JP2008048670A - Method for simultaneously culturing food poisoning bacteria, and method for detecting them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for culturing aerobic bacteria belonging to a multiple number of genuses including major food poisoning bacteria, and a quick method for detecting the bacteria by using the same culturing method for detecting the food poisoning bacteria easily and quickly, and also an enriched medium capable of culturing aerobic bacteria belonging to the multiple number of genuses simultaneously. <P>SOLUTION: By holding pH value in the enriched medium under culturing by ≥5 and ≤10 range, at least it becomes possible to culture the aerobic bacteria belonging to the genus Bacillus and genus Listeria in one enriched medium. Also, by making the salt concentration of the enriched medium as 1 to 4%, it becomes possible to culture the aerobic/sulsuginous bacteria of the genus Vibrio simultaneously. Further, by combining the above methods for culturing the bacteria with a real time PCR method, the bacteria belonging to the multiple number of genuses can be detected simultaneously. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for simultaneously culturing bacteria of a plurality of genera on a single medium and a method for detecting bacteria using the culture method.

  Detection of food poisoning bacteria is one of inspection techniques important for ensuring food safety. Therefore, in the food industry and the like, development of simple and rapid detection methods for various food poisoning bacteria based on molecular genetic techniques and immunological techniques is progressing. Among them, the nucleic acid amplification method that amplifies and detects a specific base sequence for a specific bacterial gene is currently applied to various food poisoning bacteria detection methods because of its sensitivity, rapidity, and high specificity. .

However, even in a test method using a nucleic acid amplification method, a certain number of bacteria is required to obtain a necessary amount of nucleic acid as a template. The lower limit of the number of bacteria required for the detection is about 1 × 10 ³ cfu (colony forming unit) / ml. In order to detect bacteria from food with high sensitivity, it is necessary to culture the target bacteria in most cases. is there. However, since bacteria having different properties generally have different culture conditions, in order to detect a plurality of types of bacteria, each target bacteria must be cultured under the optimum culture conditions. Therefore, simplification and speeding up of the method for detecting food poisoning bacteria poses a major problem in labor and time required for the culture. As a means for solving such a problem, there is a method of simultaneously culturing a plurality of food poisoning bacteria in one medium. However, for this purpose, it is indispensable to develop an enrichment medium capable of simultaneously culturing a plurality of species or a plurality of genera.

As an enrichment medium capable of culturing food poisoning bacteria of a plurality of genera, UEB (Universal Enrichment Broth) (Non-patent Document 1), Salmonella genus, Listeria genus, and cultivated bacteria of Salmonella genus and Listeria genus simultaneously A medium using buffered peptone water for simultaneously culturing Staphylococcus aureus is known (Non-patent Document 2). However, any of these enrichment media can only cultivate a few food poisoning bacteria at the same time. Therefore, there is a need for an enrichment medium that can simultaneously culture more food poisoning bacteria.
Bailey JS & NACox NA, (1992) J. Am. Food Prot. , 55; 256-259 Alarcon B, Garcia-Canas V, Cities, A, Gonzalez R & Aznar R (2004) J. MoI. Agric. Food Chem. , 52; 7180-7186 Nakata HM & Halvorson HO, (1960) J. MoI. Bacteriol. , 80; 801-810 Budu-Amoako E, Toora S, Ablett RF & Smith J, (1992) Appl. Environ. Microbiol. , 58; 3177-3179 Busch SV & Donnelly CW, (1992) Appl. Environ. Microbiol. , 58; 14-20 Supervised by Shigeo Ohno and Yoshifumi Nishimura, (1997) Cell Engineering Separate Volume Experimental Protocol Series Protein Experimental Protocol Sambrook J, Fritsch EF, Maniatis T, (1989) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press. Priest FG, Goodflow M, Todd C, (1998) Journal of General Microbiology, 134; 1847-1882. Seliger HPR & Jones D, (1986) Genus Listeria, In Bergey's Manual of Systemic Bacteriology Volume 2, ed. By Sneath PHA, pp 1235-1245, Williams & Wilkins Baltimore, MD, USA.

  An object of the present invention is to provide a bacterial culture method capable of simultaneously culturing a plurality of aerobic bacteria to which main food poisoning bacteria belong in order to detect food poisoning bacteria easily and quickly, and rapid bacterial detection using the culture method Is to provide a method. Moreover, it is providing the enrichment culture medium which can culture | cultivate the aerobic bacteria of several fungi simultaneously.

  In order to solve the above-mentioned problems, the present inventors have studied culture conditions that can simultaneously culture a plurality of aerobic bacteria to which major food poisoning bacteria belong. The invention of the present application is based on such research results, and relates to the following bacterial culture method, bacterial detection method, enrichment medium, and bacterial detection kit.

  The first invention of the present application is a bacterial culture method capable of simultaneously culturing at least a Bacillus genus and a Listeria genus aerobic bacteria in one enrichment medium, wherein the enrichment medium has a pH value of more than 5 and 10 or less. This is a method for culturing bacteria by holding and culturing.

  The second invention of the present application is further a bacterial culture method capable of simultaneously culturing aerobic and halophilic bacteria belonging to the genus Vibrio in one enrichment medium, wherein the salt concentration of the enrichment medium is a mass using water as a solvent. The bacterial culture method according to the first invention, characterized in that the percentage by volume is 1% to 4%.

  The third invention of the present application is the bacteria described in the first or second invention, wherein the enrichment medium uses water as a solvent and the sugar content is 0.001% or less in terms of mass to volume percentage. It is a culture method.

  In the fourth invention of the present application, the enrichment medium is a mass to volume percentage with water as a solvent, peptone: 1% to 3%, dipotassium phosphate: 0.15% to 0.35%, sodium chloride: 1% to 4% and yeast extract: 0.01% to 0.1%, The bacterial culture method according to the third aspect of the invention.

  The fifth invention of the present application is a bacterial detection method capable of simultaneously detecting aerobic bacteria of at least two genera among at least Staphylococcus, Salmonella, Escherichia, Vibrio, Bacillus, and Listeria. It is a bacteria detection method which has the culture | cultivation process cultivated by the bacteria culture | cultivation method as described in any one of this invention from 4th invention, and the detection process of detecting desired bacteria from the bacteria obtained after a culture | cultivation process.

  In the sixth invention of the present application, the detection step further comprises a nucleic acid extraction step for extracting nucleic acids from the cells obtained after the culturing step, and the nucleic acid obtained in the nucleic acid extraction step on the genomic DNA of the desired bacteria. And a nucleic acid detection step of detecting a desired bacterium using an oligonucleotide that recognizes a base sequence unique to the bacterium.

  In the seventh invention of the present application, the nucleic acid detection step further uses a nucleic acid obtained in the nucleic acid extraction step as a template and an oligonucleotide that recognizes a base sequence peculiar to the bacterium on the genomic DNA of the desired bacterium. Bacterial detection according to the sixth invention, comprising: a nucleic acid amplification step for amplifying a nucleic acid of a desired bacterium by an amplification method; and an amplified nucleic acid detection step for detecting a desired bacterium based on the nucleic acid amplified in the nucleic acid amplification step. Is the method.

  In the eighth invention of the present application, for a bacterium with two or more nucleic acid amplification steps, oligonucleotides that recognize base sequences unique to each bacterium on the genomic DNA of each bacterium are used simultaneously in one reaction solution. The method of detecting a bacterium according to the seventh invention, wherein the nucleic acid of each bacterium is amplified.

  The ninth invention of the present application is the bacteria detection method according to the seventh invention or the eighth invention, wherein the nucleic acid detection step uses a real-time PCR method.

  The tenth invention of the present application is the nucleic acid detection step according to the sixth invention, wherein the nucleic acid detection step is carried out on the genomic DNA of the desired bacterium by a hybridization method using an oligonucleotide that recognizes a base sequence unique to the bacterium as a probe. Bacteria detection method.

  In the eleventh invention of the present application, each aerobic bacterium of the genus Staphylococcus, Salmonella, Escherichia, Vibrio, Bacillus, and Listeria is an S. aureus bacterium. aureus, Salmonella spp. , E.C. coli O157, V.I. parahaemolyticus, B. et al. cereus, and L. The method for detecting bacteria according to any one of the fifth to tenth aspects of the invention, which is monocytogenes.

  In the twelfth invention of the present application, the mass to volume percentage when water is used as a solvent is sugar: 0.001% or less, peptone: 1% to 3%, dipotassium phosphate: 0.15% to 0.35% Sodium chloride: 1% to 4%, and yeast extract: 0.01% to 0.1%.

  A thirteenth invention of the present application is the enrichment medium according to the twelfth invention, wherein the peptone contains 10% to 20% soyton with respect to its own mass.

  14th invention of this application is a kit for bacteria detection containing the enrichment culture medium as described in 12th invention or 13th invention.

  According to the bacterial culture method of the present invention, aerobic bacteria belonging to the genus Bacillus and the genus Listeria can be simultaneously cultured in a single enrichment medium. As a result, the method for culturing food poisoning bacteria can be simplified and speeded up. Along with this, it is possible to reduce the cost required for the culture such as the culture medium, the culture apparatus, and the utility cost.

  According to the bacteria detection method of the present invention, it is possible to simplify the operation until bacteria are detected from the test object. In addition, the time required for detection can be shortened. In addition, according to another bacterial detection method of the present invention, food poisoning bacteria belonging to multiple genera can be detected simultaneously. Thereby, bacteria can be detected more rapidly from the subject.

  According to the enrichment medium of the present invention, when cultivating at least Bacillus genus and Listeria genus bacteria at the same time, an appropriate pH value can be maintained during culturing. Therefore, it is not necessary to adjust the pH of the medium during the culture, and the culture operation can be further simplified and speeded up.

  The best mode for carrying out the invention will be described below. However, the invention of the present application is not limited to these embodiments, and can be carried out in various modes within the scope of the effects. The first embodiment mainly relates to claims 1 to 4 and claims 12 and 13. The second embodiment mainly relates to claims 5 to 11. The third embodiment mainly relates to claim 14.

<< Embodiment 1 >>

<Embodiment 1: Overview>
Embodiment 1 relates to a bacterial culture method and an enrichment medium. The bacterial culture method of the present embodiment is characterized in that at least a Bacillus genus and a Listeria genus aerobic bacterium can be cultured at the same time by culturing while maintaining the pH value of the enrichment medium to be greater than 5 and 10 or less. In addition, the enrichment medium of the present embodiment is characterized in that the pH value can be maintained without any special operation during the culture.

<Embodiment 1: Background to Invention>
Before going into the detailed description of the bacterial culture method of the present embodiment, first, the background to the present invention will be described. As described above, in order to solve the problem, the present inventors have studied culture conditions that can simultaneously culture a plurality of aerobic bacteria to which major food poisoning bacteria belong. At present, the major food poisoning bacteria in Japan include Staphylococcus aureus (S. aureus: hereinafter referred to as “S. aureus”), Salmonella Enteritidis (hereinafter referred to as “S. Enteritidis”), Escherichia coli O157 (pathogenic Escherichia coli O157). : Hereinafter referred to as “E. coli O157”), Vibrio parahaemolyticus (hereinafter referred to as “V. parahaemolyticus”), Bacillus cereus (hereinafter referred to as “B. cereus”), and Listeria monecium (hereinafter referred to as “B. cereus”). Listeria: hereinafter referred to as “L. monocytogenes”). As a result of simultaneously culturing these 6 genera and 6 types of bacteria using common enrichment media such as TSB under commonly used culture conditions, As a result, the growth of parahaemolyticus was suppressed. The bacterial growth suppression could be solved by increasing the salt concentration of the enrichment medium.

  In addition, when the bacteria of the above 6 genera were co-cultured, As a result, the growth of monocytogenes was suppressed. In order to find a culture condition in which Listeria can be cultured at the same time, the present inventors investigated the cause of the growth inhibition. As a result, growth inhibition was observed in L.P. monocytogenes and B.I. It was found that it occurs when co-cultured with cereus and that the growth suppression is caused by a decrease in the pH value of the enrichment medium due to the growth of the genus Bacillus. Based on this discovery, two genus Bacillus and Listeria, which were difficult to co-cultivate by culturing while maintaining the pH value of the enrichment medium at greater than 5 and less than or equal to 10, were simultaneously cultured in one enrichment medium. The culture condition was found to be possible. By increasing the salt concentration of the enrichment medium in addition to this culture condition, It has become possible to simultaneously culture all the bacteria of the above six genera, including parahaemolyticus, in one enrichment medium.

  Furthermore, the present inventors have developed an enrichment medium in which the pH value does not decrease even when Bacillus genus grows. According to Non-Patent Document 3, the decrease in the pH value of the enrichment medium due to the growth of the genus Bacillus produces acids such as organic acids during the process of metabolic decomposition of glucose in the enrichment medium and discharges it into the medium. It was thought to be the cause. Therefore, by removing as much sugar as possible from the enrichment medium and adding nutrients to complement it, the bacteria of the above 6 genera can be cultured at the same time without significantly lowering the pH value even when Bacillus grows. Succeeded in developing an enrichment medium. The invention of the bacterial culture method and enrichment medium of the present embodiment is based on this discovery.

<Embodiment 1: Configuration>
Each structure of the bacterial culture method and the enrichment medium of the present embodiment will be described below.

((Configuration of bacterial culture method))

(When culturing aerobic bacteria belonging to the genus Bacillus and Listeria)
The constituent requirement of the bacterial culture method of this embodiment in which at least Bacillus genus and Listeria genus aerobic bacteria are simultaneously cultured in one enrichment medium is that the enrichment medium has a pH value of more than 5 and not more than 10. It is to culture.

  “Aerobic bacteria” is a general term for bacteria that require oxygen for the metabolism of substances. However, in this application, facultative aerobic bacteria (or facultative anaerobic bacteria) that can grow even in a completely anaerobic state are included.

  “Aerobic bacteria belonging to the genus Bacillus” refers in particular to aerobic bacterial species that cause a decrease in the pH value of the medium due to growth. That is, it refers to an aerobic bacterial species that discharges acid generated with growth into the enrichment medium. For example, B.I. cereus and Bacillus subtilis are applicable. Regarding the growth conditions, these bacterial species have similar properties (Non-patent Document 8).

  “Aerobic bacteria of the genus Listeria” Monocytogenes, Listeria innocua, Listeria ivanovii, Listeria grayi, Listeria welshimeri, Listeria segligeri and the like. Regarding the growth conditions, these bacterial species have similar properties (Non-patent Document 9).

  “Enrichment medium” is a medium intended to grow bacteria. This will be described in detail later.

  “One enrichment medium” means a single enrichment medium in the same container.

“Simultaneous culture” refers to inoculating a plurality of bacterial species in one enrichment medium and then allowing them to grow together. The criterion for determining whether or not both are growing may be, for example, that the number of bacteria per medium volume of each bacterium is equal to or greater than a predetermined value by each stationary phase. An example of the “predetermined value” is approximately 1 × 10 ³ cfu / ml. This is because the numerical value corresponds to the detection lower limit number of bacteria of the detection method using the nucleic acid amplification method or the like, and is therefore appropriate as the lower limit value of the predetermined value. There is no limitation on the time until each bacterial species reaches a predetermined number of bacteria. For example, even if a certain bacterial species reaches a predetermined number of bacteria in 2 hours after culturing, enters a stationary phase after 1 hour, and other species reach a predetermined number of bacteria in 10 hours after culturing. Both are cultured at the same time.

  “Keeping the pH value of the enrichment medium greater than 5 to 10 or less” means keeping the pH value of the entire enrichment medium within a range of greater than 5 and 10 or less during the culture. As long as the pH value of the enrichment medium is within the range, some variation is not required. However, when the pH value is outside this range, it is not preferable for culturing bacteria belonging to the genus Bacillus and Listeria.

  The reason why it is not preferable to set the pH value of the enrichment medium to 5 or less is that the growth of Listeria bacteria is suppressed. This will be described in detail below with reference to FIG. FIG. 1 shows L. in the enrichment medium at each pH value. The number of bacteria per culture medium volume of monocytogenes is shown. The experiment of FIG. 1 was carried out using a sterilized TSB adjusted to each pH value using HCl. Monocytogenes is inoculated under aseptic conditions so that the final bacterial concentration is 1 to 10 cfu / ml. After standing at 37 ° C. for 18 hours, the cells are diluted and coated on a TSB agar medium (hereinafter “TSA”). The number is counted. As shown in this figure, when the pH value of the enrichment medium is 5 or less, L. Monocytogenes can hardly be detected. Therefore, it is not preferable to set the pH value of the enrichment medium to 5 or less because the growth of Listeria bacteria is suppressed. In consideration of efficient growth of Bacillus genus and Listeria genus, the pH value of the enrichment medium is preferably 5.2 or more, and more preferably 5.5 or more. The composition of TSB and TSA will be described in detail in Example 1.

  The reason why it is not preferable that the pH value of the enrichment medium exceeds 10 is that many bacteria except alkaliphilic bacteria are known to be inhibited from growing under strong alkali exceeding pH 10. . Although not shown, the growth of Listeria bacteria is also suppressed in an environment exceeding pH 10 (Non-patent Document 9). Therefore, it is not preferable that the pH value of the enrichment medium exceeds 10. In consideration of efficient growth of Bacillus and Listeria, the pH value of the enrichment medium is preferably 9 or less, and more preferably 8.7 or less.

  By the way, when aerobic bacteria of the genus Bacillus and Listeria are cultured at the same time, it is difficult to maintain the pH value within the above range under normal culture conditions. This is because the pH value of the enrichment medium is lowered by the growth of Bacillus bacteria as described above. FIG. cereus and L. It shows the relationship between the number of bacteria per medium volume of each bacterium and the pH value of the medium when culturing monocytogenes simultaneously. In the experiment of FIG. cereus and L. Each of monocytogenes was inoculated under aseptic conditions so that the final bacterial concentration was about 100 cfu / ml, and statically cultured at 37 ° C. Subsequently, a part of the enrichment medium solution was collected every 2 hours from immediately after inoculation until 18 hours, and then the number of colonies formed by diluting the selective medium plate of each bacterium was counted. At the same time, a part of the enrichment medium solution was centrifuged, and the pH of the supernatant was measured after filter sterilization. The composition of the selective medium is described in detail in Example 1. As shown in this figure, the pH value of the enrichment medium is as follows. It can be seen that cereus proliferates and rapidly decreases after 12 hours after culturing. In addition, as the pH value of the enrichment medium decreases, L. It has also been shown that the growth of monocytogenes is largely suppressed. In this way, in the passive culture method, Bacillus genus bacteria grow, which causes conflicting problems in co-culturing the two genera that the growth of Listeria is suppressed.

  Therefore, in the present embodiment, it is a constituent requirement that the pH value in the enrichment medium is actively maintained within the range of more than 5 and 10 or less even when Bacillus is being cultured. This makes it possible to simultaneously culture at least a Bacillus genus and a Listeria aerobic bacterium in one enrichment medium. The method for maintaining the pH value of the enrichment medium is described in the section “Embodiment 1: Method”. On the alkali side, even when aerobic bacteria of the genus Bacillus and Listeria are cultured at the same time, the pH value of the enrichment medium does not increase due to an increase in one of the bacterial species. Therefore, the pH value of the enrichment medium may be adjusted to 10 or less at the time of inoculation.

  Even if the pH value of the enrichment medium is temporarily outside the range of the pH value, if the pH value is returned to the range during the culture or if the pH value returns, The pH value of the medium is considered to be maintained within the range.

  "At least Bacillus genus and Listeria genus aerobic bacteria can be cultured simultaneously in one enrichment medium" means that when each aerobic bacterium of Bacillus genus and Listeria genus is present in the test object, For at least the two genera, it means that they can be cultured simultaneously in a single medium. Therefore, a plurality of species of the same genus may be simultaneously cultured by the bacterial culture method of the present embodiment. Moreover, when other aerobic bacteria exist in the test object, they may be cultured together with the bacteria belonging to the two genera. In particular, it is preferable that other fungi including food poisoning bacteria are cultured at the same time in order to contribute to the object of the present invention. For example, in the bacterial culture method of the present embodiment using a TSB medium, in addition to the two genera, Staphylococcus, Salmonella, Escherichia, and Vibrio (in this case, non-halophilic species such as Vibrio cholerae). Co-cultivation is possible. Other than these, Shigella genus, Aeromonas genus, Plesiomonas genus and the like can be mentioned.

(In addition to the above two genera, when aerobic and halophilic bacteria belonging to the genus Vibrio are co-cultured)
In addition to at least Bacillus genus and Listeria aerobic bacteria, Vibrio genus aerobic and halophilic bacteria are simultaneously cultured in one enrichment medium. In addition to culturing while maintaining the value greater than 5 and 10 or less, the salt concentration of the enrichment medium is 1% to 4%. Among these, the configuration requirements other than those described above will be described below.

  “Vibrio genus aerobic / halophilic bacterium” refers to an aerobic bacterial species belonging to the genus Vibrio and requiring a certain concentration of salt for growth. For example, V.I. Parahaemolyticus, Vibrio flubialis, Vibrio vulnificus, Vibrio damsela, Vibrio arginolytics are applicable. Regarding the growth conditions, these species have similar properties.

  “Salt concentration” means the percentage of salt by mass (g / ml) when water is used as a medium. The “salt” here mainly means sodium chloride, but may contain magnesium chloride, magnesium sulfate, calcium sulfate, and / or potassium chloride.

  The reason for setting the salt concentration to 1% or more and 4% or less is that many halophilic species, including the halophilic Vibrio genus, inhabit in seawater. This is because it requires an environment. The salt concentration of seawater is about 3.4%. Therefore, in this embodiment, the salt concentration is set to 1% or more and 4% or less. Outside this range, it is not preferable because the growth of the bacterial species may be suppressed.

The effect of the salt concentration on the growth of the halophilic Vibrio genus will be described with an example. FIG. cereus, L.C. monocytogenes, and V. halophilic bacteria. The number of bacteria per medium volume of each bacterium when parahaemolyticus is cultured simultaneously is shown. (A) shows the result of culturing with TSB (salt concentration of 0.5%), and (b) is the enrichment medium (salt concentration of 1.5%) in TSB with NaCl being 1.5% by mass to volume percentage. It is the result of having been cultured with. In each experiment, each sterilized enrichment medium was inoculated under aseptic conditions so that the final bacterial concentration was about 1 to 10 cfu / ml, incubated at 37 ° C. for 18 hours, and then diluted with TSA. Count the number of colonies. As shown in these figures, V.V. The growth of parahaemolyticus was almost suppressed at a salt concentration of 0.5%. On the other hand, under a salt concentration of 1.5%, the cells grew to about 1 × 10 ⁶ cfu / ml, which was sufficient for bacterial detection. In FIG. The growth of monocytogenes is suppressed because the pH value of the enrichment medium used in the experiment is not maintained within the above range.

  As described above, in the present embodiment, when the aerobic / halophilic species of the Vibrio genus are co-cultured with the Bacillus genus and the Listeria genus, the salt concentration of the enrichment medium is 1% or more and 4% or less. Is preferred.

((Enrichment medium))

In the bacterial culture method of this embodiment, it is necessary to actively maintain the pH value of the enrichment medium during the culture within a range of more than 5 and 10 or less. The composition of the enrichment medium is appropriately selected according to the method for maintaining the pH value of the enrichment medium during culture. For example, any method for forcibly adjusting the pH value of the enrichment medium (A) described in “Culture process” in the section “Embodiment 1: Method” described later is generally used for bacterial culture. A general-purpose medium TSB may be used. Similarly, TSB can be used in the case of adding a reagent having pH buffering ability to the enrichment medium of (B). However, in this case, PBS (Phosphate Buffered Saline: 137 mM HCl, 8.1 mM Na ₂ HPO ₄ · 12H ₂ O, 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ , pH 7) 4) need to be added. Furthermore, in order to simultaneously culture aerobic and halophilic bacteria belonging to the genus Vibrio in one enrichment medium, the salt concentration of the enrichment medium is the final concentration in both cases (A) and (B). Sodium chloride or the like may be added to the TSB so as to be 1% to 4%.

  By the way, as described above, under normal bacterial culture conditions, the growth of Bacillus causes a decrease in the pH value of the enrichment medium. This decrease in the pH value of the enrichment medium due to the growth of the genus Bacillus is caused by the fact that Bacillus bacteria metabolize sugars to produce acids and excrete them into the medium. Therefore, if the sugar in the enrichment medium is removed or kept to an infinitely small amount, a decrease in the pH value of the enrichment medium due to the growth of Bacillus can be suppressed. Specifically, for example, the sugar content in the enrichment medium may be 0.001% or less in terms of mass to volume percentage using water as a solvent. However, in this case, it is preferable to supplement nutritionally by adding nutrients instead of sugar to the enrichment medium. Examples of nutrients in place of sugar include yeast extract (Non-patent Documents 4 and 5), which are known to be rich in various vitamins, minerals, nucleic acid substances, and the like.

  The present inventors have developed a new enrichment medium in accordance with the purpose of the enrichment medium of the present embodiment in consideration of the above points. In the present application, the new enrichment medium is hereinafter referred to as SEB (Simultaneous Enrichment Broth) for convenience. SEB is an enrichment medium with a sugar content of 0.001% or less based on the composition of TSB and supplemented with yeast extract to complement it. The SEB is expressed in terms of mass to volume percentage using water as a solvent, peptone: 1% to 3%, dipotassium phosphate: 0.15% to 0.35%, sodium chloride: 1% to 4%, and yeast Extract: 0.01% to 0.1% is a main component. The peptone is not particularly limited as long as it is fragmented by partially decomposing the protein with a hydrolase. For example, tryptone (DIFCO) obtained by trypsin treatment of casein and bacto peptone (DIFCO) obtained by pepsin treatment of bacteria can be used. For more efficient growth of bacteria, 10 to 20% of peptone may be added with soyton (DIFCO) obtained by partially degrading soybean protein with a hydrolase to a part of the peptone. The composition of SEB is 1% to 4% of sodium chloride so that Vibrio aerobic and halophilic bacteria can be cultured at the same time, but it is not necessary to culture halophilic bacteria at the same time. In such a case, the sodium chloride content may be about 0.5%, similar to TSB. Examples of co-culture of aerobic bacteria of the genus Bacillus and Listeria using SEB are described in Examples 1 and 2.

  When the purpose is to simultaneously culture a plurality of bacterial species, the state of the enrichment medium is preferably a liquid medium in principle. However, a solid medium such as an agar medium may be used as necessary when a colony is to be formed.

<Embodiment 1: Method>
An example is given and demonstrated about the bacteria culture method of Embodiment 1. FIG. FIG. 4 is an example of a flowchart of the bacterial culture method in the present embodiment. As shown in this figure, the bacterial culture method of this embodiment includes an enrichment medium preparation step (S0401), an inoculation step (S0402), and a culture step (S0403). Each step will be described below. In addition, the following series of operations is performed in an aseptic environment in principle, and the culture medium, reagents, instruments, etc. to be used are in principle sterilized ones.

  The “enrichment medium preparation step” (S0401) is a step of preparing an enrichment medium capable of simultaneously culturing at least Bacillus genus and Listeria genus aerobic bacteria. The enrichment medium may be prepared by appropriately selecting the enrichment medium having the above-described configuration. The enrichment medium used in this step is preferably a liquid medium. The amount of the enrichment medium may be determined according to the use. For example, if a desired bacterium is detected by the nucleic acid amplification method described in Embodiment 2 using the culture solution obtained by the culture method of this embodiment, the amount of the enrichment medium may be 5 ml to 20 ml. In addition, if a desired bacterium is detected using a classic Southern hybridization method, the amount of the enrichment medium is preferably about 50 to 500 ml. The enrichment medium after preparation is used after autoclaving at 121 degrees for 15 to 20 minutes. Note that this step may be omitted if an existing medium is used such as a commercially available enrichment medium.

  The “inoculation step” (S0402) is a step of inoculating the enrichment medium with bacteria in the inspection object by adding the inspection object to the enrichment medium. The inspection object referred to here mainly corresponds to food in view of the present invention, but may be other than that. For example, wraps and packs that wrapped food, or tableware can be mentioned. The method for adding the test object to the enrichment medium is not particularly limited as long as it is a method capable of inoculating the enrichment medium with bacteria present in the test object. For example, if the food to be tested is a liquid or a fluid (such as curry roux), a part of it may be added directly to the enrichment medium. In addition, if the test object is a solid food, after homogenizing the test object, add a part of the homogenate directly into the enrichment medium, or mix the homogenate with a buffer such as sterile PBS. The suspension or supernatant may be added to the enrichment medium. Alternatively, a method may be used in which the test object is homogenized with a sterile enrichment medium and cultured as it is. Furthermore, if it is a tableware or a pack, the test site may be washed with sterile PBS or the like, and the washed PBS may be added directly to the enrichment medium.

  In the bacterial culture method of the present embodiment, a pre-culture process may be provided between the inoculation process (S0402) and the next culture process (S0403) depending on the situation. The “preculture process” is a process for increasing the number of bacteria by inoculating a relatively small amount of enrichment medium in advance with a test object in order to increase the number of bacteria to be inoculated to the enrichment medium in the culture process. is there. Whether or not to go through this step is usually determined by the volume of the enrichment medium used in the culturing step. For example, when the amount of the enrichment medium exceeds 100 ml, it is preferable to go through this step. This is because the time to the logarithmic growth phase of bacteria can be shortened. Conversely, in the case of inoculating a small amount of medium such as 50 ml or less, it is not necessary to go through the preculture step. It is desirable that culture conditions such as retention of pH value and salt concentration in the pre-culture process are the same as those in the culture process. This is because, under normal culturing conditions, there is a possibility that the growth of each bacterium will be overweight at the pre-culture stage. The pre-culture may be performed at 36 ° C. to 38 ° C. for about 6 hours to 12 hours. After the pre-culture, an appropriate amount of the culture solution is directly diluted or appropriately diluted with a sterile buffer such as PBS, and then inoculated into the enrichment medium.

  The “culturing step” (S0403) is a step of culturing the inoculated bacteria by placing the enrichment medium after inoculation under certain conditions. The fixed condition is to maintain the pH value of the enrichment medium during the cultivation at more than 5 and at most 10 when at least aerobic bacteria of the genus Bacillus and Listeria are cultured at the same time. Moreover, in order to further culture aerobic and halophilic bacteria belonging to the genus Vibrio, the salt concentration of the enrichment medium should be 1 to 4%.

  As described above, under normal culture conditions, the pH value decreases due to the growth of the genus Bacillus. Therefore, in the bacterial culture method of this embodiment, it is necessary to actively maintain the pH value of the enrichment medium during culture. Hereinafter, the method for maintaining the pH value of the enrichment medium during culture will be described with specific examples.

(A) Method for forcibly adjusting the pH value of the enrichment medium from the outside While monitoring the pH value of the enrichment medium, the pH of the enrichment medium is forcibly adjusted by adding a drug or the like from the outside. This is a method of keeping the value within a predetermined range. A specific example of the method is titration. For example, when the pH value of the enrichment medium is measured with a pH meter during culture and the pH value becomes 5 or less, a NaOH solution of about 0.1 N is titrated to be within a range of more than 5 and 10 or less. It may be adjusted so that On the contrary, when the pH value exceeds 10, an HCl solution of about 0.1N is titrated, and similarly, it may be adjusted to be within a range of more than 5 and 10 or less. The enrichment medium used in this method may be a general enrichment medium with high versatility. For example, TSB can be used. In addition, what is necessary is just to add and adjust salt concentration suitably to TSB as needed. It is convenient that these adjustments can be automatically performed by connecting or integrating the titration device and the pH meter.

(B) A method of adding a reagent having a pH buffering capacity to the enrichment medium A reagent that has a pH range of more than 5 and less than 10 and has little effect on bacterial growth is added to the enrichment medium. It is a method to add. Since the enrichment medium itself has a pH buffering capacity, even if the pH value is lowered due to the growth of the genus Bacillus, it is convenient because it is automatically adjusted to a pH value at which the aerobic bacteria of the genus Listeria can grow. A specific example is a method of adding about 1/10 volume of PBS as a solvent for the enrichment medium. When culturing by this method, an enrichment medium to which a reagent having pH buffering ability is added in the enrichment medium preparation step (S0401) may be prepared.

(C) Method of using enrichment medium in which sugar content is suppressed As described in the above section of “Enrichment medium”, culture is performed by suppressing the sugar content in the enrichment medium or removing the sugar. This is a method for preventing the pH value from decreasing. When culturing by this method, an enrichment medium in which the sugar content is suppressed in advance may be prepared in the enrichment medium preparation step (S0401). In this case, as described above, it is preferable to add nutrients replacing sugar to the enrichment medium. Specific examples of the enrichment medium include SEB described in the above section “Enrichment medium”.

  The culture temperature may be 36 ° C to 38 ° C, and the culture time may be about 6 hours to 24 hours. Usually, it is sufficient to culture at 37 ° C. for about 12 to 20 hours. The enrichment medium may be either stationary culture or shaking culture. In the case of static culture, an incubator or the like may be used, and in the case of shake culture, a constant temperature shake incubator or the like may be used.

  Through the above steps, at least a Bacillus genus and a Listeria aerobic bacterium can be cultured simultaneously. In addition, the result of the bacterial culture based on the bacterial culture method of this embodiment was shown in Examples 2 and 3.

<Embodiment 1: Effect>
According to the bacterial culture method of this embodiment, it is possible to simultaneously culture a plurality of food poisoning bacteria in a single medium, and the culture method can be simplified and speeded up. Moreover, it leads to shortening of the detection time in the bacteria detection method of Embodiment 2.

  According to the enrichment medium of the present embodiment, when at least bacteria of the genus Bacillus and the genus Listeria are cultured simultaneously, an appropriate pH value can be maintained during the culture. As a result, it is not necessary to adjust the pH of the medium during culturing, and the culturing operation can be further simplified and speeded up.

<< Embodiment 2 >>

<Embodiment 2: Overview>
Embodiment 2 relates to a bacteria detection method. The bacteria detection method of this embodiment includes a culture step of culturing bacteria by the bacteria culture method of Embodiment 1. According to the bacteria detection method of the present embodiment, aerobic bacteria of at least two genera among at least Staphylococcus genus, Salmonella genus, Escherichia genus, Vibrio genus, Bacillus genus, and Listeria genus can be detected at the same time. Therefore, it is useful as a simple and rapid detection method for bacteria including food poisoning bacteria.

<Embodiment 2: Method>
The bacteria detection method of Embodiment 2 is demonstrated. FIG. 5 shows an example of a flowchart of the present embodiment. As shown in this figure, the bacterium detection method of the present embodiment has a culture process (S0501) and a detection process (S0502) as constituent requirements. Hereinafter, each step will be described.

  The “culturing step” (S0501) is a step of culturing using the bacterial culture method of Embodiment 1. Here, the culture step refers not only to the culture step described in the section “Embodiment 1: Method” but also a series of culture steps using the culture method of Embodiment 1 widely. That is, in addition to the inoculation process, the enrichment medium preparation process and the pre-culture process are included as necessary. The basic method may be the same as the method described in the first embodiment. Bacteria in the medium obtained after the culture step are used for the next detection step.

  The “detection step” (S0502) is a step of detecting desired bacteria from the bacteria obtained after the culture step (S0501). The “desired bacterium” refers to a target bacterium for which the presence or absence is to be confirmed. The detection process can be further classified into several types depending on the detection method. The first is a method of detecting the grown live bacteria as they are. The second is a method for detecting the protein of the grown bacteria. The third is a method for detecting the nucleic acid of the grown bacteria. Hereinafter, each method will be described.

(Method using live bacteria)
This is a method for detecting bacteria grown in the culturing process in a state of viable bacteria. For example, the colony formation method using the selective culture medium of desired bacteria is mentioned. Specifically, the colony formation method is such that a buffer such as PBS is added to the culture solution after culturing to prepare a 10-fold serial dilution series, and an appropriate amount is smeared on a selective medium plate of a desired bacterium, and then a colony formed. This is a method for calculating the presence of the desired bacteria and the number of bacteria based on the number. The selective medium described in detail in Examples 1 and 2 can be used as long as it is a major food poisoning bacterium in Japan listed in “Embodiment 1: Background to the Invention”.

(Method of detecting protein)
This is a method for detecting a protein specific to a desired bacterium. For example, an immunological detection method using an antibody can be mentioned. As specific examples, an ELISA method or a Western blotting method can be used. In view of simplicity and quickness, the ELISA method is preferable. Since these methods are established and well-known techniques, a detailed description thereof will be omitted. In addition, although there exist the method of extracting from bacteria and the method of utilizing the membrane surface protein of bacteria, any method may be sufficient as protein. Since the latter can be detected as it is by the ELISA method or the like by using a bacterial membrane surface protein as an antigen, it is convenient in that it contributes to the purpose of simple and rapid bacterial detection. A method for extracting proteins from bacteria is also a well-known technique, and for example, treatment may be performed according to the method described in Non-Patent Document 6. As an antibody used in this method, an antibody against a specific protein known in a desired bacterium or a protein that is specifically expressed is used. These may use commercially available antibodies.

(Method of detecting nucleic acid)
In this method, a nucleic acid having a base sequence unique to a desired bacterium is detected after the nucleic acid is extracted from the bacterium grown in the culture process. As shown in the flowchart of FIG. 6, the method is characterized in that the detection step (S0602) further includes a nucleic acid extraction step (S0603) and a nucleic acid detection step (S0604). Hereinafter, the nucleic acid extraction step (S0603) and the nucleic acid detection step (S0603), which are characteristic steps of the present method, will be described. Note that the description of the culturing step (S0601) is omitted because it has already been described in S0501.

“Nucleic acid extraction step” (S0603) is a step of extracting nucleic acid from the cells obtained after the culturing step. As used herein, “nucleic acid” corresponds to DNA or RNA. A known technique established as a nucleic acid extraction method such as an alkali method can be used as a method for extracting nucleic acid from bacterial cells. The specific method may be based on the method described in Non-Patent Document 7.
A commercially available nucleic acid extraction kit or the like can also be used as a method for extracting nucleic acid from bacterial cells. The extracted nucleic acid is suspended in a TE buffer or the like, adjusted to an appropriate concentration, and used in the next nucleic acid detection step.

  "Nucleic acid detection step" (S0604) is the detection of a desired bacterium using an oligonucleotide that recognizes the base sequence unique to the bacterium on the genomic DNA of the bacterium for the nucleic acid obtained in the nucleic acid extraction step. It is a process to do.

  “Oligonucleotide” is composed of deoxyribonucleotides, ribonucleotides, LNA (registered trademark), PNA (peptide nucleic acid), or a combination thereof having about 10 to 50 bases. The oligonucleotide of this embodiment has a function as a probe or a primer in nucleic acid amplification.

  One or two or more nucleotides constituting the oligonucleotide may be modified with a sugar chain or the like. The oligonucleotide may be labeled with a labeling substance. The use of a labeling substance is convenient because the amplified nucleic acid fragment can be easily detected. Examples of the “labeling substance” include a fluorescent substance, a fluorescence suppressing substance, RI, a hapten such as digoxigenin (DIG), or biotin. The “fluorescent substance” refers to a substance having a property of emitting fluorescence when it is excited by absorbing excitation light having a specific wavelength and returns to the original ground state. The fluorescent substance used for labeling is not particularly limited as long as it can label oligonucleotides. Examples thereof include FAM, TET, HEX, Cy3, Cy5, Texas Red, and FITC. The “fluorescence-suppressing substance” is a so-called quencher, which means a substance that absorbs the excitation energy of the fluorescent substance. For example, BHQ1, BHQ2, BHQ3, Dabcyl, TAMRA are applicable. The position at which the oligonucleotide is labeled may be appropriately determined according to the properties of the labeling substance used, and is not particularly limited. For example, it may be the 5 'and / or 3' end, or any nucleotide within the oligonucleotide. In addition, when using a fluorescence suppression substance, it labels normally with a fluorescence substance and a pair. In this case, the fluorescent substance is generally labeled at the 5 'end of the oligonucleotide, and the fluorescence suppressing substance is generally labeled at the 3' end of the same oligonucleotide. In addition, the range of fluorescence suppression varies depending on the type of fluorescence suppressing substance. Therefore, when using a fluorescence suppression substance, it is necessary to select the kind which can suppress light emission of the said fluorescent substance used as a pair.

  The “base sequence unique to the bacterium on the genomic DNA of the desired bacterium” is a base sequence specific to the target bacterium present on the genomic DNA of the target bacterium. The specific base sequence is preferably a species-specific base sequence, but may be a subgenus-specific or genus-specific base sequence. This is because the bacteria detection method of this embodiment does not necessarily need to be detected at the species level. For example, when it is desired to confirm the degree of contamination of a food by Vibrio genus, it is sufficient if the presence or absence of Vibrio genus can be detected. It is not necessary to detect up to parahaemolyticus. In such a case, the base sequence specific to the genus Vibrio corresponds to the base sequence unique to the bacterium on the genomic DNA of the desired bacterium described here.

  Examples of the unique base sequence include a specific gene sequence and nucleotide polymorphism of the target bacterium. Examples of the specific gene here include species-specific or genus-specific enzymes. The nucleotide polymorphism includes species-specific or genus-specific SNP (Single Nucleitide Polymorphism), microsatellite, VNTR (Variable Nucleotide Tandem Repeat), RFLP (Restriction Fragment Length), etc. About a specific base sequence, it is preferable to examine and select suitably according to desired bacteria. For example, in the case of major food poisoning bacteria 6 genera 6 species, the base sequences of the genes listed in Table 2 of Example 4 can be used. However, Table 2 is one specific example, and the base sequence peculiar to the bacteria on the genomic DNA of the desired bacteria referred to in the present embodiment is not limited thereto.

  “Recognize” means being identifiable. That is, the oligonucleotide to be used does not necessarily need to recognize a unique base sequence by itself having a base sequence complementary to a base sequence unique to a desired bacterium. Even if the base sequence of the oligonucleotide itself does not contain a base sequence peculiar to the desired bacterium, if the base sequence peculiar to the desired bacterium can be identified as a result, it is considered to be recognized. . For example, a primer pair that does not have a specific base sequence but can amplify a specific region including a base sequence such as SNP specific to a desired bacterium corresponds to this.

  The nucleic acid detection step can be further divided into several methods depending on the detection method. For example, a method using a hybridization method or a method using a nucleic acid amplification method is applicable. Hereinafter, each method will be described.

(Method by hybridization method)
The nucleic acid detection step by the hybridization method is a method of detecting the presence or absence of a target bacterium by hybridization using an oligonucleotide having a base sequence complementary to a base sequence peculiar to the bacterium on the genomic DNA of the desired bacterium. It is. For example, a gel blotting method or a method using a filter or a substrate on which a nucleic acid is directly fixed can be mentioned. Specific examples of the gel blotting method include Southern blot hybridization method, Northern blot hybridization method, or blotting method based on them. In Southern blot hybridization, DNA fractionated in the gel after electrophoresis is transferred to a membrane, and then the labeled probe is hybridized to DNA having the desired base sequence on the membrane for detection. It is based on that. The Northern blot hybridization method is a method in which RNA is transferred to a membrane instead of DNA and the same treatment as the Southern blot hybridization method is performed. Any of the above methods is a very general technique established in the field of molecular biology, and detailed description thereof is omitted because it may be performed according to a protocol such as Non-Patent Document 7. A specific example of a method using a filter or substrate on which a nucleic acid is immobilized is an analysis method such as a macroarray, a microarray, or a DNA chip. These methods are desirably combined with a method using a nucleic acid amplification method described below in order to increase detection sensitivity. A method using a macroarray, a microarray, or a DNA chip is preferable because it contributes to the object of the present invention because a plurality of bacteria can be detected simultaneously. Since the detection method using these methods is a well-known technique, detailed description is abbreviate | omitted. In the method using a microarray or a DNA chip, it is more convenient to use a specialist who provides a contract analysis service in consideration of technology, labor, and cost for manufacturing a substrate and the like.

(Method using nucleic acid amplification method)
As shown in the flowchart of FIG. 7, the nucleic acid detection step (S0704) further includes a nucleic acid amplification step (S0705) and an amplified nucleic acid detection step (S0706), as shown in the flowchart of FIG. Features. The nucleic acid amplification step (S0705) and the amplified nucleic acid detection step (S0706) will be described below. Description of the other steps is omitted because it is the same as the above-described steps.

  The “nucleic acid amplification step” (S0705) is a nucleic acid amplification method using an oligonucleotide that recognizes a base sequence unique to a bacterium on the genomic DNA of the desired bacterium using the nucleic acid obtained in the nucleic acid extraction step as a template. It is a step of amplifying a desired bacterial nucleic acid. The bacteria detection method using the nucleic acid amplification process increases the number of bacteria in the culturing process, and further amplifies the nucleic acid extracted from the bacteria by the nucleic acid amplification method, so that even a bacterium present in a test object may be slightly present. Since detection becomes possible, it is useful in increasing detection sensitivity. Further, as described above, by using in combination with the hybridization method, it is possible to improve the speed and simplicity.

  The “nucleic acid amplification method” is a method of amplifying a specific region sandwiched between two primers using a nucleic acid such as DNA as a template. For example, PCR method, ICAN method, LAMP method, NASBA method, or their applied methods (for example, real-time PCR method) can be mentioned. All of these are known techniques, but the PCR method is widely used all over the world in the field of molecular biology including medicine and agriculture, and various applied techniques are also known. It is convenient from.

  The nucleic acid amplification reaction in the nucleic acid amplification method is appropriately determined according to the nucleic acid amplification method to be used. For example, the PCR method is generally based on a method in which three cycles of a heat denaturation reaction of a template, an annealing reaction of a primer, and a primer extension reaction are set as one cycle and the cycle is repeated. The temperature and time in each reaction are as follows: heat denaturation reaction at 88 ° C. to 98 ° C. for 30 seconds to 1 minute, annealing reaction at 52 ° C. to 60 ° C. for 30 seconds to 1 minute, and extension reaction at 70 ° C. to 75 ° C. for 40 seconds. It may be performed for about 3 minutes. For efficient amplification, it is preferable to determine appropriately according to the base sequence and the number of bases of the primer to be used. On the other hand, the ICAN method and the LAMP method can be amplified at a constant temperature of 50 ° C. to 65 ° C. These nucleic acid amplification methods are convenient because they do not require a temperature control device such as a thermal cycler. The number of cycles varies depending on the type of DNA polymerase used and the desired amount of nucleic acid amplification, but usually about 25 to 45 cycles are sufficient. It is preferably 28 cycles to 42 cycles, particularly preferably 30 cycles to 40 cycles.

  “Using a nucleic acid as a template” means using a nucleotide sequence of a nucleic acid as a template. Therefore, it is not always necessary to use the nucleic acid itself obtained in the nucleic acid extraction step as a template. For example, when the nucleic acid obtained in the nucleic acid extraction step is mRNA, cDNA obtained by reverse transcription from the mRNA may be used as a template.

  In the nucleic acid amplification step, a plurality of primer pairs for each of two or more target bacteria may be mixed in one reaction solution to simultaneously amplify the nucleic acid of each bacterium. Specific methods include so-called multiple plex PCR method. According to this method, a nucleic acid fragment unique to each of a plurality of target bacteria can be amplified in one nucleic acid amplification reaction. Therefore, this method is preferable because it contributes to the simplification and speed-up that are the objectives of the bacterial detection method of the present invention. It should be noted that the oligonucleotides used for each target bacterium need to take into consideration that their base sequences do not inhibit each other's nucleic acid amplification, and that labeling substances do not overlap between bacteria.

  The “amplified nucleic acid detection step” (S0706) is a step of detecting desired bacteria based on the nucleic acid amplified in the nucleic acid amplification step.

  “Based on” means using. “Based on amplified nucleic acid” means that the amplified nucleic acid fragment is used as it is, meaning that the amplified nucleic acid fragment is used after a predetermined treatment, or a phenomenon accompanying amplification of nucleic acid. Has the meaning of Specific methods in each case will be described below with examples.

  Examples of the method of using the amplified nucleic acid fragment as it is include a method of detecting a target bacterium from the base length of the nucleic acid fragment after the amplification reaction. For this, the base sequence of a primer that recognizes each bacterium may be set in advance so that an amplified fragment having a unique base length is amplified in each bacterium. As a method for visually recognizing the base length of the amplified fragment, gel electrophoresis can be used. Gel electrophoresis is an established technique frequently used in the field of molecular biology, and the detailed method may be in accordance with various protocols including Non-Patent Document 7.

  Examples of the method used after a predetermined treatment is performed on the amplified nucleic acid fragment include a method of detecting a target bacterium from a pattern of the cleaved fragment obtained by cleaving the amplified nucleic acid fragment with a predetermined restriction enzyme. In this method, a target bacterium is detected by a difference in restriction enzyme sites present in the base sequence of the amplified nucleic acid fragment. This method is convenient in that it can be detected even when the base length of the amplified nucleic acid fragment approximates between a plurality of target bacteria. The restriction enzyme may be appropriately determined based on the base sequence of the nucleic acid fragment amplified from each target bacterium. In this method, the amplified nucleic acid fragment is subjected to a normal restriction enzyme cleavage treatment and then detected by the gel electrophoresis method.

Examples of the method using a phenomenon accompanying amplification of nucleic acid include a method of detecting an increase in target nucleic acid fragments over time.
Here, “time-dependent” means time-lapse. Examples of the method for detecting the increase in nucleic acid fragments over time include a method for measuring an increase in fluorescence intensity accompanying an increase in nucleic acid fragments. More specifically, a real-time PCR method or the like can be used. As long as the increase in fluorescence intensity reflecting the increase in amplification products can be detected over time, the detection method is not limited. For example, 5 'nuclease method (TaqMan probe method) and cycling probe method are mentioned. In the 5 ′ nuclease method, a probe labeled with a fluorescent substance at the 5 ′ end and a fluorescent inhibitor at the 3 ′ end is decomposed by the 5 ′ → 3 ′ exonuclease activity of the DNA polymerase in the PCR extension reaction, and the fluorescent substance Is a method for detecting in real time the fluorescence emitted by the release of. The cycling probe method is a method for detecting, in real time, fluorescence emitted from a probe containing RNA labeled with a fluorescent substance at the end by decomposing an RNA portion by adding RNaseH. Alternatively, a nucleic acid stain such as SYBER Green may be added to the reaction solution for nucleic acid amplification, and the increase in diffusion fragments may be detected by the intensity of fluorescence emitted from the nucleic acid stain by staining together with the nucleic acid amplification reaction. . Detection by real-time PCR can be performed within 6 hours from the start of amplification to the completion of detection. Therefore, by combining the bacterial culture method of Embodiment 1 and the Multiplex real-time PCR, the process from inoculation to detection of multiple types of bacteria can be completed within one day. Therefore, the bacteria detection method by the real-time PCR method is very preferable from the viewpoint of simplicity and rapidity.

  According to any of the above methods, S.I. aureus, Salmonella spp. , E.C. coli O157, V.I. parahaemolyticus, B. et al. cereus, and L. Among the monocytogenes, two or more bacterial species can be detected simultaneously. Specific examples of the detection will be described in Examples 2 to 4.

<Embodiment 2: Effect>
According to the bacteria detection method of this embodiment, the operation until bacteria are detected from the inspection object can be simplified. In addition, the time required for detection can be shortened. In addition, according to another bacteria detection method of the present embodiment, food poisoning bacteria belonging to a plurality of genera can be detected simultaneously. Thereby, bacteria can be detected from the subject more rapidly.

  According to the bacterium detection method of the present embodiment, S. aureus, Salmonella spp. , E.C. coli O157, V.I. parahaemolyticus, B. et al. cereus, and L. From inoculation to detection of multiple genera of food poisoning bacteria such as monocytogenes can be completed within only one day. Therefore, it is possible to provide a simple and rapid detection method that is extremely useful in performing a test for ensuring food safety.

<< Embodiment 3 >>

<Embodiment 3: Overview>
Embodiment 3 relates to a kit for detecting bacteria. The kit for detecting bacteria according to the present embodiment includes the enrichment medium according to the first embodiment.

<Embodiment 3: Configuration>
The bacteria detection kit of this embodiment is configured to include the enrichment medium of Embodiment 1 in the kit. The “enrichment medium of Embodiment 1” here means the SEB. However, the SEB contained in the kit does not necessarily have to have a final concentration of each component and is not necessarily in a liquid state before use. For example, it may be packed in a concentrated liquid state so as to be diluted with sterilized water at the time of use, or may be packed in a powder state that is dissolved in a predetermined amount of sterilized water before use.

  The bacteria detection kit of this embodiment is preferably composed of reagents and the like required from culture to detection of target bacteria except for SEB. For example, lysing agents for neutralizing nucleic acids from cultured cells, neutralizing agents, DNA polymerases for performing nucleic acid amplification, buffers, dATP / dGTP / dCTP / dTTP, for detecting specific food poisoning bacteria A primer set is mentioned. In addition, you may have a required tube, a column, etc.

<Embodiment 3: Effect>
According to the bacteria detection kit of this embodiment, a person who conducts a bacteria detection test does not need to purchase various reagents necessary for the bacterial culture method of Embodiment 1 and the bacteria detection method of Embodiment 2 including SEB. Convenient.

<< Simultaneous culture of Bacillus and Listeria by SEB >>

<Purpose>
When SEB is used as the enrichment medium, it is confirmed that at least aerobic bacteria belonging to the genus Bacillus and Listeria can be cultured at the same time.

<Material>

(Used bacterial species)
・ Bacillus cereus No. 55
・ Listeria monocytogenes Lis1

(Composition and preparation of various media)

(1) TSB
TSB (DIFCO; 1.7% pancreatic digest of casein, 0.3% enzymatic digest of soybean meal, 0.25% dipotassium phosphate, 0.5% NaCl, 0.5% glucose) is dissolved in water. And autoclaved at 121 ° C. for 15 minutes.

(2) TSA plate TSA (Tryptic Soy Agar; DIFCO) was sterilized by autoclaving at 121 ° C for 15 minutes and then dispensed into a petri dish about 12 ml each.

(3) SEB
2% peptone {1.7% pancreatic digest of casein (Becton & Dickinson) + 0.3% enzymatic of soybean (DIFCO)}, 0.25% dipotassium phosphate 3% NaCl (sodium chloride; Nacalai Tesque), 0.05% yeast extract (NIHON SEIYAKU) was dissolved in water, adjusted to pH 7.3 with HCl, and then autoclaved at 121 ° C for 15 minutes. Used.

(4) B. cereus selective medium: yolk-added NGKG agar medium Yolk-added NGKG agar medium was prepared by autoclaving 15 minutes at 121 ° C for 15 minutes in NGKG agar medium (Nissui Pharmaceutical) kept at 50 ° C and 10 ml of egg yolk and sterile physiology It was prepared by adding 50 ml of saline and dispensing about 12 ml each in a petri dish.

(5) L.L. Monocytogenes selection medium: Oxford agar medium The Oxford agar medium was prepared by maintaining 500 ml of Listeria selective agar base (OXOID), which was prepared by autoclaving at 121 ° C for 15 minutes, at 50 ° C. ) One vial dissolved in 70% ethanol in 5 ml was mixed and prepared by dispensing about 12 ml each in a petri dish.

<Method>
In this example, in order to confirm that the two kinds of bacteria could be cultured simultaneously with SEB, the method was performed by inoculating SEB with the two kinds of identified bacteria. As a general rule, the following operations were performed under aseptic conditions. The reagents and instruments used were those sterilized by autoclaving at 121 ° C. for 20 minutes.

  Each bacterial cell of one platinum loop scraped from the slant was inoculated into 5 ml of TSB and cultured at 37 ° C. overnight. Next, a 10-fold serial dilution series of the preculture was prepared using PBS, and 50 μl each of 225 ml of SEB was mixed and inoculated so that the final bacterial concentration was about 100 cfu / ml. After inoculation, the enrichment medium was statically cultured in a 37 ° C. incubator, and 10 ml of the culture solution was collected every 2 hours from the time of inoculation until 18 hours after culturing. The culture solution was centrifuged at 4000 × g for 10 minutes, and the supernatant was filtered and sterilized using EB-Disk25 (pore size 2.5 μm; Kanto Chemical), and then the pH was measured. At the same time, a 10-fold serial dilution series was prepared from this culture solution using PBS, and 100 μl was smeared on the selective medium plate of each bacterium, and the viable cell count was calculated from the number of colonies after culture. The amount of each inoculum was calculated from the number of colonies after culturing 100 μl on TSA.

<Result>
The results of Example 1 are shown in FIG. As can be seen from comparison with the results of FIG. 2 corresponding to the control experiment, when SEB was used in the enrichment medium, Even after 12 hours when cereus entered the stationary phase, the pH value of the medium did not significantly decrease as shown in FIG. That is, B.I. Even if cereus grew, the pH value of the enrichment medium could be maintained in the range of pH 5 to pH 10 or less. As a result, L. Monocytogenes was also able to grow to about 1 × 10 ⁵ cfu / ml or more, enough for detection. Therefore, it was confirmed that by using SEB as the enrichment medium, at least aerobic bacteria of the genus Bacillus and Listeria can be cultured at the same time without adjusting the pH of the enrichment medium during the culture.

<< Simultaneous cultivation of food poisoning bacteria of 6 major genera and 6 species based on the bacterial culture method of Embodiment 1 >>

<Purpose>
Based on the bacterial culture method of Embodiment 1, it shows that food poisoning bacteria belonging to each of the genera Bacillus, Listeria, and Vibrio, and other major food poisoning bacteria can be simultaneously cultured in one field enrichment medium .

<Material>

(Used bacterial species)
Staphylococcus aureus IFO 3060
・ Salmonella Enteritidis IFO 3313
-Escherichia coli O157: H7 (stx1 +, stx2 +)
-Vibrio parahaemolyticus WP-1 (tdh +)
・ Bacillus cereus No. 55
・ Listeria monocytogenes Lis1

(Composition and preparation of various media)

(1) S.M. aureus selective medium: yolk-added mannitol saline medium Manifl salt medium (Nissui Pharmaceutical) prepared by autoclaving at 121 ° C for 15 minutes, warmed to 50 ° C, 50 ml of egg yolk and 50 ml of 3% sterile saline It was prepared by adding about 12 ml each to a petri dish.

(2) E.E. E. coli O157 selective medium: CT-SMAC medium CT-SMAC medium (cefixime, sorbitol McConkey agar medium supplemented with potassium tellurite) is 500 ml of Solbitol MacCONKEY medium (OXOID) at 121 ° C. for 15 minutes and then autoclaved to 50 ° C. To the cooled product, a solution obtained by dissolving 1 vial of Cefixime Tellurite Selective Supplement (OXOID) in 2 ml of sterilized water was prepared by dispensing about 12 ml each into a petri dish.

(3) Salmonella spp. Selection medium: DHL agar medium According to the prescription, DHL agar medium (Nissui Pharmaceutical Co., Ltd.) was dissolved by heating and dispensed into a petri dish about 12 ml each.

(4) V. Parahaemolyticus selective medium: TCBS agar medium TCBS agar medium (Nissui Pharmaceutical Co., Ltd.) was heated and dissolved according to the prescription, and then dispensed into a petri dish about 12 ml each.

  In this example, TSB, TSA plate, SEB, B. cereus selective medium, L. Although the monocytogenes selective medium was used, the description thereof is omitted because it is the same as in Example 1. However, for TSB and TSA plates, S.I. aureus, and / or V. When culturing parahaemolyticus, 1.5% and 3% NaCl were added to TSB at final concentrations, respectively.

<Method>
In this example, in order to show that the six types of bacteria can be simultaneously cultured in one medium, the method was performed by inoculating various kinds of identified bacteria into the enrichment medium. As a general rule, the following operations were performed under aseptic conditions. The reagents and instruments used were those that had been autoclaved at 121 ° C. for 20 minutes.

  Each bacterial cell of one platinum loop scraped from the slant was inoculated into 5 ml of TSB and cultured at 37 ° C. overnight. Next, a 10-fold serial dilution series of the preculture was prepared using PBS, and 10 μl each of 10 ml SEB was mixed and inoculated so that the final bacterial concentration was about 1 to 10 cfu / ml. After inoculation, the enrichment medium was statically cultured in a 37 ° C. incubator for 18 hours. For the measurement of the number of bacteria after the culture, a 10-fold serial dilution series of the culture solution was prepared using PBS, and 100 μl was smeared on the selective medium plate of each bacterium, and the number of colonies after the culture was calculated. The amount of each inoculum was calculated from the number of colonies after culturing 100 μl on TSA.

<Result>
The result of Example 2 is shown in FIG. As shown in this figure, all six major food poisoning bacteria, 6 genera, grew to over 10 ⁵ cfu / ml in 18 hours. That is, according to the bacterial culture method of Embodiment 1 using SEB as the enrichment medium, it was confirmed that all six major food poisoning bacteria in Japan can be cultured simultaneously in a culture time of 18 hours.

<< Simultaneous detection of food poisoning bacteria from food >>

<Purpose>
It will be shown that aerobic bacteria belonging to the genus Staphylococcus, Salmonella, Escherichia, Vibrio, Bacillus, and Listeria can be simultaneously detected from foods by the bacterial detection method of Embodiment 2.

<Material>

(Used bacterial species)
The same 6 genera and 6 species as in Example 2 were used.

(Food sample)
I purchased and used chicken, sashimi salmon fillets, and lettuce on the market at a supermarket.

(Composition and preparation of various media)
Various media similar to Example 1 and Example 2 were used.

<Method>
In this example, in order to confirm that the bacteria belonging to the 6 genera can be cultured from the food at the same time, the above 6 genera and 6 species were inoculated into the food. As a general rule, the following operations were performed under aseptic conditions. The reagents and instruments used were those sterilized by autoclaving at 121 ° C. for 20 minutes.

  A 25 g food sample was crushed for 30 seconds in a filter stomacher bag together with 225 ml of SEB, and left to stand at 37 ° C. for 18 hours in an incubator. A 10-fold serial dilution series was prepared from the disrupted solution using PBS, 100 μl was smeared on TSA, and the number of viable bacteria in the food sample was calculated from the number of colonies after culture.

  To inoculate food samples with food poisoning bacteria, first, 6 types of 1 platinum ear food poisoning bacteria scraped from the slant were inoculated into 5 ml of TSB and cultured overnight at 37 ° C. Next, a 10-fold serial dilution series of the preculture was prepared using PBS, and mixed and inoculated into chicken, sashimi fillet, and lettuce as food samples so that the final bacterial concentration was about 1 cfu / g. Bacteria collection and culture from each food sample were collected by crushing from 25 g of food sample in the same manner as described above, and then statically cultured at 37 ° C. for 18 hours. Thereafter, a 10-fold serial dilution series of the culture solution was prepared using PBS, and 100 μl was smeared on each bacterial selective medium described in Examples 1 and 2, and the viable cell count of the food sample was calculated from the number of colonies after culture. Calculated.

<Result>
Table 1 shows the results. Each chicken has 2.3 x 10 ⁵ cfu / g, sashimi salmon fillet 4.4 x 10 ⁴ cfu / g, lettuce 1.2 x 10 ⁶ cfu / g It was shown that all six types of target food poisoning bacteria can also be detected by the bacteria detection method of Embodiment 2 even in food samples in which there are bacteria).

<< Simultaneous detection of food poisoning bacteria using real-time PCR >>

<Purpose>
It shows that all 6 major food poisoning bacteria 6 genera 6 species can be detected simultaneously in Japan by the bacteria detection method of Embodiment 2 using real-time PCR method.

<Material>

(Used bacterial species)
The same 6 genera and 6 species as in Example 2 were used.

(Composition and preparation of various media)
Various media similar to Example 1 and Example 2 were used.

(PCR reaction solution)
In this example, the oligonucleotides recognizing each of the 6 types of bacteria were divided into 3 types and PCR reaction was performed with 2 reaction solutions (reaction tube I and reaction tube II). This is because the upper limit of detection of fluorescent dyes in the thermal cycler for real-time PCR used in this example was four. Therefore, the four fluorescent dyes were divided into two tubes, three for bacterial detection and one for monitoring. If two thermal cyclers for real-time PCR capable of detecting seven or more types of fluorescent dyes with one unit, it is possible to react six main food poisoning bacteria and six species in one reaction tube.

(1) Common reaction solution composition 10 × EX Taq buffer (TAKARA) 2.5 μl
dNTPs for EX Taq (TAKARA) 2.0 μl
20 mM MgCl ₂ (TAKARA) 2.5 μl
5 × Rox reference dye 0.5 μl
5 U / μl EX Taq polymerase (TAKARA) 0.125 μl
Template DNA 2.0 μl

(2) Reaction tube I primer and probe 20 pmol / μl nucA forward primer 0.25 μl
20 pmol / μl nucA reverse primer 0.25 μl
20 pmol / μl nucA probe 0.25 μl
20 pmol / μl eaA forward primer 0.25 μl
20 pmol / μl eaA reverse primer 0.25 μl
20 pmol / μl eaA probe 0.25 μl
20 pmol / μl hlyA forward primer 0.25 μl
20 pmol / μl hlyA reverse primer 0.25 μl
20 pmol / μl hlyA probe 0.25 μl

(3) Reaction tube II primer and probe 20 pmol / μl tl forward primer 0.25 μl
20 pmol / μl tl reverse primer 0.25 μl
20 pmol / μl tl probe 0.25 μl
20 pmol / μl invA forward primer 0.25 μl
20 pmol / μl invA reverse primer 0.25 μl
20 pmol / μl invA probe 0.25 μl
20 pmol / μl crs forward primer 0.25 μl
20 pmol / μl crs reverse primer 0.25 μl
20 pmol / μl crs probe 0.25 μl

The common reaction solution was mixed with primers and probes for each reaction tube. The tubes after mixing were used as reaction tube I and reaction tube II for real-time PCR. Table 2 shows the base sequences of the primers and probes.

<Method>

(culture)
Six kinds of food poisoning bacteria of 6 genera used in Example 2 were mixed and inoculated into 10 ml of SEB so as to be 1 to 10 cfu / ml, and statically cultured at 37 ° C. for 18 hours. The basic culture method is the same as in Example 2. The culture corresponds to the culture process in the second embodiment.

(Nucleic acid extraction)
Centrifugation is performed at 6000 × g for 10 minutes after culturing 1.0 ml of the culture medium after the culture, and using DNA Tissue Kit (QIAGEN) according to the attached protocol (extraction method from Gram-positive bacteria). Prepared. However, 2 μl of 10 mg / ml N-acetylmuramidase was added during lysis. The culture corresponds to the nucleic acid extraction step in the second embodiment.

(Real-time PCR reaction)
Prepare the above reaction solutions in 0.2 ml real-time PCR microtubes, add sterilized water to a total volume of 25 μl, mix well, and then use a real-time PCR thermal cycler (MX3000P Stratagene) to prepare nucleic acids. An amplification reaction was performed. The reaction conditions were (1 cycle at 95 ° C. → 1 minute at 55 ° C. → 1 minute at 72 ° C.) and 42 cycles. The intensity of each fluorescent dye was measured for each cycle, and the analysis was performed with the analysis software attached to the thermal cycler.

  Confirmation of the amplified product by PCR was performed by agarose gel electrophoresis. As the agarose gel, 3% agarose gel / TAE agarose gel was used. Since the electrophoresis method is a very general technique, the description thereof is omitted here.

<Result>
FIG. 10 shows a relative fluorescence intensity plot for each cycle in the real-time PCR of this example. As shown in this figure, amplification was confirmed with all the fluorescent dyes in the two reaction tubes, indicating that six types of food poisoning bacteria can be detected simultaneously. FIG. 10 also shows that nucleic acid amplification of each bacterium can be sufficiently detected in about 30 cycles. Therefore, after setting the thermal cycler, a detection result can be obtained in about 2 to 2.5 hours. As shown in Example 1, since the culture time by the bacterial culture method is sufficient from 12 hours to 18 hours, it was also demonstrated that the inoculation to detection can be performed within 24 hours. In addition, as shown in FIG. 11, the amplification of the target size was confirmed also in the agarose gel electrophoresis.

L. in the enrichment medium at each pH value. Figure showing the number of bacteria per medium volume of monocytogenes B. cereus and L. The figure which shows the relationship between the microbe number per culture medium volume of each bacteria in each culture time, and pH value of a culture medium when monocytogenes is cultured simultaneously. B. cereus, L.C. monocytogenes, and V.I. The figure which shows the number of bacteria per culture medium volume of each bacterium when parahaemolyticus is cultured at the same time The flowchart figure of the bacteria culture method in Embodiment 4. Example (1) of flowchart of embodiment 2 Example (2) of flowchart of embodiment 2 Example (3) of flowchart of embodiment 2 The figure which shows the result of Example 1 The figure which shows the result of Example 2 The figure which shows the result of real-time PCR in Example 4 The figure which shows the result of the agarose electrophoresis which shows the nucleic acid amplification of PCR in Example 4

Explanation of symbols

FIG. 2 BC: B. cereus, LM: L. monocytogenes
FIG. 3 BC: B. cereus, LM: L. monocytogenes, VP: V. parahaemolyticus
FIG. 8 BC: B. cereus, LM: L. monocytogenes
FIG. 9 BC: B. cereus, LM: L. monocytogenes, VP: V. parahaemolyticus, SA: S. aureus, SE: S. Enteritidis, O157: E. E. coli 0157
FIG. 10 BC: B. cereus, LM: L. monocytogenes, VP: V. parahaemolyticus, SA: S. aureus, SE: S. Enteritidis, O157: E. E. coli 0157
11: M: 100 pb marker, 1: Reaction tube I (positive control), 2: Reaction tube II (positive control), 3: Reaction tube I (culture solution sample), 4: Reaction tube II (culture solution sample), 5 : Reaction tube I (negative control), 6: Reaction tube II (negative control)

Claims (14)

A bacterial culture method capable of simultaneously culturing at least an aerobic bacterium of the genus Bacillus and an aerobic bacterium of the genus Listeria in one enrichment medium,
A bacterial culture method for culturing while maintaining the pH value of the enrichment medium between 5 and 10 or less. Furthermore, a bacterial culture method capable of simultaneously culturing aerobic and halophilic bacteria of the genus Vibrio in one enrichment medium,
The bacterial culture method according to claim 1, wherein the salt concentration of the enrichment medium is 1% to 4% in terms of mass to volume percentage using water as a solvent.   The bacterial culture method according to claim 1 or 2, wherein the enrichment medium uses water as a solvent and the sugar content is 0.001% or less in terms of mass to volume percentage.   The enrichment medium contains water as a solvent in weight to volume percentage, peptone: 1% to 3%, dipotassium phosphate: 0.15% to 0.35%, sodium chloride: 1% to 4%, and yeast extra. The bacterial culture method according to claim 3, comprising: 0.01% to 0.1%. A bacterial detection method capable of simultaneously detecting aerobic bacteria of two or more genera among at least Staphylococcus, Salmonella, Escherichia, Vibrio, Bacillus, and Listeria,
A culture step of culturing by the bacterial culture method according to any one of claims 1 to 4, a detection step of detecting desired bacteria from the bacteria obtained after the culture step;
A method for detecting bacteria. The detection step further includes a nucleic acid extraction step of extracting nucleic acid from the cells obtained after the culture step,
A nucleic acid detection step of detecting a desired bacterium using an oligonucleotide that recognizes a base sequence unique to the bacterium on the genomic DNA of the bacterium with respect to the nucleic acid obtained in the nucleic acid extraction step;
The method for detecting bacteria according to claim 5, wherein The nucleic acid detection step further includes
Nucleic acid amplification step of amplifying nucleic acid of desired bacteria by nucleic acid amplification method using oligonucleotide obtained by recognizing base sequence peculiar to the bacteria on genomic DNA of desired bacteria using nucleic acid obtained in nucleic acid extraction step as template When,
The method for detecting bacteria according to claim 6, further comprising an amplified nucleic acid detection step for detecting desired bacteria based on the nucleic acid amplified in the nucleic acid amplification step.   In the nucleic acid amplification step, for two or more bacteria, the nucleic acid of each bacterium is simultaneously used in one reaction solution by using an oligonucleotide that recognizes a base sequence unique to each bacterium on the genomic DNA of each bacterium. The method for detecting a bacterium according to claim 7, which is amplified.   The method for detecting bacteria according to claim 7 or 8, wherein the nucleic acid detection step uses a real-time PCR method.   The method of detecting a bacterium according to claim 6, wherein the nucleic acid detection step is detected by a hybridization method using an oligonucleotide recognizing a base sequence peculiar to the bacterium on the genomic DNA of the desired bacterium.   The Staphylococcus genus, Salmonella genus, Escherichia genus Vibrio spp, Bacillus spp, and the aerobic bacteria of the Listeria genus, respectively Staphylococcus aureus, Salmonella Enteritidis, Escherichia coli O157, Vibrio parahaemolyticus, Bacillus cereus, and claim a Listeria monocytogenes The method for detecting bacteria according to any one of 5 to 10. The mass to volume percentage when using water as a solvent is sugar: 0.001% or less, peptone: 1% to 3%, dipotassium phosphate: 0.15% to 0.35%, sodium chloride: 1% to 4% and yeast extract: 0.01% to 0.1% enrichment medium. The enrichment medium according to claim 12, wherein the peptone contains 10% to 20% of soyton with respect to the mass of peptone. A bacteria detection kit comprising the enrichment medium according to claim 12 or 13.