ml) were prepared. 50 ⁇ l of the above Staphylococcus aureus live cell suspension (9.4 ⁇ 10 7 cfu / ml) or dead cell suspension (9.4 ⁇ 10 7 cells / ml) was used for the following test.
  • each cisplatin physiological saline solution was added to 50 ⁇ l of the above live cell suspension or dead cell suspension, and kept in a constant temperature bath at 37 ° C. for 10 minutes. Then, cool and centrifuge (4 ° C, 10,000 x G, 5 minutes), remove the supernatant, suspend the pellet in 150 ⁇ l of sterilized water, stir well, and then perform the same cooling centrifuge. Qing was removed. The obtained pellet (corresponding to 5 ⁇ l of cell suspension) was used as a sample for PCR amplification.
  • PCR amplification A master mix for direct real-time PCR was prepared with the composition shown in Table 8 below. Specifically, SYBRPremix Ex Taq TM PCR Master Mix (2 ⁇ ) (Takara-Bio Co., Ltd, Otsu, Japan) is used as a qPCR buffer, and Bacteria Screening PCR Kit (Takara as a reverse primer for PCR amplification and reverse primer). -Bio) Attached Primer Mix BS (5 ⁇ M each) was used. This primer mix is a primer that enables detection of both Staphylococcus and Bacillus, and the length of the amplified gene is about 380 bp. Real-time PCR was performed twice.
  • real-time PCR was performed under the following PCR thermal cycle conditions. 5) 95 °C, 1 minute (1 cycle) 6) 95 °C, 10 seconds; 59 °C, 30 seconds; 72 °C, 30 seconds (40 cycles)
  • Staphylococcus aureus a Gram-positive bacterium, can also distinguish between live and dead cells by cisplatin treatment.
  • Example 6 Discrimination of living and dead cells of Cronobacter sakazaki by cis-diammine (pyridine) chloroplatinum (II) chloride
  • cis-diammine (pyridine) chloro is used as a platinum complex other than cisplatin.
  • live platinum (II) chloride cis-diammine (pyridine) chloroplatium (II) chloride; cis-DPCP
  • live and dead cells of Cronobacter sakazaki were identified.
  • Test Material and Culture Method 1-1) Live cell suspension (1.0 ⁇ 10 6 cfu / ml) and dead cell suspension of Cronobacter sakazaki ATCC 29544 using physiological saline in the same manner as in Example 3 above. A liquid (1.0 ⁇ 10 6 cells / ml) was prepared, and 90 ⁇ l of each was used for the following test.
  • each sample was placed under light shielding as in the case of cisplatin, but may be placed in a normal laboratory environment.
  • PCR amplification As in Example 2 above, TaqManFast Universal PCR Master Mix (2 ⁇ ) (Applied Biosystems Pty Ltd.) was used as a real-time PCR buffer, and a predetermined amount of cDBC (10 ⁇ DBC) was used as the buffer. Real-time PCR amplification (40 cycles) was performed twice using the master mix for direct real-time PCR prepared by addition.
  • a positive control 5 ⁇ l of C. sakazakii live cell suspension (1.2 ⁇ 10 8 CFU / ml) was used as a template.
  • As a negative control 5 ⁇ l of sterilized water was used as a template.
  • Example 7 Identification of living and dead cells of Cronobacter sakazaki by Dichloro (ethylenediamine) platinum (II) In Example 7, Dichloro (ethylenediamine) platinum (II) was used as a platinum complex. We identified live and dead Sakazaki cells.
  • Test Material and Culture Method 1-1) Live cell suspension of Cronobacter sakazaki ATCC 29544 (1.5 ⁇ 10 9 cfu / ml) and damaged cells / death using physiological saline in the same manner as in Example 2 above. A cell suspension (1.5 ⁇ 10 9 cells / ml, hereinafter collectively referred to as “dead cell suspension”) was prepared. The above-mentioned Chronobacter / Sakazaki live cell suspension or dead cell suspension was diluted 100-fold with sterilized water (1.5 ⁇ 10 7 cells / ml), and 90 ⁇ l of each was subjected to the following test.
  • the supernatant was removed by cooling centrifugation (15,000 ⁇ G, 10 minutes, 4 ° C.), the inside of the microtube was washed with 1000 ⁇ l of 70% cold ethanol, and then cooled and centrifuged in the same manner. After removing the supernatant, ethanol was removed by vacuum drying (5 minutes), 20 ⁇ l of TE buffer was added to the precipitate, and the mixture was allowed to stand at 37 ° C. for 1 hour. Thereafter, the sample was gently stirred, and 5 ⁇ l thereof was used as a PCR amplification sample.
  • Example 8 Identification of living and dead cells of microorganisms by platinum chloride
  • platinum chloride which is defined as a covalently bonded macromolecule, was used to determine Cronobacter Sakazaki's We examined the discrimination between live and dead cells.
  • Cronobacter sakazaki ATCC29544 was cultured at 37 ° C. for 16 hours using BHI broth. 1 ml of the above culture solution is cooled and centrifuged (4 ° C., 3,000 ⁇ G, 10 minutes). After removing the supernatant, 1 ml of sterilized water is added to the pellet, and further diluted 100-fold with sterilized water. A cell suspension (9.4 ⁇ 10 6 CFU / ml) was prepared. Further, 1 ml of the above live cell suspension was dispensed into a 1.5 ml microtube (Eppendorf, Hamburg, Germany), immersed in boiling water for 3 minutes, and then rapidly cooled.
  • a 1.5 ml microtube Eppendorf, Hamburg, Germany
  • each platinum (II) chloride or platinum (IV) chloride solution was added to 90 ⁇ l of a Chronobacter / Sakazaki live cell suspension or dead cell suspension, and kept in a constant temperature bath at 37 ° C. for 30 minutes. After that, cool and centrifuge (4 ° C, 3,000 x G, 5 minutes), remove the supernatant, suspend the pellet in 150 ⁇ l of sterilized water, stir well, and perform the same cooling and centrifuge. Qing was removed. The obtained pellet (corresponding to 5 ⁇ l) was used as a sample for PCR amplification.
  • Example 9 Identification of living and dead cells of Escherichia coli by cis-bis (benzonitrile) dichloroplatinum (II), tetrakis (triphenylphosphine) platinum (II), chloroplatinic acid hexahydrate, and (ethylenediamine) iodoplatinum (II) dimer dinitrate
  • a ligand capable of coordinating bonding via a nitrogen atom with respect to benzonitrile a platinum complex having a phenylphosphine ligand, a chloroplatinic acid hexahydrate that is a hexacoordination complex, and further Using a dimer of a platinum complex (a dimer having two platinum elements in one complex), a new study was made to distinguish live and dead cells of E.coli. It was decided to test whether clear identification was possible.
  • Test materials and culture method 1-1) Using E. coli JCM1649 strain, sterilized water and live cell suspension (2.1 x 10 7 CFU / ml) and dead cell suspension (2.1 x 10 7 cells / ml) ; Immersed in boiling water for 2 minutes), and each 90 ⁇ l was subjected to the following test.
  • the JCM1649 strain can be obtained from RIKEN BioResource Center, Microbial Materials Development Department (3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture 305-0074).
  • tetrakis (triphenylphosphine) platinum (II) (Sigma) 8.97 mg (7.21 ⁇ mol) was accurately weighed and dissolved in 144.2 ⁇ l of DMSO to prepare a 50 mM solution. This solution was diluted with physiological saline to prepare 100 ⁇ M, 250 ⁇ M, and 1000 ⁇ M solutions.
  • chloroplatinic acid hexahydrate (Sigma) 10.51 mg (20.29 ⁇ mol) was accurately weighed and dissolved in 405.8 ⁇ l DMSO to prepare a 50 mM solution. This solution was diluted with physiological saline to prepare 20 ⁇ M, 250 ⁇ M, and 1000 ⁇ M solutions.
  • (ethylenediamine) iodoplatinum (II) dimer dinitrate (Sigma) 5.60 mg (6.31 ⁇ mol) was accurately weighed and dissolved in 631 ⁇ l of DMSO to prepare a 10 mM solution. This solution was diluted with physiological saline to prepare 100 ⁇ M, 500 ⁇ M, 1000 ⁇ M, 2 ⁇ m, and 4 ⁇ mM solutions.
  • PCR amplification A master mix for direct real-time PCR was prepared with the composition shown in Table 13 below. Specifically, Taq DNA Polymerase with Standard Taq Buffer (New England Biolabs Japan Inc .; M0273S) was used as a real-time PCR buffer, and Taq polymerase was added 4 times as much as usual, and cDBC (10 x DBC) was added to this buffer. ) was added in a predetermined amount to prepare a master mix for direct real-time PCR. 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.
  • NEB New England Biolabs product
  • Primer ENT-16S forward Enterobacteriaceae-specific 16S rRNA gene detection forward primer (5'-GTTGTAAAGCACTTTCAGTGGTGAGGAAGG-3 ': SEQ ID NO: 4)
  • Primer ENT-16S reverse Intestine A reverse primer (5′-GCCTCAAGGGCACAACCTCCAAG-3 ′: SEQ ID NO: 5) for detecting 16S rRNA gene specific for Enterobacteriaceae was used as a PCR amplification primer (both primers were outsourced to Nippon Gene).
  • the fragment length of the rRNA gene to be amplified was 424 bp.
  • an oligonucleotide having the sequence of 5 '-/ 56-FAM / AACTGCATC / ZEN / TGATACTGGCAGGCT / 3lABkFQ / -3' (SEQ ID NO: 6) was used. .
  • This probe was commissioned by Integrated DNA Technologies with specifications that a fluorescent material 56-FAM was placed at the 5 ′ end of the oligonucleotide, a ZEN at the center and a quenching dye 31ABkFQ at the 3 ′ end.
  • the base sequence information on the primers of SEQ ID NOs: 4 and 5 was obtained from Nakano, S. et al., J. Food Prot.
  • 16S rRNA gene information of each enterobacteriaceae group selected the complementary region in the enterobacteriaceae group from the GenBank database (http://www.ebi.ac.uk/genbank/) .
  • real-time PCR was performed twice under the following PCR thermal cycle conditions. 1) 95 °C, 20 seconds (1 cycle) 2) 95 °C, 5 seconds; 60 °C, 1 minute (40 cycles)
  • Foods and clinical specimens contain components that can be ligands for platinum complexes.
  • bovine somatic cells and bovine mammary epithelial cells are highly likely to retain nucleic acids such as chromosomes and rRNA in dead cells, although they are dead cells.
  • nucleic acids such as chromosomes and rRNA
  • platinum complexes such as cisplatin have already been suggested to use proteins (amino acids containing sulfur such as cysteine residues, or each amino acid can be a candidate as a ligand) as a ligand.
  • Some platinum complexes may also be coordinated with various casein and whey proteins of the above (Briplatin Injection 10 mg, Briplatin Injection 25 mg, Briplatin Injection 50 mg BRIPLATIN INJECTION Product Information Overview ”, (See Stall Myers, December 2007). Therefore, it was examined whether live cells and dead cells contained in sterilized milk can be distinguished using a platinum complex.
  • Test Material and Culture Method 1-1) Prepare a viable cell suspension (2.0 ⁇ 10 9 CFU / ml) of E. coli JCM1649 strain using physiological saline, and further 10 times, 10 3 with physiological saline. fold, diluted 10 4 fold to prepare a live cell-suspension liquid. A 10-fold diluted solution was dispensed into a microtube, immersed in boiling water for 3 minutes, and then rapidly cooled to prepare a dead cell suspension (2.0 ⁇ 10 8 cells / ml).
  • Collect 12 ml of commercially available milk (sterilized: hereinafter referred to as “sterilized milk”), inoculate 20 ⁇ l of the 10 3 fold and 10 4 fold dilutions / live cell suspension, 4.0 ⁇ 10 4 CFU each / 12 ml sterilized milk, 4.0 ⁇ 10 3 CFU / 12 ml sterilized milk to be inoculated with live cells was prepared.
  • 20 ⁇ l of the 10-fold diluted dead cell suspension was inoculated into 12 ml of sterilized milk to prepare dead cell inoculated sterilized milk to be 4.0 ⁇ 10 6 CFU / 12 ml sterilized milk.
  • the commercially available milk (sterilized milk) used in the test was confirmed by a culture method to confirm that E. coli live cells were below the detection limit ( ⁇ 1 CFU / 2.22 ml).
  • this pasteurized milk is inoculated with dead cells of known concentration of E. coli, and only the step of recovering bacteria from the milk described later without any chemical treatment is performed. Thereafter, the same direct real time as in Example 9 described above is performed. What confirmed by preliminary examination that the dead cells detected by PCR were 10 3 cells / ml at the maximum was used as a test material.
  • the dead cell inoculated sterilized milk inoculated with E. coli dead cells at a concentration of 4.0 x 10 6 CFU / 12 ml sterilized milk has negligible levels of E. coli dead cells originally contained. .
  • E. coli and other coliforms including Enterobacteriaceae, including Salmonella
  • the dead cells are 4.0 ⁇ 10 6 CFU / Since the level is 12 ml of pasteurized milk, in this method, PCR amplification from the dead cells needs to be completely suppressed.
  • Each of the sterilized milk inoculated with each live cell and the sterilized milk inoculated with a dead cell is cooled and centrifuged (4 ° C., 3,000 ⁇ G, 5 minutes), and the supernatant is removed by decantation to give a live cell milk pellet (a), Dead cell milk pellet (a) was collected.
  • Each collected milk pellet (a) is suspended in 10 ml of PBS, sabinase (Protease from Bacillus sp .; Sigma; ⁇ 16 U / g) 30 ⁇ l is added, and then in a 37 ° C.
  • the primer for PCR amplification used in Example 10 is a primer targeting 16S rRNA gene that enables comprehensive detection of Enterobacteriaceae (Enterobacteriaceae), It can be used not only for identification of dead cells, but also for test for determining whether live or dead cells of Enterobacteriaceae in pasteurized milk.
  • living cells of microorganisms can be detected and distinguished from dead cells or damaged cells by a simple process.
  • simple and rapid food and biological samples, wiped samples, industrial water, environmental water, wastewater, and other environmental microorganisms can be easily distinguished by nucleic acid amplification methods. .
  • platinum complexes used in the present invention for example, cisplatin is used as a clinical medicine and is considered to be less dangerous than drugs such as EMA. Furthermore, a preferable platinum complex is cheaper than drugs such as EMA and is industrially advantageous.

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Abstract

Selon l'invention, des cellules vivantes d'un micro-organisme dans un échantillon de test sont distinguées des cellules mortes ou des cellules endommagées et détectées par les étapes suivantes : a) une étape dans laquelle un complexe du platine est ajouté à l'échantillon de test ; b) une étape dans laquelle un domaine cible de l'ADN ou de l'ARN du micro-organisme dans l'échantillon de test est amplifié par une technique d'amplification d'acide nucléique ; et c) une étape dans laquelle le produit d'amplification est analysé.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015081891A1 (fr) 2013-12-06 2015-06-11 Baikang (Suzhou) Co., Ltd Pro-fragments bioréversibles pour médicaments contenant de l'azote et de l'hydroxyle
JP2015167531A (ja) * 2014-03-10 2015-09-28 日清オイリオグループ株式会社 飼料中のサルモネラの迅速検査方法及びそのシステム
JP2016220628A (ja) * 2015-05-29 2016-12-28 森永乳業株式会社 微生物検出法及び微生物検出キット
WO2017009999A1 (fr) * 2015-07-16 2017-01-19 森永乳業株式会社 Procédé de détection de micro-organismes et kit de détection de micro-organismes
WO2017010001A1 (fr) * 2015-07-16 2017-01-19 森永乳業株式会社 Procédé de détection de micro-organismes et kit de détection de micro-organismes
CN109207616A (zh) * 2018-11-14 2019-01-15 北京工业大学 一种检测活性污泥中死菌或休眠菌的方法
WO2019188552A1 (fr) * 2018-03-27 2019-10-03 森永乳業株式会社 Procédé de mesure de cellules de micro-organismes et/ou de virus

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WO2007094077A1 (fr) * 2006-02-17 2007-08-23 Kyushu University Procede et kit de detection de microorganismes

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WO2007094077A1 (fr) * 2006-02-17 2007-08-23 Kyushu University Procede et kit de detection de microorganismes

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015081891A1 (fr) 2013-12-06 2015-06-11 Baikang (Suzhou) Co., Ltd Pro-fragments bioréversibles pour médicaments contenant de l'azote et de l'hydroxyle
JP2015167531A (ja) * 2014-03-10 2015-09-28 日清オイリオグループ株式会社 飼料中のサルモネラの迅速検査方法及びそのシステム
JP2016220628A (ja) * 2015-05-29 2016-12-28 森永乳業株式会社 微生物検出法及び微生物検出キット
WO2017009999A1 (fr) * 2015-07-16 2017-01-19 森永乳業株式会社 Procédé de détection de micro-organismes et kit de détection de micro-organismes
WO2017010001A1 (fr) * 2015-07-16 2017-01-19 森永乳業株式会社 Procédé de détection de micro-organismes et kit de détection de micro-organismes
WO2019188552A1 (fr) * 2018-03-27 2019-10-03 森永乳業株式会社 Procédé de mesure de cellules de micro-organismes et/ou de virus
CN109207616A (zh) * 2018-11-14 2019-01-15 北京工业大学 一种检测活性污泥中死菌或休眠菌的方法

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