JP2005124409A - Method for detecting emetic type bacillus cereus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for specifically detecting emetic type Bacillus cereus. <P>SOLUTION: An oligonucleotide for detecting emetic type Bacillus cereus has a sequence containing a part or all of a specific region or its complementary sequence in a strain of a chromosomal DNA of the food poisoning emetic type Bacillus cereus. A method for detecting the emetic Bacillus cereus comprises using the oligonucleotide. A kit for detecting the emetic type Bacillus cereus comprises the oligonucleotide. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention mainly relates to a means for detecting emetic Bacillus cereus. In particular, the present invention relates to a method for specifically detecting emetic Bacillus cereus in a sample using a sequence characteristic of vomiting Bacillus cereus.

  The Bacillus genus Bacillus is an aerobic spore-forming rod that is widely distributed in the natural environment such as soil and river water, and in foods and feeds such as agricultural and marine products and livestock products. This bacterium has many opportunities to contaminate foods, causes food to rot and deteriorate, and also causes food poisoning. In recent years, it has been regarded as important in food hygiene (Non-patent Document 1).

  There are two types of Bacillus cereus food poisoning depending on clinical symptoms: vomiting and diarrhea. In 1971, an outbreak of vomiting-type food poisoning caused by cooked rice was reported in the United Kingdom, and in Japan, an outbreak of vomiting-type food poisoning caused by an anchovy in Kumamoto was reported in 2001. In 1960, Okayama Prefecture reported the occurrence of diarrhea-type food poisoning caused by nonfat dry milk. With these reports, in 1982, Bacillus cereus was administratively treated as a food poisoning bacterium. At present, Bacillus cereus is generally detectable in commercially available selective media, but this selective media cannot distinguish vomiting from diarrhea.

  Diarrhea-type food poisoning is known to be caused by a toxin protein produced by diarrhea-type Bacillus cereus, and a method for detecting the toxin protein using an antibody has been commercialized (Cereus enterotoxin detection kit) CRET-RPLA "Seiken", Denka Seikensha). A method for detecting the gene of the toxin protein has also been reported (Non-patent Document 2).

  On the other hand, vomiting-type food poisoning is known to be caused by vomiting toxins composed of heat-resistant cyclic peptides produced by emetic Bacillus cereus, and is named cereulide.

  Conventional detection methods for emetic Bacillus cereus include a method of chemically analyzing cereulide, a thermostable cyclic peptide produced by the bacterium, using a mass spectrometer, and the pharmacological effects of cereulide appear inside animal culture cells. A vacuolation test for observing vacuoles is known (Non-patent Document 3).

  The chemical analysis method using a mass spectrometer is a method in which a sample extracted from a bacterial culture solution with methanol or n-pentane is separated by high performance liquid chromatography, and then a mass spectrum peculiar to cereulide is directly detected by the mass spectrometer. It is a method (nonpatent literature 4 and nonpatent literature 5).

  In addition, the vacuolation test refers to the mitochondrial mitochondrion appearing in animal culture cells such as HEp-2 to which a heat-treated bacterial culture supernatant or a sample extracted from the bacterial culture with methanol or n-pentane is added. This is a method for observing the presence or absence of vacuoles due to swelling, and a method for analyzing the pharmacological effects of cereulide (Non-patent Documents 3 and 6).

  On the other hand, attempts have been made to detect vomiting-type Bacillus cereus by nucleic acid analysis by analyzing the genotype of Bacillus cereus using various methods and obtaining characteristic gene sequences. No such method has been found (Non-Patent Document 7). For example, various PCR amplification products generated from multiple sites of DNA obtained by the RAPD (Random Amplified polymorphic DNA) method are separated as multiple characteristic band patterns on an agarose gel, and the characteristic band pattern of emetic Bacillus cereus Although a method for detecting emetic Bacillus cereus bacteria by examining the presence or absence of such a substance is also envisaged, it is very difficult to always control the reproducibility of the RAPD method, so the reliability of the results obtained is generally low. Therefore, it is difficult to say that detecting vomiting type Bacillus cereus only by such a method using RAPD is a preferable method.

  Furthermore, there are reports that emetic Bacillus cereus has a negative starch resolution and serotype is H1, but these characteristics are also observed in Bacillus cereus, so only vomiting cereus is identified. It is not sufficient as an index for this (Non-Patent Document 8).

  The above-described method of chemically analyzing with a mass spectrometer has a problem that it is not necessarily a versatile analysis method because it requires a very expensive analyzer. In addition, in the vacuolation test, there is always the complexity of subcultured the cultured animal cells used for the test, and the interpretation of the results requires skill, and for those other than researchers who have various observation experiences. Has the problem that it is very difficult to use. Furthermore, the above two methods require a culturing step for producing cereulide from the test bacterium and a step for preparing the test sample from the culture solution, so that it takes about 2 to 3 days to obtain the analysis result. It also has the problem.

These two methods of chemically analyzing with a mass spectrometer and the vacuolation test are currently considered the best methods for detecting cereulide itself, but have the above-mentioned problems. Therefore, it is possible to easily and rapidly screen emetic Bacillus cereus as a method for detecting vomiting Bacillus cereus more easily and quickly, or as an analysis method in the previous stage of performing these mass spectrometry methods or vacuolation tests A detection method has been awaited (Non-Patent Document 3).
Ueda S .; Bokin Bobai; 25: 659-669 (1997) Hsieh YM, SJ Sheu, YL Chen, and HY Tsen; Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B. cereus strains from foods and food-borne outbreaks; J. Appl. Microbiol., 87: 481-490 (1999). Ueda S .; Bokin Bobai; 30: 511-524 (2002) Haggblom MM, C. Apetroaie, MA Andersson, and MS Salkinoja-Salonen; Quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, produced under various conditions; Appl.Environ. Microbiol. 68: 2479-2483 (2002) Hansen BM, and NB Hendriksen; Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis; Appl. Environ. Microbiol. 67: 185-189 (2001) Andersson MA, R. Mikkola, J. Helin, MC Andersson, M. Salkinoja-Salonen; A novel sensitive bioassay for detection of Bacillus cereus emetic toxin and related depsipeptide ionophores; Appl. Environ. Microbiol. 64: 1338-1343 (1998) Ripabelli G., J. McLauchlin, V. Mithani, EJ Threlfall; Epidemiological typing of Bacillus cereus by amplified fragment length polymorphism; Lett. Appl. Microbiol. 30: 358-363 (2000) Pirttijarvi TS, MA Andersson, AC Scoging, and MS Salkinoja-Salonen; Evaluation of methods for recognising strains of the Bacillus cereus group with food poisoning potential among industrial and environmental contaminants; Syst. Appl. Microbiol. 22: 133-144 (1999)

  The main object of the present invention is to provide a detection means capable of simply screening for emetic Bacillus cereus.

  In order to achieve the above object, the present inventors have intensively studied a method for detecting emetic Bacillus cereus from a molecular biological viewpoint. As a result, we found a sequence characteristic of emetic Bacillus cereus, and found that it was possible to easily detect vomiting cereus by conducting nucleic acid analysis using this sequence. The invention has been completed.

  That is, the present invention relates to the following oligonucleotide for detecting emetic Bacillus cereus, a method for detecting emetic Bacillus cereus, and a kit for detecting emetic Bacillus cereus.

  1. An oligonucleotide for detecting emetic Bacillus cereus having a sequence containing a part or all of the region from position 220 to position 275 or position 370 to position 468 in the base sequence represented by SEQ ID NO: 1 or a complementary sequence thereof. Preferably, a vomiting-type Bacillus cereus detection oligonucleotide having a sequence consisting of part or all of the region of positions 220 to 275 or positions 370 to 468 in the base sequence represented by SEQ ID NO: 1 or a complementary sequence thereof.

  2. An oligonucleotide for vomiting-type Bacillus cereus having a base sequence represented by any one of SEQ ID NOs: 2 to 4 or a complementary sequence thereof.

3. a) amplifying a target nucleotide sequence in DNA in a sample using a primer set comprising at least one oligonucleotide for detecting emetic Bacillus cereus according to item 1 or item 2,
b) measuring the product amplified in a) to detect whether a sequence characteristic of emetic Bacillus cereus is present in the sample;
For detecting vomiting type Bacillus cereus.

4). a ′) a step of amplifying a target nucleotide sequence in DNA in a sample using a primer set comprising at least one oligonucleotide for detecting emetic Bacillus cereus according to item 1 or item 2;
b ′) The product amplified in a ′) is hybridized with a probe labeled with the oligonucleotide according to item 1 or item 2, and a signal derived from the labeled probe is measured, and an emetic type is detected in the sample. Detecting whether a sequence characteristic of Bacillus cereus is present,
For detecting vomiting type Bacillus cereus.

5). a ″) a step of labeling the vomiting-type Bacillus cereus oligonucleotide for detection according to item 1 or item 2 into a labeled probe;
b '') the step of hybridizing the labeled probe of a '') with the DNA in the sample;
c ″) detecting the presence or absence of hybridization in b ″) based on the signal derived from the labeled probe;
For detecting vomiting type Bacillus cereus.

  6). 3. A vomiting-type Bacillus cereus detection kit comprising a primer comprising the oligonucleotide for vomiting-type cereus bacteria detection according to item 1 or 2 or a labeled primer obtained by labeling the oligonucleotide. Preferably, a vomiting-type Bacillus cereus detection kit comprising a primer comprising the oligonucleotide for vomiting-type Bacillus cereus detection according to item 1 or 2, or a labeled primer obtained by labeling the oligonucleotide, deoxyribonucleotide and DNA polymerase.

  7). 3. A kit for detecting emetic Bacillus cereus, comprising a labeled probe labeled with the oligonucleotide for detecting emetic Bacillus cereus according to Item 1 or Item 2.

  Hereinafter, the present invention will be described more specifically.

Oligonucleotide for detecting emetic Bacillus cereus The oligonucleotide for detecting emetic Bacillus cereus of the present invention has a base sequence characteristic of emetic Bacillus cereus. Therefore, emetic Bacillus cereus can be specifically detected using a method such as nucleic acid analysis.

  Here, nucleic acid analysis is effective for grasping the species of a living organism by analyzing the presence or absence of a specific base sequence by utilizing the existence of a specific base sequence for each species. This means is a method usefully used for detecting a specific microorganism or identifying a biological species.

  Examples of the nucleic acid include DNA and RNA.

  The nucleotide sequence peculiar to emetic Bacillus cereus can be identified by using, for example, RFLP, PCR, RAPD (Random Amplified polymorphic DNA) (hereinafter referred to as RAPD) method and the like. Among them, the RAPD method is appropriate from the viewpoint that various PCR amplification products can be obtained.

  Unlike normal PCR, RAPD is a method that uses DNA polymorphisms of various PCR amplification products from multiple sites obtained by arbitrarily changing the PCR reaction conditions, base sequence and length of synthetic primers, etc. is there. This method uses an oligonucleotide consisting of a relatively small number of base sequences such as 8-12 bases as an amplification primer, and the primer annealing temperature in PCR is also low temperature conditions such as 30 ° C-40 ° C. Including the disadvantage that it is very difficult to always control the reproducibility of the image. However, reproducibility can be improved by strictly controlling the experimental conditions. DNA polymorphism can be grasped by the band pattern obtained by electrophoresis of the obtained PCR amplification product, thereby determining the base sequence of the band characteristic of emetic Bacillus cereus and specific to the same Can be found.

  Specifically, PCR amplification products that are specifically confirmed only for emetic Bacillus cereus by RAPD analysis of genomic DNA of emetic Bacillus cereus and its related bacteria are selected from the band pattern of electrophoresis. A band that is stably detected and has good reproducibility is selected, and a base sequence is determined for the obtained band. For example, the band is subcloned into an appropriate plasmid, and the base sequence corresponding to the band is determined.

  Specific examples of sequences characteristic of emetic Bacillus cereus thus obtained include the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing.

  The resulting sequence characteristic of emetic Bacillus cereus is further searched for homology on the DDBJ / EMBL / GenBank database, and the homology is low with a known DNA sequence in a relatively wide range. A region that is considered to be characteristic only in the region can be selected and used as a sequence region for nucleic acid analysis.

  As a sequence region characteristic only to emetic Bacillus cereus thus obtained, specifically, regions 220 to 275 and 370 to 468 in the base sequence represented by SEQ ID NO: 1 in the sequence listing Can be mentioned.

  By selecting a sequence that specifically hybridizes with the DNA of the emetic Bacillus cereus from the sequence region characteristic of the obtained emetic Bacillus cereus, and designing various oligonucleotides, the emetic Bacillus cereus Can be obtained.

  The selected sequence is a sequence comprising at least a part or all of a sequence selected from the regions of positions 220 to 275 and positions 370 to 468 in the base sequence represented by SEQ ID NO: 1 in the sequence listing or its complementary sequence. If there is, it will not be specifically limited, You may be comprised including the arrangement | sequence of area | regions other than 220th-275th and 370th-468th, or some other arbitrary arrangement | sequences.

  Specific oligonucleotides designed as such include, for example, oligonucleotides having a base sequence represented by any one of SEQ ID NOs: 2 to 4 in the sequence listing. The base sequence represented by SEQ ID NO: 2 is a portion corresponding to positions 239 to 258 of the sequence represented by SEQ ID NO: 1. The base sequence represented by SEQ ID NO: 3 is a complementary sequence of a portion corresponding to positions 450 to 472 of the sequence represented by SEQ ID NO: 1. The base sequence represented by SEQ ID NO: 4 is a complementary sequence of a portion corresponding to positions 373 to 396 of the sequence represented by SEQ ID NO: 1.

  The region of positions 220 to 275 and positions 370 to 468 in the base sequence represented by SEQ ID NO: 1 is a sequence characteristic only to emetic Bacillus cereus, which is different from diarrhea Bacillus cereus, Bacillus anthracis and other organisms. It is. By using the sequence of a region characteristic only for such emetic Bacillus cereus, it is possible to specifically detect emetic Bacillus cereus.

  The length of the sequence selected from the region is not particularly limited, but it is preferable to set the length so that it can be surely hybridized with the sample. Usually, it is 10 bases or more, preferably 17 bases or more.

  The detection oligonucleotide of the present invention can be suitably used as a probe or primer. In the case of a probe, the base length of the oligonucleotide is about 10-25. In the case of a primer, it is about 15-30, preferably about 17-24.

  In addition, the oligonucleotide of the present invention can be used with an appropriate label if necessary. For example, it can be used as a labeled probe for detection of bacterial DNA, a labeled probe for detection of PCR amplification products, an internal sequence detection label probe of amplification products used in real-time PCR and the like.

Detection Method By using the oligonucleotide for detecting emetic Bacillus cereus of the present invention described above, nucleic acid such as DNA in the sample is analyzed, whereby emetic Bacillus cereus in the sample can be specifically detected.

The type of detection method (nucleic acid analysis) is not particularly limited, and a known method can be used as appropriate. For example, a method of PCR amplification using a primer, a method of detecting a PCR amplification product with a probe, a method of detecting DNA in a sample with a probe, etc. can be exemplified, for example, as a method of PCR amplification using a primer , By using a primer set comprising at least one oligonucleotide for detecting emetic Bacillus cereus of the present invention, the target nucleotide sequence in DNA in the sample is selectively amplified, and the presence or absence of the amplified product is measured or amplified A method for detecting whether or not a characteristic sequence of emetic Bacillus cereus is present in a sample by detecting and measuring the product with a probe can be mentioned.

  The primer set may be a combination of the above-mentioned oligonucleotide for detecting emetic Bacillus cereus of the present invention. Further, the oligonucleotide for detecting emetic Bacillus cereus of the present invention and other oligonucleotides, for example, the oligonucleotide for detecting emetic Bacillus cereus of the present invention and the sequence represented by SEQ ID NO: 1, positions 220 to 275 and 370 A combination with an oligonucleotide having a sequence other than positions 468 to 468 may also be used.

  Confirmation of the presence or absence of DNA amplification can usually be performed by separating PCR products by agarose gel electrophoresis and staining them with nucleic acid such as ethidium bromide or Cybergreen I. When PCR is performed using a real-time PCR apparatus, the presence or absence of DNA amplification can be automatically confirmed in a short time by the detection system of the apparatus. For example, when Cyber Green I is added to a PCR reaction solution, the amount of amplified product can be monitored for each cycle from the fluorescence value emitted when Cyber Green I binds to double-stranded DNA. Furthermore, it is possible to confirm whether the PCR amplification product is the target amplification product by performing a melting curve analysis after PCR. If the determination is difficult, the size of the amplified product may be confirmed by agarose gel electrophoresis.

  In addition, as a method for detecting a PCR amplification product with a probe, as represented by the Taq Man method, a fluorescent substance-labeled probe that hybridizes the PCR amplification product with the amplification product is added to the reaction solution, and the amplification product As a fluorescence intensity value by the probe, a method of confirming the presence or absence of DNA amplification by automatically detecting with a real-time PCR apparatus can be mentioned. For example, the oligonucleotide having the base sequence represented by SEQ ID NO: 2 and the oligonucleotide having the base sequence represented by SEQ ID NO: 3 are used as PCR primers, and all or part of the sequence in the base sequence represented by SEQ ID NO: 4 Is used as a probe for detecting an amplification product.

  Specifically, the following methods are mentioned.

  For example, in the case of the Taq Man method, an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 2 and an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 3 are used as PCR primers, and SEQ ID NO: 4 located between the two sequences. Can be used as a labeled probe for detecting a PCR amplification product, that is, a Taq Man probe. The presence or absence of DNA amplification can also be confirmed by automatically detecting the fluorescence intensity value generated when the probe hybridized to the PCR amplification product is degraded by DNA Taq polymerase using a real-time PCR apparatus.

  In the case of the Molecular Beacon method, for example, an oligonucleotide having the base sequence represented by SEQ ID NO: 2 and an oligonucleotide having the base sequence represented by SEQ ID NO: 3 are used as PCR primers, and a sequence located between the two sequences A labeled probe for detecting a PCR amplification product using an oligonucleotide containing the whole or a part of the number 4 sequence can be used as a Molecular Beacon probe. The presence or absence of DNA amplification can also be confirmed by automatically detecting the fluorescence intensity value of the labeling substance of the probe generated when the probe is hybridized with the PCR amplification product with a real-time PCR apparatus.

  In the case of the CycleavePCR method, for example, an oligonucleotide having the base sequence represented by SEQ ID NO: 2 and an oligonucleotide having the base sequence represented by SEQ ID NO: 3 are used as PCR primers, and the SEQ ID NO located between the two sequences Oligonucleotides containing all or a part of the 4 sequences can be used as labeled probes for detecting PCR amplification products, that is, DNA-RNA chimeric nucleotide probes used in CycleavePCR. The presence or absence of DNA amplification can also be confirmed by automatically detecting the fluorescence intensity value generated when the probe hybridized with the PCR amplification product is degraded by RNase H with a real-time PCR apparatus.

  When a hybrid probe is used, for example, an oligonucleotide having the base sequence represented by SEQ ID NO: 2 and an oligonucleotide having the base sequence represented by SEQ ID NO: 3 are used as PCR primers and located between the two sequences. Two adjacent sets of labeled oligonucleotides containing all or part of the sequence of SEQ ID NO: 4 can be used as hybrid probes. The presence or absence of DNA amplification can also be confirmed by automatically detecting the fluorescence intensity value generated when the two probes hybridized with the PCR amplification product are hybridized so as to be adjacent to each other.

  Moreover, as a method for detecting DNA in a sample with a probe, an oligonucleotide for detecting emetic Bacillus cereus containing a sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or the like of the present invention is used as a labeled probe. Thus, it is possible to detect vomiting type Bacillus cereus. More specifically, the oligonucleotide for detection of emetic Bacillus cereus is labeled to form a labeled probe, and then the labeled probe and DNA in the sample are hybridized, and hybridization signals are formed by signals derived from the labeled probe. A method of detecting the presence or absence of emetic Bacillus cereus in a sample can be mentioned.

  That is, whether there is a specific nucleotide sequence specific to a known emetic Bacillus cereus in the nucleic acid of an unknown test bacterium or sample depending on the presence or absence of hybridization with a probe containing a sequence characteristic of the emetic Bacillus cereus If it has a specific base sequence, it can be determined that it is likely to belong to the same species or strain as the known vomiting type Bacillus cereus. On the other hand, if it does not have a specific sequence, it can be determined that it is highly likely that it belongs to the known vomiting type Bacillus cereus or belongs to a different strain.

  The type of oligonucleotide label and the method for detecting the signal derived from the label are not particularly limited, and known methods can be used as appropriate. For example, 1) a method of labeling with a radioisotope and visualization by autoradiography, 2) a method of labeling by digoxigenin and visualization by an immunohistochemistry method using an anti-digoxigenin antibody, 3) labeling by biotin, Examples include a method of detecting with a biotin antibody, a method of detecting using streptavidin or avidin, and 4) a method of detecting a fluorescent signal using a fluorescent label such as fluorescein.

  In addition, in order to obtain a more accurate test result, another known detection test may be performed after the method for detecting emetic Bacillus cereus of the present invention as described above. For example, when a DNA fragment that is specifically PCR-amplified is detected by the detection method of the present invention, or when a specific DNA is detected by a probe, a positive result of emetic Bacillus cereus is obtained. Then, a test for chemically analyzing cereulide with a mass spectrometer (LC-MS analysis), a vacuolation test for detecting cereulide pharmacologically, and the like can be carried out. Thereby, the productivity (ability) of cereulide of the test bacterium can be determined more accurately.

  In the method for specifically detecting the strain of emetic Bacillus cereus according to the present invention, the type of the test sample as a target is not particularly limited.

  For example, food samples, clinical samples, and the like can be given as test samples. More specifically, examples of foods include foods with high starch content and milk. Examples of foods with a high starch content include cooked rice, rice cakes, Japanese confectionery, noodles, bread and the like. Moreover, as a clinical sample, the sample prepared from the patient who may be affected, the strain isolate | separated from the sample, the strain isolate | separated from the food which the patient ingested, etc. are mentioned.

  The form of the sample is not particularly limited. Usually, the detection of Bacillus cereus is often carried out on a dedicated selective medium, such as egg yolk-added NGKG agar basal medium (Nissui Pharmaceutical Co., Ltd.). The DNA extract obtained by performing the DNA extraction treatment can be applied to the above detection method. In addition, a DNA extract obtained by subjecting a specimen containing food or clinical samples to a suitable culture solution for a short time and then DNA extraction from bacteria grown in the culture solution should also be applied to the above detection method. Can do.

Detection Kit The present invention also relates to a vomiting-type Bacillus cereus detection kit. The configuration of the kit is not particularly limited as long as it includes at least one oligonucleotide for vomiting detection according to the present invention.

  Specifically, a vomiting-type Bacillus cereus detection oligonucleotide having a sequence containing part or all of the region of positions 220 to 275 or positions 370 to 468 in the base sequence represented by SEQ ID NO: 1 or a complementary sequence thereof Alternatively, a vomiting type Bacillus cereus detection kit containing a labeled nucleic acid probe obtained by labeling the oligonucleotide can be mentioned. Or a primer comprising a vomiting-type Bacillus cereus detection oligonucleotide having a sequence containing part or all of the region of positions 220 to 275 or positions 370 to 468 in the nucleotide sequence represented by SEQ ID NO: 1 or a complementary sequence thereof Another example is a vomiting type Bacillus cereus detection kit containing a labeled primer labeled with the oligonucleotide.

  The kit can contain appropriate reagents, detection means, various containers and the like according to the purpose of use.

  For example, polymerization reagents for synthesizing primer extension products, such as enzymes, means for detecting labels, means for separating double-stranded DNA strands contained in samples, deoxyribonucleotides, etc. be able to.

  Examples of the enzyme include DNA polymerase.

  The present invention has an advantageous effect that emetic Bacillus cereus can be detected more quickly and easily than the conventional method.

  In addition, the present invention enables screening of emetic Bacillus cereus with high accuracy by nucleic acid analysis using PCR and the like, and detection and confirmation of vomiting Bacillus cereus can be easily performed even in relatively small analysis institutions. There is an effect that it is possible.

  Furthermore, after performing the detection method of the present invention, in order to obtain a more accurate result, additional tests such as LC-MS analysis and vacuolation test for confirming cereulide itself can be performed. In the case of the above result, an additional test may be performed, and in the case of a negative result, the result can be obtained with high accuracy without performing a large-scale confirmation test such as LC-MS analysis or vacuolation test. Thus, the detection method using the oligonucleotide for detecting emetic Bacillus cereus according to the present invention can be effectively used as a primary screening means to be carried out before the examination such as LC-MS analysis or vacuolation test.

  In order to describe the present invention more specifically, the present invention will be described below using examples or test examples, but the present invention is not construed as being limited to these examples. The present invention can be changed as appropriate without departing from the gist of the present invention.

Example 1
Determination of nucleotide sequence specific to emetic Bacillus cereus and design of primer set for detecting the bacillus Bacillus cereus DSM4312, 13 foods, 55 foods, 98 foods, food RAPD analysis was performed for the purpose of detecting common and specific nucleotide sequences in the genomic DNA of 112 strains, 138 strains in food, 149 strains in food, and 160 strains in food. During food, it indicates that it is a cereus strain derived from food poisoning, which is managed by the Women's Nutrition University, Department of Hygiene (Associate Professor Ueda).

  RAPD analysis was performed using several 10-mer primers, and an amplification band specific to emetic Bacillus cereus was selected with high reproducibility. After determining the base sequence of the band, emetic cereus was determined from this nucleotide sequence. A new primer for detecting the fungus was designed. PCR was performed using the designed sense / antisense primers, and whether or not the DNA of emetic Bacillus cereus was specifically detected was examined by the band pattern of electrophoresis. Specifically, it was performed as follows.

  Bacillus cereus bacteria shown in Fig. 1 and other bacterial strains of Bacillus cereus group were cultured in BHI liquid medium and each prepared using PUREGENE DNA Isolation Kit (Gentra, Minneapolis, USA) DNA was used for RAPD analysis. Here, the Bacillus cereus group includes Bacillus sulingensis, Bacillus mycoides, Bacillus viencephanesis, Bacillus anthracis (Bacillus anthracis), which have similar genetic and biological properties to Bacillus cereus. It is a group that includes Bacillus cereus.

A PCR reaction solution for RAPD analysis was prepared using Gene Taq FP of Nippon Gene. That is, 0.5 μL Taq DNA polymerase (5 units / μL) (Gene Taq FP), 5 μL 10 × Gene Taq Universal Buffer (Gene Taq FP), 4 μL dNTP Mixture (2.5 mM each) (Gene Taq FP), 10 μL A 50 μL reaction solution containing 5 mM MgCl ₂ , 10 μL of a primer for RAPD (5 pg / μL), 4 μL of a test DNA sample (5 ng / μL), and 16.5 μL of distilled water was prepared. In PCR, initial denaturation was performed at 95 ° C for 5 minutes, followed by 45 cycles of 95 ° C for 1 minute, 36 ° C for 1 minute, and 72 ° C for 2 minutes, followed by incubation at 72 ° C for 1 minute. Thereafter, the temperature was lowered to 4 ° C.

  The reaction mixture was subjected to agarose electrophoresis, and the amplification band pattern obtained was examined for the presence of a characteristic and relatively reproducible amplification band in emetic Bacillus cereus. The 5'-CCCGTCAGCA-3 'primer , The formation of an amplification band characteristic of emetic Bacillus cereus and relatively reproducible was confirmed. The amplification band having a size of about 0.95 kb was selected as an amplification band characteristic of emetic Bacillus cereus and having relatively good reproducibility.

FIG. 1 shows an electrophoretogram showing the results of RAPD analysis when a 5′-CCCGTCAGCA-3 ′ primer is used, and selected amplification bands. Lanes 1 to 8 are vomiting type Cereus strains, which are Bacillus cereus DSM 4312, 13 foods, 55 foods, 98 foods, 112 foods, 138 foods, 149 foods, Shows 160 strains in the diet. Lanes 9 to 17 are non-emetic Cereus strains, Bacillus cereus DSM 8438, ATCC 7004, ATCC 10987, ATCC 11778, ATCC 33018, ATCC 14579 ^T , ATCC 10876, DSM 4313, DSM 4384 strain is shown. Lanes 18 to 20 are Bacillus thuringiensis strains, which represent Bacillus thuringiensis ATCC 10792 ^T strain, ATCC 13366 strain, and ATCC 33679 strain, respectively. Lane 21 shows Bacillus mycoides NCIMB 13305 ^T , and lane 22 shows Bacillus weihenstephanensis strain DSM11821. M represents a DNA molecular weight marker, and lane B represents a negative control.

  The band indicated by □ in FIG. 1 shows an amplification band of about 0.95 kb selected as a characteristic band for the emetic Cereus strain.

  Derived from each vomiting cereus strain (Bacillus cereus DSM 4312, 13 in food, 55 in food, 98 in food, 112 in food, 138 in food, 149 in food, 160 in food) A purified product of an amplified band of about 0.95 kb was cloned using TOPO TA Cloning kit (Invitrogen, California, USA), and the base sequence corresponding to the band was determined by the cycle sequence method. The determined nucleotide sequences were all the same in each emetic type Cereus strain. The base sequence of the amplification band excluding the amplification primer sequence used in RAPD analysis is shown in SEQ ID NO: 1 in the sequence listing.

  A homology search of the nucleotide sequence of SEQ ID NO: 1 is performed on the DDBJ / EMBL / GenBank database, and a region having a low homology with a known DNA sequence in a relatively wide range (220 to 275 bases and 370 to 468 bases) ) Was selected and used as a primer region for PCR. From these primer regions, a sense primer (SEQ ID NO: 2) and an antisense primer (SEQ ID NO: 3, SEQ ID NO: 4) that specifically hybridize with emetic Bacillus cereus DNA were designed and synthesized artificially (Qiagen). Sawasdee).

  In the PCR described below, a primer set using an oligonucleotide consisting of the nucleotide sequences represented by SEQ ID NO: 2 and SEQ ID NO: 3 and an oligonucleotide consisting of the nucleotide sequences represented by SEQ ID NO: 2 and SEQ ID NO: 4 were used. A primer set was used.

A PCR reaction solution with a total volume of 20 μL was prepared according to the instructions of Takara Ex Taq R-PCR Version (Takara Bio, Otsu, Japan), which is a kit for PCR reaction. 2 μL of 10 × R-PCR Buffer (Mg ²⁺ free), 0.16 μL of 250 mM Mg ²⁺ Solution for R-PCR, 0.4 μL of dNTP Mixture (10 mM each), 0.2 μL of TaKara EX Taq ^™ R-PCR (5 units) / μL), 0.8 μL of 0.125% SYBR ^™ Green I (Fmc Bioproducts, Rockland, USA), 1.0 μL of DNA primer solution (5 μM each of DNA primer of SEQ ID NO: 2 and DNA primer of SEQ ID NO: 3, or A PCR reaction solution was prepared by adding 1 μL of the test DNA solution to 19 μL of a preparation solution containing 5 μM each of the DNA primer of SEQ ID NO: 2 and the DNA primer of SEQ ID NO: 4) and 16.44 μL of distilled water.

  PCR was performed as follows using a LightCycler from Roche Diagnostics. In other words, initial denaturation was performed at 95 ° C for 1 minute, then 95 ° C for 5 seconds, 54 ° C for 10 seconds, and 72 ° C for 20 seconds, with fluorescence measurement (measurement of amplification products by PCR) as one cycle. 35 cycles followed by melting curve analysis of amplification products, after 0 second at 97 ° C, 10 seconds at 60 ° C, then ramped up to 97 ° C at a rate of 0.2 ° C / second and simultaneously with continuous fluorescence measurement The amount of double-stranded DNA of the amplification product at each temperature was measured, and finally the temperature was lowered to 40 ° C.

  After completion of PCR, melting curve analysis was performed with the software of the PCR apparatus, and the presence or absence of a specific amplification product was determined by determining the Tm value of the amplification product. Furthermore, using a Mupid mini-gel electrophoresis tank (ADVANCE), perform electrophoresis on a 1.8% agarose gel, stain the gel with an ethidium bromide solution, and check for the presence of the target amplification band. confirmed.

Vomiting Bacillus cereus DSM 4312 strain DNA (50 pg) and Bacillus cereus ATCC14579 ^T strain DNA (50 pg), which is a diarrhea Bacillus cereus, were used as test DNAs. PCR was performed as described above using a primer set consisting of a DNA primer and a primer set consisting of a DNA primer of SEQ ID NO: 2 and a DNA primer of SEQ ID NO: 4.

FIG. 2 shows a gel obtained by electrophoresis of the reaction solution after PCR. Lanes 1 and 3 show the Bacillus cereus DSM 4312 strain, and lanes 2 and 4 show the Bacillus cereus ATCC14579 ^T strain. Lane M shows a DNA molecular weight marker, and lane B shows a negative control. Lanes 1, 2 and B on the left of Lane 1 show the results when a primer set consisting of the DNA primer of SEQ ID NO: 2 and the DNA primer of SEQ ID NO: 3 was used. Lanes 3, 4 and B on the left of Lane 3 show the results when using a primer set consisting of the DNA primer of SEQ ID NO: 2 and the DNA primer of SEQ ID NO: 4.

As shown in the results of Fig. 2, in the PCR using the DNA primer of SEQ ID NO: 2 and the DNA primer set of SEQ ID NO: 3, a 234 bp amplification product was formed in Bacillus cereus strain DSM 4312, an emetic Bacillus cereus However, the Bacillus cereus ATCC14579 ^T strain, which is a diarrhea-type Bacillus cereus, did not form an amplification product. That is, in the detection method by PCR using the DNA primer of SEQ ID NO: 2 and the DNA primer set of SEQ ID NO: 3, the vomiting Bacillus cereus DSM 4312 strain DNA is detected, but the diarrhea Bacillus cereus Bacillus cereus is detected. It was confirmed that ATCC14579 ^T strain DNA was not detected.

Similarly, in the PCR using the DNA primer of SEQ ID NO: 2 and the DNA primer of SEQ ID NO: 4, a 158 bp amplification product was formed in the Bacillus cereus DSM 4312 strain, which is an emetic Bacillus cerevisiae. No amplification product was formed in the Bacillus cereus ATCC14579 ^T strain. That is, even in the detection method by PCR using the DNA primer of SEQ ID NO: 2 and the DNA primer set of SEQ ID NO: 4, Bacillus cereus DSM 4312 strain DNA, which is an emetic Bacillus bacterium, is detected, but Bacillus, a diarrhea Bacillus cerevisiae, is detected. It was confirmed that cereus ATCC14579 ^T strain DNA was not detected.

Example 2
Confirmation of reactivity by various detection methods for emesis type Bacillus cereus and other bacteria (1) Bacillus cereus group bacteria belonging to (Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus weihenstephanensis) and Bacillus bacterium which does not belong to the same group ( A method for chemically analyzing cereulide production ability of Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus) using a mass spectrometer (hereinafter referred to as LC-MS analysis), and an empty pharmacologically effective detection of cereulide This was confirmed by a spore formation test (hereinafter referred to as a vacuolation test).

  Each test bacterium was inoculated in a BHI medium and cultured at 32 ° C. for 20 hours with shaking. Using this culture solution, inoculated into 10% skim milk medium (BD Difco) at 100 to 500 CFU / mL, and aerobic main culture was performed by shaking culture at 21 ° C. for 72 hours. The cultured broth after the culture was extracted twice with an equal amount of n-pentane, and the collected extract was dried at room temperature under a stream of nitrogen gas. A cereulide extract was prepared by dissolving the dried product in an equal amount of methanol.

LC-MS analysis is based on the method of Ueda et al. (Bokin Bobai, 30, 511-524 (2002)), YMC-Pack ODS-AM (C18 5μm 2.0 × 250mm), guard column (2.0 × 20mm) ( Using a mass spectrometer (TSQ7000, (Finnigan mat)) online with a high performance liquid chromatograph (Waters 600 series, Waters) equipped with YMC (95% acetonitrile, 4.9%). Using a mobile phase consisting of water and 0.1% trifluoroacetic acid, 10 μL of cereulide extract was separated by the above high performance liquid chromatograph under the flow rate condition of 0.2 mL / min, and then introduced online into the mass spectrometer. ESI (positive) mode conditions were: heat gas 70 psi, aux gas 10 unit, spray volts 4.5 kv, capillary temperature 250 ° C, and the mass range to be measured was 1128.2-1129.2 m / z 1170.3-1171.3 m / z The obtained ion chromatogram was attached to the instrument. Is analyzed by analysis software was, single ion of 1128.7 The (NH ⁴⁺ adducts) valinomycin, single ion (NH ⁴⁺ adducts). Valinomycin was monitored as cereulide of 1170.8 as a reference substance analysis system before and after the test sample measurement Since it was difficult to obtain a standard product of cereulide, the obtained results are listed as qualitative results in Table 1. In the table, + (plus) indicates that cereulide was detected, and-(minus) indicates Indicates that cereulide was not detected.

The vacuolation test was performed as follows based on the method of Ueda et al. (Bokin Bobai, 30, 511-524 (2002)). As described above, the supernatant after centrifugation of the main culture was subjected to a heat treatment at 100 ° C. for 10 minutes as a sample for the vacuolation test. Prepared a 2-fold dilution series (final concentration 1/5 to 1/1280 times) of the sample for vacuolation test with PBS (without calcium and magnesium), and dispensed this into a 96-well plate at 25 μl / well . To this, 1% fetal bovine serum-containing MEM (minimum essential medium; manufactured by GIBCO) containing 1 × 10 ⁵ cells / mL HEp-2 cells (Dainippon Pharmaceutical Co., Ltd.) was added in a volume of 100 μl / well. Incubated with% CO ₂ for 24 hours. After completion of the culture, vacuolation of the cells was observed with a phase-contrast microscope, and cells having 10 or more vacuoles in one cell accounted for 30% or more per well were regarded as positive. The reciprocal of the final dilution factor showing positive was used as the emetic toxicity titer (titer) of the vomiting toxin test sample. The numerical values of the obtained emetic toxin titer are shown in Table 1 as an index indicating the strength of the pharmacological effect of emetic toxin.

  (2) To confirm the characteristics of bacteria belonging to the Bacillus cereus group (Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus weihenstephanensis) and Bacillus bacteria not belonging to the group (Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus) Diarrhea toxin productivity test and starch degradability test were conducted.

  The diarrhea-type toxin productivity test was carried out using a kit for detection of Bacillus cereus enterotoxin (CRET-RPLA “Seiken”, Denka Seiken Co., Ltd.) by reverse passive latex agglutination reaction according to the instructions of the kit. The obtained results are listed in Table 1 as qualitative results. In the table, + (plus) indicates that diarrhea-type toxin productivity is positive, and-(minus) indicates negative.

  The starch degradability test was performed as follows based on the method of Ueda et al. (Bokin Bobai, 30, 511-524 (2002)). The test strain was streaked on a standard agar medium containing 1% soluble starch, cultured at 30 ° C., and Lugol's solution (0.33% iodine, 0.66% potassium iodide) was sprayed onto the cultured agar medium. The case where the periphery of the smeared part and the part directly under the smeared line part were transparent and clearly distinguished from other purple-brown parts was positive, and the case where the whole agar medium was purple-brown was considered negative. The obtained results are listed in Table 1 as qualitative results. In the table, + (plus) indicates that starch degradability is positive, and-(minus) indicates negative.

  (3) Next, against various bacteria including bacteria belonging to the Bacillus cereus group (Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus weihenstephanensis) and Bacillus bacteria not belonging to the group (Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus) The reaction specificity was confirmed as follows by PCR using oligonucleotides having sequences represented by SEQ ID NO: 2 and SEQ ID NO: 3 as primer sets.

  Each bacterial strain was cultured using a suitable medium, and a DNA solution was prepared in the same manner as in Example 1. The amount of DNA in each DNA solution was measured, and a test DNA solution in which the DNA concentration of each bacterial strain was about 50 pg / μL was prepared. In order to examine the reactivity of the DNA primer sets of SEQ ID NO: 2 and SEQ ID NO: 3 against each of these bacterial strain DNAs, PCR using the above DNA primer set for 1 μL of each of the above test DNA solutions was carried out in the same manner as in Example 1. Carried out. The presence or absence of a PCR amplification product was confirmed by electrophoresis on an agarose gel in the same manner as in Example 1.

  In order to confirm the presence of each bacterial strain DNA, a primer set (prbac) (J. Dent. Res. 78: 850-856 (1999) that can detect bacteria in general can be used instead of the primer sets of SEQ ID NOs: 2 and 3. ) Rupf, S., K. Merte, and K. Eschrich; Quantification of bacteria in oral samples by competitive polymerase chain reaction). However, PCR was performed by substituting the following reaction conditions. That is, initial denaturation was performed at 95 ° C for 1 minute, then 95 ° C for 5 seconds, 59 ° C for 10 seconds, and 72 ° C for 20 seconds, and fluorescence measurement (measurement of amplified product by PCR) as one cycle. 30 cycles were performed. The results of these PCRs are listed in Table 1. In the table, + (plus) indicates that a band was detected by electrophoresis, and-(minus) indicates that no band was detected.

  Table 1 (Table 1-1 and Table 1-2) describing the results obtained in the above (1) to (3) is shown below. Note that NA in Table 1 indicates analysis and inspection not yet performed. In addition, in the Bacterial strain column, foods indicate cereus strains derived from food poisoning managed by Women's Nutrition University, Department of Hygiene (Associate Professor Ueda).

(4) From the results in Table 1, all the Cereus strains that were confirmed to produce cereulide in LC-MS analysis and vacuolation tests were negative for diarrhea toxin production and negative for starch solubility. It has been clarified that there are Bacillus cereus bacteria that are negative in diarrhea toxin production but negative in cereulide production, and cereus bacteria that are negative in starch solubility but negative in cereulide production. That is, it was suggested that indicators such as diarrhea toxin production and starch degradability are not sufficient indicators for identifying emetic Bacillus cereus. On the other hand, PCR analysis using the DNA primer sets of SEQ ID NO: 2 and SEQ ID NO: 3 detected all Cereus strains in which production of cereulide was confirmed. However, one strain (Bacillus cereus ATCC 7004) in which cereulide production was not observed was detected as a false positive. That is, in the case of the detection method using the DNA primer sets of SEQ ID NO: 2 and SEQ ID NO: 3, in the case of a negative reaction, it can be determined that the test bacterium is not the bacterium, and before the LC-MS analysis or the vacuolation test It was confirmed that it is highly useful as a stepwise screening tool.

Example 3
Detection of emetic Bacillus cereus on colonies on NGKG agar by PCR
To examine the effectiveness of PCR analysis using the DNA primer set of SEQ ID NO: 2 and SEQ ID NO: 3 and the DNA primer set of SEQ ID NO: 2 and SEQ ID NO: 4 when colonies on NGKG agar are used as test samples In addition, the following experiment was conducted.

Inoculate Bacillus cereus strain DSM 4312 and 13 in food, 160 in food, ATCC 14579 ^T strain, DSM 4313 strain, ATCC 10987 strain into egg yolk-added NGKG agar so that it becomes about 50 CFU / agar medium And cultured at 30 ° C. for 24 hours. Two colonies were arbitrarily selected from each of the appeared colonies, and an extremely small amount of each colony was added to 200 μL of 10% Chelex solution [10% (W / V) Kilex (Bio-Rad), 10 mM Tris-HCl, 0.1 After suspension in mM EDTA, pH 8.0], the mixture was heated at 99 ° C. for 5 minutes using a heat block, and centrifuged at 13,000 × g for 3 minutes to collect the supernatant. The supernatant contained bacterial DNA that was simply extracted, and was used as a DNA solution for PCR. Using 1 μL of the obtained DNA solution as a sample, PCR was performed in the same manner as described in Example 1, and the presence or absence of PCR amplification products was confirmed. In addition, in order to confirm the presence of each bacterial strain DNA in a DNA solution simply extracted from colonies, a primer set (prbac) (J. Dent. Res. 78: 850-856 (1999) Rupf, S., K. Merte, and K. Eschrich; Quantification of bacteria in oral samples by competitive polymerase chain reaction). However, PCR was performed under the following reaction conditions. That is, initial denaturation was performed at 95 ° C for 1 minute, then 95 ° C for 5 seconds, 59 ° C for 10 seconds, and 72 ° C for 20 seconds, and fluorescence measurement (measurement of amplified product by PCR) as one cycle. 30 cycles were performed.

The electrophoresis pattern by agarose gel is shown in FIG. FIG. 3 (A) shows the case where the DNA primer sets of SEQ ID NOs: 2 and 3 are used, FIG. 3 (B) shows the case where the DNA primer sets of SEQ ID NOs: 2 and 4 are used, and FIG. 3 (C) shows the prbac primer set. The case where it is used is shown. In FIG. 3 (A), (B) and (C), lanes 1 and 2 show Bacillus cereus DSM 4312 strain, lanes 3 and 4 show 13 foods, lanes 5 and 6 show 160 foods are shown, lanes 7 and 8 show the ATCC 14579 ^T strain, lanes 9 and 10 show the DSM 4313 strain, lane 11 and 12 show the ATCC 10987 strain. Lane M shows a DNA molecular weight marker, and lane B shows a negative control.

  From the results in FIG. 3 (C), it was confirmed that bacterial DNA was present in all test DNA solutions.

From the results shown in FIG. 3 (A), PCR analysis using the DNA primer sets of SEQ ID NO: 2 and SEQ ID NO: 3 showed vomiting when bacterial cell DNA prepared simply from colonies on NGKG agar was used as the DNA solution. Bacillus cereus strain DSM 4312 and 13 strains in food and 160 strains in food are detected, but Bacillus cereus strain ATCC 14579 ^T , DSM 4313 strain and ATCC 10987 strain are not detected I confirmed that I did not.

Similarly, from the results of FIG. 3 (B), PCR analysis using the DNA primer sets of SEQ ID NO: 2 and SEQ ID NO: 4 used microbial cell DNA prepared simply from colonies on NGKG agar as a DNA solution. In this case, Bacillus cereus DSM 4312 strain, which is an emetic Bacillus cereus strain, and 13 strains in food, 160 strains in food, but Bacillus cereus strain ATCC 14579 ^T and DSM 4313 strain, ATCC 10987, which are not emetic Bacillus cereus It was confirmed that no strain was detected.

Example 4
Detection of vomiting-type Bacillus cereus in foods by PCR Commercial steamed noodles (low-temperature distribution product) and commercial milk (130 ° C, 2 seconds sterilization, low-temperature distribution product) that have been confirmed in advance to be free from contamination with live bacteria 2 were prepared, and Bacillus cereus DSM 4312 strain, which is an emetic Bacillus cerevisiae, was used as an indicator strain to prepare 20 to 50 CFU per gram of each food and those without addition were prepared. . BHI liquid medium of 9 times the amount of food was added to each, and aseptically stirred and mixed using a stomacher.

  10 mL of these diluted food mixtures were transferred to a 15 mL culture tube and cultured at 35 ° C. with shaking. To prepare a DNA solution for use in PCR, 1.3 mL of the culture solution after 0 hours and 7 hours were collected.

  The collected culture solution was centrifuged at 100 × g for 1 minute, and the supernatant was further centrifuged at 13,000 × g for 5 minutes to obtain a precipitate. In order to wash this precipitate, it was suspended in 0.5 mL of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0), and then centrifuged at 13,000 × g for 5 minutes to obtain a precipitate. To this precipitate, 200 μL of 10% Chelex solution [10% (W / V) Kilex (Bio-Rad), 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0] was added to suspend the precipitate, and then 99 using a heat block. The supernatant was collected by heating at 1 ° C. for 5 minutes and centrifuging at 13,000 × g for 3 minutes. The supernatant contained DNA, which was used as a DNA solution for PCR.

  Using 1 μL of the obtained DNA solution as a sample, PCR was performed in the same manner as described in Example 1 using the DNA primer sets of SEQ ID NOs: 2 and 3, and the presence or absence of PCR amplification products was confirmed. FIG. 4 shows an electrophoresis pattern by an agarose gel.

  4A is an electrophoresis pattern in the case of commercially available fried noodles, and FIG. 4B is an electrophoresis pattern in the case of commercially available milk. In both FIGS. (A) and (B), lanes 1, 2, 3 and 4 are samples in which the culture time of food was 0, 0, 7 and 7 hours, respectively, without adding bacteria. Lanes 5, 6, 7 and 8 are samples in which the culture time before extraction of DNA after adding bacteria to food is 0, 0, 7 and 7 hours, respectively.

  As shown in Fig. 4 (A), when there is about 20-50 CFU of indicator strain of Bacillus cereus DSM 4312 per gram of commercially available steamed fried noodles, if the food is cultured for 7 hours before DNA extraction, the PCR Was confirmed to be able to detect the bacterial DNA. In addition, as shown in FIG. 4 (B), when 20 to 50 CFU of indicator bacteria of Bacillus cereus DSM 4312 strain per gram of commercially available milk is present, if the food is cultured for 7 hours before DNA extraction, It was confirmed that the bacterial DNA could be detected by PCR.

  The present invention can be effectively used as a means for detecting emetic Bacillus cereus bacteria in samples such as foods and clinical samples. It is also effective as a simple and rapid primary screening method for emetic Bacillus cereus, which is a method for chemically analyzing cereulide, which is an indicator of vomiting Bacillus cereus, using a mass spectrometer or before performing a vacuolation test. Can be used.

It is an electrophoretic diagram showing a band specific to emetic Bacillus cereus obtained from RAPD analysis. It is a figure which shows the PCR product of emetic Bacillus cereus by the primer set using the oligonucleotide of this invention. It is a figure which shows the PCR product of the vomiting type | mold Bacillus cereus with respect to the colony on a NGKG agar medium by the primer set using the oligonucleotide of this invention. It is a figure which shows the PCR product of the emetic Bacillus cereus when the short-term culture solution of the emetic bacillus cereus bacteria addition food by the primer set using the oligonucleotide of this invention is used as a test sample.

Claims (7)

An oligonucleotide for detecting emetic Bacillus cereus having a sequence containing a part or all of the region from position 220 to position 275 or position 370 to position 468 in the base sequence represented by SEQ ID NO: 1 or a complementary sequence thereof. An oligonucleotide for vomiting-type Bacillus cereus having a base sequence represented by any one of SEQ ID NOs: 2 to 4 or a complementary sequence thereof. a) Amplifying a target nucleotide sequence in DNA in a sample using a primer set comprising at least one oligonucleotide for detecting emetic Bacillus cereus according to claim 1 or claim 2,
b) measuring the product amplified in a) to detect whether a sequence characteristic of emetic Bacillus cereus is present in the sample;
For detecting vomiting type Bacillus cereus. a ′) a step of amplifying a target nucleotide sequence in DNA in a sample using a primer set comprising at least one oligonucleotide for detecting emetic Bacillus cereus according to claim 1 or 2;
b ′) The product amplified in a ′) is hybridized with a probe labeled with the oligonucleotide according to claim 1 or 2, and a signal derived from the labeled probe is measured. Detecting whether a sequence characteristic of emetic Bacillus cereus is present,
For detecting vomiting type Bacillus cereus. a ″) a step of labeling the vomiting type Bacillus cereus detection oligonucleotide according to claim 1 or claim 2 into a labeled probe;
b '') the step of hybridizing the labeled probe of a '') with the DNA in the sample;
c ″) detecting the presence or absence of hybridization in b ″) based on the signal derived from the labeled probe;
For detecting vomiting type Bacillus cereus. A kit for detecting emetic Bacillus cereus, comprising a primer comprising the oligonucleotide for detecting emetic Bacillus cereus according to claim 1 or 2, or a labeled primer obtained by labeling the oligonucleotide. A kit for detecting emetic Bacillus cereus, comprising a labeled probe labeled with the oligonucleotide for detecting emetic Bacillus cereus according to claim 1 or 2.