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WO2017009999A1 - Procédé de détection de micro-organismes et kit de détection de micro-organismes - Google Patents

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

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Publication number
WO2017009999A1
WO2017009999A1 PCT/JP2015/070406 JP2015070406W WO2017009999A1 WO 2017009999 A1 WO2017009999 A1 WO 2017009999A1 JP 2015070406 W JP2015070406 W JP 2015070406W WO 2017009999 A1 WO2017009999 A1 WO 2017009999A1
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Prior art keywords
iridium
nucleic acid
cells
amplification
test sample
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English (en)
Japanese (ja)
Inventor
隆志 副島
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Morinaga Milk Industry Co Ltd
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Morinaga Milk Industry Co Ltd
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Priority to PCT/JP2015/070406 priority Critical patent/WO2017009999A1/fr
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Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a method for 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 as described above enter the cell, they bind to or intercalate with DNA to inhibit the action of topoisomerase or DNA gyrase (enzyme), or DNA As a result, 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.
  • the PCR target region is a region having a certain length or more, for example, a length of 900 bases (bp) or more. Is generally used. However, in order to amplify a region of 900 bp or more by PCR compared with a target region of about 80 to 200 bp currently used in general quantitative PCR, each cycle takes several times longer, and It cannot be said that there is no problem in quantitativeness.
  • Non-Patent Document 1 discloses an iridium complex that binds to histidine and emits fluorescence.
  • the luminescent material includes DNA, a conductive polymer having at least one amino group that can be protonated in the main chain or side chain, intertwined with DNA, and a cationic functional group that binds to the anion portion of DNA.
  • a luminescent material is known that includes a lipid compound having a fluorescent compound and a metal complex such as fluorescent iridium that binds to the anion portion of DNA (Patent Document 5). In this luminescent material, the metal complex is bonded to the anion portion of DNA to which no conductive polymer or lipid compound is bonded by electrostatic interaction.
  • the present inventors can distinguish between live cells and dead cells by nucleic acid amplification method by using platinum complexes (Patent Document 6) and palladium complexes (Patent Document 7) as in EMA and the like. Is disclosed.
  • An object of the present invention is to provide a method capable of detecting living cells of a microorganism in a simple process and, in a preferred embodiment, even if the 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.
  • an iridium complex is used, it is possible to detect living cells of microorganisms without requiring light irradiation, cooling, and light-shielding environment, and in a preferred embodiment, a region having a relatively short chain length is used as a target region. Even when set, 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 living cells of microorganisms in a test sample by distinguishing them from dead cells and / or damaged cells, and includes the following steps: a) adding an iridium 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. Further, in the above method, it is preferable that the amplification of the target region is performed by adding an agent that suppresses the action of a nucleic acid amplification inhibitor, a magnesium salt, and an organic acid salt or phosphate to the test sample. It is said. Moreover, 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 method has a preferred embodiment in which the nucleic acid amplification method is a PCR method, a LAMP method, an ICAN method, an SDA method, an LCR method, a TMA method, a TRC method, an HC method, an SMAP method, or a microarray method.
  • 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 and / or a damaged cell by a nucleic acid amplification method, and includes the following elements: 1) Iridium complex or An iridium compound that forms an iridium complex when dissolved 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; 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 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 iridium complex includes 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 2 — includes a ligand selected from the group (provided that the expression “R” represents a saturated or unsaturated organic group).
  • the ligand is 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 ⁇ , R 2 S, From R 2 P ⁇ , R 3 P, RS ⁇ , CN ⁇ , RSH, RNC, (RS) 2 PO 2 ⁇ , (RO) 2 P (O) S ⁇ , SCN ⁇ , CO, H ⁇ , and R ⁇ It is a preferred embodiment to be selected from the group consisting of
  • the iridium complex is di- ⁇ -chlorobis [( ⁇ -cycloocta-1,5-diene) iridium (I)] and 2-hydroxy-N-pyridine (pentamethylcyclopenta
  • a preferred embodiment is selected from dienyl) iridium (III) dichloride.
  • 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 and / or damaged cells, and includes the following steps. . a) adding an iridium 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, blister contents, and the like.
  • 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, or foods made from milk or 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 an action of an iridium complex that binds to DNA or RNA on a microorganism is a living cell or 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, such as Staphylococcus aureus and Staphylococcus epidermidis; Micrococcus; Streptococcus spp., Streptococcus spp. Bacillus genus Bacillus (subtilis), Bacillus Basubtilis, Bacillus licheniformis (preferably vegetative cells); Clostridium genus such as Clostridium botulinum and Clostridium perfringens; Mycobacterium tuberculosis and M.
  • Mycobacterium genus mycobacteria and atypical mycobacteria group
  • Mycobacterium intracellulare Mycobacterium abium; Rae bacteria; Actinomyces; Nocardia; Nocardiopsis; Actinomadura; Streptomyces Genus Derumatofirusu genus; Eubacterium; Corynebacterium; Propionibacterium genus, and the like.
  • Gram-negative bacteria include Legionella spp .; Salmonella spp .; Enterohemorrhagic Escherichia coli including O-157, O-26, O-11, O-145; Campylobacter spp .; Alcobacter spp .; Helicobacter spp.
  • Pseudomonas genus such as fungi; Burkholderia genus; Acinetobacter genus; Alcaligenes genus; Chryseobacterium genus; Moraxella genus; Cochella genus; Haemophilus influenzae and other Haemophilus genus; Pasteurella genus; Chromobacterium genus; Streptobacillus genus Bacter, Hafnia, Prezio Enterobacteriaceae such as Monas, Proteus, Providencia, Morganella, Serratia, etc .; Vibrio; Eromonas; Bacteroides; Prevotella; Porphyroonas; Fusobacterium; Leptotricia; Genus Treponema; 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 .
  • Examples 1 and 3 below it was shown that treatment with an iridium complex makes it possible to distinguish between live and dead cells of Escherichia coli, which is a Gram-negative bacterium. Further, as shown in Examples 2 and 4, it was shown that live cells and dead cells of Staphylococcus aureus can be distinguished by iridium complexes. From these results, it is considered that the iridium complex can be used to distinguish between living cells and dead cells for all microorganisms. Furthermore, it is considered that a virus having the outermost envelope of the same component as the outer cell membrane of Gram-negative bacteria can also be used for distinguishing between live cells and dead cells.
  • virus particles are also referred to as “cells” for convenience.
  • PMA propidium monoazide
  • enteric viruses without envelopes.
  • PMA sodium monoazide
  • PMA is originally a drug that allows clear distinction between bacterial live and dead cells (A. Nocker et al., J. Microbiol. Methods.
  • nucleocapsids are available.
  • the iridium complex is similar to PMA in intestinal viruses that have only nucleocapsids (the enveloped viruses are morphologically very similar to gram-negative bacteria). Therefore, there is a high possibility that the infectious type and the non-infectious type can be clearly determined.
  • 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. A microorganism with almost no damage to the cell wall.
  • 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). 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. “Dead cells” with respect to viruses refers to 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 present invention is not limited to detection of living cells, but living cells It is intended to provide a method for detecting microorganisms that can be distinguished from dead cells and / or damaged cells.
  • the unit of the number of living cells, damaged cells, and dead cells is usually expressed by the number of cells / ml.
  • the number of viable cells can be approximated by the number of colonies formed (cfu / ml or CFU / ml (colony forming units / ml)) when cultured under suitable culture conditions on a suitable plate medium.
  • a standard sample of damaged cells and / or dead cells can be prepared, for example, by subjecting a living cell suspension to heat treatment, for example, heat treatment in boiling water, in which case damaged cells and / or dead cells are prepared.
  • the number of cells can be approximated by cfu / ml of the live cell suspension before the heat treatment.
  • the heating time in boiling water for preparing damaged cells and / or dead cells varies depending on the type of microorganism. For example, in the case of bacteria described in the examples, it is possible to prepare damaged cells in about 50 seconds. it can. If the heating time is lengthened, the ratio of dead cells becomes higher than damaged cells.
  • a standard sample of damaged cells and / or dead cells can also be prepared by antibiotic treatment.
  • the number of damaged cells and / or dead cells is determined by using a live cell suspension as an antibiotic. After removing the antibiotic, 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. It can be approximated by the number of colonies formed (cfu / ml) when cultured under suitable culture conditions on a suitable plate medium.
  • 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 distinguishing it from a dead cell and / or a damaged cell” means that a living cell is selectively detected as compared with a dead cell and / or a damaged cell. The “discrimination between live cells and dead cells and / 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) An iridium 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. The drug is presumed to inhibit the PCR reaction of the target region by binding or interfering directly with nucleic acid (DNA or RNA) or indirectly via protein or the like.
  • the drug has a different action on living cells and damaged or dead cells, bovine leukocytes and other somatic cells, leukocytes, platelets, etc., more specifically, than the cell walls of live cells. It is preferable that it is highly permeable to cell walls of damaged cells or dead cells, or somatic cells such as bovine leukocytes, and cell membranes such as leukocytes and platelets.
  • the iridium complex is not particularly limited as long as it has different permeability to the cell wall between living cells, damaged cells, and / 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 ⁇ , 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 ⁇ ,
  • 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 benzyl group (benzene ring), naphthyl group (naphthalene ring), allyl group, cyclooctadienyl group, cyclooctene group, pentamethylcyclopentadienyl group, and indenyl 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 (—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 ⁇ , R 2 S, R 2 P ⁇ , R 3
  • a halogen element Cl, F, Br, I, At
  • carboxylate —CO—O—
  • iridium complex examples include the following compounds. Di- ⁇ -chlorobis [( ⁇ -cycloocta-1,5-diene) iridium (I)] (Di- ⁇ -chlorobis [( ⁇ -cycloocta-1,5-diene) iridium (I)]) C 16 H 24 Cl 2 Ir 2 2-hydroxy-N-pyridine (pentamethylcyclopentadienyl) iridium (III) dichloride C 15 H 20 Cl 2 IrNO (Acetylacetonato) dicarbonyliridium (I)) C 7 H 7 IrO 4 (Acetylacetonato) (1,5-cyclooctadiene) iridium (I) C 13 H 19 IrO 2 (Acetylacetonato) (1,5-cyclooctadiene) iridium (I) C 13 H 19 IrO 2 (Acetylacetonato) (1,5-cycloocta
  • Preferred examples of the complex include the following compounds.
  • Di- ⁇ -chlorobis [( ⁇ -cycloocta-1,5-diene) iridium (I)] also known as: bis (1,5-cyclooctadiene) diiridium (I) dichloride (bis (1,5-cyclooctadiene) diiridium (I) dichloride)) (Chemical Formula 1, Molecular Weight (MW) or Formula Weight (FW): 671.70) 2-Hydroxy-N-pyridine (pentamethylcyclopentadienyl) iridium (III) dichloride (Chemical Formula 2, Molecular Weight (MW) or Formula Weight (FW): 493.45)
  • the iridium complex examples include an iridium complex formed 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 such an iridium compound include an iridium compound that forms a macromolecule by a covalent bond between iridium 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 iridium compound include iridium chloride, iridium bromide, iridium fluoride, iridium iodide, iridium hydroxide, iridium nitrate, iridium carbonate, iridium acetate, dimethoxyiridium, iridium methoxyphosphate, iridium phosphate, and chloride.
  • Examples include iridium acid, disulfmethyl iridium, dicyano iridium, dithiocyanate iridium, iridium dihydride, methyl iridium, iridium oxide, iridium pentachloride (IV) diammonium (diammonium hexachloroiridium (IV)), and the like.
  • preferable compounds include iridium chloride, iridium bromide, iridium fluoride, and iridium iodide, and particularly preferable compounds include iridium chloride.
  • Examples of iridium chloride include iridium (III) chloride and iridium (IV) chloride.
  • Examples of the organic solvent include dimethyl sulfoxide (DMSO) and benzonitrile.
  • Examples of the complex obtained by dissolving iridium chloride in DMSO include dichlorobis (dimethylsulfoxide) iridium (III).
  • a harmaline solution for example, an aqueous solution of harmaline hydrochloride
  • a diferrocenyl-phosphine solution for example, DMSO of diferrocenyl phosphine And the like.
  • the iridium complex may be a multimer such as a dimer (a dimer having two iridium elements in one complex).
  • dimer a dimer having two iridium elements in one complex.
  • dimer include Di- ⁇ -chlorobis [( ⁇ -cycloocta-1,5-diene) iridium (I)].
  • iridium complexes and iridium compounds that form macromolecules by covalent bonding of iridium and other elements or groups are commercially available (for example, Wako Pure Chemical Industries, Sigma), and can be used.
  • Wako Pure Chemical Industries, Sigma Wako Pure Chemical Industries, Sigma
  • 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, after adding various concentrations of the drug to the suspension of living and dead cells and / or damaged cells of the microorganism to be detected, and for various times, the cells are separated by centrifugation or the like, By analyzing by a nucleic acid amplification method, it is possible to determine conditions that make it easy to distinguish between live cells and dead cells and / or damaged cells. 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.
  • the final concentration is 20 to 3000 ⁇ M, preferably 25 to 300 ⁇ M, 4 to 43 ° C. Examples are 5 minutes to 2 hours.
  • 2-Hydroxy-N-pyridine (pentamethylcyclopentadienyl) iridium (III) dichloride the final concentration is 20 to 3000 ⁇ M, preferably 50 to 300 ⁇ M, 4 to 43 ° C., 5 minutes to 2 hours.
  • conditions can be set in accordance with these iridium complexes.
  • 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 wall of dead cells and / or damaged cells than the cell wall of living cells. Therefore, within the action time shown above, the cell wall / cell membrane of living cells of microorganisms does not substantially permeate, and the cell membrane of damaged cells or dead cells of microorganisms or somatic cells that are dead cells permeate. Conceivable. As a result, the drug enters the somatic and microbial dead cells as well as the damaged cells and subsequently binds or interferes directly or indirectly with chromosomal DNA or RNA, resulting in It is presumed that DNA or RNA to which a drug is bound does not serve as a template for nucleic acid amplification reaction.
  • chromosomal DNA or RNA When drugs permeate preferentially to damaged or dead cells over live cells, the target region of chromosomal DNA or RNA is amplified by nucleic acid amplification in live cells, whereas in damaged or dead cells, chromosomal DNA or The drug binds or interferes with RNA directly or indirectly, and the nucleic acid amplification reaction is inhibited. Therefore, 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 unreacted drug may be added between the previous drug process and the subsequent drug process.
  • 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.
  • 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.
  • a surfactant it is particularly preferable to add a surfactant to the nucleic acid amplification reaction solution containing the test sample.
  • a surfactant, a magnesium salt, or an organic acid salt or phosphate to the amplification reaction solution in addition to the agent that suppresses the action of the nucleic acid amplification inhibitor, Alternatively, any two or more of them can be used in combination, and it is particularly preferable to add all of them.
  • the order of addition of the agent that suppresses the action of the nucleic acid amplification inhibitor, the surfactant, the magnesium salt, and the organic acid salt or phosphate is not limited, and they may be added simultaneously. If necessary, the nucleic acid elongation enzyme may be added at a concentration higher than that used in the normal PCR method, for example, 2 to 10 times.
  • 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 methylammonium chloride, lysozyme, phosphorylase, and lactate dehydrogenase.
  • 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%, preferably 0.01 to 1%, more preferably 0.2 to 0.6%.
  • 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.
  • the 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.
  • Non-ionic surfactants such as Triton (registered trademark of Union Carbide), Nonidet (registered trademark of Shell), Tween (registered trademark of ICI), Brij (registered trademark of ICI), etc.
  • anionic surfactants such as SDS (sodium dodecyl sulfate) and cationic surfactants such as stearyldimethylbenzylammonium chloride.
  • Triton is Triton X-100 (polyethylene glycol tert-octylphenyl ether), Nonidet is Nonidet P-40 (octylphenyl-polyethylene glycol), etc.
  • Tween is Tween 20 (polyethylene glycol sorbitan monolaurate), Tween 40 (polyethylene glycol sorbitan monopalmitate), Tween 60 (polyethylene glycol sorbitan monostearate), Tween 80 (polyethylene glycol sorbitan monooleate), etc.
  • Brij as Brij56 (polyoxyethylene (10) cetyl ether), Brij58 (polyoxyethylene (20) cetyl ether) and the like.
  • the kind and concentration of the surfactant in the nucleic acid amplification reaction solution are not particularly limited as long as they promote the penetration of the PCR reagent into the cells of the microorganism and substantially inhibit the nucleic acid amplification reaction.
  • the range of 0.0005 to 0.01% is preferable when an anionic surfactant is used, and the range of 0.0005 to 0.01% is preferable when a cationic surfactant is used.
  • SDS for example, it is usually 0.0005 to 0.01%, preferably 0.001 to 0.01%, more preferably 0.001 to 0.005%, more preferably 0.00. 001 to 0.002%.
  • the range of 0.001 to 1.5% is preferable.
  • Nonidet P-40 it is usually in the range of 0.001 to 1.5%, preferably 0.002 to 1.2%, more preferably 0.9 to 1.%. 1%.
  • Tween 20 Tween 40, Tween 60, or Tween 80, it may be usually in the range of 0.001 to 1.5%, preferably 0.002 to 1.2%, more preferably 0.9. -1.1%.
  • Brij56 or Brij58 it is usually in the range of 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 a test sample, DNA or RNA present in cells in the presence of a drug that suppresses the action 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 (Polymerase chain reaction: White, TJet et al., Trends Genet, 5, 185 (1989)), LAMP method (Loop-Mediated Isothermal Amplification: New gene amplification method (LAMP method)) Principles and Applications, Nobutomi Tsuyoshi, Hase Satoshi, BIO INDUSTRY, Vol.18, No.2, 15-23, 2001), ICAN method (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids: Masamitsu Hamada et al., Bedside ICAN Development of Chlamydia / Phosphorus Gene Detection Reagents by Method, 2002, 51st Annual Meeting of the Japanese Society for Medical Examination, 121, Hiroyuki Mukai, Development and Application of the ICAN Method, 2002, 14th Hokkaido Transfusion Symposium, 20), SDA method (Strand Displacement Amplification: Edward L.
  • HC method Hybrid Capture: Nazarenko I., Kobayashi L. et al., J. Virol. Methods, vol.154: p. 76-81, 2008
  • SMAP method Smart Amplification Process, Smart Amp method; Mitani Y., et al., Nature Methods, vol.4, No.3, p.257-262 (2007)
  • microarray method Richard P. Spence, et al., J. Clin. Microbiol., Vol.46, No.5, .p.1620-1627, 2008
  • PCR methods include quantitative PCR methods (Quantitative PCR or Real-TimeilPCR: VanGuilder HD, et al., Biotechniques, 2008, Apr; 44 (5), 619-26, Spackman E., et al., Methods Mol Biol ., 2008, Vol.436, p.19-26), RT-PCR method (Reverse Transcription PCR: Freeman WM, et al., Biotechniques, 1999, Jan; 26 (1), 112-22, 124-5) Real-time PCR method (Nogva et al., Appl. Environ.
  • 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 a preferred embodiment of the method of the present invention, it is possible to distinguish between live cells and dead cells and / or damaged cells even if the target region is shorter than the conventional method, for example, about 400 bases in length.
  • 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 genus or Cronobacter genus outer-membrane-protein) A (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 (invasion plasmid antigen gene) and invE gene (invasion gene) , Staphylococcus aureus enterotoxin gene, Bacillus Reus bacteria cereulide (emetic toxin) gene and enterotoxin gene, various toxin genes such as Clostridium botulinum and the like.
  • ompA Enterobacter genus or Cronobacter genus outer-me
  • 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 nucleic acid amplification reaction conditions are specific according to the principle of each nucleic acid amplification method (PCR method, LAMP method, ICAN method, SDA method, LCR method, TMA method, TRC method, HC method, SMAP method, microarray method, etc.) As long as general amplification occurs, there is no particular limitation, and it 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). For example, in the case of real-time PCR, 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.
  • 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 living cells of microorganisms in a test sample from dead cells and / or damaged cells by a nucleic acid amplification method. Including complexes.
  • 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 may be a PCR method (including quantitative PCR method, RT-PCR method, real-time PCR method), LAMP method, ICAN method, SDA method, LCR method, TMA method, TRC method, HC method, SMAP method, or The microarray method is preferred.
  • the drug such as a crosslinking agent is the same as that described in the method of the present invention.
  • kits 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 can further contain a diluent, a reaction solution for reaction with an iridium complex, an enzyme and reaction solution for nucleic acid amplification, instructions describing the method of the present invention, and the like.
  • Example 1 Identification of living and dead cells of Escherichia coli by Di- ⁇ -chlorobis [( ⁇ -cycloocta-1,5-diene) iridium (I)]
  • an iridium complex is used. E. coli live cells and dead cells were identified.
  • Di- ⁇ -chlorobis [( ⁇ -cycloocta-1,5-diene) iridium (I)] which is an iridium complex dimer (a dimer having two iridium elements in one complex), was used as the iridium complex. .
  • test Materials and Methods 1-1) A live cell suspension (1.2 ⁇ 10 7 cfu / ml) of E. coli JCM1649 strain was prepared using sterilized water. 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 ⁇ l of each of these live cell suspensions and dead cell suspensions was subjected to the following test.
  • the JCM1649 strain can be obtained from JCM (National Institute of Physical and Chemical Research, BioResource Center, Microbial Materials Development Office (3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture 305-0074)).
  • test sample 10 ⁇ l of each of the above iridium complex solutions was added to 90 ⁇ l of the live cell suspension or 90 ⁇ l 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 ⁇ l of cell suspension) was used as a PCR amplification sample.
  • 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 solution is used as a concentrated direct component, cDBC Was prepared).
  • 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 1, and cDBC was mixed. Prepared.
  • Brij 58, MgCl 2 and TSC were dissolved in sterilized water, autoclaved (121 ° C., 20 minutes), cooled to room temperature, returned 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 shown in Table 2 without performing extraction of nucleic acids from cells is hereinafter referred to as “direct real-time PCR”.
  • a master mix master mix for direct real-time PCR
  • the above-mentioned Taq DNA Polymerase with Standard Taq Buffer is used as a qPCR buffer, 4 times the amount of Taq polymerase is used as usual, and a predetermined amount of cDBC (10 x DBC) is added to this buffer.
  • a 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.
  • NEB New England Biolabs product
  • Primer ENT-16S forward Enterobacteriaceae-specific 16S rRNA gene detection forward primer (5'-GTTGTAAAGCACTTTCAGTGGTGAGGAAGG-3 ': SEQ ID NO: 1)
  • Primer ENT-16S reverse Intestine A reverse primer (5′-GCCTCAAGGGCACAACCTCCAAG-3 ′: SEQ ID NO: 2) for detection of 16S rRNA gene specific for Enterobacteriaceae was used as a PCR primer (both primers were outsourced to Nippon Gene).
  • the fragment length of the amplified rRNA gene is 424 bp.
  • an oligonucleotide having the sequence of SEQ ID NO: 3 (5 '-/ 56-FAM / AACTGCATC / ZEN / TGATACTGGCAGGCT / 3lABkFQ / -3') was used. .
  • This probe was consigned and manufactured by Integrated DNA Technologies with a specification that a fluorescent substance 56-FAM was placed at the 5 ′ end of the oligonucleotide, a quencher dye of 31ABkFQ was placed at the center and ZEN at the center, and 31ABkFQ.
  • the nucleotide sequence information on the primers of SEQ ID NOs: 1 and 2 was obtained from Nakano, S. et al., J. Food Prot. 66: 1798-1804, 2003, and the nucleotide sequence of ENA-16S TaqMan probe of SEQ ID NO: 3 Sequence information 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 °C, 20 seconds (1 cycle) 2) 95 °C, 5 seconds; 60 °C, 1 minute (40 cycles) As a negative control, 5 ⁇ l of sterilized water was used as a template.
  • Table 3 shows the results of real-time PCR.
  • No Agent indicates a negative control.
  • Example 2 Identification of live and dead cells of Staphylococcus aureus by Di- ⁇ -chlorobis [( ⁇ -cycloocta-1,5-diene) iridium (I)] Using iridium complex dimers, it has been shown that it is possible to clearly distinguish between live and dead cells of E. coli, a representative gram-negative bacterium. In this example, it was examined whether live and dead cells of S. aureus, which is a gram-positive bacterium, can be clearly distinguished by the same iridium complex dimer.
  • Test Materials and Methods 1-1) A live cell suspension (4.5 ⁇ 10 7 cfu / ml) of S. aureus ATCC 6538P strain was prepared using sterilized water. A part of this live cell suspension was immersed in boiling water for 3 minutes to prepare a dead cell suspension (4.5 ⁇ 10 7 cells / ml). 90 ⁇ l of each of these live cell suspensions and dead cell suspensions was subjected to the following test.
  • test sample with iridium complex 10 ⁇ l of each of the above iridium complex solutions is added to 90 ⁇ l of the live cell suspension or 90 ⁇ l of the dead cell suspension, and 15 ° C. at 37 ° C. in a constant temperature bath. Hold for a minute. Thereafter, the mixture was cooled and centrifuged (4 ° C., 15,000 ⁇ G, 5 minutes), and the supernatant was removed. The pellet was washed with 1 ml of sterile water. The washed pellet (corresponding to 5 ⁇ l of cell suspension) was used as a PCR amplification sample.
  • PCR amplification A direct real-time PCR (DqPCR) master mix was prepared with the composition shown in Table 4 below. Specifically, SYBRPremix Ex Taq TM PCR Master Mix (2 ⁇ ) (Takara-Bio Co., Ltd, Otsu, Japan) is used as a real-time PCR buffer, and Bacteria Screening PCR Kit as a PCR amplification forward primer and reverse primer. Primer Mix BS (5 ⁇ M each) attached to (Takara-Bio) 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. 1) 95 °C, 1 minute (1 cycle) 2) 95 °C, 10 seconds; 59 °C, 30 seconds; 72 °C, 30 seconds (40 cycles)
  • a negative control 5 ⁇ l of sterilized water was used as a template.
  • Table 5 shows the results of real-time PCR.
  • S. aureus which is a gram-positive bacterium
  • iridium complex (dimer) treatment
  • Example 3 Identification of live and dead cells of E. coli by 2-Hydroxy-N-pyridine (pentamethylcyclopentadienyl) iridium (III) dichloride E. coli and S. aureus gram-negative bacteria, gram-positive bacteria live and dead cells can be clearly distinguished. In this example, it was examined whether live cells and dead cells of E. coli can be distinguished using an iridium complex monomer.
  • Test Material and Method 1 A live cell suspension (2.1 ⁇ 10 7 cfu / ml) of E. coli JCM1649 strain was prepared using sterilized water. A part of this live cell suspension was immersed in boiling water for 3 minutes to prepare a dead cell suspension (2.1 ⁇ 10 7 cells / ml). 90 ⁇ l of each of these live cell suspensions and dead cell suspensions was subjected to the following test.
  • DqPCR Direct real-time PCR
  • Table 6 shows the results of real-time PCR.
  • Example 4 Discrimination of live and dead cells of S. aureus by 2-Hydroxy-N-pyridine (pentamethylcyclopentadienyl) iridium (III) dichloride
  • Example 3 live cells of E. coli were treated with an iridium complex monomer. It was shown that it was possible to clearly distinguish between the cells and dead cells. In this example, it was examined whether live cells and dead cells of S. aureus could be clearly distinguished by the same iridium complex monomer.
  • Test Materials and Methods 1-1) A live cell suspension (6.6 ⁇ 10 7 cfu / ml) of S. aureus ATCC 6538P strain was prepared using sterilized water. A portion of this live cell suspension was immersed in boiling water for 3 minutes, and 90 ⁇ l of each of these live cell suspensions or dead cell suspensions prepared for dead cell suspensions was subjected to the following test. .
  • test sample with iridium complex 10 ⁇ l of each of the above iridium complex solutions is added to 90 ⁇ l of the live cell suspension or 90 ⁇ l of the dead cell suspension, and 15 ° C. at 37 ° C. in a constant temperature bath. Hold for a minute. Thereafter, the mixture was cooled and centrifuged (4 ° C., 15,000 ⁇ G, 5 minutes), and the supernatant was removed. The pellet was washed with 1 ml of sterile water. The washed pellet (corresponding to 5 ⁇ l of cell suspension) was used as a PCR amplification sample.
  • Table 7 shows the results of real-time PCR.
  • S. aureus which is a gram-positive bacterium
  • iridium complex monomer
  • living cells of microorganisms can be detected and distinguished from dead cells and / 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. .
  • the compound represented by Chemical Formula 2 described above is used as a metal catalyst in the field of organic chemistry or organic synthetic chemistry, and is less dangerous than a drug such as EMA. Conceivable. Furthermore, preferred iridium complexes are cheaper than drugs such as EMA and are industrially advantageous.

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  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne la détection de cellules vivantes de micro-organismes dans un échantillon test, et leur distinction par rapport à des cellules mortes et/ou des cellules endommagées au moyen des étapes suivantes : a) une étape durant laquelle un complexe d'indium est ajouté à l'échantillon test ; b) une étape durant laquelle des régions cibles d'ADN ou d'ARN des micro-organismes inclus dans l'échantillon test sont amplifiées à l'aide d'un procédé d'amplification d'acide nucléique ; et c) une étape durant laquelle le produit d'amplification est analysé.
PCT/JP2015/070406 2015-07-16 2015-07-16 Procédé de détection de micro-organismes et kit de détection de micro-organismes Ceased WO2017009999A1 (fr)

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WO2017009999A1 true WO2017009999A1 (fr) 2017-01-19

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014021352A1 (fr) * 2012-08-03 2014-02-06 森永乳業株式会社 Procédé de détection de micro-organismes et trousse de détection de micro-organismes
WO2014021351A1 (fr) * 2012-08-03 2014-02-06 森永乳業株式会社 Procédé de détection de micro-organismes et trousse de détection de micro-organismes
JP2015136340A (ja) * 2014-01-23 2015-07-30 森永乳業株式会社 微生物検出法及び微生物検出キット
JP2015139434A (ja) * 2014-01-30 2015-08-03 森永乳業株式会社 微生物検出法及び微生物検出キット

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014021352A1 (fr) * 2012-08-03 2014-02-06 森永乳業株式会社 Procédé de détection de micro-organismes et trousse de détection de micro-organismes
WO2014021351A1 (fr) * 2012-08-03 2014-02-06 森永乳業株式会社 Procédé de détection de micro-organismes et trousse de détection de micro-organismes
JP2015136340A (ja) * 2014-01-23 2015-07-30 森永乳業株式会社 微生物検出法及び微生物検出キット
JP2015139434A (ja) * 2014-01-30 2015-08-03 森永乳業株式会社 微生物検出法及び微生物検出キット

Non-Patent Citations (2)

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Title
HISAMATSU, Y. ET AL.: "Design and Synthesis of Amphiphilic and Luminescent Tris-Cyclometalated Iridium(III) Complexes Containing Cationic Peptidesas Inducers and Detectors of Cell Death via a Calcium-Dependent Pathway", BIOCONJUGATE CHEM., vol. 26, no. 5, May 2015 (2015-05-01), pages 857 - 879, XP055344971 *
TAKASHI SOEJIMA ET AL.: "Rapid methods to distinguish live from dead microorganism", BULLETIN OF JAPAN DAIRY TECHNICAL ASSOCIATION, vol. 62, 2013, pages 1 - 25 *

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