JP2008079617A - Method of detecting microorganism and microorganism detection kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of detecting microorganism included in food or clinical specimen. <P>SOLUTION: This invention is a method of detecting live bodies of microorganism and comprises following steps: (a) the specimen is treated with ethidiummonoazide and irradiated with visible light, and the specimen is treated with a topoisomerase inhibitor and/or DNA gyrase inhibitor selected from amsacrine, camptothecin, ellipticin, etoposide, mitoxantrone, and/or ciprofloxacin; (b) DNA is extracted from the specimen and the target area of the extracted DNA is amplified; and (c) the amplification product is analyzed. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for detecting microorganisms contained in foods and clinical samples, and a microorganism detection kit. More specifically, the present invention relates to a detection method and a microorganism detection kit capable of selectively detecting viable microorganisms contained in foods and clinical samples.

  Conventionally, a plate culture method has been used to measure the number of general viable bacteria in foods, clinical samples, or the environment. However, the plate culture method requires about 2 days until results are obtained.

  With the improvement of food sterilization technology and processing technology, there is a growing need to identify the life-and-death state of microorganisms present in a test sample even in a trace amount. Especially in the food hygiene inspection and clinical laboratory areas, as a rapid detection method of bacteria, an attempt is made to amplify the specific gene of each bacteria to an amount that can be visually captured by PCR method, and to determine and quantify the presence of each bacteria Has been made. However, when a target is applied to bacterial DNA, the background of dead bacteria originally contained in the test sample is detected, so if a positive determination is made by PCR, it always indicates the presence of live bacteria. It was not always. Therefore, in the field of food hygiene and clinical examination, PCR is not yet widespread although it is highly sensitive and rapid.

Recently, attempts have been made to detect and quantify only viable bacteria in a test sample by preparing mRNA using reverse transcriptase as a target and then performing PCR using specific primers of each bacterium. However, this method does not inhibit reverse transcription of mRNA of dead bacteria, and if dead bacteria of 10 ⁴ cfu / ml or 10 ⁴ cfu / g or more are contained in the test sample, Since the background is detected, it cannot be said that it is sufficient as a method for distinguishing between life and death.

  Specifically, the method described in Patent Document 1 or Patent Document 2 is disclosed as a method for determining the life and death of microorganisms such as bacteria using the PCR method. However, the following problems remain in the method for determining the viability of microorganisms such as bacteria using these PCR methods.

The technique of the above-mentioned patent document 1 is that killed bacteria in some boiled (boiled) foods that have been sterilized at high temperature for 10 to 30 minutes at 100 ° C., or in foods that have been sterilized with ethanol or formaldehyde. Although the identification of microorganisms is illustrated, there is no food that has been subjected to such a sterilization treatment, particularly for the latter. In addition, detection of only living microorganisms in foods that have been subjected to low temperature preservative sterilization (LTLT sterilization), high temperature short time (HTST sterilization) or ultra high temperature instantaneous heat sterilization (UHT sterilization), which are currently the main sterilization methods in the food industry. In addition, detection of live specific pathogens in clinical specimens of infectious disease patients who have been administered antibiotics is not expected. Further, in the technique of Patent Document 1, in the case of foods or clinical specimens in which the background of dead bacteria is present at a concentration of 10 ⁴ cfu / ml or more in the test sample, the amount of PCR final amplification product derived from dead bacteria exceeds the detection limit. Thus, it is impossible to identify whether the PCR positive reaction of the test sample is derived from live bacteria or dead bacteria.

The technique of Patent Document 2 discloses a method for discriminating between live bacteria and dead bacteria using the fact that the RNA / DNA molar ratio of dead cells is relatively lower than that of living cells. . In this method, total RNA is extracted, complementary DNA is prepared using reverse transcription reaction, PCR is then performed to calculate its Ct value, and the molar concentration of RNA is determined using a separately prepared calibration curve. The region of the chromosomal DNA corresponding to this RNA is amplified by PCR to obtain the Ct value, and the molar concentration of the chromosomal DNA is calculated from the calibration curve, whereby RN
The molar ratio of A / DNA is obtained. That is, the above operation requires complicated total RNA extraction, and involves two steps of reverse transcription reaction-PCR, and therefore is inferior to PCR targeting normal DNA in terms of quantification and rapidity. Furthermore, while live bacteria produce RNA continuously, dead bacteria-derived RNA is degraded early and lacks stability. In addition, in foods and clinical specimens containing high concentrations of dead bacteria, only 1/10 concentration of viable bacteria can be detected. Therefore, it has been difficult to apply in food hygiene inspections and clinical inspections that require rapid, high sensitivity and accuracy.
Special Table 2003-530118 International Publication No. 2002/052034 Pamphlet

  In the present invention, live cells (Viable-and-Culturable cells) of microorganisms contained in foods and clinical samples are killed (Dead cells) or damaged (Injured cells or Viable-but-Non Culturable cells); It is an object of the present invention to provide a method for selective detection as compared with “VNC cell”), that is, a rapid alternative detection method that inherits the characteristics of the culture method as it is, and a kit for carrying out the method.

  The present inventors are applicable to various sterilization methods, a method for distinguishing between viability and death of microorganisms suitable for food hygiene inspection with high detection sensitivity, and a method capable of detecting specific pathogens in patients with infectious diseases in hospitals and clinical settings As a result of intensive studies, a method for discriminating viable and damaged microorganisms in a test sample was treated with a topoisomerase inhibitor and / or a DNA gyrase inhibitor, or ethidium monoazide. Treatment with visible light irradiation, and topoisomerase inhibitors and / or DNA gyrase inhibitors other than ethidium monoazide can selectively amplify viable bacterial chromosomal DNA by PCR reaction. The inventors have found that this is a quick alternative method and have completed the present invention.

That is, the present invention provides a method for detecting viable microorganisms in a test sample, which includes the following steps.
a) treating the test sample with a topoisomerase inhibitor and / or a DNA gyrase inhibitor;
b) extracting DNA from the test sample, amplifying the target region of the extracted DNA by PCR, and c) analyzing the amplified product.

  The method is preferably characterized in that the amplification product is analyzed using a standard curve showing the relationship between the amount of microorganisms prepared using a standard sample of microorganisms and the amplification product.

  In addition, the method preferably includes performing the PCR by a real-time PCR method, and simultaneously analyzing the PCR and the amplification product.

  In the method, the test sample is preferably milk, dairy product, food made from milk or dairy product, blood sample, urine sample, spinal fluid sample, synovial fluid sample, or pleural fluid sample. It is an aspect.

  In the method, the microorganism is preferably a bacterium.

In the method, the target region is preferably a 23S rRNA gene. In this embodiment, the PCR is preferably performed using the primer set shown in SEQ ID NOs: 1 and 2, or the primer set shown in SEQ ID NOs: 3 and 4.

  In the method, the microorganism is preferably a pathogenic bacterium. In this embodiment, the target region is preferably a pathogenic gene. Furthermore, in this embodiment, the PCR is preferably performed using the primer sets shown in SEQ ID NOs: 7 and 8.

  In the method, the topoisomerase inhibitor is selected from the group consisting of Amsacrine, Camptothecin, Doxorubicin, Ellipticine, Etoposide, Mitoxantrone, Sintopin, Topotecan, Topotecan) and CP-115,953 are preferred embodiments.

  In the method, the DNA gyrase inhibitor is selected from the group consisting of ciprofloxacin, ofloxacin, enoxacin, pefloxacin, fleroxacin, norfloxacin, nalidixic acid It is preferable that the acid is selected from acid, oxolinic acid, and pyromidic acid.

  Further, the method preferably includes a step of irradiating a test sample to which ethidium monoazide is added with visible light, wherein the topoisomerase inhibitor and / or DNA gyrase inhibitor is ethidium monoazide. .

  Moreover, the said method makes it a preferable aspect to process a test sample with ethidium monoazide and topoisomerase inhibitors other than ethidium monoazide, and / or a DNA gyrase inhibitor.

Moreover, the said method makes it a preferable aspect to perform the following process before the process of said a).
d) A step of treating the test sample with topoisomerase and / or DNA gyrase.

The method also provides a kit for detecting live microorganisms in a test sample by PCR, comprising the following elements:
A topoisomerase inhibitor and / or DNA gyrase inhibitor, and primers for amplifying the target region of the microbial DNA to be detected by PCR.
In addition, the kit preferably includes topoisomerase and / or DNA gyrase.

  In the kit, the topoisomerase inhibitor includes Amsacrine, Camptothecin, Doxorubicin, Ellipticine, Etoposide, Mitoxantrone, Sintopin, Topotecan, Topotecan) and CP-115,953 are preferred embodiments.

  In the kit, the DNA gyrase inhibitor includes ciprofloxacin, ofloxacin, enoxacin, pefloxacin, fleroxacin, norfloxacin, nalidixic acid It is preferable that the acid is selected from acid, oxolinic acid, and pyromidic acid.

  In addition, the kit preferably includes ethidium monoazide and a topoisomerase inhibitor and / or DNA gyrase inhibitor other than ethidium monoazide.

  Moreover, the said kit makes it a preferable aspect that the said primer is the primer set shown to sequence number 1 and 2, or the primer set shown to sequence number 3 and 4.

  Moreover, the said kit makes it a preferable aspect that the said primer is a primer set shown to sequence number 7 and 8.

  Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following preferred 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 viable microorganisms in a test sample, and includes the following steps.
a) treating the test sample with a topoisomerase inhibitor and / or a DNA gyrase inhibitor;
b) extracting DNA from the test sample, amplifying the target region of the extracted DNA by PCR, and c) analyzing the amplified product.

  In the present specification, the “test sample” is a target for detecting viable microorganisms present therein, and can detect the presence by amplification of a specific region of chromosomal DNA by PCR. Although there is no particular limitation as long as it is present, foods such as milk, dairy products, foods made from milk or dairy products, blood samples, urine samples, spinal fluid samples, synovial fluid samples, pleural effusion samples and the like can be mentioned. In particular, milk, dairy products, foods made from milk or dairy products are preferred. In the present invention, the test sample may be the product or biological sample itself as described above, which is diluted or concentrated, or pretreated other than the treatment according to the method of the present invention. Also good. Examples of the pretreatment include heat treatment, filtration, and centrifugation.

  A “microorganism” is an object to be detected by the method of the present invention, can be detected by the PCR method, and the action of the topoisomerase inhibitor or DNA gyrase inhibitor on the microorganism is viable and dead. As long as it is different from those of damaged bacteria, it is not particularly limited, but preferably, bacteria, filamentous fungi, yeast 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 bacteria, Listeria monocytogenes such as Listeria monocytogenes, Bacillus cereus ( Bacillus cereus), Mycobacterium, and the like. In addition, Gram-negative bacteria include Escherichia bacteria such as Escherichia coli, Enterobacter sakazakii such as Enterobacter sakazakii, and Citrobacter koseri such as Citrobacter koseri. Intestinal bacteria group represented by Klebsiella oxytoca such as Klebsiella oxytoca, Salmonella bacteria, Vibrio bacteria, Pseudomonas bacteria and the like.

In the present invention, a “live cell” generally means a state that can grow when cultured under suitable culture conditions and exhibits the metabolic activity of the microorganism (Viable-and-Cultura).
ble state) and refers to a microorganism with little cell wall damage. The metabolic activity mentioned here can be exemplified by ATP activity and esterase activity.

  A “dead cell” is a microorganism that is incapable of growing even when cultured under suitable culture conditions and does 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.

  “Injured cell or Viable-but-Non Culturable cell” is damaged by human or environmental stress, so it can proliferate even when cultured under suitable culture conditions. Although the metabolic activity of the microorganism is lower than that of live bacteria, it is significantly more active than that of dead bacteria.

  In particular, in food hygiene inspections and clinical tests, attention has been paid to the detection of bacteria that show the state of damaged bacteria by mild heat treatment and administration of antibiotics. It is an object of the present invention to provide a method for detecting microorganisms that can also distinguish between bacteria and dead or damaged bacteria.

  In addition, the number of cells of live bacteria, damaged bacteria, and dead bacteria is usually represented by the number of cells / ml. The number of viable cells can be approximated by the number of colonies formed when cultured on a suitable plate medium under suitable conditions (cfu / ml (colony formatting units / ml)). In addition, a standard sample of damaged bacteria can be prepared by, for example, heat-treating a viable cell suspension, for example, by heating in boiling water. In this case, the number of cells of damaged bacteria is heat-treated. It can be approximated by the cfu / ml of the previous viable cell suspension. In addition, although the heating time in boiling water for preparing damaged bacteria changes with kinds of microorganisms, in the case of the bacteria described in the Example, damaged bacteria can be prepared in about 50 seconds. In addition, a standard sample of damaged bacteria can also be prepared by antibiotic treatment, in which case the number of cells of damaged bacteria can be determined by treating the viable cell suspension with antibiotics and then removing the antibiotics. By measuring the transmittance of visible light (wavelength 600 nm), that is, turbidity, and comparing it with the turbidity of a viable cell suspension in which the viable cell count concentration is known in advance, the preferable conditions on a suitable plate medium Can be approximated by the number of colonies formed (cfu / ml).

  The method of the present invention is intended to detect live bacteria, and the microorganisms distinguished from live bacteria may be damaged or dead.

In the present invention, “detection of viable bacteria” includes both determination of the presence or absence of viable bacteria in the test sample and determination of the amount of viable bacteria. Further, the amount of viable bacteria is not limited to an absolute amount, and may be an amount relative to a control sample.
Hereinafter, the method of the present invention will be described step by step.

(1) Step a)
A test sample is treated with a topoisomerase inhibitor and / or a DNA gyrase inhibitor.
The topoisomerase inhibitor (Topoisomerase poison) and the DNA gyrase poison (DNA Gyrase poison) used in the present invention do not inhibit the activity of cleaving DNA by topoisomerase (Topoisomerase) and DNA gyrase (DNA Gyrase), respectively. Those that inhibit recombination of DNA or those that promote the rate of cleaving DNA. Preferably, the topoisomerase inhibitor and DNA gyrase inhibitor are covalently linked to chromosomal DNA of microorganisms or intercalated to chromosomal DNA and covalently linked to chromosomal DNA by irradiation with visible light, or simply intercalated to chromosomal DNA. Or a complex with topoisomerase or DNA gyrase.

  Both topoisomerase inhibitors or DNA gyrase inhibitors may be used, or only one of them may be used.

  The topoisomerase inhibitor and the DNA gyrase inhibitor are preferably those that have different effects on live cells, damaged cells or dead cells, bovine leukocytes and other somatic cells, leukocytes, platelets and the like, more specifically, It is preferable that it is more permeable to cell walls of damaged or dead cells, somatic cells such as bovine leukocytes, and cell membranes such as leukocytes and platelets than cell walls of live bacteria.

  Examples of the topoisomerase inhibitor include Amsacrine, Camptothecin, Doxorubicin, Ellipticine, Etoposide, Mitoxantrone, Sintopin, Topotecan, Topote, and Topotecan CP-115, 953 etc. are mentioned. One type of topoisomerase inhibitor may be used alone, or two or more types may be used in combination.

  Examples of the DNA gyrase inhibitor include ciprofloxacin, ofloxacin, enoxacin, pefloxacin, fleroxacin, norfloxacin, nalidixic acid, oxolin Examples include acids (Oxolinic acid) and pyromidic acid. One DNA gyrase inhibitor may be used alone, or two or more DNA gyrase inhibitors may be used in combination.

  Conditions for treatment with a topoisomerase inhibitor or a DNA gyrase inhibitor can be set as appropriate. For example, various concentrations of topoisomerase can be added to suspensions of live and dead or damaged microorganisms to be detected. Inhibitors or DNA gyrase inhibitors are added for various periods of time, and then the cells are separated by centrifugation, etc., and analyzed by PCR to make it easy to distinguish between live and dead or damaged bacteria. Can be determined. Furthermore, after adding a topoisomerase inhibitor of various concentrations to suspensions of living cells of microorganisms to be detected and somatic cells such as bovine leukocytes or platelets, and leaving them for a predetermined time, the cells and By separating the various cells and analyzing them by PCR, it is possible to determine conditions for easily distinguishing live cells from various cells. As such conditions, specifically, a final concentration of 1 to 100 μg / ml for amsacrine, 25 to 37 ° C., 5 minutes to 48 hours, a final concentration of 0.05 to 5 μg / ml for ellipticine, 25 to 37 ° C., 10 Min-48 hours, final concentration of 1-100 μg / ml for camptothecin, 25-37 ° C., 10-48 hours, final concentration of 0.4-40 μg / ml for ciprofloxacin, 25-37 ° C., 10 minutes-48 In time, etoposide has a final concentration of 1 to 100 μg / ml, 25 to 37 ° C., 5 minutes to 48 hours, and mitoxantrone has a final concentration of 0.1 to 10 μg / ml, 25 to 37 ° C., 10 minutes to 48 hours. . After the test sample is processed under predetermined conditions, the processing is preferably stopped by dilution and / or removal by centrifugation or the like.

The above-described topoisomerase inhibitor and DNA gyrase inhibitor are more permeable to the cell walls of dead and damaged bacteria than the cell walls of live bacteria. Therefore, it is considered that the cell wall of viable bacteria does not substantially permeate within the action time shown above, and the cell membrane of somatic cells that are damaged or dead or dead cells permeate. In addition, it is considered that the inhibitor permeates the living somatic cell because it has only a cell membrane and no cell wall. As a result, topoisomerase inhibitors or DNA gyrase inhibitors enter dead and dead cells of somatic cells and cells of damaged bacteria, followed by random covalent linkage or intercalation with chromosomal DNA, Alternatively, it forms a complex with topoisomerase and further inhibits DNA rebinding by topoisomerase II or topoisomerase I in somatic cells, topoisomerase IV or topoisomerase I, III, or DNA gyrase in dead or damaged cells. Or DN
It is presumed that chromosomal DNA is fragmented by promoting the A cleavage rate.

  When chromosomal DNA of damaged or dead bacteria is preferentially fragmented over live bacteria, the target region of chromosomal DNA is amplified by PCR in live bacteria, whereas the target region is cleaved in damaged or dead bacteria As a result, PCR amplification is inhibited. For example, amsacrine and camptothecin inhibit PCR amplification due to further cross-linking. Therefore, live bacteria can be selectively detected by PCR compared with damaged or dead bacteria.

  In dead bacteria, intracellular topoisomerase and / or DNA gyrase activity may be lost. In addition, even in damaged bacteria, the activity of these enzymes may be reduced or lost. Therefore, in a preferred embodiment of the present invention, prior to step a), the test sample is treated with topoisomerase and / or DNA gyrase (step d)). Step d) will be described in detail later.

  In another preferred embodiment of the present invention, the topoisomerase inhibitor or DNA gyrase inhibitor is ethidium monoazide and includes a step of irradiating a test sample to which ethidium monoazide is added with visible light. Ethidium monoazide (EMA) is more likely to penetrate the cell walls of damaged and dead bacteria than the cell walls of living microorganisms. Therefore, it is considered that EMA does not substantially permeate the cell walls of viable bacteria, but permeates the cell walls of damaged or dead cells or somatic cells that are dead cells. In addition, when leukocytes and platelets in the blood are living cells, EMA permeates the cell membrane of the cells under sterile water or a hypotonic salt solution. EMA enters dead cells and damaged bacteria of somatic cells and cells of dead bacteria and randomly intercalates into nuclear DNA, and then only EMA intercalated by visible light irradiation is converted to nitrene, Covalently binds to internal DNA. Then, it is presumed that chromosomal DNA is fragmented by inhibiting DNA recombination by topoisomerase II in somatic cells, topoisomerase IV in damaged or dead cells, or DNA gyrase.

  The conditions for treatment with EMA can be set as appropriate. For example, EMA of various concentrations is added to a suspension of living microorganisms to be detected and damaged or dead bacteria for various times. After placing the sample, irradiate visible light, and if necessary, separate the cells by centrifugation, etc., and analyze them by PCR to determine conditions that make it easy to distinguish between live and dead and damaged bacteria. Can do. Moreover, the conditions for visible light irradiation can be determined by changing the irradiation time and conducting the above experiment. Specifically, the treatment with EMA preferably has a final concentration of 0.5 to 100 μg / ml, 4 to 10 ° C., and 5 minutes to 48 hours. The EMA treatment is preferably performed under light shielding. As visible light, visible light containing a component of 500 to 700 nm is preferable. Specific examples of the conditions for irradiation with visible light include conditions for irradiating 100 to 750 W of visible light for 5 minutes to 2 hours from a distance of 10 to 50 cm from the test sample. The visible light irradiation is preferably performed at a low temperature, for example, by cooling the sample with ice.

  In a particularly preferred embodiment of the present invention, the test sample is subjected to EMA treatment and visible light irradiation treatment, and treatment with a topoisomerase inhibitor and / or DNA gyrase inhibitor other than EMA. In this case, the order of the EMA treatment and the visible light irradiation treatment and the treatment with a topoisomerase inhibitor and / or a DNA gyrase inhibitor other than EMA is not particularly limited, and these treatments may be performed simultaneously.

(2) Step b)
DNA is extracted from the test sample treated in step a), and the target region of the extracted DNA is amplified by PCR (White, TJ et al., Trends Genet., 5, 185 (1989)).

  Extraction of DNA from a test sample is not particularly limited as long as the extracted DNA can function as a template in PCR, and can be performed according to a commonly used method for extracting DNA of a microorganism.

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

  In the present invention, the “target region” is a region of the chromosomal DNA that can be amplified by PCR using the primer used in the present invention, and is not particularly limited as long as it can detect the microorganism to be detected. It can be set as appropriate according to the purpose. 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 chromosomal DNA of a wide range of microorganisms, the amount of living microorganisms of the detection target microorganisms and the number of living microorganisms of many types of microorganisms Can be measured simultaneously. The length of the target region is usually 80 to 3000 bases, preferably 900 to 3000 bases, particularly preferably 2000 to 3000 bases. Specific examples include 16S rRNA gene and 23S rRNA gene. Of these, the 23S rRNA gene is preferred.

  The primer used for PCR is not particularly limited as long as it can specifically amplify the target region. Specifically, examples of the primer corresponding to the 23S rRNA gene include the primer sets shown in SEQ ID NOs: 1 and 2, or the primer sets shown in SEQ ID NOs: 3 and 4. Moreover, as a primer corresponding to a 16S rRNA gene, the primer set shown to sequence number 5 and 6 is mentioned.

  In addition, when the microorganism to be detected is a pathogenic bacterium, the target region includes a pathogenic gene. The pathogenic genes include Listeria ricin O (hlyA) gene of Listeria bacterium, enterotoxin gene and invA gene of Salmonella bacterium, verotoxin gene of pathogenic E. coli O-157, MMS gene of Enterobacter bacterium Sakazaki bacteria), Staphylococcus aureus enterotoxin gene, Bacillus cereus cereulide (vomiting toxin) gene, enterotoxin gene, botulinum various toxin genes, and the like. Moreover, as a primer corresponding to the same gene, the primer set shown to sequence number 7 and 8 is mentioned.

  When primers common to a plurality of types of microorganisms are used, viable bacteria 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 viable bacterium of a specific bacterial species in a test sample can be detected.

  The PCR conditions are not particularly limited as long as specific amplification in accordance with the principle of PCR occurs, and can be set as appropriate.

  Among the embodiments of the present invention, in the embodiment in which the EMA treatment and the visible light treatment are performed, when the target region is long, for example, 2000 or more, viable bacteria are effectively detected only by the EMA treatment and the visible light irradiation treatment alone. be able to. On the other hand, when the target region is short, for example, 200 or less, it is preferable to use EMA treatment and visible light treatment together with treatment with a topoisomerase inhibitor and / or DNA gyrase inhibitor other than EMA.

(3) Step c)
Subsequently, the PCR amplification product is analyzed. The analysis method is not particularly limited as long as the PCR amplification product can be detected or quantified, and electrophoresis method, real-time PCR method (Nogva et al./Application of 5'-nuclease PCR for quantitative detection of Listeria monocytogenes in pure cultures, water, skim milk, and unpasteurized whole milk.Appl.Environ.Microbiol., vol.66, 2000, pp.4266-4271, Nogva et al./ Application of the 5'-nuclease PCR assay in evaluation and development Appl. Environ. Microbiol., vol. 66, 2000, pp. 4029-4036). According to the electrophoresis method, the amount and size of the PCR 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. The fluorescence value multiplied by 10 is defined 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 viable bacteria are detected by the method of the present invention, PCR amplification products are analyzed 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 products. The presence or absence of bacteria 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. In addition, if the correlation between the amount of microorganism and the amount of DNA is examined in advance, DNA isolated from the microorganism can be used as a standard sample.

(4) Step d)
As described above, in dead bacteria, intracellular topoisomerase and / or DNA gyrase activity may be lost, and even when treated with a topoisomerase inhibitor or DNA gyrase inhibitor, chromosomal DNA cleavage does not occur. Sometimes. Even in such a case, if the test sample is treated with topoisomerase and / or DNA gyrase prior to step a), the DNA is selectively cleaved to dead bacteria. Amplification can be inhibited.

Both topoisomerase and DNA gyrase may be used, or only one of them may be used. Moreover, each may be used alone or in combination of two or more.
The reaction conditions by topoisomerase or DNA gyrase are specifically exemplified by the conditions shown in the reference examples described later, but in general, a supernatant containing microorganisms collected from clinical specimens such as food and blood On the other hand, after cooling and centrifugation at 14,000 × g for 10 minutes at 4 ° C., the supernatant was removed, and then a DNA cleavage buffer (10 mM Tris-HCl buffer pH 7.9, 50 mM potassium chloride, 50 mM sodium chloride). 5 mM magnesium chloride, 0.01 mM
1 ml of EDTA (2.5% glycerol) is added, topoisomerase or DNA gyrase is added to a final concentration of 1 to 50 mM, and ATP is further added to a final concentration of 1 to 50 mM. And react for 5 to 30 minutes. In addition, since ATP is required for the activity of topoisomerase and DNA gyrase, it is preferable to add an ATP or ATP synthesis system when a test sample is treated with these enzymes.

<2> Kit of the Present Invention The kit of the present invention is a kit for detecting viable microorganisms in a test sample by PCR, comprising a topoisomerase inhibitor and / or a DNA gyrase inhibitor, and a detection target. Primers for amplifying the target region of microbial DNA by PCR are included.

  In the above kit, the topoisomerase inhibitor and / or the DNA gyrase inhibitor are the same as those described in the method of the present invention.

  A preferred embodiment of the kit of the present invention contains, as a topoisomerase inhibitor and / or DNA gyrase inhibitor, other topoisomerase inhibitors and / or DNA gyrase inhibitors other than EMA and EMA.

  The kit of the present invention contains topoisomerase and / or DNA gyrase in addition to the above-described elements.

  The kit of the present invention can further contain a diluent, a reaction solution for topoisomerase and / or DNA gyrase reaction, instructions describing the method of the present invention, and the like.

  EXAMPLES Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.

[Example 1]
Each of living microorganisms and damaged microorganisms was treated with a topoisomerase inhibitor, and the degree of cleavage in each chromosomal DNA was examined.

1. Preparation of Sample 1-1) Preparation of Suspension of Live and Damaged Bacteria Listeria monocytogenes JCM 2873 (hereinafter sometimes abbreviated as “Listeria”) is a BHI broth. Was cultured at 30 ° C., and 40 ml of the logarithmic growth medium was centrifuged at 8,000 × g for 15 minutes at 4 ° C., and the supernatant was removed. After adding 40 ml of physiological saline to the cells and stirring well, similarly cooling and centrifuging and removing the supernatant, 10 ml of physiological saline was added to the cells to obtain a viable cell suspension. When the viable cell count of this viable cell suspension was measured with a standard agar plate medium, it was 1.2 × 10 ⁹ cfu / ml.

  Moreover, 1 ml of the above-mentioned viable cell suspension was placed in a 1.5 ml microtube, immersed in boiling water for 50 seconds, and then rapidly cooled with ice water to prepare a damaged cell suspension. In addition, although it is thought that a small number of living bacteria and a part of dead bacteria are included in the cell in this suspension, since they are mainly damaged bacteria, they are simply described as “damaged bacteria”. In addition, the method of this invention is a method of detecting a living microbe originally, and it does not ask | require whether the microbe distinguished from a live microbe is a damaged microbe or a dead microbe. The same applies to other bacteria described below.

1-2) Treatment with Topoisomerase Inhibitor 1 ml each of the live and injured suspensions of Listeria prepared above (each 1.2 × 10 ⁹ cfu / ml) was added to 9 ml of BHI broth prepared newly. In addition, the number of viable and damaged bacteria in the medium is 1.2 × 10 ⁸ cfu / ml, and 5 mg dissolved in DMSO
100 μl of / ml amsacrine solution was added. The final concentration of amsacrine was 50 μg / ml, and the final concentration of DMSO was 1%. Then, it incubated at 30 degreeC for 24 hours, 48 hours, or 72 hours, respectively.

  For damaged bacteria, in order to examine the effect of DNase having the activity remaining in the damaged bacteria on the chromosomal DNA of the damaged bacteria, 1 ml of the damaged bacteria suspension was added to 9 ml of BHI broth prepared newly, and then Instead of the amsacrine solution, 100 μl of DMSO was added (DMSO final concentration 1%) and incubated at 30 ° C. for 72 hours. In addition, 1 ml of viable cell suspension and 1 ml of damaged cell suspension were used as controls.

1-3) Extraction of DNA Each suspension was centrifuged at 8,000 × g for 15 minutes at 4 ° C., and the supernatant was completely removed. Add 0.5 ml of 5 mM EDTA solution to the pellet, add 20 μl of achromopeptidase solution (manufactured by Wako Pure Chemicals, catalog number: 014-09661) previously prepared to 5 mg / ml with 10 mM sodium chloride aqueous solution, and add it at 50 ° C. Left for 30 minutes. Thereafter, 0.5 ml of 10 mM Tris-HCl buffer (pH 8.0) was added, and 20 μl of 1250 U / ml proteinase K (Sigma, EC 3.4.21.64) was added, and 10% (w / v) in advance with sterile water. 400 μl of the prepared SDS solution was added and treated overnight at 50 ° C.

  Half of each treatment solution is divided into two 2 ml microtubes, 1M Tris-HCl buffer (pH 8.0) / saturated phenol 0.5 ml is added, and after gently mixing for 15 minutes, 0.5 ml of chloroform is added. And gently mixed for 5 minutes. Centrifuge at 6,000 × g for 10 minutes at 4 ° C., transfer the upper aqueous layer to a freshly prepared 2 ml microtube, and add 70 μl of 3M sodium acetate buffer (pH 5.2), 99.5% cold. 1.21 ml each of ethanol was added and gently mixed. After cooling and centrifuging at 15,000 × g for 10 minutes at 4 ° C., the supernatant was removed, followed by washing with 0.4 ml of 70% cold ethanol. To the pellet, 0.5 ml of TE buffer (10 mM Tris-HCl buffer solution, 1 mM EDTA · 2Na) was added and left overnight at 4 ° C. to dissolve the DNA.

  5 μl of an RNase (Sigma: EC.3.1.27.5) solution previously prepared to 10 mg / ml with sterilized water was added and incubated at 37 ° C. for 1 hour. 0.25 ml of a phenol / chloroform (1/1) solution was added and mixed gently for 10 minutes, and further 0.25 ml of chloroform was added and gently mixed for 5 minutes. Centrifuge at 6,000 × g for 10 minutes at 4 ° C., transfer the upper aqueous layer to a new 2 ml microtube, add 50 μl of 3M sodium acetate aqueous solution and 1 ml of 99.5% cold ethanol, and mix gently. did. After cooling and centrifuging at 15,000 × g for 10 minutes at 4 ° C. and removing the supernatant, the pellet was dried by washing with 0.4 ml of 70% cold ethanol.

125 μl of TE buffer was added to the dried pellet and allowed to stand at 4 ° C. overnight to dissolve the DNA, thereby obtaining extracted DNA. The absorbance values of the purified DNA solution at 260 nm and 280 nm (OD ₂₆₀ , OD ₂₈₀ : OD ₂₆₀ = 1.0 at a DNA solution of 50 μg / ml, cell length 1 cm) were measured, the DNA concentration was calculated from OD ₂₆₀ , and the purified DNA solution Purity was evaluated by OD ₂₆₀ / OD ₂₈₀ .

2. Test results The chromosomal DNA extracted according to 1-3) was electrophoresed using a 0.8% agarose gel. After completion of electrophoresis, the agarose gel was immersed in an aqueous solution of 1 μg / ml ethidium bromide for 20 minutes, then washed twice with ion-exchanged water, and the degree of chromosomal DNA cleavage was observed with a UV transilluminator (wavelength 254 nm). .

  Each of the extracted DNAs was analyzed by electrophoresis. The results are shown in FIG. FIG. 1 shows the effect of DNase retained in damaged cells on the chromosomal DNA of Listeria (damaged bacteria) and the effect of amsacrine on the chromosomal DNA of Listeria (live and damaged bacteria).

  As a result, there was a significant difference between the very long DNA fragment staying in the well and the long DNA fragment located around 19,329 bp even after incubation for 72 hours without adding amsacrine to the suspension of damaged Listeria. The chromosomal DNA of Listeria-damaged bacteria was hardly cleaved by DNase whose activity remained in the damaged cells. However, when amsacrine is added to the damaged bacteria and incubated for up to 72 hours, the extremely long DNA fragments that stay in the wells are clearly reduced, and active DNA gyrase or topoisomerase remaining in the damaged bacteria of Listeria It was suggested that amsacrine cuts chromosomal DNA everywhere.

  In addition, when amsacrine was added to viable suspension of Listeria and incubated for up to 72 hours, extremely long DNA fragments staying in the wells in 24 hours and long DNA fragments located around 19,329 bp were temporarily reduced, Thereafter, when the incubation was continued, both DNA fragments increased in proportion to the incubation time. As a result, amsacrine permeates the cell wall of living bacteria after 24 hours, and cleaves the chromosomal DNA through DNA gyrase or topoisomerase in the living bacteria. It was suggested that the remaining viable bacteria of the Listeria that had not been received or received substantially grew by re-incubation.

  In addition, since amsacrine is a yellow-colored substance and a very hydrophobic substance, in the case of a short time action of 1 to 30 minutes, the cell wall of a highly hydrophilic living cell without damage to the cell wall hardly penetrates and is a pellet of the living cell Is white, but the damaged bacteria have cell wall damage and increased hydrophobicity, so the cell walls of the damaged bacteria are permeated and the pellet of the damaged bacteria is yellow. According to the present example, it was confirmed that even if viable or damaged, if amsacrine penetrates the cell wall, the chromosomal DNA is cut everywhere via the DNA gyrase and / or topoisomerase in the cell. However, if it acts for a short time, the chromosome of the damaged bacterium is mild but will be selectively cleaved at random, and additional treatment with a short-time topoisomerase inhibitor such as amsacrine (for example, treatment with a topoisomerase inhibitor such as EMA) Then, if additional processing is performed, it is possible to discriminate between live bacteria, damaged bacteria, or dead bacteria with high sensitivity using the PCR method.

[Example 2]
Analysis was carried out by PCR using the 23S rRNA gene as a target, using live and damaged chromosomal DNA treated with a DNA gyrase inhibitor.

1. Preparation of Sample 1-1) Preparation of Live and Damaged Suspensions Gram-negative bacteria Enterobacter sakazakii ATCC 51329 (hereinafter sometimes abbreviated as “Enterobacter”) may be used as BHI broth. Was cultured at 37 ° C., and 40 ml of the culture solution at the late logarithmic growth was cooled and centrifuged at 8,000 × g for 15 minutes at 4 ° C., and the supernatant was removed. After adding 40 ml of physiological saline to the cells and stirring well, similarly cooling and centrifuging and removing the supernatant, 10 ml of physiological saline was added to the cells to obtain a viable cell suspension. When the viable cell count of this viable cell suspension was measured with a standard agar plate medium, it was 4.4 × 10 ⁸ cfu / ml.

Moreover, 1 ml of the above-mentioned viable cell suspension was placed in a 1.5 ml microtube, immersed in boiling water for 50 seconds, and then rapidly cooled with ice water to prepare a damaged cell suspension.
A viable cell suspension and a damaged cell suspension of Listeria monocytogenes JCM 2873 were prepared in the same manner as in Example 1. The viable cell count in the viable cell suspension was 4.0 × 10 ⁸ cfu / ml.

1-2) DNA gyrase inhibitor (ciprofloxacin) treatment 1 ml each of the suspension of Enterobacter (live and damaged) suspension and Listeria (live and damaged) suspension , In addition to 9 ml of freshly prepared BHI broth (the numbers of viable and damaged Enterobacter in the medium are 4.4 × 10 ⁷ cfu / ml, 4.4 × 10 ⁷ cfu / ml, Listeria The number of viable bacteria and damaged bacteria is 4.0 × 10 ⁷ cfu / ml, 4.0 × 10 ⁷ cfu / ml), and 2 ml of ciprofloxacin solution (130 μg / ml; dissolved in physiological saline). Added to.

For the damaged bacteria, in order to examine the effect of DNase having the activity remaining in the damaged bacteria on the chromosomal DNA of the damaged bacteria, 9 ml of BHI broth prepared freshly from each 1 ml of each suspension of damaged bacteria of Enterobacter and Listeria Then, 2 ml of physiological saline was added in place of the ciprofloxacin solution.
Thereafter, Enterobacter (live bacteria and damaged bacteria) were each prepared at 37 ° C. for 1 hour 30 minutes, 3 hours 30 minutes, 5 hours or 72 hours. In addition, Listeria (live bacteria and damaged bacteria) were respectively prepared as suspensions cultured at 30 ° C. for 1 hour 30 minutes, 3 hours 30 minutes, 5 hours, or 72 hours. Furthermore, as a control corresponding to 0-hour culture, each suspension of viable bacteria and damaged bacteria of Enterobacter and Listeria was suspended in viable suspension without addition of ciprofloxacin and suspension of damaged bacteria without addition of ciprofloxacin. Liquid.

  Each suspension was cooled and centrifuged at 8,000 × g for 15 minutes at 4 ° C., and the supernatant was completely removed to recover the pellet. For each of Enterobacter and Listeria, DNA was extracted by the following method.

1-3) Extraction of DNA Extraction of Enterobacter DNA was performed by the following method.
0.5 ml of 10 mM Tris-HCl buffer (pH 8.0) was added to the pellet, 10 μl of 1250 U / ml proteinase K (Sigma: EC.3.4.21.64) was added, and 10% (w / 200 μl of the prepared SDS solution was added to v) and treated at 50 ° C. overnight. 1 M Tris-HCl buffer (pH 8.0) / saturated phenol 0.5 ml was added to this treatment solution, and after gently mixing for 15 minutes, 0.5 ml of chloroform was added and gently mixed for 5 minutes. Centrifuge at 6,000 × g for 10 minutes at 4 ° C., transfer the upper aqueous layer to a freshly prepared 2 ml microtube, and add 70 μl of 3M sodium acetate buffer (pH 5.2), 99.5% cold. Add 1.29 ml of ethanol and mix gently. After cooling and centrifuging at 15,000 × g for 10 minutes at 4 ° C., the supernatant was removed, followed by washing with 0.4 ml of 70% cold ethanol. The pellet was charged with 0.5 ml of TE buffer (10 mM Tris-HCl buffer, 1 mM EDTA · 2Na) and left overnight at 4 ° C. to dissolve the DNA.

  5 μl of an RNase (Sigma: EC.3.1.27.5) solution prepared in advance to 10 mg / ml with sterilized water was added and incubated at 37 ° C. for 1 hour. 0.25 ml of a phenol / chloroform (1/1) solution was added and mixed gently for 10 minutes, and further 0.25 ml of chloroform was added and gently mixed for 5 minutes. Centrifuge at 6,000 × g for 10 minutes at 4 ° C., transfer the upper aqueous layer to a new 2 ml microtube, add 50 μl of 3M sodium acetate aqueous solution and 1 ml of 99.5% cold ethanol, and mix gently. did. After cooling and centrifuging at 15,000 × g for 10 minutes at 4 ° C. and removing the supernatant, the pellet was dried by washing with 0.4 ml of 70% cold ethanol.

125 μl of TE buffer was added to the dried pellet and allowed to stand at 4 ° C. overnight to dissolve the DNA, thereby obtaining extracted DNA. The absorbance values of the purified DNA solution at 260 nm and 280 nm (OD ₂₆₀ , OD ₂₈₀ : OD ₂₆₀ = 1.0 at a DNA solution of 50 μg / ml, cell length 1 cm) were measured, the DNA concentration was calculated from OD ₂₆₀ , and the purified DNA solution Purity was evaluated by OD ₂₆₀ / OD ₂₈₀ .

  The extraction of Listeria DNA was performed according to the method of “1-3) DNA extraction” in Example 1.

1-4) Electrophoresis of PCR amplification product The chromosomal DNA extracted in 1-3) was electrophoresed using a 0.8% agarose gel. After completion of electrophoresis, the agarose gel was immersed in an aqueous solution of 1 μg / ml ethidium bromide for 20 minutes, then washed twice with ion-exchanged water, and the degree of chromosomal DNA cleavage was observed with a UV transilluminator (wavelength 254 nm). .

2. Test method (PCR and electrophoresis targeting the 23S rRNA gene)
2-1) Preparation of PCR Master Mix A master mix (total amount 50 μl) was prepared according to the following composition.
・ Ex-Taq (Takara Shuzo, catalog number: RR001B): 0.25 μl
・ 10 × Ex-Taq Buffer (Takara Shuzo, catalog number: RR001B): 5 μl
・ DNTP mixture (Takara Shuzo, catalog number: RR001B): 4μl
5 pmol / μl, SEQ ID NO: 1 (23S-F) DNA: 2.5 μl
5 pmol / μl, SEQ ID NO: 2 (23S-R) DNA: 2.5 μl
5 pmol / μl, SEQ ID NO: 3 (23S-MF) DNA: 2.5 μl
5 pmol / μl, SEQ ID NO: 4 (23S-MR) DNA: 2.5 μl
2 × SYBA Green (manufactured by BMA, catalog number: 50513): 10 μl
・ Sterile water: 15.75 μl
Template DNA (15 ng / μl): 10 μl
The primers of SEQ ID NOs: 1 and 2 are primers for amplifying almost the entire region of 23S rRNA, and an amplified fragment of about 2840 bp is obtained mainly from gram-negative bacteria. The primers of SEQ ID NOs: 3 and 4 correspond to the central region of 23S rRNA, and an amplified fragment of about 900 bp is obtained from a gram positive bacterium (also amplifies a gram negative bacterium).

2-2) PCR thermal cycle profile for 23S rRNA gene amplification The PCR thermal cycle profile for 23S rRNA gene amplification of Enterobacter is as shown in Table 1.

  The PCR thermal cycle profile for 23S rRNA gene amplification of Listeria is as shown in Table 2.

2-3) PCR reaction Each DNA solution prepared in 1-3) was diluted to 15 ng / μl with TE buffer, and 10 μl thereof was used as the template DNA in 2-1). That is, 150 ng of template DNA is contained in 50 μl of the PCR reaction solution. As a negative control, 10 μl of TE buffer was used.

  In accordance with the PCR thermal cycle profile shown in 2-2) above, PCR reaction and TM analysis (melting temperature analysis) of amplification products are performed using a real-time PCR device i Cycler (manufactured by Bio-Rad, model number: iQ). It was. The threshold value (boundary value) of real-time PCR was a value obtained by multiplying the standard deviation SD of the fluorescence amount by SYBA Green from 0 to 10 cycles by 10. In each real-time PCR amplification curve, the number of cycles exceeding the threshold value is hereinafter abbreviated as “Ct value”.

3. Test Results Test results in this example are shown in FIGS. FIG. 2 shows the effect of intracellular DNase on the chromosomal DNA of Enterobacter (damaged bacteria). FIG. 3 shows the effect of ciprofloxacin on the chromosomal DNA of the same bacterium (live and damaged). FIG. 4 shows the effect of damaged DNase retained on the chromosomal DNA of Listeria (damaged bacteria). FIG. 5 shows the effect of the drug on the chromosomal DNA of the same bacteria (live bacteria and damaged bacteria).

  FIG. 6 shows a real-time PCR curve targeting the 23S rRNA gene of viable Enterobacter treated with ciprofloxacin. FIG. 7 shows a TM pattern analysis of the amplification product in FIG. Similarly, FIG. 8 and FIG. 9 show the real-time PCR curve and the TM pattern analysis result of the amplification product of the same-damaged bacterium treated with the same drug, respectively. Furthermore, real-time PCR curves of live Listeria and damaged bacteria treated with the same drug are shown in FIGS. 10 and 11, respectively.

From FIG. 2, it was confirmed that the damaged chromosomal DNA was slightly cleaved over 72 hours by DNase retained in Enterobacter damaged cells. However, from FIG. 3, even when ciprofloxacin was allowed to act on damaged bacteria for 1.5 to 5 hours, a DNA cleavage phenomenon much stronger than that of DNase was observed. For live bacteria, long chromosome D around 19329 bp
On the contrary, the NA fragment increased, and it was confirmed that the chromosomal DNA of the damaged bacterium was cut more than the living bacterium through bacterial DNA gyrase by allowing ciprofloxacin to act for a short time.
As shown in FIG. 4, the damaged bacterial chromosomal DNA was a long fragment derived from chromosomal DNA near 19329 bp, and DNA cleavage by DNase retained in the Listeria-damaged bacterial body was hardly observed even over 72 hours. However, as shown in FIG. 5, when ciprofloxacin was allowed to act on damaged bacteria for 72 hours, a much stronger DNA cleavage phenomenon was observed due to the DNase. The effect of ciprofloxacin on live bacteria is interpreted as well.

  In addition, when ciprofloxacin was allowed to act for 72 hours for both enterobacter live bacteria and damaged bacteria, PCR was greatly suppressed, but Ct (live bacteria; Cip 72 hours) −Ct (live bacteria; Cip not) = 13 and Ct (damaged bacteria; Cip 72 hours) −Ct (damaged bacteria; Cip not) = 17, PCR was suppressed more in the damaged bacteria. That is, when PCR was terminated in 27 cycles, it was judged that PCR derived from live bacteria was positive, and PCR derived from damaged bacteria was negative. Thus, it was confirmed that ciprofloxacin can distinguish live and damaged bacteria. Similarly, for Listeria, as shown in FIGS. 10 and 11, when PCR is terminated in 20 cycles, it is determined that PCR derived from live bacteria is positive and PCR derived from damaged bacteria is negative. As a result, it was confirmed that live bacteria and damaged bacteria could be distinguished from Listeria.

  As shown in FIG. 7 and FIG. 9, the PCR amplification product of the living bacteria was determined by the TM pattern analysis of the PCR amplification products of the chromosomal DNA of the enterobacter Sakazaki viable bacteria and the damaged bacteria treated with ciprofloxacin for 72 hours. Although it corresponds to the target 23S rRNA gene, it is presumed that the PCR amplification product of the damaged bacterium is not the target amplification product. The PCR amplification product of the damaged bacterium is considered to have amplified a part of the 23S rRNA as a result because a plurality of cleavages occurred within the 23S rRNA gene of the template DNA. As described above, it was shown that viable bacteria and damaged bacteria can be distinguished by PCR by ciprofloxacin treatment.

Example 3
Using the chromosomal DNA of microorganisms and damaged bacteria treated with EMA, analysis was performed by PCR method targeting 16SrRNA gene or 23SrRNA gene.

1. Preparation of Sample 1-1) Preparation of Gram-Negative Bacteria (Live and Damaged Bacteria) Suspension Escherichia coli DH5α (hereinafter sometimes abbreviated as “Escherichia coli”), Citrobacter koseri JCM 1658; "Citrobacter" may be abbreviated), Salmonella enteritidis IID 604 (hereinafter abbreviated as "Salmonella"), Klebsiella oxytoca JCM 1665; After culturing at 37 ° C. using BHI broth, 40 ml of the late-logarithmic growth medium was cooled and centrifuged at 8,000 × g for 15 minutes at 4 ° C., and the supernatant was removed. Then, 40 ml of physiological saline was added, stirred well, centrifuged in the same manner, and the supernatant was removed. Then, 10 ml of physiological saline was added to the cells to obtain a viable cell suspension. The viable cell counts of these viable cell suspensions were E. coli: 3.2 × 10 ⁸ cfu / ml, Citrobacter: 6.7 × 10 ⁷ cfu / ml, Salmonella: 1.9 × 10 ⁸ cfu / ml, Klebshella: 4.8 × 10 ⁸ cfu / ml.

  Moreover, 1 ml of each viable cell suspension was put into a 1.5 ml microtube, immersed in boiling water for 50 seconds, and then rapidly cooled with ice water to prepare a damaged cell suspension.

1-2) Preparation of suspension of Gram-positive bacteria (live and damaged bacteria) Bacillus cereus (Bacillus cereus JCM 2152; hereinafter, may be abbreviated as “Bacillus”), and Staphylococcus epidermidis KD; hereinafter abbreviated as “staphylococcus”) at 37 ° C., and Listeria monocytogenes JCM 2873; hereinafter abbreviated as “listeria”) at 30 ° C. Each was cultured in BHI broth. 40 ml of each culture solution in the logarithmic growth phase (vegetative cells for Bacillus) was centrifuged at 8,000 × g for 15 minutes at 4 ° C., the supernatant was removed, and 40 ml of physiological saline was added and stirred well. Similarly, after cooling and centrifuging and removing the supernatant, 10 ml of physiological saline was added to the cells to obtain a viable cell suspension. The viable cell count of this viable cell suspension was Bacillus: 3.0 × 10 ⁷ cfu / ml, Listeria: 1.3 × 10 ⁸ cfu / ml, Staphylococcus: 1.1 × 10 ⁶ cfu / ml there were.
Moreover, 1 ml of each viable cell suspension was put into a 1.5 ml microtube, immersed in boiling water for 50 seconds, and then rapidly cooled with ice water to prepare a damaged cell suspension.

2. Test Method 2-1) EMA Treatment and Visible Light Irradiation Step EMA (Sigma, catalog number: E 2028) was dissolved in sterilized water so as to be 1000 μg / ml, and filtered through a 0.45 μm microfilter. 10 μl of the aqueous solution was added to 1 ml of each suspension of the gram-negative bacteria (live bacteria and damaged bacteria) and gram-positive bacteria (live bacteria and damaged bacteria), and allowed to stand at 4 ° C. for 30 minutes in the dark. Thereafter, each suspension was placed on ice, and irradiated with 500 W visible light (FLOOD PRF: 100 V, 500 W, manufactured by Iwasaki Electric Co., Ltd.) placed at a distance of 20 cm from the suspension for 10 minutes. The process of adding the above EMA solution and irradiating visible light may be abbreviated as “EMA treatment”. Separately, 10 μl of sterilized water is added instead of the EMA solution to each 1 ml of the suspension of the Gram negative bacteria (live bacteria and damaged bacteria) and the Gram positive bacteria (live bacteria and damaged bacteria). Processed.

2-2) DNA extraction A microtube containing live and damaged bacteria of gram-negative and gram-positive bacteria (untreated with EMA and treated with EMA, respectively) was cooled and centrifuged at 15,000 × g for 10 minutes at 4 ° C. . After adding 990 μl of physiological saline to each microtube and stirring well, the whole amount was transferred to a 2 ml microtube, and cooled and centrifuged at 15,000 × g for 10 minutes at 4 ° C., and the supernatant was removed. Thereafter, DNA was extracted in the same manner as in Example 1, “1-3) DNA extraction”, and the DNA concentration was measured and the purity was evaluated.

3.16S rRNA gene or PCR targeting 23S rRNA gene
3-1) Preparation of PCR master mix

A master mix (total amount 50 μl) was prepared with the following composition.
・ Ex-Taq (Takara Shuzo, catalog number: RR001B): 0.25 μl
・ 10 × Ex-Taq Buffer (Takara Shuzo, catalog number: RR001B): 5 μl
・ DNTP mixture (Takara Shuzo, catalog number: RR001B): 4μl
5 pmol / μl, SEQ ID NO: 1 (23S-F) DNA: 2.5 μl
5 pmol / μl, SEQ ID NO: 2 (23S-R) DNA: 2.5 μl
5 pmol / μl, SEQ ID NO: 3 (23S-MF) DNA: 2.5 μl
5 pmol / μl, SEQ ID NO: 4 (23S-MR) DNA: 2.5 μl
5 pmol / μl, SEQ ID NO: 5 (16S-F) DNA: 2.5 μl
5 pmol / μl, SEQ ID NO: 6 (16S-R) DNA: 2.5 μl
2 × SYBA Green (manufactured by BMA, catalog number: 50513): 10 μl
・ Sterile water: 15.75 μl
Template DNA (15 ng / μl): 10 μl

3-2) PCR thermal cycle profile for 16S rRNA gene amplification PCR thermal cycle profile for 16S rRNA gene amplification of each bacterium is as shown in Table 3.

3-3) PCR thermal cycle profile for 23S rRNA gene amplification PCR thermal cycle profile for 23S rRNA gene amplification of each bacterium is as shown in Table 4 (Gram negative bacteria) and Table 5 (Gram positive bacteria).

3-4) PCR reaction Each DNA solution prepared in 2-2) above was diluted to 15 ng / μl with TE buffer, and 10 μl thereof was used as the template DNA in 3-1). That is, 150 ng of template DNA is contained in 50 μl of the PCR reaction solution. As a negative control, 10 μl of TE buffer was used.

In accordance with the PCR thermal cycle profile shown in 3-2) or 3-3) above, PCR analysis and TM analysis of amplification products using a real-time PCR apparatus iCycler (Biorad, model number: iQ) ( Melting temperature analysis). The calculation of threshold values (boundary values) and Ct values for real-time PCR is the same as in Examples 2 and 2-3).
3-5) Agarose gel electrophoresis of PCR amplification products

  A 0.8% agarose gel was prepared using Seakem GTG agarose (manufactured by FMC, catalog number: 50070) and TAE buffer (Tris 4.84 g / l, acetic acid 1.142 ml / l, EDTA · 2Na 0.149 g / l), and marker Λ-EcoT14 I digest (Takara Shuzo Co., Ltd., Code: 3401) and 100 bp DNA Ladder (Takara Shuzo Co., Ltd., Code: 3407A) were used, and 10 μl of each PCR reaction solution was added to each well for Gram-negative bacteria and Gram-positive bacteria. The gel was injected and subjected to electrophoresis, and the electrophoresis was terminated when bromphenol blue (BPB) migrated about 90% of the gel.

  The gel after electrophoresis was immersed in 1 μg / ml ethidium bromide aqueous solution for 20 minutes, washed twice with ion-exchanged water, and then the PCR amplification product was observed with a UV transilluminator (254 nm).

4). Test Results FIG. 12 shows the change in temperature over time when a 1.5 ml microtube containing 1 ml of bacterial suspension is immersed in boiling water. From FIG. 12, the soaking of boiling water for 50 seconds in the preparation of the damaged bacteria suspension is a method of heat treatment slightly more intense than the high-temperature short-time sterilization (HTST sterilization) at 72 to 75 ° C. and 15 to 16 seconds. It can be seen that this corresponds to heat treatment equivalent to ultra-high temperature instant sterilization (UHT sterilization).

  FIG. 13 shows the identification results of live bacteria and damaged bacteria targeting 16SrRNA gene, and FIGS. 14 and 15 show the identification results of live bacteria and damaged bacteria targeting 23SrRNA gene, respectively. 13 to 15, the PCR reaction solution of each bacterium is a live cell suspension / EMA untreated, a live cell suspension / EMA treated, a damaged cell suspension / EMA untreated, a damaged cell suspension / EMA The gel is loaded in the order of processing.

  From FIG. 13, when the target was a 16S rRNA gene, in Citrobacter and Klebsiella, no PCR amplification product was seen in EMA-treated damaged bacteria, and it was possible to clearly distinguish between live bacteria and damaged bacteria. As for other bacteria, amplified products were observed in damaged bacteria treated with EMA, and the distinction between live bacteria and damaged bacteria was unclear.

On the other hand, FIG. 14 and FIG. 15 show that when the target is a 23S rRNA gene, viable bacteria and damaged bacteria can be clearly distinguished by EMA treatment in both gram-negative bacteria and positive bacteria. It was done. Even when 150 ng of template DNA was subjected to 50 μl of PCR using a test sample containing 10 ⁸ cfu / ml level of bacterial damage as a sample background, the PCR reaction of damaged bacteria was completely suppressed, PCR was not almost completely suppressed. Furthermore, in milk containing 10 ⁵ to 10 ⁷ cfu / ml damaged bacteria, it is considered that PCR of damaged bacteria is completely suppressed and viable bacteria can be detected at a low concentration. It can be applied as a screening test for general bacteria in the test. Furthermore, in the case of a patient with sepsis due to hepatic dysfunction, viable bacteria and damaged bacteria may be present in the blood at a high concentration of 10 ⁴ cfu / ml or more. Only can be detected quickly.

Example 4
Pathogenic bacteria were treated with EMA and topoisomerase inhibitor or DNA gyrase inhibitor, and live bacteria and damaged bacteria were identified by PCR method targeting pathogenic genes.

1. Preparation of Bacteria (Live Bacterium) Culture Solution In the same manner as in Example 1, a live cell suspension and a damaged cell suspension of Listeria (Listeria monocytogenes JCM 2873) were prepared. The viable cell count of the viable cell suspension was 1.3 × 10 ⁸ cfu / ml.

2. Test Method 2-1) EMA Treatment and Visible Light Irradiation Step In the same manner as in Example 3, EMA treatment and visible light irradiation were performed on the viable suspension and damaged fungus suspension of Listeria prepared above. . In the case of Gram-positive bacteria that do not have an outer membrane on the cell wall, EMA tends to permeate even viable bacteria without cell wall damage. The time was shortened to 5 minutes, and the visible light irradiation time was also shortened to 5 minutes. In addition, 10 μl of sterilized water was added instead of adding EMA to the viable cell and damaged cell suspension, and the same treatment was performed.

2-2) Topoisomerase inhibitor or DNA gyrase inhibitor treatment step After finishing the EMA treatment and visible light irradiation step, the microtube containing each treatment solution was placed at 4 ° C for 15,000 xg for 10 minutes. The supernatant was removed by cooling and centrifuging. 1 ml of physiological saline was added to the cells and stirred well. After cooling and centrifuging in the same manner, after removing the supernatant, 1 ml of saline was added to the cells and stirred well. Thus, 1 ml × 1 viable EMA untreated, 1 ml × 7 live EMA treated, 1 ml × 1 untreated EMA untreated, and 1 ml × 7 treated EMA treated damaged bacteria were prepared.

  Each of the EMA-treated live bacteria and damaged bacteria was divided into 6 sets. The first set was 8 μl of the DNA gyrase inhibitor ciprofloxacin (0.5 mg / ml; dissolved in physiological saline), the second set was the topoisomerase inhibitor camptothecin (1 mg / ml; dimethyl sulfoxide) 10 μl of Topoisomerase inhibitor etoposide (1 mg / ml; dissolved in dimethyl sulfoxide) for the third set, 10 μl of Topoisomerase inhibitor ellipticine (0.1 mg / ml; dimethyl) for the fourth set 5 μl of sulfoxide (dissolved in sulfoxide), 10 μl of mitoxantrone (0.1 mg / ml; dissolved in dimethyl sulfoxide) as a topoisomerase inhibitor in the fifth set, amsacrine (1 mg as a topoisomerase inhibitor) in the sixth set / Ml; dissolved in dimethyl sulfoxide) μl each was added. Each sample was incubated at 30 ° C. for 30 minutes, then transferred to a 2 ml microtube, and centrifuged at 15,000 × g for 10 minutes at 4 ° C. to remove the supernatant.

2-3) DNA extraction step DNA was extracted from each sample prepared as described above in the same manner as in Example 1, “1-3) DNA extraction”.

3. PCR targeting various genes of Listeria monocytogenes
3-1) Pathogenic gene Listeria listeriolysin O (hlyA) gene amplification 3-1-1) Preparation of PCR master mix

A master mix (total amount 50 μl) was prepared with the following composition.
・ Ex-Taq (Takara Shuzo, catalog number: RR001B): 0.25 μl
・ 10 × Ex-Taq Buffer (Takara Shuzo, catalog number: RR001B): 5 μl
・ DNTP mixture (Takara Shuzo, catalog number: RR001B): 4μl
5 pmol / μl, SEQ ID NO: 7 (hlyA-F) DNA: 2.5 μl
5 pmol / μl, SEQ ID NO: 8 (hlyA-R) DNA: 2.5 μl
・ 2 × SYBA Green (manufactured by BMA, catalog number: 50513): 10 μl
・ Sterile water: 15.75 μl
Template DNA (15 ng / μl): 10 μl
3-1-2) PCR thermal cycle profile for hlyA gene amplification

3-1-3) PCR reaction Each DNA solution prepared in 2-3) above was diluted to 15 ng / μl with TE buffer, and 10 μl thereof was used as template DNA in 3-1-1). That is, 150 ng of DNA is contained in 50 μl of the PCR reaction solution. As a negative control, 10 μl of TE buffer was used.

  According to the PCR thermal cycle profile shown in 3-1-2), PCR reaction was performed using a real-time PCR apparatus i Cycler (manufactured by Bio-Rad, model number: iQ).

3-2) 16S rRNA and 23S rRNA gene amplification 3-2-1) Preparation of PCR master mix Example 3, 3-1) A master mix (total amount 50 μl) was prepared in the same manner as “Preparation of PCR master mix”.
3-2-2) PCR thermal cycle profile for 16S rRNA gene amplification Example 3, 3-2) According to “PCR thermal cycle profile for 16S rRNA gene amplification” (Table 3).

3-2-3) PCR Thermal Cycle Profile for Gram-Positive Bacteria 23SrRNA Gene Amplification Example 3, 3-3) The PCR thermal cycle profile for Gram-positive bacteria 23SrRNA gene amplification (Table 5).
3-2-4) PCR reaction Each DNA solution prepared in 2-3) above is diluted to 15 ng / μl with TE buffer, and 10 μl thereof is the template of 3-1-1) or 3-2-1). Used as DNA. That is, a template of 150 ng of DNA is contained in a 50 μl PCR reaction solution. As a negative control, 10 μl of TE buffer was used.

  According to the PCR thermal cycle profile shown in 3-2-2) or 3-2-3), a PCR reaction was performed using a real-time PCR apparatus i Cycler (manufactured by Bio-Rad, model number: iQ).

3-3) Agarose gel electrophoresis of PCR amplification products Electrophoresis was performed in the same manner as in Example 2, 1-4) “Electrophoresis of PCR amplification products”. For detection of the hlyA gene amplification product, a 3% agarose gel was used.

4). Test Results The results when the hlyA gene is the target are shown in FIG. 16, and the results when the 16S rRNA gene and the 23S rRNA gene are the targets are shown in FIGS. 17 and 18, respectively.

From FIGS. 16 and 17, when hlyA and 16S rRNA genes were targeted, DNA amplification of damaged bacteria was only slightly inhibited by EMA treatment. However, when treated with a topoisomerase inhibitor or DNA gyrase inhibitor with EMA, DNA amplification of the damaged bacteria was further inhibited. In particular, when the hlyA gene is targeted, etoposide, mitoxantrone, or amsacrine, and when the 16S rRNA gene is targeted, camptothecin, etoposide, ellipticine, or amsacrine are used in combination with EMA, respectively. DNA amplification was significantly inhibited.
Further, as shown in FIG. 18, in the case of viable bacteria targeting the 23S rRNA gene, PCR was significantly inhibited by using EMA in combination with other topoisomerase inhibitors or DNA gyrase inhibitors.

When focusing on specific pathogenic bacteria and quickly distinguishing between live and dead or damaged bacteria, there is a tendency to use a short gene region of 100 to 200 bp as a target region in order to increase the specificity of the pathogenic gene. When the target region is so short, even if EMA permeates the cell wall of the damaged bacterium and the DNA is cleaved, the target region is amplified without being cleaved. As a result, PCR is not completely suppressed. On the other hand, by using EMA in combination with a topoisomerase inhibitor or DNA gyrase inhibitor, even when targeting an extremely short hlyA gene of 113 bp, PCR of 10 ⁸ cfu / ml level Listeria-damaged bacteria is almost completed. It was completely suppressed, and PCR of viable bacteria was hardly suppressed.
This is because, among the cleavage-recombination of DNA gyrase, bacterial topoisomerase I, III, and IV in which the topoisomerase inhibitor randomly performs DNA cross-linking at a position different from EMA and the activity in the damaged cells remains. By inhibiting recombination, it is considered that the DNA cleavage state is reached more severely at every location than the DNA cleavage state by EMA alone, and the cleavage also occurs in a short target gene of 100 bp to 200 bp.
Therefore, it is possible to clearly distinguish between live bacteria of specific pathogenic bacteria and damaged bacteria or dead bacteria in foods and various clinical samples containing a background of damaged bacteria of high specific pathogenic bacteria. However, to suppress PCR amplification products of dead bacteria, unlike to damaged bacteria, topoisomerase or DNA gyrase may be inactivated, so ATP and Mg ²⁺ and topoisomerase or DNA gyrase ( It is more preferable to add an enzyme).
For example, in a tuberculosis patient who has been administered an anti-tuberculosis agent, tuberculosis-infecting bacteria are present at a concentration of 10 ⁸ to 10 ⁹ cfu / ml due to the anti-tuberculosis agent in the sputum of the test material in the later stage of treatment. However, even in such a case, it is possible to detect only viable bacteria by the method of the present invention.

[Reference Example 1] Preparation of test sample from food As an example of the test sample of the present invention, a sample preparation method assuming that microorganisms are mixed in milk is shown below. To do.

  EDTA solution is added to the milk so that the final concentration is 1 to 5 mM, particularly 2 mM, and Tween 80 is added so that the final concentration is 0.1 to 0.5%, particularly 0.1%, and 10,000 is added. The mixture was cooled and centrifuged at 4 ° C. for 10 minutes at × g. In addition, after adding lipase (EC3.1.1.3, manufactured by Sigma) at a final concentration of 10 to 20 U / ml and treating at 30 to 37 ° C. for 30 minutes to 1 hour, proteinase K so that the final concentration becomes 20 U / ml. (EC3.4.21.64 manufactured by Sigma) is preferably added and allowed to stand for 30 minutes to 1 hour. The fat layer on the liquid surface and the middle aqueous layer were removed, and the sediment was collected. This sediment contains somatic cells such as bacteria, mammary epithelial cells, bovine leukocytes and the like. When centrifuged at 10,000 × g or more, a micellar protein degradation product (casein) degraded incompletely by proteinase K. Incomplete micelle degradation products) are also included. Here, the casein micelle incompletely decomposed product is considered to be a highly hydrophobic α, β-casein sub-micelle.

  After adding the same volume of physiological saline as the initial suspension to the sediment and suspending it, the mixture was cooled and centrifuged at 100 × g for 5 minutes at 4 ° C., and the supernatant containing microorganisms was collected.

[Reference Example 2] Preparation of test sample from blood (1)
The same volume of physiological saline was added to heparinized blood, centrifuged at 10,000 × g for 5 minutes at 4 ° C., the supernatant was removed, and the sediment was collected. The sediment contained bacteria, platelets, monocytes such as monocytes and lymphocytes, granulocytes, and red blood cells.

[Reference Example 3] Preparation of test sample from blood (2)
Add the same volume of physiological saline to heparinized blood, and centrifuge at 100 xg for 5 minutes at 4 ° C. Plasma and blood cell components (monocytes such as monocytes and lymphocytes, granulocytes and The red blood cells were separated, and the plasma in which microorganisms were present was collected.

[Reference Example 4] Preparation of test sample from blood (3)
The same volume of physiological saline was added to heparinized blood. First, Ficoll-Paque (manufactured by Amersham Biosciences; Ficoll 400 (Ficoll 400)) is 5.7 g / 100 ml, and diatrizoate sodium is 9 g / 100 ml. It is. Specific gravity 1.077 g / ml] was filled, and then the heparinized blood diluted 2-fold was layered slowly while tilting the test tube at an angle. Subsequently, the mixture was centrifuged at 100 × g for 5 minutes at 4 ° C., and the supernatant in which microorganisms were present was collected. Before adding heparinized blood diluted 2-fold to Ficoll-Paque, a lipase solution (EC3.1.1.3 manufactured by Sigma) having a final concentration of 10-20 U / ml was added, and then 10-50 U / ml was added. After adding ml of deoxyribonuclease I (Sigma, EC 3.1.21.1) solution and treating at 30-37 ° C. for 30 minutes to 1 hour, proteinase K (manufactured by Sigma) to a final concentration of 10-20 U / ml. EC3.4.21.64) is preferably added and treated at 30 to 37 ° C. for 30 minutes to 1 hour.

[Reference Example 5] Preparation of test sample from blood (4)
Monopoly ^™ [Amersham Biosciences; Ficoll and Metrizoate mixed solution with a specific gravity of 1.115 g / ml] is added to a sterile test tube in advance so that it becomes 1/2 volume of heparinized blood. Then, heparinized blood was slowly layered while tilting the test tube. Thereafter, the mixture was cooled and centrifuged at 100 × g for 5 minutes at 4 ° C., and the supernatant containing bacteria was collected.

According to the present invention, viable-and-culturable microorganisms contained in foods and clinical samples can be replaced as dead or damaged bacteria (Injured) as a rapid alternative method that inherits the characteristics of the culture method.
or Viable-but-Non Culturable state).

Electrophoresis showing the effect of damaged DNase on chromosomal DNA of Listeria (damaged bacteria), the effect of amsacrine on chromosomal DNA of Listeria (damaged bacteria), and the effect of amsacrine on chromosomal DNA of Listeria (live bacteria) Photo. Untreated: Untreated; amsacrine (-): no amsacrine added; amsacrine (+): amsacrine added; 1: 30 ° C for 24 hours incubation; 2: 30 ° C for 48 hours incubation; 3: 30 ° C for 72 hours incubation; Electrophoresis photograph showing the effect of damaged bacterial DNase on the chromosomal DNA of Enterobacter (damaged bacteria). Not: Untreated; 1: 37 ° C, 24 hours incubation; 2: 37 ° C, 48 hours incubation; 3: 37 ° C, 72 hours incubation; Electrophoresis photograph showing the effect of ciprofloxacin on the chromosomal DNA of Enterobacter (live and damaged bacteria). Not: Untreated; 1: 37 ° C, 1.5 hour incubation; 2: 37 ° C, 3.5 hour incubation; 3: 37 ° C, 5 hour incubation; 4: 37 ° C, 72 hour incubation; Electrophoretic diagram showing the effect of damaged bacterial DNase on chromosomal DNA of Listeria (damaged bacteria). Not: Untreated; 1: 30 ° C, 24 hours incubation; 2: 30 ° C, 48 hours incubation; 3: 30 ° C, 72 hours incubation; Electrophoresis photograph showing the effect of ciprofloxacin on chromosomal DNA of Listeria (live and damaged). Not: Untreated; 1: 30 ° C, 1.5 hours incubation; 2: 30 ° C, 3.5 hours incubation; 3: 30 ° C, 5 hours incubation; 4: 30 ° C, 72 hours incubation; Real-time PCR amplification curve targeting the 23S rRNA gene of ciprofloxacin-treated Enterobacter (live bacteria) (picture of halftone image). TM pattern of a real-time PCR amplification product targeting the 23S rRNA gene of citrofloxacin-treated enterobacter (live bacteria) (picture of halftone image). Real-time PCR amplification curve targeting the 23S rRNA gene of citrofloxacin-treated Enterobacter (damaged bacteria) (half-tone image photo). TM pattern of real-time PCR amplification products targeting the 23S rRNA gene of ciprofloxacin-treated Enterobacter (damaged bacteria) (picture of halftone image). Real-time PCR amplification curve targeting the 23S rRNA gene of Listeria treated with ciprofloxacin (photo of halftone image). Real-time PCR amplification curve targeting the 23S rRNA gene of citrufloxacin-treated Listeria (damaged bacteria) (picture of halftone image). The figure which shows the relationship between the immersion time to the boiling water of the microtube containing bacteria suspension, and liquid temperature. Electrophoresis photograph showing the results of PCR gene amplification targeting 16S rRNA genes of 7 types of bacteria (live bacteria / damaged bacteria) untreated or treated with EMA. For each bacterium, the order of the live cell suspension / EMA untreated, the live cell suspension / EMA treatment, the damaged cell suspension / EMA untreated, and the damaged cell suspension / EMA treatment, respectively. The same applies to FIGS. 14 and 15. Electrophoresis photograph showing the results of PCR gene amplification targeting the 23S rRNA gene of four types of bacteria (live bacteria / damaged bacteria) untreated or treated with EMA. Electrophoresis photograph showing the result of PCR gene amplification targeting 23S rRNA gene of Listeria (live bacteria / damaged bacteria) that has not been or has been treated with EMA. Electrophoresis photograph showing the result of PCR gene amplification targeting the hlyA gene of Listeria (live bacteria / damaged bacteria) treated with DNA gyrase inhibitor or topoisomerase inhibitor after EMA treatment or after EMA treatment. Un: Untreated; E: EMA; 1: EMA-ciprofloxacin; 2: EMA-camptothecin; 3: EMA-etoposide; 4: EMA-ellipticine; 5: EMA-mitoxantrone; 6: EMA-amsacrine Electrophoresis photograph showing results of PCR gene amplification targeting 16S rRNA gene of Listeria (live bacteria / damaged bacteria) treated with DNA gyrase inhibitor or topoisomerase inhibitor after EMA treatment or after EMA treatment. Un: Untreated; E: EMA; 1: EMA-ciprofloxacin; 2: EMA-camptothecin; 3: EMA-etoposide; 4: EMA-ellipticine; 5: EMA-mitoxantrone; 6: EMA-amsacrine Electrophoresis photograph showing the result of PCR gene amplification targeting 23S rRNA gene of Listeria (live bacteria / damaged bacteria) treated with DNA gyrase inhibitor or topoisomerase inhibitor after EMA treatment or after EMA treatment. Untreated: Untreated; E: EMA; 1: EMA-ciprofloxacin; 2: EMA-camptothecin; 3: EMA-etoposide; 4: EMA-ellipticine; 5: EMA-mitoxantrone; 6: EMA-amsacrine;

Claims (11)

A method for detecting viable microorganisms in a test sample, comprising the following steps:
a) treating the test sample with ethidium monoazide and irradiating with visible light; a topoisomerase inhibitor selected from amsacrine, camptothecin, ellipticine, etoposide, mitoxantrone, and / or ciprofloxacin; A process comprising a step of treating a test sample with a DNA gyrase inhibitor;
b) extracting DNA from the test sample, amplifying the target region of the extracted DNA by PCR, and c) analyzing the amplified product.   The method according to claim 1, wherein the analysis of the amplification product is performed using a standard curve showing the relationship between the amount of microorganisms created using a standard sample of microorganisms and the amplification product.   3. The method according to claim 2, wherein the PCR is performed by a real-time PCR method, and the PCR and the amplification product are analyzed simultaneously.   The test sample is any one of milk, dairy products, foods using milk or dairy products, blood samples, urine samples, spinal fluid samples, synovial fluid samples, or pleural fluid samples. The method according to one item.   The method according to any one of claims 1 to 4, wherein the microorganism is a bacterium.   The method according to claim 5, wherein the target region is a 23S rRNA gene.   The method according to claim 6, wherein the PCR is performed using a primer set shown in SEQ ID NOs: 1 and 2, or a primer set shown in SEQ ID NOs: 3 and 4. The method according to claim 1, wherein the following step is performed before the step a):
d) A step of treating the test sample with topoisomerase and / or DNA gyrase. A kit for detecting a living microorganism of a microorganism in a test sample by PCR, comprising the following elements:
A primer for amplifying, by PCR, ethidium monoazide, at least one selected from amsacrine, camptothecin, ellipticine, etoposide, mitoxantrone, and ciprofloxacin, and a target region of microbial DNA to be detected.   The kit according to claim 9, further comprising topoisomerase and / or DNA gyrase.   The kit according to claim 9 or 10, wherein the primer is a primer set shown in SEQ ID NOs: 1 and 2, or a primer set shown in SEQ ID NOs: 3 and 4.

Images