JPWO2014021352A1 - Microorganism detection method and microorganism detection kit - Google Patents

Abstract

Through the following steps, the living cells of the microorganism in the test sample are identified and detected as dead cells or damaged cells. a) a step of adding a palladium complex to the test sample, b) a step of amplifying a DNA or RNA target region of a microorganism contained in the test sample by a nucleic acid amplification method, and c) a step of analyzing an amplification product.

Description

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

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

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

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

  As the PCR template, nucleic acids extracted from microbial cells contained in the test sample have been conventionally used, but the function of the nucleic acid amplification inhibitor is suppressed without extracting the nucleic acids from the cells. A method of rapidly detecting viable bacteria by performing PCR in the presence of a drug has been disclosed (Patent Document 4).

  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.

  In the conventional methods, in order to distinguish viable bacteria from dead bacteria and damaged bacteria, 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. 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.

  On the other hand, after a sample is treated with a crosslinking agent, a medium is added to the sample and heated, and then the treatment with the crosslinking agent is performed again (Patent Document 3), or 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.

  By the way, platinum complexes such as cisplatin (cisplatin, cis-diammineplatinum (II) dichloride, cis-diammineplatinum (II) dichloride) and 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. In particular, 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.

  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 Document 3).

  In addition, regarding palladium, which is a platinum group element, basic studies have been made on palladium complexes such as diamminedichloro-palladium (II) in anticipation of antiviral and antitumor effects ( Non-patent document 4), so far there has been no actual use in clinical practice.

Patent No. 4340734 International Publication No. 2007/094077 International Publication No. 2009/022558 International Publication No. 2011/010740

Rosenberg, B. et al., Nature, 205: 698-699, 1965 Lovejoy. K.S. et al., PNAS, 105 (26): 8902-8907, 2008 “Briplatin Injection 10 mg, Briplatin Injection 25 mg, Briplatin Injection 50 mg BRIPLATIN INJECTION Product Information Summary”, Bristol-Myers KK, December 2007 Graham, R.D. et al., J. Inorg. Nucl. Chem., 41: 1245-1249, 1979

  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 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. Was examined. And, when a palladium complex is used, 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 a target region However, the present inventors have found that living cells of microorganisms can be detected with high accuracy and have completed the present invention.

That is, the present invention is a method for detecting a living cell of a microorganism in a test sample by distinguishing it from a dead cell or a damaged cell, and includes the following steps:
a) adding a palladium 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.

In the method, the amplification of the target region is preferably performed without extracting nucleic acid from cells.
Moreover, the said method makes it a preferable aspect that amplification of the said target area | region is performed within microbial 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 | region is performed in presence of surfactant.

Moreover, the said method makes it the 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.
Moreover, the said method makes it the preferable aspect that the said target region is a 50-5000 base target region.
In addition, 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. .
In the above method, 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.
Further, the method is preferably such that the PCR method is performed by a real-time PCR method, and the analysis of the PCR and the amplification product is performed simultaneously.
Moreover, 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.

Further, the present invention is a kit for detecting a living cell of a microorganism in a test sample by distinguishing it from a dead cell or a damaged cell by a nucleic acid amplification method, and includes the following elements:
1) Palladium complex or
A palladium compound which forms a palladium complex when dissolved in an organic solvent capable of binding to palladium as a ligand, or a solution containing a substance capable of binding to palladium as a ligand;
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 palladium 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.
In addition, the kit preferably includes a surfactant.

In the method and kit of the present invention, the palladium complex may be NH ₃ , RNH ₂ , halogen element, carboxylate group, H ₂ O, CO ₃ ²⁻ , OH ⁻ , NO ₃ ⁻ , ROH, N ₂ H _{4. ^{, PO 4 3-, R 2 O}} , RO -, ROPO 3 2-, (RO) 2 PO 2 -, R 2 S, R 3 P, RS -, CN -, RSH, RNC, (RS) 2 PO 2 _{^{-, (RO) 2 P (}} O) S -, SCN -, CO, H -, R - ( provided that the designation "R" represents either saturated or unsaturated organic group), NO ₂ ^-, It is preferable to include a ligand selected from Ar—NH ₂ , Ar—CN (Ar is an unsaturated organic group), N ₂ , SO ₃ ²⁻ , an imidazole ring, an unsaturated cyclic organic group, and N ₃ ^−. It is in form.
In the method and kit, the ligand is preferably selected from the group consisting of NH ₃ , RNH ₂ , a halogen element, a carboxylate group, R ₃ P, and Ar—CN.

  In the method and kit, the palladium complex may be dichloro (η-cycloocta-1,5-diene) palladium (II), bis (benzonitrile) dichloropalladium (II), diamminedichloropalladium (II), dichloro ( A preferred embodiment is selected from the group consisting of (ethylenediamine) palladium (II) and bis (triphenylphosphine) palladium (II) diacetate.

  In addition, the method and kit are generated by dissolving the palladium complex in a solution containing an organic solvent capable of binding palladium as a ligand or a substance capable of binding palladium as a ligand. A preferred form is a palladium complex.

In the method and kit, the palladium compound may be palladium chloride, palladium fluoride, palladium bromide, palladium iodide, palladium hydroxide, palladium dinitrate (II), palladium tetranitrate (IV), palladium acetate, phosphorus acetate. The preferred form is selected from the group consisting of palladium acid, dimethoxypalladium, palladium methoxyphosphate, palladium sulfite, dinitropalladium, and palladium diazide.
In the method and kit, the palladium compound is preferably palladium (II) chloride or palladium (II) acetate.

  In the method and kit of the present invention, the organic solvent is preferably dimethyl sulfoxide.

  Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be freely changed within the scope of the present invention. In the present specification, percentages are expressed by mass unless otherwise specified.

<1> Method of the Present Invention The method of the present invention is a method for detecting living cells of microorganisms in a test sample by distinguishing them from dead cells or damaged cells, and includes the following steps.
a) adding a palladium 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.

  In the method of the present invention, 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.

  In the present specification, the “test sample” is a target for detecting living cells of microorganisms present therein, and the presence is detected by amplification of a specific region of chromosomal DNA or RNA by a nucleic acid amplification method. Although it will not be restrict | limited if it is possible, A foodstuff, biological sample, drinking water, industrial water, environmental water, drainage, soil, or a wipe sample etc. are mentioned.

  In particular, as food, beverages such as soft drinks, carbonated drinks, nutrient drinks, fruit juice drinks, lactic acid bacteria drinks (including concentrated concentrates and powders for preparation of these drinks); ice confectionery such as ice cream, ice sherbet, shaved ice; Dairy products such as milk, milk drinks, fermented milk, butter; enteral nutrition foods, liquid foods, milk for childcare, sports drinks; functional foods such as foods for specified health use and health supplements are preferred.

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

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

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

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

  Examples of the lipolytic enzyme include lipase and phosphatase, examples of the proteolytic enzyme include serine protease, cysteine protease, proteinase K, and pronase, and examples of the saccharide-degrading enzyme include amylase, cellulase, and N-acetylmuramidase.

  “Microorganism” is an object to be detected by the method of the present invention, can be detected by a nucleic acid amplification method, and acts on a microorganism of a palladium complex that binds to a DNA-binding protein or an RNA-binding protein. Is not particularly limited as long as it is different between live cells and dead cells or damaged cells, but preferably bacteria, filamentous fungi, yeasts, viruses, and the like can be mentioned. 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 pyogenes, etc. Bacteria, Listeria monocytogenes such as Listeria monocytogenes, Bacillus cereus, Bacillus anthracis such as Bacillus anthracis, Mycobacterium tuberculosis ( Mycobacteria such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium intracellulare Bacteria, botulinum (Clostridium botulinum (Clostridium botulinum)), Clostridium perfringens (Clostridium perfringens (Clostridium perfringens)) Clostridium bacteria, etc., and the like.

  Gram-negative bacteria include Escherichia bacteria such as Escherichia coli, Enterobacter sakazakii (formerly Enterobacter sakazakii), and Citrobacter. -Citrobacter bacteria such as Citrobacter koseri, enterobacteria group typified by Klebsiella oxytoca such as Klebsiella oxytoca, Salmonella bacteria, Vibrio bacteria, Pseudomonas bacteria, Legionella bacteria, etc. Is mentioned.

  Examples of the virus include viruses such as influenza viruses having an envelope, and noroviruses, rotaviruses, adenoviruses and the like that do not have an envelope and have only a nucleocapsid.

  Regarding viruses, as shown in Examples 1-4 and 6-8 below, dichloro (η-cycloocta-1,5-diene) palladium (II) (dichloro (η-cycloocta-1,5 -diene) palladium (II)), bis (benzonitrile) dichloropalladium (II), diamminedichloropalladium (II), dichloro (ethylenediamine) palladium (II) ) (Dichloro (ethylenediamine) palladium (II)), bis (triphenylphosphine) palladium (II) diacetate (bis (triphenylphosphine) palladium (II) diacetate) or palladium chloride in dimethyl sulfoxide (DMSO) It is possible to distinguish between live and dead cells of the Gram-negative bacterium Cronobacter Sakazaki or Escherichia coli by treatment with the resulting complex. As a result, the active virus (living virus) having an envelope that maintains the integrity of the outermost envelope such as influenza virus, which is the same component as the outer membrane of the gram-negative bacterial cell wall, is palladium as described above. It can be inferred that the complex does not permeate, and that the inactive virus (dead virus) having a damaged envelope easily permeates the palladium complex. Furthermore, as shown in Example 5, it was shown that the living cells and dead cells of Staphylococcus aureus can be distinguished by means of diamminedichloropalladium (II). From these results, it is considered that the palladium complex can be used for distinguishing between living cells and dead cells for all microorganisms.

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

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

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

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

The unit of 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. In addition, 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. In addition, although the heating time in the boiling water for preparing damaged cells changes with kinds of microorganisms, in the case of bacteria described in the examples, damaged cells can be prepared in about 50 seconds.
In addition, 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).
In viruses, 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.

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

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

(1) Step a)
A palladium complex (hereinafter also referred to as “agent of the present invention” or simply “agent”) is added to a test sample. That is, the microorganism in the test sample is treated with the drug.
In a microbial cell, the drug does not substantially bind to a nucleic acid, and a DNA-binding protein (for example, HU histone-like protein) or an RNA-binding protein (for example, Synaptotagmin-binding, cytoplasmic RNA-interacting protein “SYNCRIP” molecular weight 66 kDa (JP 2002-101890 A) and nuclear RNA-protein complex, paraspeckle (Sasaki, YT et. al., (2009), Proc. Natl. Acad. Sci. USA, 106: 2525-2530) or a protein such as a ribosomal protein, it is presumed to inhibit the PCR reaction of the target region, and therefore a protein such as a DNA-binding protein or a ribosomal protein and a nucleic acid are present. In the cell, it is mainly bound to the same protein. Binding of the drug and the protein possible. The present invention with an agent that may be a coordinate bond (covalent bond).

  It is preferable that 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.

Palladium complexes have different permeability to the cell wall of living cells, damaged cells, or dead cells, and directly bind to intracellular nucleic acid binding proteins (DNA binding proteins or RNA binding proteins) to perform PCR reactions in the target region. The ligand is not particularly limited as long as it can be inhibited. For example, as a ligand, at least NH ₃ , RNH ₂ , halogen element (Cl, F, Br, I, At), carboxylate group, H ₂ O, CO ₃ ^{2- _{, OH -, NO 3 -,}} ROH, N 2 H 4, PO 4 3-, R 2 O, RO -, ROPO 3 2-, (RO) 2 PO 2 -, R 2 S, R 3 P, RS - ^{, CN -, RSH, RNC,} (RS) 2 PO 2 -, (RO) 2 P (O) S -, SCN -, CO, H -, R - ( provided that the notation "R" are both Represents a saturated or unsaturated organic group), NO ₂ ⁻ , Ar—NH ₂ , Ar—CN (Ar is an unsaturated organic group such as an aromatic ring), N ₂ , SO ₃ ²⁻ , N ₃ ⁻ , an imidazole ring, selected from ^- unsaturated cyclic organic group, or N ₃ One example is a palladium complex containing one. The palladium complex may contain one of the above ligands and may contain a plurality of the same or different ligands.

  Examples of the saturated organic group include alkyl groups such as methyl group, ethyl group, propyl group, isopropyl group, butyl group, cyclobutyl group, pentyl group, cyclopentyl group, and cyclohexyl group. Examples of the unsaturated organic group include unsaturated cyclic organic groups such as benzyl group (benzene ring), naphthyl group (naphthalene ring), allyl group, and 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, 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, Ar—CN, and an unsaturated cyclic organic group are preferable. Among these, NH ₃ , RNH ₂ , halogen element, carboxylate group, R ₃ P, and Ar—CN are particularly preferable.
As the halogen element, Cl (chlorine) is preferable.

Lippard et al. (Bioinorganic Chemistry Lippard, SJ and Berg, JM; supervised by Kazuko Matsumoto, pages 21-23 “Coordination Chemistry Principles in Bioinorganic Chemistry Research”) Tables 2 and 1 show metals (soft, intermediate , Hard Lewis acids) and ligands (soft, intermediate, hard Lewis bases), with individual exceptions, but with soft Lewis acids such as palladium bound to each Lewis base Sex is [soft Lewis base] (such as protein-constituting amino acids such as cysteine [sulfhydryl group] and methionine [thioether moiety])> [intermediate Lewis base] (such as adenine (A) and guanine (G))> [ hard Lewis base] be in the order of (Cl such and NH ₃ group) has been suggested (hard-soft acid-base theory ; Lippard et al., pp. 21-22).
Thus, since palladium prefers cysteine and methionine, which are soft Lewis bases, as coordination partners, a palladium complex in which palladium is coordinated in advance with a hard or intermediate Lewis base is used in cells. It is presumed to be stabilized by coordination with amino acids in the protein. Therefore, from the viewpoint of the ability to suppress nucleic acid amplification via a nucleic acid binding protein, the agent of the present invention is preferably a compound in which a hard or intermediate Lewis base and palladium are coordinated, and a hard Lewis base and palladium are coordinated. Bound compounds are more preferred.
Soft Lewis bases such as R ₂ S, R ₃ P, RS ⁻ , CN ⁻ , RSH, RNC, (RS) ₂ PO ₂ ⁻ , (RO) ₂ P (O) S ⁻ , SCN ⁻ , CO, Even a palladium complex containing H ⁻ and R ⁻ (wherein “R” represents a saturated or unsaturated organic group) as a ligand can be used in the present invention.

  On the other hand, dead cells are generally more morphologically damaged than living cells (damaged cells are mildly damaged), and complexes containing a hard or intermediate Lewis base as a ligand are generally Dead cells (including damaged cells) are considered to be more permeable. In addition, it is generally said that 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. However, as shown in the examples, even if the compound as a whole is nonpolar, it can be nonpolar because permeation into living cells can be significantly suppressed by shortening the action time to some extent.

Specific examples of the palladium complex include the following compounds.
Dichloro (η-cycloocta-1,5-diene) palladium (II), bis (benzonitrile) dichloropalladium (II), diamminedichloropalladium (II), dichloro (ethylenediamine) palladium (II), chloro (η ² -P , C (-) tris (2,4-di-tert-butylphenyl) phosphite) (tricyclophenylphosphine) palladium (II), di-μ-chloro-bis- [5-hydroxy-2- [1- (Hydroxyimino-κN) -ethyl] -phenyl-κC] -palladium (II) dimer, bis (triphenylphosphine) palladium (II) dichloride, bromo (tri-tert-butylphosphine) palladium (I) dimer, 1, 2-bis (phenylsulfinyl) ethane palladium (II) acetate, chloro [2- (dicyclohexylphosphino) -3,6-dimethoxy-2 ', 4', 6'-triisopropyl-1,1'-biphenyl] [ 2- (2-Aminoethyl) phenyl] palladium (II), chloro- (2-disic Hexylphosphino-2 ', 6'-diisopropoxy-1,1'-biphenyl) [2- (2-aminoethyl) phenyl] palladium (II), chloro [2- (di-tert-butylphosphino) -2 ', 4', 6'-triisopropyl-1,1'-biphenyl] [2- (2-aminoethyl) phenyl] palladium (II), chloro (2-dicyclohexylphosphino-2 ', 4', 6'-triisopropyl-1,1'-biphenyl) [2- (2-aminoethyl) phenyl] palladium (II), chloro (2-dicyclohexylphosphino-2 ', 6'-dimethoxy-1,1'- Biphenyl) [2- (2-aminoethylphenyl)] palladium (II), chloro (2-dicyclohexylphosphino-2 ', 6'-dimethoxy-1,1'-biphenyl) [2- (2-aminoethylphenyl) )] Palladium (II), chloro (2-dicyclohexylphosphino-2 ', 6'-dimethoxy-1,1'-biphenyl) [2- (2-aminoethylphenyl)] palladium (II), bis [(dicyclohexyl) ) (4-Dimethylaminophene Nyl) phosphine] palladium (II) chloride, bis [di- (tert-butyl) (4-trifluoromethylphenyl) phosphine] palladium (II) chloride, 2- (dimethylaminomethyl) ferrocene-1-yl-palladium ( II) Chloride dinorbornylphosphine, 1,3-bis (2,4,6-trimethylphenyl) imidazol-2-ylidene (1,4-naphthoquinone) palladium (0) dimer, salicylaldehyde thiosemicarbazone palladium ( II) Chloride, 2- [bis (2,4-di-tert-butyl-phenoxy) phosphinoxy] -3,5-di (tert-butyl) phenyl-palladium (II) chloride dimer, di-μ-chlorobis [5 -Chloro-2-[(4-chlorophenyl) (hydroxyimino-κN) methyl] phenyl-κC] palladium dimer, palladium (II) acetate-S-Phos (Pd: P 1: 2), tris [dibenzylideneacetone] Dipalladium (0) -S-Phos (Pd: P 1: 2), 2- (dimethyl) Amino) -2-biphenylyl-palladium (II) chloride dinorbornylphosphine complex, palladium pivalate, bis (benzonitrile) palladium (II) chloride, palladium (II) trifluoroacetate, palladium (π-cinnamyl) chloride dimer, Bis (triphenylphosphine) palladium (II) diacetate, 1,4-bis (diphenylphosphino) butane-palladium (II) chloride, palladium (II) cyanide, palladium (II) propionate, (2-methylallyl) palladium ( II) Chloride dimer, [2,6-bis [(di-1-piperidinylphosphino) amino] phenyl] palladium (II) chloride, [1,2-bis (dicyclohexylphosphino) ethane] palladium (II) Chloride, Triphenylphosphine Palladium (II) Dichloride Phosphaadamantane Ethyl Silica, Di Lorobis (tricyclophenylphosphine) palladium (II), dichloro (1,5-cyclooctadiene) palladium (II), (1,3-bis (diphenylphosphino) propane) palladium (II) chloride, 1,1 ' -Bis (di-isopropylphosphino) ferrocene palladium dichloride, dichlorobis (triphenylphosphine) palladium (II), chloro (2-dicyclohexylphosphino-2 ', 4', 6'-triisopropyl-1,1'-biphenyl ) [2- (2′-amino-1,1′-biphenyl)] palladium (II), bis (3,5,3 ′, 5′-dimethoxydibenzylideneacetone) palladium (0), dichloro (1,10 -Phenanthroline) palladium (II), dichlorobis (tri-o-tolylphosphine) palladium (II), trans-benzyl (chloro) bis (triphenylphosphine) palladium (II), tris (3,3 ', 3''-Phosphinidin tris (benzenesulfonato) palladium (0) Nine Thorium salt nonahydrate, [(R)-(+)-2,2'-bis (diphenylphosphino) -1,1'-binaphthyl] -diaquo-palladium (II) bis (triflate), trans -Bis (dicyclohexylamine) palladium (II) acetate, (R) -1-[(S _P ) -2- (dicyclohexylphosphino) ferrocenyl] ethyldi-tert-butylphosphine palladium (II) dichloride, dichloro [2- ( 4,5-dihydro-2-oxazolyl) quinoline] palladium (II), polymer modified bis [(diphenylphosphanyl) methyl] amine palladium (II) dichloride, trans-dibromobis (triphenylphosphine) palladium (II), [ (R)-(+)-2,2'-bis (diphenylphosphino) -1,1'-binaphthyl] palladium (II) chloride, 2- [bis (triphenylphosphine) palladium (II) bromide] benzyl Alcohol, [(R)-(+)-2,2'-bis (di-p-tolylphosphino) -1,1'-binaphthyl] paradiu (II) chloride, [bis [((R)-(+)-2,2′-bis (diphenylphosphino) -1,1′-binaphthyl) palladium (II)] bis (μ-hydroxo)] bis ( Triflate), dichlorobis (triethylphosphine) palladium (II), [(S)-(-)-2,2'-bis (diphenylphosphino) -1,1'-binaphthyl] -diaquo-palladium (II) bis (Triflate), diacetbis (triphenylphosphine) palladium (II), [bis [((S)-(-)-2,2'-bis (diphenylphosphino) -1,1'-binaphthyl) palladium (II )] Bis (μ-hydroxo)] bis (triflate), [(S)-(−)-2,2′-bis (di-p-tolylphosphino) -1,1′-binaphthyl] palladium (II) chloride , Chloro [(1,2,5,6-η) -1,5-cyclooctadiene] (2,2-dimethylpropyl) -palladium, chloro [(tricyclophenylphosphine) -2- (2'-amino Biphenyl)] palladium (II), chloro (triphenylphosphine) [2- (2'- Mino-1,1'-biphenyl)] palladium (II), dichloro [(S)-(-)-2,2'-bis (diphenylphosphino) -1,1'-binaphthyl] palladium (II), bis (Benzonitrile) palladium (II) bromide, chloro [(tri-tert-butylphosphine) -2- (2-aminobiphenyl)] palladium (II), dibromo [1,1'-bis (diphenylphosphino) ferrocene] Palladium (II), dibromo (1,5-cyclooctadiene) palladium (II), dichloro [1,1'-bis (diphenylphosphino) ferrocene] palladium (II) acetone adduct, dichloro [8- (di- tert-butylphosphinoxy) quinoline] palladium (II), dichloro (chloro-tert-butylcyclohexylphosphine) palladium (ii) dimer, dichloro (chlorodi-t-butylphosphine) palladium (ii) dimer, dichloro (chlorodicyclohexylphosphine Palladium (ii) Dimer, Dicro Lobis (chloro tert-butylcyclohexylphosphine) palladium (ii), dichlorobis (chlorodi-tert-butylphosphine) palladium (ii), dichlorobis (methyldiphenylphosphine) palladium (II) Phosphine)] palladium (II), tris [μ-[(1,2-η: 4,5-η)-(1E, 4E) -1,5-bis (4-methoxyphenyl) -1,4-pentadiene -3-one]] di-palladium, [1,3-bis (2,6-diisopropylphenyl) imidazol-2-ylidene] chloro [3-phenylallyl] palladium (II), chloro (2-dicyclohexylphosphino- 2 ', 6'-diisopropoxy-1,1'-biphenyl) [2- (2'-amino-1,1'-biphenyl)] palladium (II), dichloro (N, N, N', N ' -Tetramethylethylenediamine) palladium (II), chloro (2-dicyclohexylphosphino-2 ', 6'-dimethoxy-1,1'-biphenyl) [2- (2'-amino-1,1'-biphenyl)] Radium (II), [1,3-bis (2,6-di-isopropylphenyl) -4,5-dihydroimidazol-2-ylidene] chloro) [3-phenylallyl] palladium (II), dichloro [1, 3-bis (2,6-diisopropylphenyl) -1,3-dihydro-2H-imidazol-2-ylidene] palladium (II) dimer, dichloro- [1,3-bis (diisopropylphenyl) imidazolidene-2-ylidene ] Palladium (II) dimer, bis [tris (4- (1H, 1H, 2H, 2H-perfluorodecyl) phenyl) phosphine] palladium (II) dichloride, bromo [(2- (hydroxy-κO) methyl) phenylmethyl -κC] (triphenylphosphine) palladium (II), [(2-dicyclohexylphosphino-3,6-dimethoxy-2 ′, 4 ′, 6′-triisopropyl-1,1′-biphenyl) -2- ( 2'-amino 1,1'-biphenyl)] palladium (II) methanesulfonate, (2-dicyclohexylphosphino-2 ', 6'-diisopropoxy-1 , 1'-biphenyl) [2- (2'-amino-1,1'-biphenyl)] palladium (II) methanesulfonate, [(4,5-bis (diphenylphosphino) -9,9-dimethylxanthene) -2- (2'-amino-1,1'-biphenyl)] palladium (II) methanesulfonate, [(di (1-adamantyl) -butylphosphine) -2- (2'-amino-1,1'-biphenyl) )] Palladium (II) methanesulfonate, allylchloro [1,3-bis (2,4,6-trimethylphenyl) imidazol-2-ylidene] palladium (ii), bis [tris (4- (heptadecafluorooctyl) phenyl ) Phosphine] palladium (II) dichloride, chloro [(1,3,5,7-tetramethyl-5-phenyl-2,4,8-trioxa-6-phosphaadamantane) -2- (2-aminobiphenyl) ] Palladium (II), chloro [(4,5-bis (diphenylphosphino) -9,9-dimethylxanthene) -2- (2'-amino-1,1'-biphenyl)] palladium (II), chloro [(Di (1-adama Til) -N-butylphosphine) -2- (2-aminobiphenyl)] palladium (II), chloro (sodium-2-dicyclohexylphosphino-2 ', 6'-dimethoxy-1,1'-biphenyl-3' -Sulfonate) [2- (2'-amino-1,1'-biphenyl)] palladium (II), dichloro [(S) -N, N-dimethyl-1-[(R) -2- (diphenylphosphino ) Ferrocenyl] ethylamine] palladium (II), dichloro [bis (2- (diphenylphosphino) phenyl) ether] palladium (II), dichlorobis (3,7-diacetyl-1,3,7-triaza-5-phospha Bicyclo [3.3.1] nonane) palladium (II), [(2- [bis [3,5-bis (trifluoromethyl) phenyl] phosphine] -3,6-dimethoxy-2 ', 4', 6'- Triisopropyl-1,1'-biphenyl) -2- (2'-amino-1,1'-biphenyl)] palladium (II) methanesulfonate, chloro [(2-dicyclohexylphosphino-2 ', 6'-bis (N, N-dimethylamino) -1,1'-biphenyl)- 2- (2′-amino-1,1′-biphenyl)] palladium (II), allyl [1,3-bis (2,6-diisopropylphenyl) imidazol-2-ylidene] chloropalladium (II), (1 , 3-Bis (2,6-diisopropylphenyl) imidazolidene) (3-chloropyridyl) palladium (II) dichloride, 2- (2'-di-tert-butylphosphine) biphenylpalladium (II) acetate, tetrachloropalladium Acid (II) sodium, bis (acetonitrile) dichloropalladium (II), tetrachloropalladate (II) ammonium, hexachloropalladate (IV) ammonium, tetrachloropalladate (II) potassium, hexachloropalladate (IV) potassium, (2,2'-bipyridine) dichloropalladium (II), (bicyclo [2.2.1] hepta-2,5-diene) dichloropalladium (II), allyl [1,3-bis (2,6-diisopropylphenyl) -2-Imidazolidinylidene] chloro Palladium (II), di-μ-chlorobis [1-[(1R) -1- (dimethylamino) ethyl] -2-naphthyl-C, N] dipalladium (II), (2,2'-bipyridine) dichloro Palladium (II), (bicyclo [2.2.1] hepta-2,5-diene) dichloropalladium (II), allyl [1,3-bis (2,6-diisopropylphenyl) -2-imidazolidinylidene] chloropalladium (II), di-μ-chlorobis [1-[(1R) -1- (dimethylamino) ethyl] -2-naphthyl-C, N] dipalladium (II), bis (di-tert-butyl (4- Dimethylaminophenyl) phosphine) dichloropalladium (II), trans-bis (acetato) bis [o- (di-o-tolylphosphino) benzyl] dipalladium (II), allyl [1,3-bis (mesityl) imidazole-2 -Ilidene] chloropalladium (II), [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II), potassium tetrachloropalladate (II), hexachloropalladium Acid (IV) potassium, allyl palladium (II) chloride dimer, [1,1'-
Bis (di-tert-butylphosphino) ferrocene] dichloropalladium (II), (2-butenyl) chloropalladium dimer (English: dichloro (η-cycloocta-1,5-diene) palladium (II), bis (benzonitrile) dichloropalladium (II), diamminedichloropalladium (II), dichloro (ethylenediamine) palladium (II), chloro (η ² -P, C (-) tris (2,4-di-tert-butylphenyl) phosphite) (tricyclohexylphosphine) palladium (II ), Di-μ-chloro-bis- [5-hydroxy-2- [1- (hydroxyimino-κN) -ethyl] -phenyl-κC] -palladium (II) dimer, bis (triphenylphosphine) palladium (II) dichloride, bromo (tri-tert-butylphosphine) palladium (I) dimer, 1,2-bis (phenylsulfinyl) ethane palladium (II) acetate, chloro [2- (dicyclohexylphosphino) -3,6-dimethoxy-2 ', 4', 6 '-triisopropyl-1,1'-biphenyl] [2- (2-aminoethyl) phenyl] palladium (II), chloro- (2-Dicyclohexylphosphino-2', 6'-diisopropoxy-1,1'-biphenyl) [2 -(2-aminoethyl) phenyl] palladium (II), chloro [2- (di-tert-butylphosphino) -2 ', 4', 6'-triisopropyl-1,1'-biphenyl] [2- (2-aminoethyl ) phenyl] palladium (II) , Chloro (2-dicyclohexylphosphino-2 ', 4', 6'-triisopropyl-1,1'-biphenyl) [2- (2-aminoethyl) phenyl] palladium (II), chloro (2-dicyclohexylphosphino-2 ', 6 '-dimethoxy-1,1'-biphenyl) [2- (2-aminoethylphenyl)] palladium (II), chloro (2-dicyclohexylphosphino-2', 6'-dimethoxy-1,1'-biphenyl) [2- ( 2-aminoethylphenyl)] palladium (II), chloro (2-dicyclohexylphosphino-2 ', 6'-dimethoxy-1,1'-biphenyl) [2- (2-aminoethylphenyl)] palladium (II), bis [(dicyclohexyl) (4-dimethylaminophenyl) phosphine] palladium (II) chloride, bis [di- (tert-butyl) (4-trifluoromethylphenyl) phosphine] palladium (II) chloride, 2- (dimethylaminomethyl) ferrocen-1-yl-palladium (II) chloride dinorbornylphosphine, 1,3-bis (2,4,6-trimethylphenyl) imidazol-2-ylidene (1,4-naphthoquinone) palladium (0) dimer, salicylaldehyde thiosemicarbazone palladium (II) chloride, 2- [bis (2, 4-di-tert-butyl-phenoxy) phosphinooxy] -3,5-di (tert-butyl) phenyl-palladium (II) chloride dimer, di-μ-chlorobis [5-chloro-2-[(4-chlorophenyl) (hydroxyimino-κN) methyl] phenyl-κC] palladium dimer palladium (II) acetate-S-Phos (Pd: P 1: 2), tris [dibenzylideneacetone] dipalladium (0) -S-Phos (Pd: P 1: 2), 2- (dimethylamino) -2-biphenylyl-palladium (II) chloride dinorbornylphosphine complex, palladium pivalate, bis (benzonitrile) palladium (II) chloride, palladium (II) trifluoroacetate, palladium (π-cinnamyl) chloride dimer, bis (triphenylphosphine) palladium (II) diacetate, 1,4-bis (diphenylphosphino) butane-palladium (II) chloride, palladium (II) cyanide, palladium (II) propionate, (2-methylallyl) palladium (II) chloride dimer, (2,6-bis [(di-1-piperidinylphosphino) amino ] phenyl] palladium (II) chloride, [1,2-bis (dicyclohexylphosphino) ethane] palladium (II) chloride, triphenylphosphine palladium (II) dichloride phosphaadamantane ethyl silica, dichlorobis (tricyclohexylphosphine) palladium (II), dichloromethane (1,5 -cyclooctadiene) palladium (II), (1,3-bis (diphenylphosphino) propane) palladium (II) chloride, 1,1'-bis (di-isopropylphosphino) ferrocene palladium dichloride, dichlorobis (triphenylphosphine) palladi um (II), chloro (2-dicyclohexylphosphino-2 ', 4', 6'-triisopropyl-1,1'-biphenyl) [2- (2'-amino-1,1'-biphenyl)] palladium (II) , Bis (3,5,3 ', 5'-dimethoxydibenzylideneacetone) palladium (0), dichloro (1,10-phenanthroline) palladium (II), dichlorobis (tri-o-tolylphosphine) palladium (II), trans-benzyl ( chloro) bis (triphenylphosphine) palladium (II), tris (3,3 ', 3''-phosphinidynetris (benzenesulfonato) palladium (0) nonasodium salt nonahydrate, [(R)-(+)-2,2'-bis ( diphenylphosphino) -1,1'-binaphthyl] -diaquo-palladium (II) bis (triflate), trans-bis (dicyclohexylamine) palladium (II) acetate, (R) -1-[(S _P ) -2- (dicyclohexylphosphino ) ferrocenyl] ethyldi-tert-butylphosphine palladium (II) dichloride, dichloro [2- (4,5-dihydro-2-oxazolyl) quinoline] palladium (II), bis [(diphenylphosphanyl) methyl] amine palladium (II) dichloride, polymer-bound, trans-dibromobis (triphenylphosphine) palladium (II), [(R)-(+)-2,2'-bis (diphenylphosphino) -1,1'-binaphthyl] palladium (II) chloride, 2- [ bis (triphenylphosphine) palladium (II) bromide] benzyl alco hol, [(R)-(+)-2,2'-bis (di-p-tolylphosphino) -1,1'-binaphthyl] palladium (II) chloride, [bis [((R)-(+)- 2,2'-bis (diphenylphosphino) -1,1'-binaphthyl) palladium (II)] bis (μ-hydroxo)] bis (triflate), dichlorobis (triethylphosphine) palladium (II), [(S)-(- ) -2,2'-bis (diphenylphosphino) -1,1'-binaphthyl] -diaquo-palladium (II) bis (triflate), diacetobis (triphenylphosphine) palladium (II), [bis [((S)-(- ) -2,2'-bis (diphenylphosphino) -1,1'-binaphthyl) palladium (II)] bis (μ-hydroxo)] bis (triflate), [(S)-(-)-2,2'- bis (di-p-tolylphosphino) -1,1'-binaphthyl] palladium (II) chloride, chloro [(1,2,5,6-η) -1,5-cyclooctadiene] (2,2-dimethylpropyl)- palladium, chloro [(tricyclohexylphosphine) -2- (2'-aminobiphenyl)] palladium (II), chloro (triphenylphosphine) [2- (2'-amino-1,1'-biphenyl)] palladium (II), dichloromethane [ (S)-(-)-2,2'-bis (diphenylphosphino) -1,1'-binaphthyl] palladium (II), bis (benzonitrile) palladium (II) bromide, chloro [(tri-tert-butylphosphine)- 2- (2-aminobiphenyl)] palladium (II), dibromo [1,1'-bis (diphenylphosphino) fe rrocene] palladium (II), dibromo (1,5-cyclooctadiene) palladium (II), dichloro [1,1'-bis (diphenylphosphino) ferrocene] palladium (II) acetone adduct, dichloromethane [8- (di-tert-butylphosphinooxy ) quinoline] palladium (II), dichloro (chloro-tert-butylcyclohexylphosphine) palladium (ii) dimer, dichloro (chlorodi-t-butylphosphine) palladium (ii) dimer, dichloro (chlorodicyclohexylphosphine) palladium (ii) dimer, dichlorobis (chloro- t-butylcyclohexylphosphine) palladium (ii), dichlorobis (chlorodi-tert-butylphosphine) palladium (ii), dichlorobis (methyldiphenylphosphine) palladium (II), trans-dibromo [bis (tri-o-tolylphosphine)] palladium (II), Tris [μ-[(1,2-η: 4,5-η)-(1E, 4E) -1,5-bis (4-methoxyphenyl) -1,4-pentadien-3-one]] di-palladium, [1,3-bis (2,6-diisopropylphenyl) imidazol-2-ylidene] chloro [3-phenylallyl] palladium (II), chloro (2-dicyclohexylphosphino-2 ', 6'-diisopropoxy-1,1'-biphenyl ) [2- (2'-amino-1,1'-biphenyl)] palladium (II), dichloro (N, N, N ', N'-tetramethylethylenediamine) palladium (II), chloro (2-dicyclohexylphosp hino-2 ', 6'-dimethoxy-1,1'-biphenyl) [2- (2'-amino-1,1'-biphenyl)] palladium (II), [1,3-bis (2,6- di-isopropylphenyl) -4,5-dihydroimidazol-2-ylidene] chloro) [3-phenylallyl] palladium (II), dichloro [1,3-bis (2,6-diisopropylphenyl) -1,3-dihydro-2H- imidazol-2-ylidene] palladium (II) dimer, dichloro- [1,3-bis (diisopropylphenyl) imidazoliden-2-ylidene] palladium (II) dimer, bis [tris (4- (1H, 1H, 2H, 2H- perfluorodecyl) phenyl) phosphine] palladium (II) dichloride, bromo [(2- (hydroxy-κO) methyl) phenylmethyl-κC] (triphenylphosphine) palladium (II), [(2-di-cyclohexylphosphino-3,6-dimethoxy- 2 ', 4', 6'-triisopropyl-1,1'-biphenyl) -2- (2'-amino-1,1'-biphenyl)] palladium (II) methanesulfonate, (2-dicyclohexylphosphino-2 ', 6 '-diisopropoxy-1,1'-biphenyl) [2- (2'-amino-1,1'-biphenyl)] palladium (II) methanesulfonate, [(4,5-bis (diphenylphosphino) -9,9-dimethylxanthene ) -2- (2'-amino-1,1'-biphenyl)] palladium (II) methanesulfonate, [(di (1-adamantyl) -butylphosphine) -2- (2'-amino-1,1'-biphenyl) )] palladium (II) methanesulfonate, allylchloro [1, 3-bis (2,4,6-trimethylphenyl) imidazol-2-ylidene] palladium (ii), bis [tris (4- (heptadecafluorooctyl) phenyl) phosphine] palladium (II) dichloride, chloro [(1,3,5 , 7-tetramethyl-5-phenyl-2,4,8-trioxa-6-phosphaadamantane) -2- (2-aminobiphenyl)] palladium (II), chloro [(4,5-bis (diphenylphosphino) -9,9 -dimethylxanthene) -2- (2'-amino-1,1'-biphenyl)] palladium (II), chloro [(di (1-adamantyl) -N-butylphosphine) -2- (2-aminobiphenyl)] palladium ( II), chloro (sodium-2-dicyclohexylphosphino-2 ', 6'-dimethoxy-1,1'-biphenyl-3'-sulfonate) [2- (2'-amino-1,1'-biphenyl)] palladium ( II), dichloro [(S) -N, N-dimethyl-1-[(R) -2- (diphenylphosphino) ferrocenyl] ethylamine] palladium (II), dichloro [bis (2- (diphenylphosphino) phenyl) ether] palladium (II), dichlorobis (3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo [3.3.1] nonane) palladium (II), [(2- [bis [3,5-bis (trifluoromethyl) phenyl] phosphine] -3,6-dimethoxy-2 ', 4', 6'-triisopropyl-1,1'-biphenyl) -2- (2'-amino-1,1'-biphenyl)] palladium (II) methanesulfonate, chloro [(2-dicyclohexylphosphino-2 ', 6'-bis (N, N- dimethylamino) -1,1'-biphenyl) -2- (2'-amino-1,1'-biphenyl)] palladium (II), allyl [1,3-bis (2,6-diisopropylphenyl) imidazol-2- ylidene] chloropalladium (II), (1,3-bis (2,6-diisopropylphenyl) imidazolidene) (3-chloropyridyl) palladium (II) dichloride, 2- (2'-di-tert-butylphosphine) biphenylpalladium (II) acetate , Sodium tetrachloropalladate (II), bis (acetonitrile) dichloropalladium (II), ammonium tetrachloropalladate (II), ammonium hexachloropalladate (IV), potassium tetrachloropalladate (II), potassium hexachloropalladate (IV), (2,2'-bipyridine) dichloropalladium ( II), (bicyclo [2.2.1] hepta-2,5-diene) dichloropalladium (II), allyl [1,3-bis (2,6-diisopropylphenyl) -2-imidazolidinylidene] chloropalladium (II), di-μ -chlorobis [1-[(1R) -1- (dimethylamino) ethyl] -2-naphthyl-C, N] dipalladium (II), (2,2'-bipyridine) dichloropalladium (II), (bicyclo [2.2.1 ] hepta-2,5-diene) dichloropalladium (II), allyl [1,3-bis (2,6-diisopropylphenyl) -2-imidazolidinylidene] chloropalladium (II), di-μ-chlorobis [1-[(1R) -1- (dimethylamino) ethyl] -2-naphthyl-C, N] dipalladium (II), bis (di-tert-butyl (4-dimethylaminophenyl) phosphine) dichloropalladium (II), trans-bis (acetato) bis [o- (di-o-tolylphosphino) benzyl] dipalladium (II), allyl [1,3-bis (mesityl) imidazol-2-ylidene] chloropalladium (II), [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II), potassium tetrachloropalladate (II) , Palladium hexachloropalladate (IV), allylpalladium (II) chloride dimer, [1,1'-bis (di-tert-butylphosphino) ferrocene] dichloropalladium (II), (2-butenyl) chloropalladium dimer) It is done.

Preferred examples of the complex include the following compounds.
Dichloro (η-cycloocta-1,5-diene) palladium (II) (dichloro (η-cycloocta-1,5-diene) palladium (II)) (chemical formula 1, molecular weight 285.51),
Bis (benzonitrile) dichloropalladium (II) (chemical formula 2, molecular weight 383.57),
Diamminedichloropalladium (II) (chemical formula 3, molecular weight 211.39),
Dichloro (ethylenediamine) palladium (II) (chemical formula 4, molecular weight 237.41),
Bis (triphenylphosphine) palladium (II) diacetate (chemical formula 5, molecular weight 749.08).

Further, examples of the palladium complex include a palladium complex formed by dissolving a palladium compound in an organic solvent capable of binding to palladium as a ligand or a solution containing a substance capable of binding to palladium as a ligand. Examples of such a palladium compound include a palladium compound that forms a macromolecule by a covalent bond between palladium and another element or group.
Examples of the element or group include halogen elements (Cl, F, Br, I, At), OH ⁻ , NO ₃ ⁻ , CH ₃ COO ⁻ , NO ₂ ⁻ , CO ₃ ²⁻ , PO ₄ ³⁻ , RO ⁻ , ROPO ₃ ²⁻ , (RO) ₂ PO ₂ ⁻ (wherein “R” represents a saturated or unsaturated organic group), SO ₃ ²⁻ , N ₃ ^{− and the} like.

  Specific examples of the palladium compound include palladium chloride, palladium fluoride, palladium bromide, palladium iodide, palladium hydroxide, palladium dinitrate (II), palladium tetranitrate (IV), palladium acetate, dinitropalladium, Palladium carbonate, palladium phosphate, dimethoxypalladium, palladium methoxyphosphate, palladium sulfite, dinitropalladium, palladium diazide and the like can be mentioned. Among these, preferable compounds include palladium chloride, palladium bromide, palladium fluoride, and palladium iodide, and particularly preferable compounds include palladium chloride. Examples of palladium chloride include palladium (II) chloride and palladium (IV) chloride. Examples of palladium acetate include palladium (II) acetate (palladium (II) acetate, chemical formula 6, molecular weight 224.5).

  Examples of the organic solvent include DMSO. Examples of the complex obtained by dissolving palladium chloride in DMSO include dichlorobis (dimethylsulfoxide) palladium (II) and tetrakis (dimethylsulfoxide) palladium (II).

Further, as a solution containing a substance capable of binding to palladium as a ligand, a harmaline solution, for example, an aqueous solution of harmaline hydrochloride, and a diferrocenyl-phosphine solution, for example, DMSO of diferrocenyl phosphine And the like. When the above-mentioned macromolecules containing palladium are dissolved in these solutions, palladium is rebound to harmaline or diferrocenyl phosphine as a ligand, and the molecular weight is reduced as a palladium complex. The palladium complex thus produced can also be used in the present invention.
The palladium complex may be a multimer such as a dimer.

  Various palladium compounds and palladium compounds that form macromolecules by covalent bonding of palladium and other elements or groups are commercially available (for example, Wako Pure Chemical Industries, Sigma), and can be used. Further, for example, it can be synthesized according to the method described in Nishiyama M, Yamamoto T. (1998) Tetrahedron Lett, 39: 617-620, J. Am. Chem. Soc. (1999) 121: 9550-9561, and the like.

  In the method of the present invention, 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.

  As such conditions, specifically, dichloro (η-cycloocta-1,5-diene) palladium (II) has a final concentration of 10 to 3000 μM, preferably 10 to 100 μM, 4 to 43 ° C., 5 minutes to 2 hours. Is exemplified. In the case of bis (benzonitrile) dichloropalladium (II), the final concentration is 10 to 3000 μM, preferably 10 to 100 μM, 4 to 43 ° C., 5 minutes to 2 hours. In diamminedichloropalladium (II), the final concentration is 10 to 3000 μM, preferably 10 to 100 μM, 4 to 43 ° C., and 5 minutes to 2 hours. In the case of dichloro (ethylenediamine) palladium (II), the final concentration is 10 to 3000 μM, preferably 10 to 250 μM, 4 to 43 ° C., 5 minutes to 2 hours. In the case of bis (triphenylphosphine) palladium (II) diacetate, the final concentration is 10 to 3000 μM, preferably 10 to 100 μM, 4 to 43 ° C., and 5 minutes to 2 hours.

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

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

In addition, in the conventional method using ethidium monoazide or the like, light irradiation with a wavelength of 350 nm to 700 nm is required in order to crosslink DNA or RNA molecules after hydrogenating these crosslinkers with DNA or RNA. However, the method of the present invention using the drug of the present invention does not require light irradiation. Therefore, cooling for preventing the sample from being heated by light irradiation, for example, immersion of the sample in ice water is not required.
In addition, in order to prevent the drug from being denatured by exposure, it is necessary to keep the sample in a dark room or the like in a dark room in order to prevent the drug from being denatured by exposure. There is no need.

  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. . As a result, the drug enters dead cells of somatic cells, dead cells of microorganisms, and cells of damaged cells, and then the drug is coordinated to the cysteine or methionine sites of the nucleic acid binding protein. Although it does not act directly on the nucleic acid itself, the nucleic acid and the nucleic acid binding protein are integrated like a complex (a thread-like nucleic acid is wrapped around a circular nucleic acid binding protein). As a result, when the nucleic acid elongation enzyme (DNA polymerase, RNA polymerase, etc.) is performing an elongation reaction, a large number of the palladium complexes are immobilized on the protein in the vicinity of the template nucleic acid. It is estimated that the elongation reaction of is significantly inhibited.

  When the drug permeates preferentially to damaged or dead cells over live cells, the target region of chromosomal DNA or RNA is amplified by the nucleic acid amplification method in live cells, whereas chromosomal DNA binding in damaged or dead cells. Since the drug binds to the protein or RNA-binding protein and inhibits the elongation reaction of the nucleic acid elongation enzyme, live cells can be selectively detected compared to damaged cells or 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. Examples of the method for removing the drug include a method of centrifuging a test sample, separating a precipitate containing a microorganism and a supernatant containing a drug, and removing the supernatant. In this case, it is possible to add a step of washing the microorganism with a cleaning agent as appropriate after removing the drug. Furthermore, since this drug binds to proteins having amino acid residues such as cysteine and methionine in preference to nucleic acids, in order to nullify the action of the first high-concentration drug, bovine serum albumin solution or yeast A medium containing a protein such as extract (lactose broth or brain heart infusion medium) may be added, and the unreacted palladium complex may be bound and adsorbed to the protein (because palladium is coordinated to the protein).
Moreover, it is preferable to remove the unreacted drug from the drug-treated sample before the following step b).

(2) Step b)
Next, 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.
When nucleic acid amplification is performed without extraction of nucleic acid from cells, 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). In addition, 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.

  Furthermore, it is also possible to add 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. These can be used either alone or in any combination of two or more. It is particularly preferred to add all of these. 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, a nucleic acid elongation enzyme may also 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. For example, a positive charge inhibitor that adsorbs to a nucleic acid (DNA or RNA) template, or a nucleic acid synthase (DNA polymerase, etc.) Examples include adsorbed negative charge inhibitors. Examples of 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.

  Examples of 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, α2-macroglobulin, tetra Examples thereof include one or more selected from phosphorylase and lactate dehydrogenase from methylammonium chloride and lysozyme.

Examples of the polyethylene glycol include polyethylene glycol 400 and polyethylene glycol 4000. Examples of betaine include trimethylglycine and its derivatives. Examples of phosphorylase and lactate dehydrogenase include glycogen phosphorylase and lactate dehydrogenase derived from rabbit muscle. As glycogen phosphorylase, glycogen phosphorylase b is preferable.
In particular, it is preferable to use albumin, dextran, T4 gene 32 protein, or lysozyme.

  Assuming blood, feces, and meat as test materials, the above-mentioned substances are added to the PCR reaction solution as an attempt to reduce the inhibitory action of nucleic acid amplification inhibitors contained in these test materials, thereby reducing the inhibitory action. Have been evaluated (Abu Al-Soud, W. et al, Journal of Clinical Microbiology, 38: 4463-4470, 2000).

It has been suggested that albumin typified by BSA (bovine serum albumin) 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.) .
In addition, BSA, T4 Gene 32 protein, and proteinase inhibitor can reduce proteolytic activity by binding to proteinase and maximize the function of nucleic acid synthase. Has been suggested. In fact, 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 α2-macroglyblin). In addition, a case where the nucleic acid amplification reaction progressed well has been introduced (Abu Al-Soud et al.).

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.).

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

  Examples of albumin include bovine serum albumin, ovalbumin, milk albumin, 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.

  Examples of dextran include dextran 40 and dextran 500. Of these, dextran 40 is preferred. The 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 it is 0.01 to 0.02%.

  An example of 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 μg / ml, preferably 6 to 15 μg / ml, more preferably 9 to 13 μg / 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) as Triton, Nonidet P-40 (octylphenyl-polyethylene glycol) etc. as Nonidet, Tween 20 (polyethylene glycol sorbitan monolaurate) as Tween, Tween 40 (polyethylene glycol sorbitan monopalmitate), Tween 60 (polyethylene glycol sorbitan monostearate), Tween 80 (polyethylene glycol sorbitan monooleate), etc., Brij 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 (the nucleic acid amplification reaction solution 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. In the case of SDS, for example, 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%.

In the case of other surfactants, for example, in the case of Nonidet P-40, it 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%, and 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%.
When the enzyme solution used for the nucleic acid amplification reaction contains a surfactant, only the surfactant derived from the enzyme solution may be used, or the same or different surfactant may be added.

  Examples of the magnesium salt include magnesium chloride, magnesium sulfate, magnesium carbonate and the like. The density | concentration of the magnesium salt in a test sample (nucleic acid amplification reaction liquid) is 1-10 mM normally, for example, Preferably it is 2-6 mM, More preferably, it is 2-5 mM.

  Examples of organic acid salts include salts of citric acid, tartaric acid, propionic acid, butyric acid, and the like. Examples of the salt include sodium salt and potassium salt. Moreover, 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).

  When nucleic acid is extracted from a test sample, 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. However, the proteolytic enzyme treatment operation is not performed in the nucleic acid extraction operation or the subsequent operation.

  For example, Maniatis T., Fritsch E.F., Sambrook, J .: Molecular Cloning: A Laboratory Manual. 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001.

  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. As a template for nucleic acid amplification, 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. .

  When nucleic acid is extracted from a test sample, 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 the nucleic acid at a high temperature, for example, 90 to 95 ° C, preferably 93 to 95 ° C, more preferably 94 to 95 ° C.

  As the nucleic acid amplification method, PCR method (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. Sci. USA, Vol. 88, p.189-193, 1991), TMA method (Transcription-Mediated) -Amplification: Sarrazin C. et al., J. Clin. Microbiol., Vol.39: p.2850-2855 (2001)), TRC (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. Methods, vol.154: p.76) -81, 2008), SMAP method (Smart Amplification Process, Smart Amp method; Mitani Y., et al., Nature Methods, v ol.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). In the present invention, the PCR method is particularly preferably used, but is not limited thereto.

  In the present invention, 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 | limit in particular, it can set suitably according to the objective. For example, when the test sample includes cells of a different type from the microorganism to be detected, the target region preferably has a sequence specific to the microorganism to be detected. Further, depending on the purpose, it may have a sequence common to a plurality of types of microorganisms. Furthermore, the target area may be single or plural.

Using a primer set corresponding to the target region specific to the detection target microorganism and a primer set corresponding to the nucleic acid of a wide range of microorganisms, the amount of living cells of the detection target microorganism and the number of living cells of many types of microorganisms can be calculated. Can be measured simultaneously. The length of the target region usually includes 50 to 5000 bases 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 bacteria.

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

  Examples of preferred target regions are various specific genes such as 5S rRNA gene, 16S rRNA gene, 23S rRNA gene, tRNA gene, and pathogenic gene. One or a part of these genes may be targeted, and a region spanning two or more genes may be targeted. For example, by using the primer sets shown in SEQ ID NOS: 1 and 2, a part of the Chronobacter / Sakazaki-specific 16S rRNA gene can be amplified. A commercially available primer for 16S rRNA gene amplification may also be used.

  In addition, when the microorganism to be detected is a pathogenic bacterium, the target region includes a pathogenic gene. Examples of the pathogenic gene include Listeria ricin O (hlyA) gene of Listeria bacterium, enterotoxin (enterotoxin) gene and invasion (invA) gene of Salmonella bacterium, pathogenic E. coli O-157, O-26, O-111, etc. Verotoxin gene, Enterobacter or Cronobacter bacterium outer-membrane-protein A (ompA) gene (Cronobacter sakazaki) and macromolecular synthesis (MMS) operon (Cronobacter sakazaki), Legionella bacterium macrophage- invasion protein (mip) gene, heat-resistant hemolytic toxin gene of Vibrio parahaemolyticus, heat-resistant hemolytic toxin-like toxin gene, ipa gene of Shigella and intestinal invasive Escherichia coli (invasion plasmid antigen gene), invE gene (invasion gene), Staphylococcus aureus enterotoxin gene, Bacillus cereu Bacteria cereulide (emetic toxin) gene and enterotoxin gene, various toxin genes such as Clostridium botulinum and the like.

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

  When primers common to a plurality of types of microorganisms are used, viable cells of a plurality of types of microorganisms in a test sample can be detected. In addition, when a primer specific to a specific bacterium is used, a living cell of a specific bacterial species in a test sample can be detected.

  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.

(3) 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. When PCR is used for nucleic acid amplification, 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) can be used.

According to the electrophoresis method, 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.
When the real-time PCR method is employed, since the change in fluorescence intensity is generally a noise level and is equal to zero for the number of amplification cycles 1 to 10, they 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). Therefore, the larger the initial DNA template amount in the PCR reaction solution, the smaller the Ct value, and the smaller the template DNA amount, the larger the Ct value. Even if the amount of template DNA is the same, the Ct value of the PCR reaction in the same region becomes larger as the ratio of cleavage in the PCR target region in the template increases.

The presence or absence of an amplification product can also be determined by analyzing the melting temperature (TM) pattern of the amplification product.
Each of the above methods can also be used in optimizing various conditions in the method of the present invention.

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

<2> Kit of the Present Invention The kit of the present invention 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 contain a primer for amplifying a target region of DNA or RNA of a microorganism to be detected by a nucleic acid amplification method.

In a preferred embodiment, the kit of the present invention may further contain a drug that suppresses the action of the 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.
In addition, an enzyme having an activity of degrading cells other than microorganisms, protein colloid particles, fat, or carbohydrates present in a test sample can be added to the kit of the present invention.

  Enzymes, palladium complexes, and agents that suppress the action of nucleic acid amplification inhibitors as needed, as well as magnesium salts, organic acid salts or phosphates, and surfactants, all contain a single composition It may be a product, or may be a plurality of solutions or compositions containing each component in any combination.

  The nucleic acid amplification reaction is preferably a 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. In the above kit, the crosslinking agent and the medium are the same as those described in the method of the present invention.

  Preferable palladium complexes contained in the kit of the present invention are the same as the compounds described for the method of the present invention.

  Examples of 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, α2-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, and magnesium carbonate.

  Examples of organic acid salts include salts of citric acid, tartaric acid, propionic acid, butyric acid, and the like. Examples of the salt include sodium salt and potassium salt. Moreover, pyrophosphate etc. are mentioned as a phosphate. These may be one kind, or a mixture of two or more kinds.

In addition, 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 | limit in particular, For example, a lipolytic enzyme, a proteolytic enzyme, and a carbohydrase are mentioned. As the enzyme, one kind of enzyme may be used alone, or two or more kinds of enzymes may be used in combination, but both lipolytic enzyme and proteolytic enzyme, or lipolytic enzyme, proteolytic enzyme It is preferable to use all of saccharide-degrading enzymes.
Examples of the lipolytic enzyme include lipase and phosphatase, examples of the proteolytic enzyme include serine protease, cysteine protease, proteinase K, and pronase, and examples of the 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 a palladium complex, an enzyme and reaction solution for nucleic acid amplification, instructions describing the method of the present invention, and the like.

EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to these Examples.
In the following examples, each operation was performed under non-light-shielding unless otherwise specified.

[Example 1] Identification of viable and dead cells of Cronobacter sakazaki by dichloro (η-cycloocta-1,5-diene) palladium (II), bis (benzonitrile) dichloropalladium (II), or diamminedichloropalladium (II) For Chronobacter and Sakazaki, we examined the conditions for clarifying the distinction between living and dead bacteria using each palladium complex.

1. Test Material and Culture Method 1-1) Cronobacter sakazaki ATCC29544 was cultured at 37 ° C. for 16 hours using BHI broth.
1 ml of the above culture broth is chilled and centrifuged (4 ° C, 3,000 x G, 10 minutes), and after removing the supernatant, 1 ml of sterilized water is added to the pellet, and a portion of that is diluted 100-fold with sterile water A live cell suspension (4.7 × 10 ^{6 to} 1.0 × 10 ⁷ 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. Confirm that cells in each suspension do not form colonies with standard agar medium (Eiken, Tokyo, Japan), and use suspension / dead cell suspension (4.7 × 10 ⁶ to 6.1 × 10 ⁷ cells / ml). Hereinafter, damaged cells and dead cells are included and expressed as dead cell suspension).

  90 μl of each of the above-mentioned Chronobacter / Sakazaki live cell suspension or dead cell suspension (only 50 μl of the suspension subjected to the treatment with diamondinedichloropalladium) was subjected to the following test.

1-2) Preparation of each palladium complex solution and sample treatment
Dichloro (η-cycloocta-1,5-diene) palladium (II) 3.11 mg (10.89 μmol) was dissolved in 218 μl of DMSO (D8418-50ML, Sigma) to prepare a 50 mM solution. Further, 4.34 mg (11.31 μmol) of bis (benzonitrile) dichloropalladium (II) was dissolved in 226 μl of DMSO (Sigma) to prepare a 50 mM solution. These 50 mM palladium complex solutions were diluted with physiological saline to prepare 25 μM, 250 μM, 500 μM, 1 mM, 5 mM, 10 mM, and 25 mM complex solutions.

  Further, diamminedichloropalladium (II) (323-04961 Wako Pure Chemical Industries, Ltd., Osaka, Japan) 2.00 mg (9.46 μmol) was dissolved in 946 μl of DMSO (Sigma) to prepare a 10 mM solution. This solution was diluted with physiological saline to prepare 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM diamminedichloro-palladium (II) physiological saline solution.

  Then, 10 μl of each of the above palladium complex solutions (diamminedichloropalladium (II) only 50 μl) is added to Chronobacter sakazaki live cell suspension or dead cell suspension 90 μl (diamminedichloropalladium (II) 50 μl), and the constant temperature bath PERSONAL -11 (TAITEC, Tokyo, Japan) at 37 ° C for 30 minutes (diamminedichloropalladium (II) for 10 minutes). Then, cool and centrifuge (4 ° C, 10,000 x G, 5 minutes), remove the supernatant, suspend the pellet in 150 µl of sterilized water, stir well, and then perform the same cooling and centrifuge. Qing was removed. The obtained pellet (corresponding to 5 μl of cell suspension) was used as a sample for PCR amplification.

1-3) PCR amplification In order to efficiently perform PCR amplification without extracting nucleic acids from cells, a concentrated solution of a drug mixture that suppresses the action of a nucleic acid amplification inhibitor (this concentrated solution is used as a concentrated direct component, cDBC)) was prepared.
Specifically, 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.

For example, for PCR amplification 200 samples, 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 μl, 200 μl, 15 μl, 15 μl, 20 μl, respectively. When mixed, 500 μl of cDBC (10 × DBC) shown in Table 1 can be prepared. It should be noted that when using TaqManFast Universal PCR Master Mix (2 ×) described later and preparing a master mix according to the manufacturer's manual, it is assumed that MgCl ₂ equivalent to 2 mM is included as the final concentration, so the total MgCl ₂ Was estimated to be equivalent to 5 mM.

Brij 58, MgCl ₂ , and TSC were dissolved in sterilized water, autoclaved (121 ° C., 20 minutes), cooled to room temperature, and used as a stock solution. For BSA and Lysozyme, a stock solution was prepared with sterilized water, and sterilized by filtration through a 0.22 μm filter to obtain a stock solution.

Next, in order to perform real-time PCR shown in Table 2 without performing extraction of nucleic acids from cells (hereinafter referred to as “direct real-time PCR”). A master mix (master mix for direct real-time PCR) was prepared.
For PCR amplification, Primer 16S forward: 16S forward primer for C. sakazakii detection (5'-TAACAGGGAGCAGCTTGCTGCTCTG-3 ': SEQ ID NO: 1), Primer 16S reverse: Reverse primer for C. sakazakii detection (5'-CGGGTAACGTCAATTGCTGCGGT-3' : SEQ ID NO: 2) was used as a primer for PCR amplification. The fragment length of the amplified rRNA gene was 426 bp. As the 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. PCR amplification was performed using the above-described primers and the PCR amplification sample as a template. Real-time PCR amplification was performed twice.

Real-time PCR was performed using the real-time PCR apparatus (StepOnePlus Real-Time PCR System; Applied Biosystems) under the following PCR thermal cycle conditions.
3) 95 ℃, 20 seconds (1 cycle)
4) 95 ℃, 5 seconds; 55 ℃, 10 seconds; 72 ℃, 30 seconds (40 cycles)

As a positive control, 5 μl of the above-mentioned C. sakazakii live cell suspension (4.7 × 10 ⁷ CFU / ml) was used as a template. As a negative control, 5 μl of sterilized water was used as a template.

2. Results The results of real-time PCR are shown in Tables 3 and 4.

3. DISCUSSION As shown in Tables 3 and 4, with dichloro (η-cycloocta-1,5-diene) palladium (II) 25 μM, the Ct value of living cells hardly increased compared to that of untreated, and PCR amplification of dead cells was completely suppressed. Similarly, in bis (benzonitrile) dichloropalladium (II) and diamminedichloropalladium (II) 10 μM, the Ct value of living cells is hardly increased or slightly increased compared to untreated, And PCR amplification of dead cells was completely suppressed. Therefore, it was clarified that the living cells and the dead cells of Cronobacter sakazaki can be distinguished by the palladium complex. This method does not require light irradiation. Moreover, even if the target region was short (426 bp), it was possible to distinguish between live cells and dead cells.

[Example 2] Examination of action of palladium complex In Example 2, Dichloro (η-cycloocta-1,5-diene) palladium (II) was used as a palladium complex, and living and dead cells of Cronobacter sakazaki were used. Was identified.

1. Test Material and Culture Method 1-1) In the same manner as in Example 1, using a physiological saline, Chronobacter sakazaki ATCC 29544 live cell suspension (1.5 × 10 ⁹ CFU / ml) and damaged / dead cell tendon A 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 μl of each was subjected to the following test.

1-2) Preparation of Dichloro (η-cycloocta-1,5-diene) palladium (II) solution and sample treatment
Dichloro (η-cycloocta-1,5-diene) palladium (II) 6.28 mg (22.0 μmol) was accurately weighed and dissolved in 440 μl of DMSO (Sigma) to prepare a 50 mM solution. The 50 mM solution was diluted with physiological saline to prepare 250 μM and 1 mM solutions (palladium complex solution).

  Add 250 μM or 1 mM Dichloro (η-cycloocta-1,5-diene) palladium (II) 10 μl to the above live cell suspension or dead cell suspension 90 μl, and in a constant temperature bath at 37 ° C. for 30 minutes Retained. Then, add 1 ml of sterilized water, cool and centrifuge (4 ° C, 3,000 x G, 5 minutes), remove the supernatant, put 10 glass beads for cell disruption into the pellet, and vortex for 30 seconds The bacterial cells were disrupted by vigorous stirring. Then add 500 μl of 10 mM Tris-HCl (pH 8.0), gently stir, cool and centrifuge (3,000 × G, 5 minutes, 4 ° C.), and transfer the supernatant (equivalent to 500 μl) to a new 2.0 ml microtube. did.

  These samples were divided into a proteinase K treatment group and a proteinase K / SDS treatment group, and 10 μl proteinase K (1,250 U / ml; Sigma) was added to the proteinase K treatment group. In addition, 5 μl of 10% SDS and 10 μl of proteinase K (1,250 U / ml; Sigma) were added to the proteinase K / SDS treatment group. These samples were kept at 50 ° C. for 14 hours in a block incubator.

Thereafter, 0.5 ml of 1 M Tris-HCl / phenol (saturated phenol) was added to each sample and gently shaken for 15 minutes. Further, 0.5 ml of chloroform was added and gently shaken for 5 minutes. Then, cool and centrifuge (4 ° C, 15,000 × G, 10 minutes), transfer about 600 μl of the supernatant to a new 2 ml microtube, add 60 μl of 3M aqueous sodium acetate solution, add 1500 μl of cold ethanol, and gently And placed on ice for 5 minutes.
After cooling and centrifuging (15,000 × G, 10 minutes, 4 ° C.), the supernatant was removed, and the inside of the microtube was washed with 1000 μl of 70% cold ethanol, and then similarly cooled and centrifuged. After removing the supernatant, ethanol was removed by vacuum drying (5 minutes), 20 μl 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 μl thereof was used as a PCR amplification sample.

  Moreover, real-time PCR (quantitative PCR) amplification was performed directly from bacteria on the ¼ amount of the pellet just before the introduction of the cell disruption glass beads without performing the DNA extraction step as described above.

1-3) PCR amplification In the same manner as in Example 1, each of the above PCR amplification samples was subjected to real-time PCR amplification twice.

2. Results The results of real-time PCR are shown in Table 5.

According to Table 5, cells were treated with Dichloro (η-cycloocta-1,5-diene) palladium (II), and PCR was performed directly on chromosomal DNA present in the cells without DNA extraction from the cells. When this was done, the Ct value of the living cells of Chronobacter sakazaki was about 1.6 higher than that of the untreated cells at a palladium complex concentration of 25 μM, but PCR amplification of dead cells was completely suppressed, and a clear viability determination was possible. Met.
On the other hand, after treating the cells with a palladium complex and then crushing the cells with glass beads, the cells are treated with proteinase K to decompose intracellular proteins (including histone-like proteins that are DNA-binding proteins), and then phenol / chloroform. When real-time PCR amplification is carried out using DNA that has been extracted and purified by extraction / ethanol precipitation and extracted and purified, live and dead cells can be detected regardless of the concentration of the palladium complex and the presence or absence of SDS. Ct values were almost constant with no significant difference observed compared to no treatment.

3. Discussion As shown in Table 5, when real-time PCR amplification was performed directly without extracting DNA from the cell pellet after treatment with palladium complex, it was possible to distinguish between live and dead cells, When the cells were disrupted and treated with protease K, live cells and dead cells could not be distinguished.

[Example 3] Study on the mechanism of action of Dichloro (η-cycloocta-1,5-diene) palladium (II) on C. sakazakii dead cells As described above, when palladium complexes are allowed to act on microbial cells, most Palladium complexes have been shown to be more likely to covalently bind to DNA binding proteins, and this possibility was further investigated.
Specifically, after a palladium complex is allowed to act on dead cells, the cells are crushed with glass beads, and then DNA is extracted without protease K treatment. ) To maintain a tightly coupled state.

1. Test Material and Culture Method 1-1) 1 ml of live cell suspension (6.7 × 10 ⁸ CFU / ml) using physiological saline in the same manner as in Example 2 using Cronobacter sakazaki ATCC29544 and Damaged cell / dead cell suspension (hereinafter referred to as dead cell suspension including damaged cells and dead cells; 6.7 × 10 ⁸ cells / ml) was prepared.

The above-mentioned Chronobacter / Sakazaki live cell suspension or dead cell suspension was diluted 100-fold with sterilized water (6.7 × 10 ⁶ cells / ml), and 90 μl of each was subjected to the following test.

1-2) Preparation of Dichloro (η-cycloocta-1,5-diene) palladium (II) solution and sample treatment
Dichloro (η-cycloocta-1,5-diene) palladium (II) 4.24 mg (14.85 μmol) was accurately weighed and dissolved in 297 μl of DMSO (Sigma) to prepare a 50 mM solution. The 50 mM solution was diluted with physiological saline to prepare 250 μM and 1 mM solutions (palladium complex solution).

Add 10 μl of 250 μM or 1 mM dichloro (η-cycloocta-1,5-diene) palladium (II) to 90 μl of the above live cell suspension or dead cell suspension and hold at 37 ° C. for 30 minutes in a constant temperature bath. did.
Then, add 1 ml of sterilized water, cool and centrifuge (4 ° C, 3,000 x G, 5 minutes), remove the supernatant, put 10 glass beads for cell disruption into the pellet, and vortex for 30 seconds The bacterial cells were disrupted by vigorous stirring. Thereafter, 200 μl of TE buffer was added, gently stirred, cooled and centrifuged (3,000 × G, 5 minutes, 4 ° C.), and the supernatant (approximately 200 μl) was transferred to a new 1.5 ml microtube.

  Thereafter, 20 μl of 3M sodium acetate aqueous solution was added to each sample, 500 μl of 99.5% cold ethanol was added, gently stirred, and placed on ice for 5 minutes. After cooling and centrifuging (15,000 × G, 10 minutes, 4 ° C.), the supernatant was removed, and the inside of the microtube was washed with 500 μl of 70% cold ethanol, followed by cooling and centrifuging in the same manner. After removing the supernatant, ethanol was removed by vacuum drying (5 minutes), 20 μl of TE buffer was added to the pellet, and the mixture was allowed to stand at 4 ° C. overnight. After returning to room temperature, the mixture was gently stirred to obtain a DNA solution. 5 μl was used as a PCR amplification sample, and the remaining 15 μl was used below.

  200 μl of TE buffer was added to 15 μl of the DNA solution, 0.5 ml of 1 M Tris-HCl / phenol (saturated phenol) was added, gently mixed for 15 minutes, and then 0.5 ml of chloroform was further added, and gently mixed for 5 minutes. Then, cool and centrifuge (4 ° C, 15,000 x G, 10 minutes), transfer approximately 300 μl of supernatant to a new 1.5 ml microtube, add 30 μl of 3M aqueous sodium acetate, and then add 750 μl of 99.5% cold ethanol. Stir gently and place on ice for 5 minutes. After cooling and centrifuging (15,000 × G, 10 minutes, 4 ° C.), the supernatant was removed, and the inside of the microtube was washed with 500 μl of 70% cold ethanol, followed by cooling and centrifuging in the same manner. After removing the supernatant, ethanol was removed by vacuum drying (5 minutes), 15 μl of sterilized water was added to the pellet, and the mixture was kept at 37 ° C. for 30 minutes to prepare a DNA solution. 5 μl thereof was used as a PCR amplification sample.

  In addition, after treatment with the palladium complex solution, real-time PCR (quantitative PCR) amplification was performed directly from bacteria without performing the DNA extraction as described above on the pellet immediately before the introduction of the cell disruption glass beads.

1-3) PCR amplification In the same manner as in Example 2, real-time PCR amplification was performed twice using each of the above PCR amplification samples.

2. Results The results of real-time PCR are shown in Table 6.

  As shown in Table 6, Dichloro (η-cycloocta-1,5-diene) palladium (II) treatment was performed, and PCR was performed directly on chromosomal DNA present in the cells without extracting DNA from the cells. In this case, it was confirmed that the viability and death of Chronobacter sakazaki can be clearly determined with 25 μM of the palladium complex.

In addition, when cells are treated with a palladium drug, the cells are crushed with glass beads, DNA is extracted directly by ethanol precipitation, and real-time PCR amplification is performed. It was possible to identify the cells.
Furthermore, even when phenol / chloroform extraction was performed before ethanol precipitation, it was possible to clearly distinguish between live and dead cells with a palladium complex of 25 μM.

3. Discussion The major difference between Example 3 (Table 6) and Example 2 (Table 5) is the presence or absence of protease K treatment after disrupting cells with glass beads. In Example 3, protease K treatment is not performed.
That is, in Example 2 in which protease K treatment was performed, live cells and dead cells could not be distinguished by real-time PCR amplification using purified dead cell DNA, whereas this example in which protease K treatment was not performed. In 3, it was possible to distinguish between live cells and dead cells.

[Example 4] Additional studies on the mechanism of action of Dichloro (ethylenediamine) palladium (II) on C. sakazakii dead cells As a palladium complex, instead of Dichloro (η-cycloocta-1,5-diene) palladium (II), Dichloro (ethylenediamine) palladium (II) was used to examine the effect on C. sakazakii dead cells.

1. Test Material and Culture Method 1-1) Using Cronobacter sakazaki ATCC29544, in the same manner as in Example 1, 1 ml of live cell suspension (6.1 × 10 ⁸ CFU / ml) and damaged cells in sterilized water / Dead cell suspension (hereinafter referred to as dead cell suspension including damaged cells and dead cells; 6.1 × 10 ⁸ cells / ml) was prepared.
The above-mentioned Chronobacter / Sakazaki live cell suspension or dead cell suspension is diluted 10-fold with sterile water (6.1 × 10 ⁷ CFU / ml or cells / ml). Provided.

1-2) Preparation of Dichloro (ethylenediamine) palladium (II) solution and sample treatment
Dichloro (ethylenediamine) palladium (II) 3.60 mg (15.16 μmol) was accurately weighed and dissolved in 303 μl DMSO (Sigma) to prepare a 50 mM solution. The 50 mM solution was diluted with physiological saline to prepare 250 μM and 1 mM solutions (palladium complex solution).

  10 μl of each dichloro (η-cycloocta-1,5-diene) palladium (II) solution was added to 90 μl of the above live cell suspension or dead cell suspension, and kept at 37 ° C. for 30 minutes in a constant temperature water bath. After that, 0.5 ml of sterilized water is added, cooled and centrifuged (4 ° C, 3,000 x G, 5 minutes), the supernatant is removed, and QuickPene SP kit DNA tissue (SP-DT) (containing a surfactant) is added to the pellet. 180 μl of MDT (Tissue Lysis Buffer; cell lysis buffer) attached with FUJIFILM, Tokyo, Japan) and 20 μl of EDT (Proteinase K) were added and incubated at 55 ° C. for 3 hours. 20 μl of 3M sodium acetate aqueous solution was added thereto, and then 500 μl of 99.5% cold ethanol was added, gently stirred, and placed on ice for 5 minutes.

  The supernatant was removed by cooling centrifugation (15,000 × G, 10 minutes, 4 ° C.), and the inside of the microtube was washed with 500 μl of 70% cold ethanol, followed by cooling centrifugation similarly. After removing the supernatant, ethanol was removed by vacuum drying (5 minutes), 50 μl of sterilized water was added to the pellet, and the mixture was kept at 37 ° C. for 1 hour to prepare a DNA solution. 5 μl thereof was used as a PCR amplification sample.

  After the treatment with the palladium complex solution, real-time PCR (quantitative PCR) amplification was directly performed from bacteria without performing the DNA extraction as described above on the pellet obtained by the centrifugal separation treatment.

1-3) PCR amplification Each sample for PCR amplification was subjected to real-time PCR amplification twice in the same manner as in the above Examples.

2. Results The results of real-time PCR are shown in Table 7.

  According to Table 7, when treated with Dichloro (ethylenediamine) palladium (II) and PCR was directly performed on the chromosomal DNA present in the cells (in the column “Cells” in the table), the palladium complex concentration was 25 μM. The Ct value of real-time PCR of Cronobacter sakazaki live cells increased by 3.6 compared to untreated, whereas the Ct value of dead cells increased by 13.4 compared to untreated. The possibility of viability of live cells and dead cells was suggested. In addition, with the palladium complex of 250 μM, PCR of dead cells was completely suppressed.

On the other hand, when cells were treated with a palladium complex solution, then treated with protease K in the presence of a surfactant, and extracted and purified by ethanol precipitation, real-time PCR was performed using DNA as a template ("PK (+)" in the table) In the column of “+ Detergent + EtOH method”, no significant increase in Ct value was observed in both dead cells and live cells.
3. Consideration

  From the above results, it is not limited to dichloro (η-cycloocta-1,5-diene) palladium (II). Palladium complexes generally pass through dead cell membranes and are mostly DNA-binding, typically HU protein. It is considered that there is a high possibility of binding to a protein.

[Example 5] Identification of live and dead cells of Staphylococcus aureus by Diamminedichloropalladium (II) In this Example 5, Staphylococcus aureus, a gram-positive bacterium, was lived with a palladium complex. We examined whether it was possible to distinguish between cells and dead cells.

1. Test Material and Culture Method 1-1) 1 ml of live cell suspension (2.1 × 10 ⁷ CFU) in sterile water using Staphylococcus aureus ATCC 6538P strain in the same manner as in Example 1. / ml) and dead cell suspension (2.1 × 10 ⁷ cells / ml) were prepared.
50 μl each of the above-mentioned Staphylococcus aureus live cell suspension or dead cell suspension was subjected to the following test.

1-2) Preparation of Diamminedichloropalladium (II) solution and sample treatment
Diamminedichloropalladium (II) 1.70 mg was weighed and dissolved in 804 μl DMSO (Sigma) to prepare a 10 mM solution. The 10 mM solution was diluted with physiological saline to prepare 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM solutions.

  50 μl of each Diamminedichloropalladium (II) solution was added to 50 μl of the above S. aureus live cell suspension or dead cell suspension and kept at 37 ° C. for 10 minutes in a constant temperature water bath. Then, cool and centrifuge (4 ° C, 3,000 x G, 5 minutes), remove the supernatant, suspend the pellet in 150 µl of sterilized water, stir well, and perform the same cooling and centrifuge. Qing was removed. The obtained pellet (corresponding to 5 μl) was used as a sample for PCR amplification.

1-3) PCR amplification In the same manner as in the above examples, each of the above PCR amplification samples was subjected to real-time PCR amplification twice.

2. Results The results of real-time PCR are shown in Table 8.

  According to Table 8, Diamminedichloropalladium (II) 1 μM, the Ct value of living cells remained at about 3.4 as compared to the untreated Ct value, whereas target gene amplification was completely suppressed in dead cells. It was. Therefore, the palladium complex could clearly distinguish between live and dead cells of S. aureus.

3. Discussion As described above, it was clarified that the use of a palladium complex makes it possible to clearly distinguish live and dead cells even in S. aureus. According to the above examples, the palladium complex can be used not only for gram-negative bacteria, and viruses such as influenza viruses that have the same composition as the outer membrane of gram-negative bacteria as an envelope, but also without any outer membrane. It was also found that live and dead cells can be clearly distinguished even in Gram-negative bacteria and Gram-positive bacteria that are different from influenza viruses. Therefore, it is considered that the palladium complex can distinguish between living cells and dead cells of all microorganisms.

[Example 6] Study on discrimination of living cells and dead cells of microorganisms by a complex obtained by dissolving palladium (II) chloride (PdCl ₂ (II)) in DMSO In Example 6, as a macromolecule by covalent bond Using the palladium complex obtained by dissolving the defined palladium chloride (II) (PdCl ₂ (II)) in DMSO, we examined the distinction between live and dead cells of Cronobacter sakazaki.

1. Test Material and Culture Method 1-1) Chronobacter 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), and 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. Each suspension liquid soaked in boiling water was confirmed to not form colonies with standard agar medium (Eiken, Tokyo, Japan), and damaged / dead cell suspensions (hereinafter included damaged and dead cells). And expressed as dead cell suspension; 9.4 × 10 ⁶ cells / ml).
50 μl of each of the above-mentioned Chronobacter Sakazaki live cell suspension or dead cell suspension was subjected to the following test.

1-2) Preparation of palladium (II) chloride solution and sample treatment Palladium (II) 4.9 mg (27.63 μmol) was dissolved in 1104 μl DMSO (Sigma) to prepare a 25 mM solution. 100 mM, 1 mM, and 10 mM solutions of the 25 mM palladium (II) chloride solution were prepared in physiological saline.
10 μl of the above palladium (II) chloride solution was added to 90 μl of Chronobacter / Sakazaki 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, 3,000 x G, 5 minutes), remove the supernatant, suspend the pellet in 150 µl of sterilized water, stir well, and perform the same cooling and centrifuge. Qing was removed. The obtained pellet (corresponding to 5 μl) was used as a sample for PCR amplification.

1-3) PCR amplification Real-time PCR amplification of each PCR amplification sample was carried out in the same manner as in the above-described Examples.

2. Results The results of real-time PCR are shown in Table 9.

3. DISCUSSION As shown in Table 9, the Ct value of real-time PCR of living cells of Chronobacter sakazaki at 20 μM palladium chloride remained elevated by 7.2 compared to untreated, whereas death In the cell-treated group, it rose about 15.7 compared to untreated, and at 30 μM, the Ct value of real-time PCR of Chronobacter sakazaki live cells stayed at a level increased by 10.2 compared to untreated, In dead cells, amplification of the target gene was completely suppressed, suggesting the possibility of determining whether live cells and dead cells are alive.
Therefore, a palladium complex that clearly distinguishes between live cells and dead cells is organically composed of palladium compounds that are defined as macromolecules by covalent bonds with other elements or groups as disclosed in Example 6. A palladium complex obtained by dissolving in a solvent is also considered to be included.

[Example 7] Identification of living and dead cells of Escherichia coli by bis (triphenylphosphine) palladium (II) diacetate or palladium (II) acetate In Example 7, a carboxylate group which is a hard Lewis base Bis (triphenylphosphine) palladium (II) diacetate with palladium as a ligand and palladium complex produced by dissolving palladium (II) acetate in DMSO were used to distinguish between live and dead cells .

1. Test Material and Culture Method 1-1) Using E. coli strain JCM1649 with sterilized water, live cell suspension (2.2 × 10 ⁷ CFU / ml) and dead cell suspension (2.2 × 10 ⁷ cells / ml; 2 minutes immersion in boiling water) was prepared, and 90 μl of each was subjected to the following test. The JCM1649 strain can be obtained from RIKEN BioResource Center, Microbial Materials Development Office (3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture 305-0074).

1-2) Preparation of palladium complex solution and sample treatment
Bis (triphenylphosphine) palladium (II) diacetate (II) (Sigma) 3.11 mg (14.15 μmol) was accurately weighed and dissolved in 415.2 μl of dimethyl sulfoxide (DMSO, Sigma) to prepare a 10 mM solution. This solution was diluted with physiological saline to prepare 100 μM, 500 μM, and 1000 μM solutions.
Similarly, palladium (II) acetate (Palladium (II) acetate recrystallized, Aldrich) 4.02 mg (17.9 μmol) was accurately weighed and dissolved in 1790.0 μl DMSO to prepare a 10 mM solution. This solution was diluted with physiological saline to prepare 10 μM, 20 μM, 250 μM, and 500 μM solutions.

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

1-3) PCR amplification A master mix for direct real-time PCR was prepared with the composition shown in Table 10 below. Specifically, Taq DNA Polymerase with Standard Taq Buffer (New England Biolabs Japan Inc .; M0273S) is used as a real-time PCR buffer, Taq polymerase is added 4 times the usual amount, and cDBC (10 x DBC) is added to the buffer. A predetermined amount was added. Direct real-time PCR amplification (40 cycles) was performed twice. Hereinafter, New England Biolabs products are referred to as NEB.

For PCR amplification, 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 primer (both primers were outsourced to Nippon Gene). The fragment length of the amplified rRNA gene is 424 bp. As the Enterobacteriaceae ENT-16S TaqMan probe, 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. 66: 1798-1804, 2003, and the base of the ENA-16S TaqMan probe of SEQ ID NO: 6 As for the sequence, 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/) .

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

2. Results The results of real-time PCR are shown in Table 11.

3. Discussion According to Table 11, when bis (triphenylphosphine) palladium (II) diacetate was allowed to act on E. coli live cells and dead cells, target gene amplification of dead cells was completely suppressed at 50 μM or more. In the case of living cells, a tendency for the Ct value to increase depending on the concentration of this drug was observed, but it was possible to clearly distinguish between live and dead cells at a concentration of 50 μM.
In addition, the DMSO solution of palladium (II) acetate completely suppresses target cell amplification from dead cells at a concentration of 1 μM, and for live cells, the concentration is up to 2 μM compared to no treatment. However, it was suppressed to a degree that the Ct value slightly increased, and it was possible to clearly distinguish between live cells and dead cells.

Example 7 shows that it is possible to distinguish between living cells and dead cells using a palladium complex in which a soft R ₃ P group (triphenylphosphine group) is coordinated to palladium metal. In addition, when a palladium complex formed by dissolving palladium (II) acetate in DMSO was used, it was possible to clearly distinguish between live cells and dead cells.
As described above, the palladium complex finally produced regardless of the progress of the process, whether the ligand is a hard Lewis base, an intermediate Lewis base, or a soft Lewis base, is excellent in living cells, damaged cells, and dead cells. Identification of cells is possible.

Example 8
Identification of E. coli live and dead cells in milk using Dichloro (η-cycloocta-1,5, -diene) palladium (II)

Food samples and clinical specimens contain components that can be ligands for palladium complexes. For example, sterilized milk contains bovine somatic cells and bovine mammary epithelial cells. Even if these cells are dead cells, DNA binding protein (for example, HU histone-like protein), RNA binding protein (for example, Synaptotagmin-binding, cytoplasmic RNA-interacting protein “SYNCRIP”), molecular weight 66 kDa ), Proteins that form nuclear RNA-protein complexes, paraspeckle (Sasaki YTet.al. (2009) Proc. Natl. Acad. Sci. USA 106: 2525-2530), and ribosomal proteins It is also possible that these proteins are shed from bovine somatic cells and bovine mammary epithelial cells into sterilized milk, and these proteins are palladium Can be a ligand of a complex (see Examples 1-7).
In addition, since it has already been suggested that the palladium complex has a protein (or amino acid containing sulfur such as cysteine residue or other amino acid) as a ligand, various caseins in milk, whey protein, Palladium complexes may also be partly coordinated (Lippard et al. (Bioinorganic Chemistry Lippard, SJ and Berg, JM; supervised by Kazuko Matsumoto, pages 21-23 “Principle of coordination chemistry in bioinorganic chemistry research)).
Therefore, it was tested whether live cells and dead cells contained in sterilized milk can be distinguished using a palladium complex.

1. Test materials and culture method 1-1) Live cell suspension (8.2 × 10 ⁸ CFU / ml) and dead cell suspension (8.2 × 10 ⁸ cells / ml) of E. coli JCM1649 strain using physiological saline (The viable cell suspension was boiled in boiling water for 3 minutes and then rapidly cooled), and further diluted 10-fold with physiological saline to prepare a viable cell suspension and a dead cell suspension.

1-2) Preparation of palladium complex solution and sample treatment
Dichloro (η-cycloocta-1,5, -diene) palladium (II) (Sigma) 3.01 mg (10.54 μmol) and Diamminedichloropalladium (II) (Wako) 2.48 mg (11.73 μmol) are accurately weighed. 211 μl and the latter were dissolved in 1173 μl DMSO to prepare 50 mM solutions respectively. This solution was diluted with physiological saline to further prepare 100 μM, 500 μM, 1 mM, 5 mM, and 10 mM solutions.

Collect 12 ml of commercially available milk (sterilized: hereinafter referred to as “sterilized milk”), inoculate 20 μl of the 10-fold diluted live cell suspension, and add 1.6 × 10 ⁶ CFU / 12 ml of pasteurized milk. Prepared. Similarly, 20 μl of the 10-fold diluted dead cell suspension was inoculated into 12 ml of sterilized milk to prepare dead cell inoculated sterilized milk of 1.6 × 10 ⁶ cells / 12 ml of sterilized milk.
In addition, the commercially available milk (sterilized milk) used for the test confirmed that E. coli live cells were below the detection limit (<1 CFU / 2.22 ml) by the culture method.
In addition, this pasteurized milk is inoculated with dead cells of known concentrations of E. coli, and only the step of recovering bacteria from the milk described later without chemical treatment is performed. Thereafter, the same direct real-time as in Example 7 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.
Therefore, the dead cell-inoculated sterilized milk inoculated with E. coli dead cells at a concentration of 1.6 × 10 ⁶ CFU / 12 ml of sterilized milk has a negligible level of E. coli dead cells originally contained.
In addition, E. coli and other coliforms (including Enterobacteriaceae, including Salmonella) that are expected to be mixed at relatively high concentrations in pasteurized milk, the dead cells are 1.6 × 10 ⁶ CFU / Since the level of 12 ml of pasteurized milk is sufficiently envisaged, the dead cell-derived PCR needs to be completely suppressed in this method.

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.
PBS 10 ml was added to each collected milk pellet (a), stirred well, cooled and centrifuged (4 ° C., 3,000 × G, 5 minutes), and the supernatant was removed by decantation to collect each pellet (b) . 990 μl of sterilized water was added to each collected pellet (b) to make it suspend. To each of the pellets (b) suspension, 10 μl of the 100 μM, 500 μM, 1 mM, 5 mM, 10 mM, or 50 mM Dichloro (η-cycloocta-1,5, -diene) palladium (II) solution is added. μl was added, and the mixture was kept in a 37 ° C. constant temperature bath for 30 minutes while stirring (constant temperature water bath PERSONAL-11; TAITEC constant temperature bath 60 rpm).
Separately, a test was carried out using a diamminedichloropalladium (II) solution in the same dilution series as Dichloro (η-cycloocta-1,5, -diene) palladium (II).
Then, 10 ml PBS was added to each sample, stirred well, cooled and centrifuged (4 ° C, 3,000 x G, 5 minutes), and the supernatant was removed by decantation to obtain each pellet (c) . Furthermore, the same process was performed again. Add 100 μl of sterilized water to each pellet (c) to suspend, transfer to a 100 μl PCR tube from ABI, cool and centrifuge (4 ° C, 3,000 × G, 5 minutes), and remove the supernatant with a pipette. Each pellet (d) was recovered by removal. Each collected pellet (d) (corresponding to 5 μl) was used as a PCR amplification sample.

1-3) PCR amplification Direct real-time PCR amplification (40 cycles) was performed twice using the same master mix for direct real-time PCR as in Table 10 of Example 7 described above.

2. Results The results of real-time PCR are shown in Table 12.

3. Discussion According to Table 12, in the dead cell inoculated sterilized milk, the Ct value in the case of no drug treatment (no addition of palladium complex) was 30.9, whereas Dichloro (η-cycloocta-1,5, -diene ) No PCR amplification was observed at 10-500 μM when treated with palladium (II) and 50-500 μM when treated with diamminedichloropalladium (II).
On the other hand, in the case of sterilized milk inoculated with live cells, PCR (Dichloro (η-cycloocta-1,5, -diene) palladium (II) at 0-100 μM and Diamminedichloropalladium (II) at 0-50 μM, PCR Amplification was observed, and there was almost no difference even when compared with the Ct value when no palladium complex was added.
That is, it was shown that it is possible to distinguish between live cells and dead cells in sterilized milk using two types of palladium complexes.

[Example 9] Detection limit of specific identification of E. coli living cells in milk using Dichloro (η-cycloocta-1,5, -diene) palladium (II) The detection limit of specific identification of live E. coli was investigated.

1. Test Material and Culture Method 1-1) Prepare a live cell suspension (1.5 × 10 ⁹ CFU / ml) of E. coli JCM1649 strain using physiological saline, and then add 10, 100, 1000 in physiological saline. , and 10 ^four-fold diluted, was prepared live cell-suspension liquid.

1-2) Preparation of palladium complex solution and sample treatment
Dichloro (η-cycloocta-1,5, -diene) palladium (II) (Sigma) 3.01 mg (10.54 μmol) was accurately weighed and dissolved in 211 μl of DMSO to prepare a 50 mM solution. This was diluted with physiological saline to prepare a 5 mM solution.

Commercially available milk (sterilized:. Hereinafter, described as "sterilization milk") and 12 ml were collected and inoculated with the 10, 100, 1000, and 10 ⁴ fold dilutions cells tendon Nigoeki 10 [mu] l, 1.5 × 10 ^{6 ~1.5 × 10 3 CFU / 12} ml sample of milk was prepared.
In addition, the commercially available milk (sterilized milk) used for the test confirmed that E. coli live cells were below the detection limit (<1 CFU / 2.22 ml) by the culture method.
In addition, this pasteurized milk is inoculated with dead cells of known concentrations of E. coli, and only the step of recovering bacteria from the milk described later without chemical treatment is performed. Thereafter, the same direct real-time as in Example 7 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 sterilized milk inoculated with each live cell was cooled and centrifuged (4 ° C., 3,000 × G, 5 minutes), the supernatant was removed by decantation, and the milk pellet (a) was recovered. Add 10 ml of PBS to the collected milk pellet (a), stir well, cool and centrifuge again (4 ° C, 3,000 x G, 5 minutes), remove the supernatant by decantation, and remove the pellet (b). It was collected. The recovered pellet (b) is suspended in 990 μl of sterilized water, 10 μl of the 5 mM Dichloro (η-cycloocta-1,5, -diene) palladium (II) solution is added, and this is maintained in a 37 ° C. constant temperature bath. The mixture was kept for 30 minutes while stirring (constant temperature bath PERSONAL-11; TAITEC constant temperature bath 60 rpm).
Thereafter, 10 ml of PBS was added and stirred well, followed by cooling and centrifugation (4 ° C., 3,000 × G, 5 minutes), and the supernatant was removed by decantation to obtain a pellet (c). Furthermore, the same process was performed again. Suspend pellet (c) in 100 μl of sterile water, transfer to a 100 μl PCR tube from ABI, cool and centrifuge (4 ° C, 3,000 × G, 5 minutes), remove the supernatant with a pipette, and pellet (d) was recovered. The recovered pellet (d) (corresponding to 5 μl) was used as a PCR amplification sample.

1-3) PCR amplification PCR amplification (40 cycles) was performed twice using the same master mix for direct real-time PCR as in Table 10 of Example 7 described above.

2. Results The results of real-time PCR are shown in Table 13.

3. Discussion According to Table 13, dichloro (η-cycloocta-1,5, -diene) palladium (II) 50 μM, live E. coli cells in sterilized milk (1.5 × 10 ⁴ CFU / 12 ml milk = 1.3 × 10 ³ CFU / ml) was confirmed for target gene amplification.

  Since the primer for direct real-time PCR used in this example is a primer that targets the 16S rRNA gene that enables comprehensive detection of Enterobacteriaceae (Enterobacteriaceae), live cells of E. coli・ It can be used not only for identification of dead cells, but also for testing for determination of live or dead cells of Enterobacteriaceae in pasteurized milk.

In this example, even if E. coli is present in the pasteurized milk at a concentration of 10 ³ CFU / ml, or even if the E. coli live cells are contaminated at a lower concentration, 10 ³ CFU / ml It was shown that only viable cells can be detected by culturing for a short time until the concentration is reached. Therefore, it is easy to analogize the distinction between live and dead cells of a target cell clearly even in clinical specimens such as blood having a composition similar to milk.

  As shown in the above examples, it is concluded that the use of palladium complexes enables clear discrimination of living and dead cells (including damaged cells) of target cells in foods, environmental samples, and clinical specimens. Is done.

  According to the method of the present invention, living cells of microorganisms can be detected and distinguished from dead cells or damaged cells by a simple process. According to the present invention, 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. .

  Among the palladium complexes used in the present invention, for example, diamminedichloro-palladium (II) has no clinical experience, but basic studies have been conducted on antiviral and antitumor effects. It is considered that the risk is low compared to drugs such as EMA. Furthermore, a preferable palladium complex is cheaper than drugs such as EMA and is industrially advantageous.

Claims (30)

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, comprising the following steps:
a) adding a palladium 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 palladium complex is NH ₃ , RNH ₂ , halogen element, carboxylate group, H ₂ O, CO ₃ ²⁻ , OH ⁻ , NO ₃ ⁻ , ROH, N ₂ H ₄ , PO ₄ ³⁻ , R ₂ O, ^{_{RO -, ROPO 3 2-, (}} RO) 2 PO 2 -, R 2 S, R 3 P, RS -, CN -, RSH, RNC, (RS) 2 PO 2 -, (RO) 2 P (O) S ⁻ , SCN ⁻ , CO, H ⁻ , R ⁻ (where “R” represents a saturated or unsaturated organic group), NO ₂ ⁻ , Ar—NH ₂ , Ar—CN (Ar is an unsaturated organic group) The method according to claim 1, comprising a ligand selected from ^{: a} group), N ₂ , SO ₃ ²⁻ , an imidazole ring, an unsaturated cyclic organic group, and N ₃ ⁻ . The method according to claim 2, wherein the ligand is selected from the group consisting of NH ₃ , RNH ₂ , a halogen element, a carboxylate group, R ₃ P, and Ar—CN.   The palladium complex is dichloro (η-cycloocta-1,5-diene) palladium (II), bis (benzonitrile) dichloropalladium (II), diamminedichloropalladium (II), dichloro (ethylenediamine) palladium (II), and The process according to claim 1, wherein the process is selected from the group consisting of bis (triphenylphosphine) palladium (II) diacetate.   The palladium complex is a palladium complex produced by dissolving a palladium compound in a solution containing an organic solvent capable of binding to palladium as a ligand or a substance capable of binding to palladium as a ligand. The method described in 1.   The palladium compound is palladium chloride, palladium fluoride, palladium bromide, palladium iodide, palladium hydroxide, palladium (II) dinitrate, palladium (IV) tetranitrate, palladium acetate, palladium phosphate, dimethoxy palladium, methoxy phosphorus. 6. The method of claim 5, wherein the method is selected from the group consisting of palladium acid palladium, palladium sulfite, dinitropalladium, and palladium diazide.   The method according to claim 6, wherein the palladium compound is palladium chloride or palladium acetate, the palladium chloride is palladium (II) chloride, and the palladium acetate is palladium (II) acetate.   The method according to claim 5, wherein the organic solvent is dimethyl sulfoxide.   The method according to any one of claims 1 to 8, wherein the amplification of the target region is performed without extraction of nucleic acid from cells.   The method according to claim 9, wherein the amplification of the target region is performed in a microbial cell.   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, 10. The method according to 10.   The method according to any one of claims 9 to 11, wherein the amplification of the target region is performed in the presence of a surfactant.   The method according to any one of claims 1 to 12, wherein the target region is a target region of 50 to 5000 bases.   The method according to claim 13, wherein 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 according to any one of claims 1 to 14, wherein the test sample is any one of a food, a biological sample, drinking water, industrial water, environmental water, drainage, soil, or a wiping sample.   The method according to any one of claims 1 to 15, wherein the microorganism is a bacterium or a virus.   The method according to claim 16, wherein the bacterium is a gram-negative bacterium or a gram-positive bacterium.   The nucleic acid amplification method is 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. The method according to item.   The method according to claim 18, wherein the PCR method is performed by a real-time PCR method, and PCR and amplification product analysis are performed simultaneously.   The analysis of the amplification product is performed using a standard curve showing a relationship between the amount of microorganisms created using a standard sample of microorganisms and the amplification product, 20. The method described in 1. 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, comprising the following elements:
1) Palladium complex or
A palladium compound which forms a palladium complex when dissolved in an organic solvent capable of binding to palladium as a ligand, or a solution containing a substance capable of binding to palladium as a ligand;
2) A primer for amplifying a target region of DNA or RNA of a microorganism to be detected by a nucleic acid amplification method. The palladium complex is NH ₃ , NH ₂ , halogen element (Cl, F, Br, I, At), carboxylate group, H ₂ O, CO ₃ ^2- , OH ⁻ , NO ₃ ⁻ , ROH, N ₂ H ^{_{4, PO 4 3-, R 2}} O, RO -, ROPO 3 2-, (RO) 2 PO 2 -, R 2 S, R 3 P, RS -, CN -, RSH, RNC, (RS) 2 PO ^{_{2 -, (RO) 2 P}} (O) S -, SCN -, CO, H -, R - ( wherein "R" both represent a saturated or unsaturated organic ^{_{group), NO 2 -, Ar-}} NH The kit according to claim 21, comprising a ligand selected from ₂ (Ar is an unsaturated organic group), N ₂ , SO ₃ ²⁻ , an imidazole ring, an unsaturated cyclic organic group, and N ₃ ⁻ . It said ligand, NH _3, RNH _2, halogen, carboxylate groups, selected from the group consisting of R ₃ P, and Ar-CN, A method according to claim 22.   The palladium complex is dichloro (η-cycloocta-1,5-diene) palladium (II), bis (benzonitrile) dichloropalladium (II), diamminedichloropalladium (II), dichloro (ethylenediamine) palladium (II), and The kit according to claim 21, wherein the kit is selected from the group consisting of bis (triphenylphosphine) palladium (II) diacetate.   The palladium compound is palladium chloride, palladium fluoride, palladium bromide, palladium iodide, palladium hydroxide, palladium (II) dinitrate, palladium (IV) tetranitrate, palladium acetate, palladium phosphate, dimethoxypalladium, methoxyphosphorus. The kit of claim 21, selected from the group consisting of palladium acid palladium, palladium sulfite, dinitropalladium, and palladium diazide.   The kit according to claim 25, wherein the palladium compound is palladium chloride or palladium acetate, wherein the palladium chloride is palladium (II) chloride and the palladium acetate is palladium (II) acetate.   Furthermore, the kit of Claim 21, 25 or 26 containing the organic solvent which can couple | bond with palladium as a ligand.   28. The kit according to claim 27, wherein the organic solvent is dimethyl sulfoxide.   Furthermore, the kit as described in any one of Claims 21-28 containing the chemical | medical agent which suppresses the effect | action of a nucleic acid amplification inhibitor, magnesium salt, and organic acid salt or phosphate.   Furthermore, the kit as described in any one of Claims 21-29 containing surfactant.