JP5692489B2 - Microbial detection method - Google Patents

Description

  The present invention relates to a method of detecting only living microorganisms targeting microorganisms present in food or the environment.

  As a method for inspecting microorganisms in food, a medium culture method that complies with the food hygiene inspection guidelines has been conventionally used. However, in the culture medium culture method, it takes 2 days or more to detect microorganisms, and in the case of performing a plurality of types of tests, it is necessary to perform different culture mediums, which is troublesome and expensive.

  On the other hand, as a technique for determining the presence or absence of microorganisms in a shorter time, there is a method of amplifying a specific gene of microorganisms by PCR method to determine the presence or absence of microorganisms, which is one of food hygiene tests, environmental tests and infectious disease tests. (See Non-Patent Document 1 and Non-Patent Document 2). However, it can be detected in a short time and has good detection sensitivity. However, since microbial DNA is used, dead bacteria contained in the sample are also detected at the same time. However, it could not be said that living microorganisms were necessarily present in the sample.

  As a method of discriminating between live and dead microorganisms, a test sample is treated with ethidium monoazide, the test sample is irradiated with visible light, DNA is extracted from the test sample, and the extracted DNA target A method for discriminating between live and dead bacteria by amplifying the region by PCR has been proposed (see Patent Documents 1 and 2).

Japanese Patent No. 4127847 JP 2008-173132 A

Tuberculosis Bacteria Test Guideline 2007 Food Safety Supervision No. 1102004

However, in the above-described method for distinguishing between live and dead bacteria, the time for irradiating the test sample with visible light differs depending on the type of microorganism, that is, between gram-positive and gram-negative bacteria. A longer irradiation time was required to distinguish between the bacterium and the dead bacteria. Therefore, this method cannot simultaneously detect viable bacteria of gram positive bacteria and gram negative bacteria.
An object of this invention is to provide the detection method of the living microbe of microorganisms which can discriminate | determine a living microbe and a dead microbe about a Gram-negative microbe in shorter irradiation time.

The present invention is a method for detecting viable microorganisms in a test sample, in which a) the test sample is treated with a surfactant and b) the test sample treated with the surfactant is treated with ethidium monoazide or Treated with propidium monoazide, c) then irradiates the test sample with visible light, d) extracts DNA from the test sample irradiated with visible light, and e) any of steps a) to d) Treatment with a topoisomerase inhibitor or DNA gyrase inhibitor in
f) The detection method is characterized in that the target region of the extracted DNA is amplified by a nucleic acid amplification method.
In the detection method of the present invention, the test sample contains Gram-positive bacteria and Gram-negative bacteria as microorganisms, and simultaneously detects live Gram-positive and Gram-negative bacteria.
The topoisomerase inhibitor or DNA gyrase inhibitor is amsacrine,
Is preferred.

  According to the present invention, it is possible to provide a method for detecting a live bacterium of a gram-negative bacterium and a gram-positive bacterium at the same time by distinguishing a live bacterium and a dead bacterium from a gram-negative bacterium with a shorter irradiation time.

It is an electrophoretic photograph in examination of the standing time and light irradiation time after EMA addition. It is an electrophoretic photograph in examination of the standing time and light irradiation time after EMA addition. It is an electrophoretic photograph in examination of the addition effect of surfactant. It is an electrophoretic photograph in examination of the addition effect of surfactant. It is an electrophoretic photograph in examination of the addition effect of surfactant. It is an electrophoretic photograph in examination of the addition effect of surfactant. It is an electrophoretic photograph in examination of the addition effect of surfactant. It is an electrophoresis photograph in examination of the addition effect of surfactant at the time of using a propidium monoazide solution. It is an electrophoresis photograph in examination of the addition effect of surfactant at the time of using a propidium monoazide solution.

The method for detecting viable microorganisms in a test sample of the present invention comprises: a) treating a test sample with a surfactant; b) treating the test sample treated with the surfactant with ethidium monoazide or propidium monoazide C) Then, the test sample is irradiated with visible light, d) DNA is extracted from the test sample irradiated with visible light, and e) topoisomerase inhibition is performed in any of the steps from a) to d). Treatment with an agent or DNA gyrase inhibitor,
f) The target region of the extracted DNA is amplified by a nucleic acid amplification method.

  As test samples, for example, beverages such as milk, water, green tea, acidic beverages, and a wide range of foods such as meat, chilled foods, fresh foods (meat, vegetables, fish), lunch boxes and prepared dishes, and feeds can be used. . In addition, environmental samples such as food production equipment and air in the production environment can be used as the test sample of the present invention. The property of the test sample may be any of solid, liquid and gas. The test sample may be a product obtained by diluting or concentrating the product or the biological sample itself, or a pre-treatment other than the treatment according to the method of the present invention. Examples of the pretreatment include heat treatment, filtration, and centrifugation.

  Examples of the microorganism include bacteria, filamentous fungi, and yeast. Bacteria include both gram-positive bacteria and gram-negative bacteria. Examples of Gram-positive bacteria include Streptococcus bacteria, Listeria monocytogenes such as Listeria monocytogenes, Bacillus subtilis such as Bacillus subtilis, and Mycobacterium bacteria. Gram-negative bacteria include Escherichia bacteria such as Escherichia coli, Enterobacter sakazakii such as Enterobacter sakazakii, Salmonella bacteria, Vibrio bacteria, and Pseudomonas bacteria .

Viable bacteria are generally viable-and-culturable states that can grow when cultured under suitable culture conditions, and that have little cell wall damage. Say. Examples of the metabolic activity include ATP activity and esterase activity.
Dead bacteria 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.

In the detection method of the present invention, a test sample is treated with a surfactant (step a)). Either an ionic surfactant or a nonionic surfactant can be used as the surfactant.
Examples of ionic surfactants include anionic surfactants such as fatty acid sodium, fatty acid potassium, sodium alphasulfo fatty acid ester, sodium alkylbenzene sulfonate, sodium alkyl sulfate, sodium alkyl ether sulfate, sodium alpha olefin sulfonate, alkyl sulfone. Examples thereof include sodium acid, alkyltrimethylammonium salt, dialkyldimethylammonium salt, alkylbenzyldimethylammonium salt of cationic surfactant, alkylcarboxybetaine of amphoteric surfactant, alkylamine oxide, alkylamino fatty acid salt and the like.
Examples of the nonionic surfactant include polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, fatty acid sorbitan ester, fatty acid alkanolamide, alkyl polyglucoside, and alkyl monoglyceryl ether.
Treatment of the test sample with the surfactant is performed by adding a surfactant to the test sample. The addition amount of the surfactant is preferably 0.1 to 0.00001% by weight, more preferably 0.01 to 0.0001% by weight based on the weight of the test sample after adding the surfactant. %.

  In the detection method of the present invention, the test sample treated with the surfactant is then treated with ethidium monoazide or propidium monoazide (step b)). Treatment of the test sample with ethidium monoazide or propidium monoazide is performed by adding ethidium monoazide or propidium monoazide to the test sample and allowing to stand for a certain period of time. The amount of ethidium monoazide or propidium monoazide added is preferably 0.5 to 100 μg / ml in the final concentration. The temperature of ethidium monoazide or propidium monoazide to be added is preferably 4 to 10 ° C. Furthermore, the temperature of the test sample is preferably 4 to 10 ° C. Moreover, the standing time after adding ethidium monoazide or propidium monoazide to the test sample is preferably 3 to 60 minutes, more preferably 5 to 20 minutes. The treatment with ethidium monoazide or propidium monoazide is preferably performed under light shielding.

  In the detection method of the present invention, the test sample is then irradiated with visible light (step c)). The visible light is preferably visible light including light having a wavelength of 550 to 700 nm. For the irradiation with visible light, for example, visible light of 100 to 750 W is irradiated for 3 minutes to 1 hour from a distance of 5 to 50 cm from the test sample. Note that the visible light irradiation is preferably performed at a low temperature, for example, by cooling the sample with ice.

  In the detection method of the present invention, DNA is then extracted from the test sample irradiated with visible light (step d)). Examples of a method for extracting DNA from a test sample include a phenol extraction method, a phenol / chloroform extraction method, an alkali dissolution method, and a boiling method. Moreover, the method of extracting DNA using a commercially available DNA extraction reagent and a nucleic acid automatic extraction apparatus is mentioned.

  In the detection method of the present invention, the test sample is treated with a topoisomerase inhibitor or a DNA gyrase inhibitor in any of steps a) to d) (step e)). Topoisomerase inhibitors and DNA gyrase inhibitors are those that inhibit the recombination of DNA without inhibiting the DNA cleavage activity of topoisomerase (Topoisomerase) and DNA gyrase, or the rate at which DNA is cleaved. It means something to promote. It is preferable that the topoisomerase inhibitor and the DNA gyrase inhibitor have different effects on live bacteria and dead bacteria. More specifically, it is preferable that it is more permeable to the cell wall of dead bacteria than the cell wall of live bacteria.

  Conditions for treatment with a topoisomerase inhibitor or a DNA gyrase inhibitor can be set as appropriate. For example, various concentrations of a topoisomerase inhibitor are added to a suspension of living and dead microorganisms to be detected. After various periods of time, by extracting the DNA of the microorganism and analyzing it by PCR, it is possible to determine conditions that make it easy to distinguish between live and dead bacteria. Such conditions include a final concentration of 1 to 100 μg / ml, 25 to 37 ° C., and 5 minutes to 36 hours for amsacrine.

  In the detection method of the present invention, the target region of the extracted DNA is then amplified by a nucleic acid amplification method (step f)). The target region is a region of the chromosomal DNA that can be amplified by the nucleic acid amplification method, and is not particularly limited as long as it can detect the microorganism to be detected, and can be appropriately set 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. The length of the target region is usually 80 to 1000 bases, preferably 100 to 500 bases. Nucleic acid amplification methods include PCR (polymerase chain reaction), SDA (strand displacement amplification), LCR (ligase chain reaction), LAMP (loop-mediated isothermal amplification), ICAN (isothermal and chimeric primer-initiated amplification) of nucleic acids), among which the PCR method is preferably used.

The PCR primer used in the present invention is not particularly limited as long as it can specifically amplify the target region. Specifically, as a primer corresponding to 16S rRNA gene, the primer set shown to sequence number 1 and 2 or the primer set shown to sequence number 3 and 2 is mentioned.
In the detection method of the present invention, PCR amplification is performed using a known method. For example, using the extracted DNA as a template, heat-denaturing the DNA into a single strand (eg, 94 ° C.), annealing the primer (eg, 60 ° C.), and then using a heat-resistant DNA polymerase to form the complementary strand By repeating the cycle of synthesis (for example, 72 ° C.) 20 to 40 times, for example, only the target gene region is amplified.
The obtained PCR amplification product can be analyzed by a known method such as electrophoresis, chromatography, or a DNA chip (microarray).

1. Used gram-negative bacteria: Escherichia coli ATCC 8739
Gram-positive bacteria: Bacillus subtilis ATCC 6633

2. Preparation of live bacterial solution and dead bacterial solution Cultured at 35 ° C. using LB medium, and the culture solution in the logarithmic growth phase was taken out. Then, it diluted stepwise using sterilization dilution liquid Pro-media MT-11 (Elmex Corporation), and was made into the living microbe suspension of each density | concentration. 1 ml of the viable cell suspension was weighed and measured with an AC plate for measuring the viable cell count of Petrifilm medium (Sumitomo 3M).
In addition, 1 ml of the viable cell suspension is put into a 1.5 ml tube and heated in a heat block at 100 ° C. for 3 minutes (for E. coli) or at 100 ° C. for 10 minutes (for Bacillus subtilis) and then rapidly cooled with ice This was used as a dead bacterial suspension.

3. reagent

4). Experimental Method (1) Examination of Standing Time and Light Irradiation Time after EMA Addition A live and dead suspension of Escherichia coli, and a live and dead suspension of Bacillus subtilis 100 μl each at × 10 ⁷ cfu / ml was taken into a 1.5 ml tube, and 1 μl of 1 mg / ml amsacrine solution was added. 1 μl of 1 mg / ml ethidium monoazide solution was added under light shielding. This was allowed to stand at 4 ° C. under light shielding for 5, 10 or 30 minutes, and then left on ice. 100V-500W light (Toshiba Lighting & Technology AL-UF-6-2) installed at a distance of 25 to 30 cm from each suspension was irradiated for 5 or 10 minutes.
Then, after standing at 30 ° C. for 30 minutes in the dark, add 100 μl of 0.2N sodium hydroxide solution, heat at 100 ° C. for 10 minutes, quench rapidly in ice and sterilize with 1M Tris-HCl (pH 8.0). DNA was extracted by adding water and centrifuging at 16000 × g for 5 minutes at 4 ° C. Using the extracted DNA solution, PCR targeting 16S rRNA gene was performed. The PCR composition and reaction conditions are shown below.

PCR composition (E. coli)
Reaction conditions

PCR composition (Bacillus subtilis)
Reaction conditions

After PCR, electrophoresis was performed using 1.5% (w / v) agarose gel. After completion of electrophoresis, the agarose gel was stained by dipping in a SYBR Green I (TaKaRa) solution diluted 10,000 times with TAE buffer for 30 minutes, and the PCR amplification product was observed with a UV transilluminator. The results are shown in FIGS. As is clear from FIGS. 1 and 2, in order to detect only the DNA of live Escherichia coli, which is a gram-negative bacterium, a 4 ° C. incubation time of 30 minutes and a light irradiation of 10 minutes are required. When Bacillus subtilis is kept at 4 ° C. for 30 minutes and irradiated with light for 10 minutes, the DNA of live bacteria is also degraded, and under these conditions, live bacteria of Gram-negative and Gram-positive bacteria are detected simultaneously. I couldn't.
On the other hand, in order to detect only DNA of live bacteria of Bacillus subtilis which is a Gram-positive bacterium, it is necessary to set the incubation time at 4 ° C. for 5 minutes and light irradiation for 5 minutes. When 5 minutes of exposure time and 5 minutes of light irradiation were performed, both live and dead bacteria were detected, and even under this condition, live bacteria of gram-negative and gram-positive bacteria could not be detected simultaneously. .

(2) Examination of effect of addition of surfactant A live cell suspension and a dead cell suspension of Escherichia coli, a live cell suspension and a dead cell suspension of Bacillus subtilis, and 1 × 10 ⁷ cfu / ml and 100 μl of each at 1 × 10 ³ cfu / ml was taken into a 1.5 ml tube, and 1 μl of 1 mg / ml amsacrine solution was added. Furthermore, 1 μl or 10% by weight of surfactant (SDS, SDS, CTAB and Triton X-100) at each concentration (10% by weight, 1% by weight, 0.1% by weight and 0.01% by weight) was added. , CTAB and Triton X-100) were added. 1 μl of 1 mg / ml ethidium monoazide solution was added under light shielding. This was allowed to stand at 4 ° C. for 5 minutes under light shielding, and then left on ice. A 100V-500W light (Toshiba Lighting & Technology AL-UF-6-2) placed at a distance of 25 to 30 cm from each suspension was irradiated for 5 minutes.
Then, after standing at 30 ° C. for 30 minutes in the dark, add 100 μl of 0.2N sodium hydroxide solution, heat at 100 ° C. for 10 minutes, quench rapidly in ice and sterilize with 1M Tris-HCl (pH 8.0). DNA was extracted by adding water and centrifuging at 16000 × g for 5 minutes at 4 ° C. Using the extracted DNA solution, PCR targeting 16S rRNA gene was performed. The sample added with CTAB was used after further diluting the DNA solution 100 times in order to avoid the influence of residual CTAB during PCR. The PCR composition and reaction conditions are the same as those used in (1) above.
After PCR, electrophoresis was performed using 1.5% (w / v) agarose gel. After completion of the electrophoresis, the sample was immersed in a SYBR Green I (TaKaRa) solution diluted 10,000 times with TAE buffer for 30 minutes to stain the agarose gel, and the PCR amplification product was observed with a UV transilluminator. The results are shown in FIGS. As apparent from FIGS. 3 to 7, only the DNA of Escherichia coli, which is a Gram-negative bacterium, and viable bacteria of Bacillus subtilis, which is a Gram-positive bacterium, was detected by light irradiation for 5 minutes. A summary of these results is shown in Table 1.

(3) Examination of addition effect of surfactant when propidium monoazide solution is used Live cell suspension and killed cell suspension of Escherichia coli, live cell suspension and killed cell suspension of Bacillus subtilis 100 μl each of 1 × 10 ⁷ cfu / ml and 1 × 10 ³ cfu / ml was taken in a 1.5 ml tube, and 1 μl of 1 mg / ml amsacrine solution was added. Further, 1 μl of 1% by weight of a surfactant (SDS, Triton X-100) was added. 1 μl of 1 mg / ml propidium monoazide solution was added under shading. This was allowed to stand at 4 ° C. for 5 minutes under light shielding, and then left on ice. A 100V-500W light (Toshiba Lighting & Technology AL-UF-6-2) placed at a distance of 25 to 30 cm from each suspension was irradiated for 5 minutes.
Then, after leaving still for 30 minutes at 30 ° C. under light shielding, DNA was extracted by an alkali dissolution method. Using the extracted DNA solution, PCR targeting 16S rRNA gene was performed. The PCR composition and reaction conditions are the same as those used in (1) above.
After PCR, electrophoresis was performed using 1.5% (w / v) agarose gel. After completion of electrophoresis, the agarose gel was stained by dipping in a SYBR Green I (TaKaRa) solution diluted 10,000 times with TAE buffer for 30 minutes, and the PCR amplification product was observed with a UV transilluminator. The results are shown in FIGS. Note that the sample numbers 1 to 16 in FIGS. 8 and 9 represent the following.
1: PMA, AMSA not added
2: PMA, AMSA added
3: PMA, AMSA, 0.01% SDS added
4: PMA, AMSA, 0.01% TritonX-100 added
5: PMA, AMSA not added
6: PMA, AMSA added
7: PMA, AMSA, 0.01% SDS added
8: PMA, AMSA, 0.01% TritonX-100 added
9: PMA, AMSA not added
10: PMA, AMSA added
11: PMA, AMSA, 0.01% SDS added
12: PMA, AMSA, 0.01% TritonX-100 added
13: PMA, AMSA not added
14: PMA, AMSA added
15: PMA, AMSA, 0.01% SDS added
16: PMA, AMSA, 0.01% TritonX-100 added

Claims (9)

A method for simultaneously detecting live Gram-positive and Gram-negative bacteria in a test sample under the same reaction conditions ,
a) treating a test sample with a surfactant;
b) treating a test sample treated with a surfactant with ethidium monoazide or propidium monoazide;
c) Next, the test sample is irradiated with visible light,
d) DNA is extracted from the test sample irradiated with visible light,
e) treating with a topoisomerase inhibitor or DNA gyrase inhibitor in any of steps a) to d);
f) The detection method, wherein the target region of the extracted DNA is amplified by a nucleic acid amplification method. The detection method according to claim 1, wherein the surfactant is contained in an amount of 0.0001 to 0.1% by weight in the step of treating the test sample with the surfactant. The detection method according to claim 1, wherein in the step of treating the test sample with a surfactant, the surfactant is an anionic surfactant and contained at 0.0001 to 0.01% by weight. The detection method according to claim 3, wherein the anionic surfactant is sodium dodecyl sulfate. The detection method according to claim 1, wherein in the step of treating the test sample with a surfactant, the surfactant is a cationic surfactant and contained at 0.001 to 0.01% by weight. The detection method according to claim 5, wherein the cationic surfactant is hexadecyltrimethylammonium bromide. The detection method according to claim 1, wherein in the step of treating the test sample with a surfactant, the surfactant is a nonionic surfactant and is contained at 0.0001 to 0.1% by weight. The detection method according to claim 7, wherein the nonionic surfactant is Triton X-100.   The detection method according to claim 1, wherein the topoisomerase inhibitor or DNA gyrase inhibitor is amsacrine.