JP2009284853A - Method for extracting genome dna from spore-forming bacterial spore - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently extracting and acquiring a genome DNA under a mild condition through extraction and acquisition of genome DNA from spores without carrying out preparation of nutritive cells by germination. <P>SOLUTION: The spores of spore-forming bacteria are incubated in the presence of a bacteriostatic emulsifying agent in a solution to directly elute and extract a genome from spores without carrying out preparation of nutritive cells by germination of spores. A bacteriostatic surfactant such as a sucrose fatty acid ester, a glycerol fatty acid ester, a polyglycerol fatty acid ester, a sugar alcohol fatty acid ester etc., may be cited as a bacteriostatic surfactant to be used in a method for extracting and acquiring genome DNA from spores. Since the method does not use a means such as a means for grinding spores, a genome is directly extracted by a simple method without causing degeneration such as fractionation of genome DNA. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for extracting and obtaining genomic DNA from spores in order to prepare a sample for genetic analysis of spores of spore-forming bacteria, and in particular, extraction and acquisition of genomic DNA from spores. The present invention relates to a method for extracting and obtaining genomic DNA that is efficiently performed under mild conditions without preparation.

  Conventionally, in order to analyze bacteria, a culture method in which the bacteria are transferred to a medium, cultured, grown and observed has been the mainstream. However, since it takes days to cultivate, in recent years, genetic testing methods have been introduced in which bacterial genes are extracted, amplified, and detected to detect the presence or absence of the desired bacteria. In these genetic test methods, it is necessary to separate and obtain genes from bacterial samples, that is, genomic DNA, and prepare samples for gene analysis. However, in spore-forming bacteria such as Bacillus, spore is formed when the surrounding water is low and nutrients are depleted, so it is difficult to directly extract genomic DNA for gene analysis. That is, the spore is covered with a hard spore shell and has a strong resistance to heat, chemical substances, etc., and it is very difficult to directly extract the chromosomal genomic DNA inside it.

  Therefore, in the conventional method, in order to extract a gene from a spore of a spore-forming bacterium and perform a molecular biological analysis, firstly, the spore is germinated, then propagated as a vegetative cell, and the genomic DNA is extracted after culturing. It was necessary. In the method of taking out a gene after germination of such a spore, since it takes days for germination of the spore, a method using a germination promoter of spore such as bouillon containing alanine, adenosine and glucose is disclosed ( JP, 2005-253365, JP, 2006-345727, A).

  In addition, in order to easily separate and purify DNA from cells with high purity, surfactants such as sodium dodecyl sulfate (SDS) and sodium N-lauroyl sarcosinate, and cell wall polysaccharides such as lysozyme and chromopeptidase A method of cell disruption using a degrading enzyme (Japanese Patent Laid-Open No. 7-143879) or an ionic surfactant such as 3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonic acid Has disclosed a method (Japanese Patent Laid-Open No. 2000-350595) for elution of cell contents by disrupting cell membranes without performing disruption operations such as ultrasonic disruption or freeze-thaw.

  However, these methods require many days for the treatment, and it is difficult to efficiently obtain DNA from cells, or it is difficult to separate and purify DNA from the obtained cell contents. Furthermore, there are problems such as enzymatic degradation of components to be separated / acquired. For example, it has not been sufficient as a means for separating / acquiring genes from spores of spore-forming bacteria. Therefore, conventionally, in order to separate and obtain genomic DNA from spore-forming bacteria, a method of physically destroying and extracting spores of spore-forming bacteria and preparing vegetative cells has been employed.

  As a method for separating and obtaining genes from spore-forming bacteria, as a method for physically destroying spore-forming bacteria, use "freeze crushing method", "ultrasonic destruction method", "milling agent, pressure, or homogenizer". The “physical destruction method” was known. Freezing and crushing is a method in which a sample is frozen at a very low temperature, and a strong external pressure is applied to a weakened area to destroy the cells and extract the contents. There is a problem that difficult-to-use substances are difficult to use. The ultrasonic disruption method destroys cells by applying ultrasonic waves. However, since the destructive force is strong, there is a problem that DNA and the like are broken into small pieces together with spores. The physical destruction method using a grinding agent is a method of destroying cells and the like by mixing glass beads or alumina powder and a sample and mechanically rubbing them. The physical destruction method using pressure is In order to destroy cells and the like by injecting a sample at a high pressure from a small pressure or through a small hole, the physical destruction method using a homogenizer destroys cells and the like using a homogenizer. However, there is a problem of safety and equipment, or it is difficult to apply when the amount of the sample is very small, or there is a problem of fragmentation of DNA or the like, and it cannot be separated and obtained directly from the spores.

  Attempts to solve these problems of physical destruction methods have also been disclosed. For example, in Japanese Patent Application Laid-Open No. 2006-141292, in cell disruption processing using beads, the size of beads for cell disruption is adjusted, the space capacity of a container at the time of cell disruption, A method of increasing the nucleic acid extraction efficiency by crushing cells in a simple, rapid and high-efficiency manner by performing cell disruption treatment in the absence, adding a surfactant after cell disruption, and performing nucleic acid extraction operations, etc. Is disclosed. However, these methods have not been able to extract and obtain genomic DNA simply, quickly and effectively from spores of spore-forming bacteria under mild conditions.

Japanese Patent Laid-Open No. 7-143879. JP 2000-350595 A. JP-A-2005-253365. Japanese Patent Laid-Open No. 2006-141292. JP 2006-345727 A.

  Traditionally, methods used to extract and collect genetic genome samples from bacterial cells use a culture method to germinate spores when extracting and obtaining genomic DNA from spores of spore-forming bacteria. If you need to prepare vegetative cells and take time, for example, if you want to directly determine whether spore bacteria are present in a sample, direct genome extraction from spores that do not require a culture method is essential, There was a problem that the culture method alone could not be judged directly. In addition, in the method using physical disruption, it has been introduced that efficiency is improved by selecting beads, etc., but setting and operation of processing conditions is complicated, and if the number of bacteria is small, genomes can be efficiently generated. There was a problem that it could not be extracted. Furthermore, even with methods using cell wall lytic enzymes such as lysozyme and achromopeptidase, there are problems that the setting and operation of processing conditions are complicated, effective bacteria are limited, and enzymes are mixed. In addition, in the method using a surfactant, denaturing cells is extremely effective for Gram-negative bacteria, but exhibits sufficient lytic effects on Gram-positive bacteria, especially spore-forming bacteria such as Bacillus There was a problem that I could not.

  Therefore, the subject of the present invention can be applied when genomic DNA is extracted / obtained from spores of spore-forming bacteria. When genomic DNA is extracted / separated from such spores, means such as spore crushing means An object of the present invention is to provide a method for extracting and obtaining genomic DNA from spores in a simple and rapid manner under mild conditions.

  As a result of observation by a nanosearch microscope, the present inventor has intensively studied to solve the above problems, and that the appearance of spores of spore-forming bacteria changes to a swollen state by contacting with a bacteriostatic emulsifier. From the headline. Furthermore, the present inventor has found that the spore contacted with the bacteriostatic emulsifier changes to a state in which the contents such as genomic DNA are easily eluted, and has completed the present invention. That is, the present invention elutes and extracts genomic DNA from spores under mild conditions in which spores of spore-forming bacteria are incubated in a solution in the presence of a bacteriostatic emulsifier. Thus, genomic DNA can be extracted and obtained from spores simply and quickly. The method for extracting and obtaining genomic DNA from the spore according to the present invention can be performed directly from the spore of a spore-forming bacterium, which was difficult to extract and obtain by the conventional method, without causing degeneration such as fragmentation of genomic DNA. Can be extracted by a simple means.

  Examples of the bacteriostatic emulsifier used in the method for extracting and obtaining genomic DNA from the spore of the present invention include sucrose fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, and sugar alcohol fatty acid ester. In the present invention, typical spore-forming bacteria of Gram-positive bacteria include bacteria belonging to the order of Bacillus.

  In the present invention, the incubation of the spore of the spore-forming bacterium in the solution in the presence of the bacteriostatic emulsifier is carried out at room temperature in a stationary state. By using the method for extracting and obtaining genomic DNA from the spore of the present invention, genomic DNA from the spore of a spore-forming bacterium is isolated and obtained, a sample of genomic DNA is prepared, genetic analysis is performed, and spore formation is performed. Genetic analysis of bacterial spores can be performed.

  Specifically, the present invention provides [1] a method for extracting and obtaining genomic DNA from spores characterized by incubating spores of spore-forming bacteria in a solution in the presence of a bacteriostatic emulsifier, [2] From the spore according to [1], wherein the bacteriostatic emulsifier is one or more selected from sucrose fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, and sugar alcohol fatty acid ester. And [3] The method for extracting and obtaining genomic DNA from spores according to [1] or [2] above, wherein the spore-forming bacterium is a Gram-positive bacterium. .

  The present invention also provides [4] the method for extracting and obtaining genomic DNA from a spore according to the above [3], wherein the spore-forming bacterium is a bacterium belonging to the order of the Bacillus, Incubation in a solution in the presence of a bacteriostatic surfactant is carried out at room temperature in a stationary state, from the spore according to any one of [1] to [4] above Genomic DNA from spore of spore-forming bacteria can be obtained by using a method for extracting and acquiring genomic DNA, or [6] a method for extracting and acquiring genomic DNA from spore according to any one of [1] to [5] above. It comprises a method for preparing a sample for genetic analysis of spores of spore-forming bacteria characterized by separation and acquisition.

  The method for extracting / acquiring genomic DNA from spores of the present invention can be applied to the case of extracting / acquiring genomic DNA from spores of spore-forming bacteria, for which effective extraction / acquisition was difficult in the conventional method. it can. In addition, when genomic DNA is extracted and separated from such spores, it is possible to obtain genomic DNA from spores under mild conditions without using means such as spore crushing means. The sample can be extracted and acquired for accurate genetic analysis of the sample without causing denaturation of the sample such as fragmentation. Furthermore, since the genomic DNA extraction / acquisition method of the present invention can be carried out by a processing operation of incubating spores of spore-forming bacteria in a solution in the presence of a bacteriostatic emulsifier, The present invention provides a practical means that eliminates the problem of operational complexity and enables simple and rapid extraction and acquisition of genomic DNA from spores.

  In preparing a sample for genetic analysis or the like of spores of spore-forming bacteria, the present invention can be performed by directly incubating spore of spore-forming bacteria in a solution in the presence of a bacteriostatic surfactant. It consists of a method for extracting and obtaining genomic DNA by eluting and extracting the genome.

  Examples of the bacteriostatic emulsifier used in the method of the present invention include one or more selected from sucrose fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters, and sugar alcohol fatty acid esters. Examples of the glycerin fatty acid ester include monoglycerin fatty acid ester, diglycerin fatty acid ester, and triglycerin fatty acid ester. Examples of fatty acids of sucrose fatty acid esters and glycerin fatty acid esters include saturated or unsaturated linear fatty acids having 8 to 22 carbon atoms such as octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, and oleic acid. be able to. As polyglycerol fatty acid ester, polyglycerol (degree of condensation is preferably 2 to 30) fatty acid ester is preferable, as sugar alcohol fatty acid ester, erythritol, xylitol, arabitol, sorbitol, mannitol as fatty alcohol, C7-20 saturated or unsaturated fatty acids such as lauric acid, myristic acid and palmitic acid can be mentioned as suitable fatty acids.

  In the present invention, incubation in a solution in the presence of a bacteriostatic emulsifier of spores of spore-forming bacteria can be performed in a stationary state at room temperature (room temperature), preferably 1 to 72 hours. It is preferable to incubate in a stationary state to extract genomic DNA from spores. In the present invention, a means such as spore crushing means is not used, and the spore is changed to a state in which contents such as genomic DNA are easily eluted under mild conditions. By applying the purification means, genomic DNA can be efficiently separated and purified.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

(Test strain)
Bacillus subtilis IFO3134 strain was used as a test strain.

(Preparation of spores)
(1) A spore solution of Bacillus subtilis was smeared on a standard agar medium (Nissui Pharmaceutical), and cultured at 35 ° C. for 1 week.
(2) The colony was scraped off and suspended in a 50 ml culture tube with an appropriate amount of sterilized water.
(3) The cells were collected by centrifugation at 5000 rpm × 10 minutes.
(4) 20 ml of lysozyme solution [10 mM Tris-HCl (buffer pH 7.6) lysozyme concentration 10 mg / ml] was added to suspend the cells.
(5) It left still at 37 degreeC for 5 minutes.
(6) 20 ml of Wash solution [10 mM Tris-HCl (buffer pH 7.6), 500 ml NaCl] was added, mixed by vortexing, and centrifuged at 10,000 rpm × 10 minutes.
(7) The supernatant was removed, and the operation of (6) was repeated 3 times.
(8) Suspended in 30-40 ml of stock solution.
(9) A dilution series was prepared, and the cell concentration was confirmed on a standard agar medium (Nissui Pharmaceutical).
(10) It was confirmed by Gram staining that vegetative cells were removed and only spores were formed. The prepared spore is shown in FIG.

(Extraction process)
The genomic DNA was extracted from the spores according to the following procedure:
(1) A small amount of sterilized water was added to a 50 ml culture tube, and the following four types of emulsifiers were dispersed, and then adjusted to a final concentration of 1000 ppm (30 mg / 30 g sterilized water). The emulsifiers used are shown in Table 1.
(2) Heated at 100 ° C. for 10 minutes in an autoclave to dissolve the surfactant.
(3) The test spore was inoculated so that the sample was 10 ⁴ order CFU / ml.
(4) Incubated at room temperature for 72 hours.
(5) Genomic DNA was extracted and separated using a commercially available genome extraction kit PrepMan Ultra Sample Preparation Reagent (Applied Biosystems).

(PCR reaction)
Primers for amplifying the entire 16S rRNA chain length were prepared, and gene amplification was performed by PCR reaction according to a conventional method.

(Electrophoresis and gel photography)
Electrophoresis was performed using a 2% agarose gel and photographed using a UV illuminator.

(result)
The electrophoresis results are shown in FIG. 2 (gel photograph). The shade of the band is shown in Table 2. A clear band was confirmed in P1670, TRP97RF and S570 which are bacteriostatic emulsifiers. Bands could not be confirmed in the Triton X-100 and W-60 contact groups and the negative control surfactant non-contact group. It was found that by adding a bacteriostatic emulsifier, genomic DNA extraction efficiency was improved and gene amplification by PCR reaction could be confirmed.

  [Reference experiment example: Change of spore by contact with bacteriostatic emulsifier]

(Test strain)
Geobacillus stearothermophilus DSM5394 strain was used as a test strain.

(Preparation of spores)
Spores were prepared in the same manner as described above (Preparation of spores) in this example.

(Contact treatment with bacteriostatic emulsifier)
(1) A small amount of sterilized water was added to a culture tube for 50 ml, and after adding and dispersing a bacteriostatic emulsifier (P-1670), the final concentration was adjusted to 500 ppm (30 mg sample / 30 g sterilized water). As a comparative control, a sample to which no bacteriostatic emulsifier was added was also prepared. (All operations were performed in a clean bench to avoid contamination.)
(2) Heated at 100 ° C. for 10 minutes in an autoclave to dissolve.
(3) The test strain was inoculated so that the sample was 10 ⁴ order CFU / ml.
(4) Incubated at room temperature for 3 days.

(Observation of spores)
The surface shape of the spore and the state of dispersion / aggregation were observed with a nanosearch microscope (SFT3500, Shimadzu Corporation).

(result)
When the spores of Geobacillus stearothermophilus contacted with a bacteriostatic emulsifier were observed with an atomic force microscope, swelling of the spores was observed (FIG. 3). As shown in FIG. 3, in the group (3-a) to which no bacteriostatic emulsifier is added, the spores (size: 0.2 to 0.3 μm) are aggregated and show a state of clumping. In the group (3-b) of the addition (P1670 500 ppm addition), the spores (size: 0.3 to 0.5 μm) swelled and showed a dispersed state.

It is a photograph which shows the result (confirmation that a vegetative cell is removed and it becomes only a spore) as a result of Gram dyeing | staining of the spore prepared in the Example of this invention. In the Example of this invention, it is a photograph which shows the result of having electrophoresed the sample extracted and acquired by making it contact with a bacteriostatic emulsifier using 2% agarose gel, and image | photographing using UV illuminator. In the Example of this invention, it is a photograph which shows the result of having observed the spore of Geobacillus stearothermophilus which contacted the bacteriostatic emulsifier with the atomic force microscope. In the figure, 3-a is a photograph of a group with no bacteriostatic emulsifier added, and 3-b is a photograph of a group with a bacteriostatic emulsifier added (P1670 500 ppm added).

Claims (6)

A method for extracting and obtaining genomic DNA from spores, comprising incubating spores of spore-forming bacteria in a solution in the presence of a bacteriostatic emulsifier. 2. The genomic DNA from spores according to claim 1, wherein the bacteriostatic emulsifier is one or more selected from sucrose fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, and sugar alcohol fatty acid ester. Extraction / acquisition method. 3. The method for extracting and obtaining genomic DNA from spores according to claim 1, wherein the spore-forming bacteria are gram-positive bacteria. 4. The method for extracting and obtaining genomic DNA from spores according to claim 3, wherein the spore-forming bacteria are bacteria belonging to the order of Bacillus. From the spore according to any one of claims 1 to 4, wherein the incubation in the solution in the presence of a bacteriostatic surfactant of the spore-forming bacterium is performed at room temperature in a stationary state. Extraction and acquisition method of genomic DNA. A genomic DNA from a spore of a spore-forming bacterium is separated and obtained by using the method for extracting and acquiring genomic DNA from a spore according to any one of claims 1 to 5. Sample preparation for genetic analysis.