KR20060076786A - Oligonucleotide to be used in determining sporogenous aerobic bacterium and determination method and determination kit for sporogenous aerobic bacerium using the same - Google Patents

Abstract

An oligonucleotide which contains a base sequence specific to 16S ribosomal RNA gene of a sporogenous aerobic bacterium or a sequence complementary to the above base sequence. The specific base sequence as described above is a base sequence represented by any of SEQ ID NOS:1 to 6. This oligonucleotide is employed as a primer in a PCR reaction wherein a nucleic acid prepared from a subject bacterium to be determined is used as a specimen and the specimen serves as a template. Thus, it can be determined whether a subject bacterium is a sporogenous aerobic bacterium or not based on the amplification of a specific DNA fragment as an indication.

Description

OLIGONUCLEOTIDE TO BE USED IN DETERMINING SPOROGENOUS AEROBIC BACTERIUM AND DETERMINATION KIT FOR SPOROGENOUS AEROBIC BACERIUM USING THEA

The present invention relates to oligonucleotides that can be used in PCR for determining whether a flap bacterium to be judged is a diffuser aerobic bacterium, and the use thereof. Specifically, the oligonucleotide and the oligonucleotide The present invention relates to a method for determining whether a flap bacterium is a diffuser aerobic bacterium by performing PCR using a primer and detecting a DNA fragment specific for the diffuser aerobic bacterium, and a judgment kit for performing the determination method.

Conventionally, methods for classifying and identifying bacteria include methods based on physiological biochemical properties, methods based on quinone composition, cell fatty acid composition, cell wall components, etc., methods based on G + C content of DNA, and methods based on DNA homology. , A method using a DNA probe, and a method of analyzing the base sequence of a 16S ribosomal RNA gene are known.

In particular, in recent years, it is common to classify and identify bacteria by using groups (clusters) obtained by phylogeny based on the nucleotide sequence of 16S ribosomal RNA gene as an index.

On the other hand, diffuser aerobic bacteria, including the genus Bacillus, is widely distributed in the natural world such as soil, water, air. Among the genus Bacillus, anthrax (Bacillus anthracis), Bacillus cereus, which is a bacterium that causes food poisoning, and the like are known as pathogenic to humans. In addition, for the phylogenetic classification and classification system of the diffuser aerobic bacteria containing Bacillus genus, see, for example, Non Patent Literature 1 (Osamda Shida, "Classification and Identification of Aerobic Diffuser Bacteria", Soft Drink Article 3, 2001, 221-227), a variety of genera are known in addition to the genus Bacillus. Such diffuser aerobic bacteria have a characteristic of forming apo, but since the apo is high in heat resistance and drug resistance, it is difficult to sterilize and hardly completely removed from the manufacturing process of a food factory. Therefore, the diffuser aerobic bacteria are often separated by the process inspection of the food factory.

Thus, for example, in a food manufacturing plant, when bacteria are separated from a product or a manufacturing process, it is important to determine whether or not the separated bacteria form follicles in order to identify the cause of contamination. Therefore, particularly in the food industry, it is required to quickly determine whether or not bacteria isolated from a product or a manufacturing process form apo.

In general, whether or not the isolated bacteria are diffuser aerobic bacteria is determined by culturing a target bacterium (a test bacterium) to be judged on a follicle-forming medium, and confirming by microscopically whether or not the cells form follicles. Is taking. According to this method, it is necessary to cultivate the bacteriostatic bacterium on the follicle-forming medium, and therefore, it takes quite a long time from several days to one week until the determination. In such a general determination method, although a certain period can be shortened by shortening the detection determination time (cultivation time), in this case, there is a possibility that a strain having a slow growth rate cannot be detected, and thus the risk is high.

In this manner, in the above general determination method, it is necessary to culture for a long time in order to reliably determine the diffuser aerobic bacteria. Therefore, the determination method is not particularly suitable for pre-shipment inspection of the food industry, which requires promptness.

As a technique for identifying bacteria belonging to a diffuser aerobic bacterium in addition to the above general determination method, for example, Patent Document 1 (Japanese Patent Application Laid-Open No. 9-94100) (published: April 8, 1997) ) And Non-Patent Document 2 (Shida, O et al., Proposal for Two New Genera, Brevibacillus gen.nov., And Aneurinibacillus gen.nov., International Journal of Systematic Bacteriology, 939-946 (1996)). Methods are known. In this technique, it has a specific nucleotide sequence present in the 16S ribosomal RNA gene of bacteria belonging to the diffuser aerobic bacteria such as genus Brevibacillus, Aneurinibacillus, Paenibacillus. DNA is used as a primer, chromosomal DNA obtained from the identified bacteria is used as a template, and PCR is applied, and specific DNA amplification is used as an index to identify at the genus level of the identified bacteria.

In addition, as a technique for classifying Bacillus bacteria using 16S ribosomal RNA gene, Non-Patent Document 3 (Goto, K et al., Application of the partial 16S rDNA sequence as an index for rapid identification of species in the genus Bacillus, The Journal) of General and Applied Microbiology, 46, 1-8 (2000)) are known. Since about 275bp on the 5 'side, called the hypervariant region (HV region) of the 16S ribosomal RNA gene, is a species-specific region, this technique is capable of classifying Bacillus bacteria by performing base sequence analysis of this region. .

In addition, Patent Document 2 (Japanese Patent Laid-Open Publication No. 7-89956) (Date: October 4, 1995, corresponding Japanese Laid-Open Patent Publication No. 3-49696) (Published: 1991) On March 4, 2008), a method for detecting cereus bacteria is disclosed by detecting the gene using a primer for detecting β-lacutase gene derived from cereus.

However, at present, there is no effective method for rapidly and comprehensively determining whether or not a diffuser is an aerobic bacterium that forms an apo, and therefore, there is a demand for the development of a technology that can be used for pre-shipment inspection of the food industry, which particularly requires rapidity.

Specifically, first, the method disclosed in Patent Document 1 and Non-Patent Document 1 is not a technology originally used for pre-shipment inspection of the food industry, but a technology focused on the identification of bacteria. Therefore, this method can guarantee the rapidity because it is detected by the PCR method, but since it is a specific method, it is necessary to employ primers corresponding to each genus, and the operation becomes complicated and cannot be detected. Problems that can not be applied to the bacteria in the genus occurs.

In addition, the method described in Non-Patent Document 3 is effective for identifying species of the genus Bacillus, but is not applicable to bacteria of other genera, and the method described in Patent Document 2 is effective only for the detection of Cereus bacteria. .

As described above, a technique for detecting (or determining) a diffuser aerobic bacteria of a specific genus is known relatively quickly, but a technique for rapidly and comprehensively determining a diffuser aerobic bacteria is not known.

The present invention has been made in view of the above problems, and an object thereof is to perform an amplification reaction using an oligonucleotide that can be suitably used for a method capable of rapidly and comprehensively determining diffuser aerobic bacteria and the oligonucleotide as a primer. The present invention provides a method for determining whether a flap bacterium is a diffuser aerobic bacterium by detecting a DNA fragment specific for a diffuser aerobic bacterium, and a kit for performing the determination method.

MEANS TO SOLVE THE PROBLEM In order to solve the said subject, the present inventors earnestly examined by taking into account the comparatively common base sequence region on the 16S ribosomal RNA gene of the genus Bacillus and its related diffuser aerobic bacteria. As a result, by PCR amplification using the oligonucleotide according to the present invention as a primer, it was found that DNA fragments of about 100 bp in size, which are specific for the diffuser aerobic bacteria including Bacillus genus, were amplified, thus completing the present invention. . That is, this invention includes the following inventions.

(1) Diffuser An oligonucleotide comprising a base sequence specific for the 16S ribosomal RNA gene of aerobic bacteria or a complementary arrangement of the base sequences, wherein the specific base sequence is any of the base sequences shown in 1 to 6.

(2) The diffuser aerobic bacteria, Bacillus genus, Paenibacillus genus, Aneurinibacillus genus, Geobacillus genus, Aricyclobacillus genus, Brevibacillus genus Genus (Brevibacillus), genus Amphibacillus, genus Virgibacillus, genus Harobacillus, genus Grachiribacillus, genus Salibacillus, genus Salibacillus, and genus Marinibacillus The oligonucleotide as described in (1) which is a bacterium which belongs to at least 1 sort (s) chosen from the bacteria of the genus Rolactobacillus (Sporolactobacillus).

(3) Amplification of a specific DNA fragment obtained by performing a PCR reaction using the sample as a template, using a nucleic acid prepared from a target bacterium that is the target of the determination, and whether the bacteriostatic bacterium is a diffuser aerobic bacterium. A method for determining a diffuser aerobic bacterium for determining whether a primer is used, and as a primer used for a PCR reaction, at least one oligonucleotide comprising a base sequence specific for a 16S ribosomal RNA gene of a diffuser aerobic bacterium or a complementary sequence of the base sequence is present. A method for determining a diffuser aerobic bacterium, wherein a species is used and the specific nucleotide sequence is any one of the nucleotide sequences shown in SEQ ID NOs: 1-6.

(4) The base sequence shown in any one of SEQ ID NOs: 3 to 6, while the base sequence shown in SEQ ID NO: 1 or 2, or the oligonucleotide including the complementary sequence of the base sequence is used as a sense primer among the primers. Or the method for determining a diffuser aerobic bacterium according to (3), wherein an oligonucleotide comprising a complementary arrangement of the nucleotide sequences is used as an antisense primer.

(5) The diffuser aerobic composition according to (3) or (4), wherein at least one of the sense primer and the antisense primer is used in two or more kinds of oligonucleotides including the specific base sequence or the complementary arrangement of the base sequence. Bacterial determination method.

(6) The method for determining the diffuser aerobic bacterium according to any one of (3) to (5), wherein the specific DNA fragment to be amplified has a size that is detected at about 0.1 kb by separation by gel electrophoresis.

(7) The diffuser aerobic bacteria, genus Bacillus, genus Paenibacillus, genus Aneurinibacillus, genus Geobacillus, Alicyclobacillus, Alicyclobacillus, Brevibacillus Genus (Brevibacillus), genus amphibacillus, genus Virgibacillus, genus Harobacillus, genus Grachiribacillus, genus Salivacillus, genus Salibacillus, and genus Marinibacillus The determination method of the diffuser aerobic bacterium in any one of (3)-(6) which is a bacterium which belongs to at least 1 sort (s) chosen from the bacteria of the genus Rolactobacillus.

(8) A judgment kit for carrying out the method for determining a diffuser aerobic bacterium according to any one of (3) to (7) above.

(9) Amplification of a specific DNA fragment obtained by performing a PCR reaction using the sample as a template, using a nucleic acid prepared from a target bacterium that is the target of the determination, and is determined as a diffuser aerobic bacterium. It is a determination kit used to determine whether or not, as a primer used in a PCR reaction, comprising a base sequence specific to the 16S ribosomal RNA gene of the diffuser aerobic bacteria or complementary arrangement of the base sequence, the specific base sequence A determination kit for a diffuser aerobic bacterium comprising at least one oligonucleotide which is any one of the nucleotide sequences shown in SEQ ID NOs: 1 to 6.

(10) used as a marker for gel electrophoresis, further comprising a DNA fragment mixture including several species of indicator DNA fragments of different lengths, wherein the DNA fragment mixture contains an indicator DNA fragment having a size of 0.1 kb. The determination kit of the diffuser aerobic bacteria as described in (9).

(11) The kit for determining a diffuser aerobic bacterium according to (9) or (10), further comprising a reagent group for preparing nucleic acid from flap bacilli.

(12) The kit for determination of diffuser aerobic bacteria according to any one of (9) to (11), further comprising a reagent group used for PCR reaction.

Still other objects, features and advantages of the present invention will be fully understood from the following description. Further benefits of the present invention will become apparent from the following description with reference to the accompanying drawings.

1 is a view showing the results of comparing the base sequences of 16S ribosomal RNA genes of various bacteria and obtaining regions of specific DNA fragments as indicators to obtain oligonucleotides according to the present invention.

Fig. 2 shows the results of electrophoresis of amplified products when PCR was carried out using the oligonucleotide according to the present invention as a primer in Example 1, and using genomic DNA of the diffuser aerobic bacteria and other bacteria as a template. Figure is shown.

Fig. 3 shows the use of the oligonucleotide according to the present invention as a primer in Example 2, with the genomic DNA of two strains (No. 1 and No. 2) of non-identifying bacteria isolated from the food production environment as a template. It is a figure which shows the electrophoresis result of the amplified product at the time of PCR.

One Embodiment of this invention is described in detail based on FIG. This invention is not limited to the content described below.

In this embodiment, an oligonucleotide according to the present invention, a method for determining a diffuser aerobic bacterium according to the present invention using the oligonucleotide (hereinafter, also referred to simply as a determination method), and a kit for a diffuser aerobic bacterium according to the present invention The present invention will be described in detail in the following order (hereinafter, referred to simply as a determination kit).

(1) oligonucleotide according to the present invention

The oligonucleotide according to the present invention includes a base sequence specific for the 16S ribosomal RNA gene of a diffuser aerobic bacterium or a complementary arrangement of the base sequence, and specifically, the specific base sequence is represented by SEQ ID NOs: 1 to 6 An oligonucleotide comprising any of the nucleotide sequences shown as the specific nucleotide sequences.

The oligonucleotide is not particularly limited as long as the oligonucleotide contains a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 6 or a complementary sequence of the nucleotide sequence. More specifically, the following six types of oligonucleotides (a)-(f) are mentioned.

(a) has the following nucleotide sequence (SEQ ID NO: 1) and consists of 24 bases.

Oligonucleotide (a): (5 ') d-ACGATGAGTGCTAAGTGTTGGGGG (3')

(b) has the nucleotide sequence shown below (SEQ ID NO: 2) and consists of 24 bases.

Oligonucleotide (b): (5 ') d-ACGATGAGTGCTAGGTGTTGGGGG (3')

(c) has the nucleotide sequence shown below (SEQ ID NO: 3) and consists of 22 bases.

Oligonucleotide (c): (5 ') d-TTGAGTTTCAGTCTTGCGAGCG (3')

(d) has the following nucleotide sequence (SEQ ID NO: 4) and consists of 22 bases.

Oligonucleotide (d): (5 ') d-TTGAGTTTCAGTCTTGCGACCG (3')

(e) has the following nucleotide sequence (SEQ ID NO: 5) and consists of 22 bases.

Oligonucleotide (e): (5 ') d-TTGAGTTTCACTCTTGCGAGCG (3')

(f) has the nucleotide sequence shown below (SEQ ID NO: 6) and consists of 22 bases.

Oligonucleotide (f): (5 ') d-TTGAGTTTCACTCTTGCGACCG (3')

The oligonucleotide according to the present invention is not limited to the above (a) to (f), and may have a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 6, and may have a complementary arrangement of such nucleotide sequences. do. In addition, the oligonucleotide according to the present invention may include an additional base sequence in addition to the base sequence shown in any one of SEQ ID NOs: 1 to 6 or their complementary sequence. In addition, the oligonucleotide according to the present invention is modified so that a base in the range of about 1 to 3 is substituted, deleted, inserted, and / or added to the base sequence shown in any one of SEQ ID NOs: 1 to 6 or their complementary arrangement. It may be.

The method for obtaining an oligonucleotide according to the present invention is not particularly limited. Specifically, the base sequence or complementary sequence of any one of SEQ ID NOs: 1 to 6 may be chemically synthesized by a known method.

The oligonucleotide according to the present invention is suitably used as a primer for PCR, and its nucleotide sequence or length (size) was determined using a BLAST database, as shown in Examples described later. Specifically, 16S ribosomal RNA gene sequences of the genus Bacillus and its related apopoietic and apopoietic bacteria were extracted from the database (BLAST). Next, alignment was performed using a gene analysis software called AlignX of VectorNTI to select a region relatively common to the diffuser aerobic bacteria.

Among these various kinds of regions, the base sequence and length were determined by satisfying each of the conditions suitable as the primers shown below as much as possible.

1. 20 ~ 25 base is suitable for length.

2. The content of G + C (guanine + cytosine) is preferably about 50%.

3. For complementarity between primers, avoid primers with complementary 3 ′ ends to prevent dimer formation.

4. In primers, to avoid forming secondary structures, do not include autocomplementary alignment.

5. Select two primers as close to the Tm value as possible.

As a result, the polynucleotides (a) to (f) were designed by alignment as shown in FIG. 1. The region shown as "DNA amplification region" in this figure corresponds to "specific DNA fragment" serving as an index for determining whether the target bacterium bacterium to be judged is a diffuser aerobic bacterium. In addition, on the left side of the figure, Paenib validus abnormality (upper than the line in the figure) shows the scientific name of the genus Bacillus and its associated apopoietic bacteria, and Staphy aureus below (lower than the line in the figure) shows the scientific name of the apotyping bacteria. The part enclosed with the line represents the base having homology. In the arrows below the figure, an arrow indicated by F is a region corresponding to oligonucleotides (a) and (b), and an arrow shown by R is a region corresponding to oligonucleotides (c) to (f). .

(2) Method of determining diffuser aerobic bacteria according to the present invention

In the method for determining a diffuser aerobic bacterium according to the present invention, PCR is carried out using the oligonucleotides (a) to (f) described in (1) as a primer to amplify a specific DNA fragment and use it as an index. It is a method of determining.

Specifically, in the determination method according to the present invention, at least the oligonucleotides (a) to (f) are used as a primer to perform a PCR reaction, and specific amplification in the PCR product obtained in the PCR process. The DNA fragment amplification confirmation step for determining whether the DNA fragment is included may be included, and other steps such as the "sample preparation step" may also be included.

<Diffuser aerobic bacteria>

The diffuser aerobic bacterium to be determined in the present invention is not particularly limited as long as it is a bacillus genus and a bacterium associated therewith, but specifically, for example, genus Bacillus and Paenibacillus Genus, Aneurinibacillus, Geobacillus, Alicyclobacillus, Brevibacillus, amphibacillus, Virgibacillus, Harro Bacteria, such as genus Bacillus (Halobacillus), Grachiribacillus (Gracillibacillus), Saribacillus (Salibacillus), Marinibacillus (Marinibacillus), Sporolactobacillus (Sporolactobacillus). The diffuser aerobic bacterium has a feature of forming apoptosis.

Each of the diffuser aerobic bacteria can be classified into a plurality of subgroups according to their molecular systems. Specifically, for example, various subgroups of the genus Bacillus, subgroups of the genus aricyclobacillus, subgroups of the genus Paenibacillus, subgroups of the genus Brevibacillus, subgroups of the genus Anerinibacillus, subgroups of the genus Genoa Bacillus, vir Subgroups of the genus Zivacillus, Harobacillus, Grachiribacillus, and Sarivacillus, and the subgroups of the genus Enpivacillus and Sporolactobacilli. The present invention can also be applied to the use of determining diffuser aerobic bacteria of any of these subgroups.

Among the diffuser aerobic bacteria, each subgroup of the genus Bacillus, Paenibacillus, Anurinibacillus, Geovacillus, Aricyclobacillus, Brevibacillus is a bacterium that is particularly well separated in food relations. The present invention is particularly suitably applied to the determination of such diffuser aerobic bacteria.

In the determination method according to the present invention, oligonucleotides comprising a base sequence specific to the 16S ribosomal RNA gene of a diffuser aerobic bacterium or a complementary sequence of the base sequence, such as oligonucleotides (a) to (f), are used as primers. use. Therefore, it becomes possible to judge comprehensively whether the flap bacilli are a diffuser aerobic bacterium.

Therefore, the determination method according to the present invention, unlike the conventional technique of specifying the individual genus, can quickly determine whether the bacteriostatic bacillus is a bacterium forming apo, and therefore, particularly in the food industry, It can be used suitably as a test | inspection method which determines rapidly whether the cell detected from the manufacturing process forms the follicle.

<PCR process>

The PCR step is a step of performing a PCR reaction using a sample prepared as a template using a nucleic acid prepared from a bacteriostatic bacterium that is the target of determination, wherein the oligonucleotide according to the present invention is used as a primer, for example, the oligonucleotide Use

Here, among the primers, oligonucleotides comprising the nucleotide sequence shown in SEQ ID NO: 1 or 2 or the complementary sequence of these nucleotide sequences are used as sense primers. Specifically, among the oligonucleotides (a) to (f), oligonucleotides (a) and / or (b) are used as sense primers. On the other hand, an nucleotide sequence shown in any one of SEQ ID NOs: 3 to 6 or an oligonucleotide comprising a complementary sequence of these base sequences is used as an antisense primer. Specifically, at least any one of oligonucleotides (c), (d), (e) and (f) of the oligonucleotides (a) to (f) is used as an antisense primer.

In addition, only one type of oligonucleotide may be used in the said sense primer, antisense primer, or any one of these, and two or more types of oligonucleotides may be used. Specifically, only the oligonucleotide (a) or (b) may be used as a sense primer, or both may be used. Similarly, as an antisense primer, any one of oligonucleotides (c)-(f) may be used, and two or more of these four types may be used.

The reagent group, apparatus, and PCR reaction conditions for the PCR process are not particularly limited, and the reagent group can be suitably used a commercially available PCR kit or apparatus, and the conditions such as reaction temperature, reaction time, cycle number, and the like. Can be appropriately set according to the type of PCR kit or device.

<DNA fragment amplification confirmation process>

The DNA fragment amplification confirmation step is not particularly limited as long as it is a step of determining whether there is a specific DNA fragment amplified in the PCR product obtained in the PCR step. In the determination method according to the present invention, the amplification of the specific DNA fragment as an index makes it possible to determine whether the target bacteriostatic bacterium is a diffuser aerobic bacterium.

Although the method of confirming the amplification of the said specific DNA fragment is not specifically limited, Generally, the method by the gel electrophoresis method is used preferably. Gel electrophoresis is a technique widely used in experiments for handling nucleic acids, and it is possible to reliably and efficiently separate nucleic acids such as DNA and RNA, as well as the size (length) by staining gels. You can check. The device for carrying out the gel electrophoresis method and the electrophoretic conditions are not particularly limited, and commercially available electrophoretic devices can be suitably used, and electrophoresis may be performed under the conditions corresponding to the above devices.

The length of the specific DNA fragment identified in the DNA fragment amplification confirmation step is about 100 bp, as can be clearly seen from the "DNA amplification region" shown in FIG. That is, the specific DNA fragment to be amplified has a size detected near 0.1 kb by separation by gel electrophoresis. For example, in the example shown in FIG. 1, the 101 base region is amplified. Depending on the type of diffuser aerobic bacteria, the deletion or addition of bases in the region is also considered, so that DNA fragments having a size in the range of at least 90 to 110 bp, preferably in the range of 100 bp to 110 bp can be amplified. have. That is, within the above range, it can be regarded as having a size of 0.1 kb. Therefore, in the DNA fragment amplification confirmation step, it is necessary to confirm whether a DNA fragment having a size detected in the vicinity of 0.1 kb is detected by separation by gel electrophoresis.

<Other processes>

In the determination method according to the present invention, the PCR step and the DNA fragment amplification confirmation step may be included, but other steps, for example, a sample preparation step, may be included.

The sample preparation step is carried out at the front end of the PCR step, and it is only necessary to prepare nucleic acids such as chromosomal DNA from a predetermined amount of the bacteriostatic bacteria by a known method. The method for preparing the nucleic acid from the cells is not particularly limited, and a known method can be suitably used, and therefore, a commercial DNA preparation kit or the like may be used.

In addition, the method may include a culture of a bacteriostatic bacterium for culturing a predetermined amount of the bacteriostatic bacterium separated from a product, a manufacturing process, a machine, or the like to be examined. Although the medium and culture conditions at this time are not specifically limited, Well-known medium and culture conditions can be selected suitably.

(3) Judgment kit of diffuser aerobic bacteria according to the present invention

The determination kit of the diffuser aerobic bacterium according to the present invention may be a determination kit for carrying out the method for determining the diffuser aerobic bacteria described in (2). Specifically, at least an oligonucleotide comprising a nucleotide sequence of any one of SEQ ID NOs: 1 to 6 or a complementary sequence of the nucleotide sequence, which is specific for 16S ribosomal RNA gene of a diffuser aerobic bacterium, is used as a primer for PCR reaction. What is necessary is just the structure containing, Preferably the structure containing the reagents etc. for performing each process mentioned above is mentioned.

For example, in the judgment kit according to the present invention, the oligonucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 6 or the complementary sequence of the nucleotide sequences is the oligonucleotide (a). At least one of (b) may be included, and at least any one of oligonucleotides (c)-(f) may be included as an antisense primer. Such oligonucleotide may be in any state as long as it can be used as a primer for PCR reaction, and may be in a solution state dissolved in a buffer, for example.

The determination kit according to the present invention may include reagents for performing the above-described steps, and the like may be prepared by preparing a reagent group for PCR reaction, a reagent group for confirming DNA fragment amplification, or nucleic acid from flap bacteria. And a reagent group for culturing the flap bacilli. These reagent groups should just contain at least 1 type. In addition, the reagent group here refers to what contains two or more types of reagents used for the said process, an experimental material, or a disposable laboratory apparatus.

Specifically as a reagent group for PCR, dNTP, Taq polymerase, Taq polymerase buffer (Taq Buffer), etc. are mentioned specifically ,. In addition, you may attach experimental materials, such as a micro centrifuge tube, as needed.

As a reagent group for confirming DNA fragment amplification, when amplification is confirmed by gel electrophoresis, agarose, a buffer for preparing an electrophoresis gel, a buffer, a marker for gel electrophoresis, and the like can be given. Among these, the gel electrophoresis marker may be a DNA fragment mixture containing several kinds of indicator DNA fragments having different lengths, but the DNA fragment mixture should contain an indicator DNA fragment having a size of 0.1 kb. As described above, since the specific DNA fragment as an indicator of determination has a size of about 100 bp, the marker for gel electrophoresis includes an index DNA fragment which can clearly know the band of this size in electrophoresis. It is because it is more preferable. In addition, the origin of the DNA fragment used for the gel electrophoresis marker is not specifically limited.

In addition, a well-known DNA preparation kit is mentioned as a reagent group which prepares nucleic acid from a bacteriostatic bacterium, and a medium or its raw material is mentioned as a reagent bacterium for cultivating a bacteriostatic bacterium.

(4) Use of the present invention

The use of the oligonucleotide, the determination method using the same, and the determination kit according to the present invention are not particularly limited, and can be used for all applications requiring determination of a diffuser aerobic bacteria. Specifically, for example, in the manufacturing process of various industrial products in which contamination by microorganisms greatly affects the quality, it is determined whether the bacteria isolated from the industrial product or its production environment or the like are diffuser aerobic bacteria. It is suitably used for.

The industrial products derived from the bacteriostatic bacterium that is the target of determination include, but are not particularly limited to, foods, beverages, medicines, reagents, quasi-drugs, disposable medical instruments, and the like. As the above-mentioned foods, more specifically, bread, Western confectionery (including ice cream, etc.), side dishes, dairy products, cereal products, tofu and milk products, some noodles, lunch boxes, seasonings, agricultural products such as flour and edible meat, nutritional supplements ( Supplements, etc.), long-term stored foods (canned food, frozen foods, retort foods, etc.), but is not particularly limited. Examples of the beverage include, but are not particularly limited to, various soft drinks, mineral water, milk / milk drinks, sake, beer, effervescent liquor, and the like. Specific examples of the medicine, reagent, and quasi-drug include, but are not particularly limited to, various medicines for doctors, various popular medicines, clinical test drugs, laboratory reagents, and other test drugs. As a disposable medical apparatus, although a needle etc. are mentioned specifically, it is not specifically limited.

In the production facilities of industrial products, airborne bacteria, surface-adhesive bacteria, and other bacteria can all be flap bacteria. For the separation of airborne bacteria, methods such as a dropping bacterium test method and an air sampler method can be used, but are not particularly limited. Although the smear test method, the stamp agar method, etc. can be used for isolation | separation of surface adhesion bacteria, It is not specifically limited.

In addition, this invention is not limited to each structure mentioned above, Various changes are possible in the range shown by a claim, and embodiment obtained by combining suitably the technical means disclosed respectively in other embodiment is also the technical scope of this invention. Included in

Hereinafter, the present invention will be described in more detail with reference to Examples 2 and 3. However, the present invention is not limited to this.

Example 1 Assay of Diffuser Aerobic Bacterial Detection of Primer

Whether to detect a diffuser aerobic bacteria and other bacteria by using a primer designed by searching for a gene sequence specific for the 16S ribosomal RNA gene of the Bacillus genus and related diffuser aerobic bacteria. Rated it.

<Sample Preparation Process>

Genus Bacillus, Paenibacillus, Aneurinibacillus, Aricyclobacillus, and Bacillus, Bacillus Forming bacteria were used. The bacteria used as the specimens are listed in Table 1. Genomic DNA was prepared using the Genomic DNA Purification Kit (manufactured by Edge Biosystems, Inc.) according to the attached protocol from the strains incubated under optimal culture conditions for each of these strains.

TABLE 1

<Bacillus genus bacteria> 1.Bacillus subtilis (IFO13719) 2.B. sphaerius (NBRC15095) 3.B. cereus (IAM12605) 4.B. coagulans (IAM12463) 5.B. megaterium (JCM2506) <A diffuser aerobic bacterium related to the genus Bacillus> 6.Paenibacillus validus (BUB75) 7.Paenibacillus illinoisensis (IFO15379) 8.Aneurinibacillus aneurinolyticus (IAM1077) 9.Alicyclobacillus acidoterrestris (ATCC49025) 10.Alicyclobacillus acidocaldarius (JCM5260) <Apo-forming bacteria> 11. Staphylococcus aureus (ATCC9144) 12. Staphylococcus capitis (ATCC27840) 13. Streptococcus salivarius (SAM0180) 14. Lactobacillus delbrueckii (IAM1928)

Primer Design and Synthesis

Genus Bacillus and its related diffuser aerobic bacteria from the database (BLAST) (eg Paenibacillus genus, Aneurinibacillus genus, Geobacillus genus, 16S ribosomal RNA gene arrays of the genus Alicyclobacillus, Brevibacillus, etc.) and apo-forming bacteria were extracted and aligned using a gene analysis software called AlignX of VectorNTI. Relatively common areas were chosen for aerobic bacteria. Among them, the sequences of SEQ ID NOs: 1 to 6 were selected, and oligonucleotides having the same sequence as those were chemically synthesized and used as primers. Hereinafter, each primer is called oligonucleotide (a)-(f), respectively.

<PCR process / DNA fragment amplification confirmation process>

Using the said genomic DNA as a template, the sample shown in Table 2 was prepared using the said oligonucleotide (a)-(f), and PCR reaction was performed. In addition, PCR reaction is performed using T GRAGIENT made from Biometra, and PCR conditions are as shown in Table 3. After completion of the PCR reaction, analysis was carried out according to the protocol attached to the Agilent 2100 Bioanalyzer (manufactured by Agilent Technologies, Inc.) to confirm the amplification band of the DNA and the size of the band.

<Result>

The results of electrophoresis by the bioanalyzer are shown in FIG. In Figure 2 lane 1 is the molecular weight marker, lane 2 is the result of Bacillus subtilis (IFO13719), lane 3 is the result of B. sphaericus (NBRC15095), lane 4 is the result of B. cereus (IAM12605), lane 5 is B. coagulans As a result of (IAM12463), lane 6 is the result of B. megaterium (JCM2506), lane 7 is the result of Paenibacillus validus (BUB75), lane 8 is the result of P. illinoisensis (IF015379), lane 9 is Aneurinibacillus aneurinolyticus (IAM1077) Lane 10 is the result of Alicyclobacillus acidoterrestris (ATCC49025), lane 11 is the result of A. acidocaldarius (JCM5260), lane 12 is the result of Staphylococcus aureus (ATCC9144), lane 13 is the result of Staphylococcus capitis (ATCC27840), lane 14 Shows the results of Streptococcus salivarius (SAM0180), and lane 15 shows the results of Lactobacillus delbrueckii (IAM1928), respectively.

The band was detected only around 100 bp by the diffuser aerobic bacteria shown in lanes 2 to 11 of FIG. 2, and in the lanes 12 to 15 showing the results of bacteria other than the diffuser aerobic bacteria, the band was not detected around 100 bp. Therefore, the DNA fragment having a size of about 100 bp is specific for the diffuser aerobic bacteria, and it is easy to determine whether the DNA fragment is a diffuser aerobic bacterium by using the presence or absence of the DNA fragment as an index.

Example 2: Assay of Unidentified Bacteria Isolated from Food Environment

The validity of the detection and determination method of the diffuser aerobic bacterium according to the present invention is determined by comparing the result of the identification of the diffuser aerobic bacterium with the presence or absence of a DNA fragment of 100 bp as an index for the unidentified bacterial strain. I decided to review.

<Sample Preparation Process>

Two strains of unidentified bacteria (called No. 1 and No. 2) isolated from the food production environment were cultured on tryticase soy agar medium (BBL Co.) at 35 ° C. under aerobic conditions. One colony of the cells on the medium was collected, DNA was collected using the Genomic DNA Purification Kit (manufactured by Edge Biosystems, Inc.) according to the attached protocol, and this was used as a specimen (sample preparation step).

PCR process and DNA fragmentation amplification confirmation process

Using the sample liquid as a template, the samples shown in Table 2 were prepared using the oligonucleotides (a) to (f) described above, and PCR was performed. In addition, PCR was performed using T GRAGIENT made from Biometra, and PCR conditions were as shown in Table 3. After completion of PCR, the Agilent 2100 bioanalyzer (manufactured by Agilent Technologies, Inc.) was analyzed in accordance with the attached protocol to confirm the amplification band of DNA and the size of the band.

TABLE 2

                                                             Μl Genomic DNA 2 sense primers (primers 1 and 2) 0.4 Antisense primers (primers 3, 4, 5, 6) 0.4 dNTP 2 x 10 Taq Buffer 2 Taq polymelase 0.5 Water 12.7

TABLE 3

 Temperature (℃) Time (seconds) Cycle Count

<Result>

The results of electrophoresis are shown in FIG. In FIG. 3, lane 1 shows the molecular weight marker, lane 2 shows the result of unidentified bacterial strain No. 1, and lane 3 shows the result of unidentified bacterial strain No. 2, respectively.

No. In 1, a band was detected around 100 bp, and it was determined that the bacterium was a diffuser aerobic bacterium. In addition, when observed under a microscope, the formation of apo was confirmed. The bacteria were identified using Api50CHB (manufactured by Bio Merieux Co., Ltd.) according to the attached protocol, and identified as Bacillus licheniformis, a diffuser aerobic bacterium. Therefore, it was consistent with the result of the judgment made using the band of 100bp as an index.

Meanwhile, No. In 2, the band was not detected around 100bp, and it was judged that it is not a diffuser aerobic bacterium. The bacterium was identified by performing a partial nucleotide sequence of the 16S ribosomal RNA gene and found to be a Methylobacterium genus rather than a diffuser aerobic bacterium. Therefore, No. Consistent with the result of judging using the band of 100 bp as an index for 2, it was found that a method of detecting and determining whether or not it is a diffuser aerobic bacterium using the presence or absence of a band around 100 bp as an index was found to be valid.

Based on the above results, amplification reactions, such as PCR amplification, were carried out using the oligonucleotides (a) to (f) according to the present invention to determine whether the bacteriostatic bacterium was amplified by specific DNA fragments (about 100 bp). It is possible to easily determine whether or not the diffuser is an aerobic bacterium. That is, the determination method according to the present invention can be said to be an effective detection and determination means of the diffuser aerobic bacteria. In addition, the kit may be provided by including the oligonucleotide, thereby providing a kit for determining a diffuser aerobic bacteria.

On the other hand, specific embodiments or examples of the invention are intended to clarify the technical details to the last, and should not be construed as limited to such specific examples only, and within the spirit of the present invention and the claims set forth below. Various changes can be made in.

Specificity present in 16S ribosomal RNA gene of Bacillus genus and related diffuser aerobic bacteria by performing amplification reaction such as PCR amplification using oligonucleotide according to the present invention as a primer and chromosomal DNA obtained from the bacteriostatic bacterium as a template DNA fragments can be amplified. As a result, whether or not the specific DNA fragment (about 0.1 kb) is amplified by the amplification reaction described above can quickly determine whether the bacteriostatic bacillus is a Bacillus genus and its associated diffuser aerobic bacterium. . Moreover, the kit for containing the said oligonucleotide can be provided and the determination kit of a diffuser aerobic bacterium can be provided.

Therefore, according to this invention, the effect which can detect the diffuser aerobic bacterium which was considered difficult until now is quick and comprehensive is achieved.

To determine whether or not it is a bacterium which forms apoptosis (a diffuser aerobic bacterium), it was necessary to cultivate the bacteriostatic bacterium on a follicle forming medium, and examine whether or not the bacterium forms follicles under a microscope. Therefore, it took quite a long time, ranging from a few days to a week, to reach a decision. According to the present invention, it is possible to determine whether or not it is a diffuser aerobic bacterium in about half a day, and it can be easily and quickly determined whether it is a diffuser aerobic bacterium.

Therefore, the present invention can be used for hygiene control, process control, and quality control in the food manufacturing industry, the food industry, the pharmaceutical industry, and the like. In addition, even when microbial contamination is detected, it is possible to respond quickly to the trouble by quickly determining whether the bacterium is a diffuser aerobic bacterium.

<110> SUNTORY LIMITED <120> Oligonucleotide used for identification of spore aerobic          bacteria, identification method and kit of spore aerobic          bacteria <130> SU0415 <150> JP 2003-346167 <151> 2003-10-03 <160> 6 <170> Patent In Ver. 2.1 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artistically Synthesized          Sequence <400> 1 acgatgagtg ctaagtgttg gggg 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artistically Synthesized          Sequence <400> 2 acgatgagtg ctaggtgttg gggg 24 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artistically Synthesized          Sequence <400> 3 ttgagtttca gtcttgcgag cg 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artistically Synthesized          Sequence <400> 4 ttgagtttca gtcttgcgac cg 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artistically Synthesized          Sequence <400> 5 ttgagtttca ctcttgcgag cg 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artistically Synthesized          Sequence <400> 6 ttgagtttca ctcttgcgac cg 22

Claims (12)

A nucleotide sequence specific for the 16S ribosomal RNA gene of a diffuser aerobic bacterium or a complementary arrangement of the nucleotide sequence, The specific nucleotide sequence is an oligonucleotide, characterized in that any one of the nucleotide sequence shown in SEQ ID NO: 1 to 6. The method of claim 1, The diffuser aerobic bacteria, Bacillus (Bacillus), Paenibacillus (Paenibacillus), Aneurinibacillus (Aneurinibacillus), Geobacillus (Geobacillus), Alicyclobacillus (Alicyclobacillus), Brevibacillus (Brevibacillus) Genus, Amphibacillus genus, Virgibacillus genus, Halobacillus genus, Graciribacillus genus, Salibacillus genus, Marinibacillus genus and Sporolactobacilli (Sporolactobacillus) oligonucleotide, characterized in that the bacteria belonging to at least one selected from bacteria. Using a nucleic acid prepared from a bacteriostatic bacterium, which is the subject of determination, as a sample, the amplification of a specific DNA fragment obtained by performing a PCR reaction using the sample as a template, as an index, determines whether the flapper bacterium is a diffuser aerobic bacterium. It is a determination method of the diffuser aerobic bacteria to judge, As the primer used for the PCR reaction, at least one oligonucleotide comprising a base sequence specific to the 16S ribosomal RNA gene of a diffuser aerobic bacterium or a complementary sequence of the base sequence is used, The specific nucleotide sequence is any one of the nucleotide sequence shown in SEQ ID NOs: 1 to 6, characterized in that the diffuser aerobic bacteria determination method. The method of claim 3, wherein Of the primers, the base sequence shown in SEQ ID NO: 1 or 2, or an oligonucleotide comprising a complementary sequence of the base sequence is used as the sense primer, A method for determining a diffuser aerobic bacterium, wherein an nucleotide sequence represented by any one of SEQ ID NOs: 3 to 6 or an oligonucleotide comprising a complementary sequence of the base sequence is used as an antisense primer. The method according to claim 3 or 4, At least one of the sense primer and the antisense primer, a method for determining a diffuser aerobic bacterium, characterized in that at least two oligonucleotides comprising the specific base sequence or the complementary arrangement of the base sequence is used. The method according to any one of claims 3 to 5, The specific DNA fragment to be amplified has a size detected in the vicinity of 0.1 kb by separation by gel electrophoresis, characterized in that the diffuser aerobic bacteria determination method. The method according to any one of claims 3 to 6, The diffuser aerobic bacteria, genus Bacillus, genus Paenibacillus, genus Anuriinibacillus, genus Bacillus, Geobacillus, Aricyclobacillus, Alicyclobacillus, Brevibacillus Genus, genus amphibacillus, genus Virgibacillus, genus Harobacillus, genus Grachiba bacillus, genus Salibacillus, genus Marinibacillus and sporolactobacillus (Sporolactobacillus) A method for determining a diffuser aerobic bacterium, characterized in that the bacteria belong to at least one species selected from among the bacteria. A judgment kit for performing the method of determining the diffuser aerobic bacteria according to any one of claims 3 to 7. Using the nucleic acid prepared from the target bacterium as the target of the judgment as a sample, the amplification of a specific DNA fragment obtained by performing a PCR reaction using the sample as a template is used as an index to determine whether the bacteriostatic bacterium is a diffuser aerobic bacterium. Judgment kit used to judge, A primer used for a PCR reaction, comprising a base sequence specific for a 16S ribosomal RNA gene of a diffuser aerobic bacterium or a complementary sequence of the base sequence, wherein the specific base sequence is shown in SEQ ID NOs: 1 to 6 Diffuser aerobic bacteria determination kit comprising at least one oligonucleotide which is any one. The method of claim 9, It is used as a marker for gel electrophoresis and further comprises a DNA fragment mixture containing several species of indicator DNA fragments of different lengths, The DNA fragment mixture comprises a DNA fragment for indicator having a size of 0.1kb, diffuser aerobic bacteria determination kit, characterized in that. The method according to claim 9 or 10, Diffuser aerobic bacteria determination kit, characterized in that it further comprises a reagent group for preparing nucleic acid from the bacteriostatic bacterium. The method according to any one of claims 9 to 11, Diffuser aerobic bacteria determination kit further comprises a reagent group used for the PCR reaction.

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