KR20210033369A - Development of primer for genetic identification and identification method to distinguish Bacillus subtilis and closely related Bacillus species - Google Patents

Abstract

The present invention relates to: a primer for molecular identification manufactured by using a DNA helicase RecQ gene base sequence; an identification method of Bacillus subtilis and allied species thereof comprising a step of manufacturing a primer pair for identification by using the DNA helicase RecQ gene base sequence, a step of amplifying the gene by performing PCR using the primer pair for identification, and a step of identifying Bacillus subtilis by comparing the amplified gene base sequences; and a kit for identifying Bacillus subtilis and allied species thereof using the identification method.

Description

Primers for molecular identification of Bacillus subtilis and closely related Bacillus species using the primers {Development of primer for genetic identification and identification method to distinguish Bacillus subtilis and closely related Bacillus species}

The present invention is related to ensuring clear identification and safety of microbial species by comparing the RecQ gene sequence amplified by PCR (Polymerase Chain Reaction).

Bacillus subtilis is a microorganism that produces various hydrolytic enzymes and functional substances, and is acting as a key fermenting microorganism in manufacturing fermented foods in East Asia including Korea and Japan.

Many companies and research institutes are carrying out selection of excellent Bacillus subtilis strains for the production of Korean cheonggukjang and meju and Japanese natto, and many strains with high industrial value are protected through patents and distributed in the market.

Since Bacillus subtilis and its related species are genetically similar, it is difficult to clearly classify them by biochemical analysis and analysis using 16S ribosomal nucleotide sequence (rRNA nucleotide sequence). Therefore, it takes a lot of time and money to accurately classify. Therefore, for Bacillus subtilis and its related species, which are difficult to accurately identify, a simple and accurate identification technology that can complement the existing identification methods including the 16S ribosomal sequence is required, and at the same time, a reliable alternative to genome analysis is required. It is necessary to develop a technology that can be done.

The present invention provides a primer for identification prepared using a DNA helicase (helicase) RecQ gene sequence.

The identification primer is a forward or reverse primer, and in the case of a forward primer, the sequence of the identification primer is 5'-TTA CAT YAC ACC RGA GCG-3', and in the case of a reverse primer, the sequence of the identification primer is 5'- MGT CGG AAA YGT KCC RTC-3'.

The present invention provides a step of preparing a primer pair for identification using a DNA helicase RecQ gene sequence, amplifying a gene by performing PCR using the primer pair for identification, and the amplified gene nucleotide sequence. By comparison, it provides a method for identifying a related species of Bacillus subtilis and a related species thereof, including the step of identifying a related species of Bacillus subtilis.

In one embodiment of the present invention, the primer is a forward or reverse primer, in the case of a forward primer, the sequence of the primer is 5'-TTA CAT YAC ACC RGA GCG-3', and in the case of a reverse primer, the sequence of the primer is 5'-MGT CGG AAA YGT KCC RTC-3'.

In an embodiment of the present invention, the Bacillus subtilis and its related species are Bacillus subtilis subtilis subspecies (Bacillus subtilis subsp. subtilis ), Bacillus subtilis stercoris subspecies (Bacillus subtilis subsp. stercoris). , Bacillus subtilis subsp. inaquosorum , Bacillus subtilis subsp. spizizenii , Bacillus tequilensis , Bacillus vallismortis , Bacillus mojavensis , Bacillus velezensis , Bacillus siamensis , Bacillus atrophaeus , Bacillus sonorensis , Bacillus sonorensis, Bacillus sonorensis It may be at least one of Miss ( Bacillus licheniformis ) and Bacillus cereus (Bacillus cereus ).

An embodiment of the present invention provides a kit for identification of Bacillus subtilis and a related species thereof provided with a primer pair for identification including a DNA helicase RecQ gene sequence.

In one embodiment of the present invention, the kit for identification of Bacillus subtilis and its relative species may further include a PCR amplifier for amplifying the RecQ gene sequence.

By using a pair of primers for identification, it is possible to quickly detect Bacillus subtilis and related microorganisms thereof and to perform a systematic analysis through sequence comparison.

It is possible to select a Bacillus strain that can be used as a food raw material and secure the stability of fermented foods through accurate identification of Bacillus subtilis and its related species.

It is possible to ensure the clear identification and safety of species for microbial agents used as eco-friendly microbial materials.

1 shows the sequence position and sequence variation of a pair of primers for identification produced from the recQ gene region of Bacillus subtilis and its related species.
2 is a result of PCR and electrophoresis of Bacillus subtilis and its related species using a primer pair for identification.
3 is a result of creating a schematic diagram through analysis of the gene sequence of the amplified PCR product.
Figure 4a is a result of a univariate linear analysis between the homology of the genome (ANI value) and 16S ribosomal sequence between Bacillus subtilis and its related species.
4B is a result of univariate linear analysis between the homology of the genome between Bacillus subtilis and its related species (ANI value) and the homology of the RecQ gene sequence.

In the present invention, various modifications may be made and various forms may be applied, and specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific form disclosed, it should be understood to include all changes, equivalents, and substitutes included in the spirit and scope of the present invention.

The terms, techniques, and the like described in the present specification are used with meanings generally used in the technical field to which the present invention belongs unless there is a specific limitation.

The present invention relates to a primer for molecular identification of Bacillus subtilis related species, and a method for identifying Bacillus subtilis related species using the same.

In more detail, a DNA helicase RecQ gene sequence capable of detecting Bacillus subtilis and its related species was used to prepare a primer pair for identification, and a gene by PCR using the identification primer pair. And amplified, and through sequence comparison of the RecQ gene amplified by PCR, it relates to a method of analyzing the phylogenetic relationship and the relationship between Bacillus subtilis and its related species.

Bacillus subtilis is a microorganism that produces various hydrolytic enzymes and functional substances, and is acting as a key fermenting microorganism in manufacturing fermented foods in East Asia, including Korea and Japan. Many companies and research institutes are carrying out selection of excellent Bacillus subtilis strains for the production of Korean cheonggukjang and meju and Japanese natto, and many strains with high industrial value are protected through patents and distributed in the market. However, among the more than 300 species belonging to the genus Bacillus, only three species, Bacillus subtilis and Bacillus amyloliquefaciens, are stable and can be used as food ingredients.

In the present invention, the term " Bacillus subtilis subsp. subtilis " is Bacillus subtilis (Bacillus subtilis) subspecies, the Bacillus subtilis (Bacillus subtilis) is Bacillus ( Bacillus ) Belongs to the genus.

The term "subspecies" is a group of phenotypically similar groups among a group belonging to a species, which is a unit of classification of organisms that is subdivided into a species.

The genus Bacillus is a gram-positive, spore-forming bacterium, an aerobic rod-shaped bacteria that can be grown by fermentation, protease, and amylase. It secretes useful enzymes in industrial fields such as (amylase) and cellulase, and is present in various fermented foods such as kimchi, soil, and the like.

In the present invention, the term "primer" is a nucleic acid sequence having a short free 3'hydroxyl group, which can form a base pair with a template of a complementary nucleic acid. It refers to a short nucleic acid sequence that functions as a starting point for strand copying. The primer can initiate DNA synthesis in the presence of a polymerase (ie, DNA polymerase) and a reagent for the polymerization reaction at an appropriate buffer and temperature.

In the PCR product of the present invention, various reagents necessary for confirming the experimental process and results necessary to detect Bacillus subtilis subspecies subtilis using the primer pair, such as PCR composition, restriction enzyme, agarose, hybridization and/or Buffer solutions required for electrophoresis may additionally be included.

More specifically, the PCR product includes an appropriate amount of DNA polymerase (eg, Thermus aquaticus ; Taq), Thermus thermophiles ; Tth, in addition to the target DNA and the PCR primer pair provided in the present invention. ), Thermostable DNA polymerase obtained from Thermus filiformis , Thermus flavus , Thermococcus literalis or Pyrococcus furiosus (Pfu)), dNTP, PCR buffer and water (dH ₂ O) may be included.

Initial identification was performed using a method of identifying microorganisms by observing the cell morphology of microorganisms using a microscope, and then a gram staining method, a method of identifying microorganisms through cell staining, was introduced. By using the above Gram staining method, microorganisms can be largely classified into Gram-positive bacteria and Gram-negative bacteria. When stained with Gram dye, it is gram-positive and red. When stained with color, it is a Gram-negative bacteria. In addition, whether or not oxygen is required for microbial growth is used to identify microorganisms, bacteria that require oxygen for growth are identified as aerobic bacteria, and bacteria that do not require oxygen for growth are anaerobic bacteria. Is sympathized with. In addition to the above, the growth inhibition test developed during the development of the diagnostic microorganism is a method of identifying microorganisms by adding a compound that inhibits the growth of a specific microorganism to the microorganism culture medium to check the growth of the microorganism. As a representative example, streptococci cannot be grown in a medium containing 6.5% sodium chloride, but enterococci can grow. In addition, other products can be synthesized by using the same nutrients depending on the type of microorganism, and there is also a biochemical test method that identifies microorganisms using these properties. For example, entericbacteria ferment glucose to produce acid end products, while non-fermentative bacteria do not.

Microbiologists have identified microorganisms as species, genus, family, etc. by combining the above identification methods. However, attempts have been made to identify species or genus in more detail with the development of microbiology. For example, Facklam et al. have been fermented with glucose to match the metabolism and growth characteristics of Viridans streptococci. By modifying the test (Glucose fermentation test), Viridans streptococci was identified in more detail (Facklam, RR et al., Manual of Clinical Microbiology, 5 ^th ed., 238-257, 1991).

A medium for identifying bacteria belonging to a specific family using the above-described microbial identification method is commercially available.Phenol red broth for identifying streptococci, Pre-Reduced Anaerobically for identifying anaerobic bacteria Sterilized) medium and chocolate medium, Cystine Trypticase Agar (CTA) medium and heart infusion broth to identify Fastidious microorganisms, non-fermenting Gram-negative Bacilli and intestines There are Oxidation-Fermentation (OF) medium for identifying bacteria (enterics) and yeast fermentation broth for identifying yeast. However, the above methods require a specific growth medium to identify specific bacteria, and when identifying highly correlated species, the accuracy is very low. Therefore, the long cultivation time required for culturing microorganisms has been pointed out as a disadvantage.

In addition, carbohydrate assimilation tests and fatty acid analysis are the representative identification methods that have been widely used in the past. Carbohydrate assimilation tests are a method that focuses on the fact that microorganisms can use different carbohydrates for growth. Usually, a carbohydrate assimilation profile of an experimental strain to be identified using more than 20 different substrates is used. This is a method of identifying by comparing the profile of the standard strain entered in the database. Currently, commercial systems such as Biolog and API are widely used for the analysis of carbohydrate assimilation, and by using these, various bacteria such as Gram-positive and Gram-negative can be identified, but the database is one or two species. Since it is prepared based on the profile of the standard strain of, the accuracy is poor when the first isolated strain is identified.

Fatty acid composition analysis is a method of identifying using a different fatty acid pattern for each species of microorganism. It is noted that even microorganisms belonging to the same genus have unique types and ratios of fatty acids depending on the species. It is a sympathy method developed with the idea of it. When identifying bacteria using the fatty acid analysis method, first, the fatty acids are separated from the bacteria, methylated, and then the fatty acid pattern is analyzed using gas liquid chromatography to identify the species according to the pattern. . For example, A. cumminsii , which has been identified as an Anthrobacter species, has C16:O i and C16:0 fatty acids that are not found in other species of the genus Anthrobacter as a result of fatty acid analysis. It was high and C14:O i and C14:0 fatty acids were detected and classified as a new species.

However, the identification method has a disadvantage in that the accuracy is poor because errors are easily included in the determination of the result according to the skill level of the experimenter, and accordingly, a more accurate method of identifying microorganisms has been required. As a result, an identification method was recently developed to compare the similarity by analyzing the nucleotide sequence of a specific gene. As a representative method, a 16S rRNA (Ribosomal ribonucleic acid) gene analysis method was developed and used to accurately analyze many species at the gene level. come. 16S rRNA is an RNA that forms a ribosome by interacting with various proteins. Because the complete sequence or gene sequence of the oligonucleotide list has been found in more than 10,000 bacteria, the gene similarity of 16S rRNA is On the basis of this, bacteria can be divided into several major groups. Since the rate of change of the 16S rRNA gene sequence is much less than that of other gene sequences in most genomes, it is recognized that the degree of similarity of the 16S rRNA gene sequence reflects the phylogenetic distance between organisms. The method of identifying microorganisms according to similarity by analyzing the gene sequence of the 16S rRNA gene fragment has been used as a representative identification method for identifying microorganisms, particularly industrially useful microorganisms, along with the above-described fatty acid analysis and carbohydrate magnetization assay method. .

Existing microbial identification methods, including carbohydrate magnetization assay, fatty acid assay, and 16S rRNA gene assay, are not suitable for accurate identification of Bacillus subtilis-related strains. First of all, not only the accuracy of the carbohydrate magnetization assay and the fatty acid analysis method is very low, but for the recently discovered Bacillus subtilis-related species, the commercial database has not been established, so it is not possible to accurately identify the Bacillus subtilis-related species. In addition, in the case of biochemical analysis and 16S rRNA gene analysis, the similarity of the 16S rRNA gene sequence between Bacillus subtilis-related species is 99% or more, and the variability is very low, making it impossible to use for identification. Due to these problems, there is a problem that microorganisms that cannot be used as food are used in food, or Bacillus subtilis and its related strains, which are patented and academically reported, are misidentified.

In order to accurately identify Bacillus subtilis-related strains, it is necessary to use gene sequences that have been relatively well preserved while being distributed at comparable phylogenetic distances in addition to biochemical analysis and 16S rRNA genes. Genes that can be used for this purpose include proton transfer ATPase (ATPase; Proton-traslocating ATPase), DNA polymerase, RNA polymerase, histone-like protein, and ferredoxin. (ferredoxin), DNA gyrase, and DNA helicase.

Among the genus Bacillus, 7 species including Bacillus subtilis, Bacillus valis motis, and Bacillus amyloliquefaciens are registered in the list of fertilizer standards, and other Bacillus genus are restricted as fertilizers. Therefore, due to the development of a method for accurate classification and identification of Bacillus subtilis and its related species, it is possible to reduce the cost for fertilizer registration according to the accurate classification identification of some of the Bacillus genus, and to use Bacillus species with secured safety. Can be utilized.

In the present invention, a primer pair that binds to the specific sequence of the DNA helicase RecQ gene sequence of Bacillus subtilis and its related species is prepared, and the Bacillus subtilis and its related species are selected through PCR analysis using the same. At the same time, it provides an identification method that can be classified at the subspecies level through gene sequencing analysis using the corresponding PCR product.

In order to prepare a primer pair for detecting Bacillus subtilis and its related species, the RecQ gene sequence of Bacillus subtilis and its related species is secured. For example, the RecQ gene sequence of Bacillus subtilis and its related species can be obtained from the genomes of standard strains registered in the NCBI (National Center for Biotechnology Information) database.

The obtained gene sequences are multi-sequenced. In this multi-sequencing process, multi-sequencing software can be used. The software may be ClusterW. Conserved regions that match from the aligned gene sequence are identified, and a region suitable for making a primer pair among the gene sequences of the corresponding region is identified. Finally, a primer pair having a size of 18 bp that can detect both Bacillus subtilis and its related species is prepared, and the positions and sequences of the designed primer pair are indicated.

PCR analysis was performed to obtain a PCR product for specific detection and gene sequencing of Bacillus subtilis and its related species using the prepared identification primer pair. As the experimental strain, a standard strain included in the genus Bacillus can be used.

The gene sequence of the obtained PCR product is decoded, and the decoded gene sequence is aligned with the RecQ gene sequence obtained from the genome of Bacillus strains obtained from the NCBI database. Based on this, it is possible to create a genealogy related to the aligned gene sequence. To increase reliability, for example, the following analysis methods (Neighbor-joining, Maximum-parsimony, Maximum-likelihood) can be used. . The topology of the tree can be bootstrapped 1,000 times.

However, the analysis method and the schematic diagram are not limited to the number of bootstraps of the topology.

It can be confirmed whether or not all species, including subspecies, are systematically branched through a phylogenetic diagram using the RecQ gene sequence obtained from Bacillus subtilis and its related species.

By analyzing the homology between the genomes of Bacillus subtilis and its related species through the average genome sequence similarity (Average Nucleotide Identity, ANI) analysis, correlation analysis is performed to determine how accurately the RecQ gene homology reflects the lineage relationship. The ANI analysis value, 16S ribosomal sequence homology value, and RecQ gene sequence homology value are compared through univariate linear analysis.

However, the nucleotide sequence similarity is not limited to the analysis method.

DNA helicase RecQ [EC: 3.6.4.12] enzyme is an enzyme that repairs damaged gene sequences along with other DNA helicase enzymes. Most microorganisms play an important role in repairing DNA damage in the cell cycle. It possesses this enzyme, and the sequence encoding this enzyme, like other housekeeping genes including the 16S ribosomal nucleotide sequence, undergoes the same molecular evolution as the lineage. similar.

The RecQ gene sequence encoding Bacillus subtilis and its related species RecQ enzyme was confirmed to be molecularly evolved at a faster rate than the 16S ribosomal nucleotide sequence or other housekeeping genes based on comparative genomic analysis results. , It can be seen that the result of phylogenetic analysis using this gene sequence is similar to the result of the average genome sequence average similarity (ANI) analysis, which is the most reliable method among species classification methods.

<Experimental Example 1: Preparation of a pair of primers for detection of Bacillus subtilis and its related species>

First, a pair of primers for detecting Bacillus subtilis and its related species was prepared. The base sequence of the RecQ gene of Bacillus subtilis and its related species was obtained from the genomes of standard strains registered in the NCBI database as shown in Table 1.

Table 1 is a table showing the base sequence of the RecQ gene of Bacillus subtilis and its related species.

Species name Strain name Accession No. Bacillus amyloliquefaciens KACC 12067 ^T GCA_000196735 Bacillus atrophaeus KACC 12090 ^T GCA_001591925 Bacillus mojavensis KACC 12096 ^T GCA_000245335 Bacillus siamensis KACC 16244 ^T GCA_000262045 Bacillus subtilis subsp . inaquosorum KACC 19623 ^T GCA_000332645 Bacillus subtilis subsp . spizizenii KACC 14394 ^T GCA_000227465 Bacillus subtilis subsp . stercoris KACC 19726 ^T GCA_000738015 Bacillus subtilis subsp . subtilis KACC 10854 ^T GCA_000009045 Bacillus tequilensis KACC 19682 ^T GCA_000507145 Bacillus vallismortis KACC 12149 ^T GCA_000245315 Bacillus velezensis KACC 19683 ^T GCA_001461825 Bacillus halotolerans ATCC 25096 ^T GCA_001517105 Bacillus nakamurai NRRL B-41091 ^T GCA_001584325 Bacillus licheniformis ATCC 14580 ^T GCA_000011645

The obtained gene sequences were aligned using ClusterW, and conserved regions that matched more than 80% from the aligned gene sequences were identified, and suitable for making a primer pair among the gene sequences of the corresponding region. Confirmed the area. Finally, a primer pair having a size of 18 bp capable of detecting both Bacillus subtilis and its related species was prepared, and the positions and sequences of the designed primer pairs are shown in FIGS. 1 and 2.

<Experimental Example 2: PCR performed using the prepared primer pair>

Table 2 is information on primers for identification to be subjected to PCR analysis.

Sequence number Primer name Primer direction Primer sequence (18 bp) Amplification size One recQ336F Forward Sense 5’-TTA CAT YAC ACC RGA GCG-3’ 1,123 bp 2 recQ1458R Reverse Antisense strand 5’-MGT CGG AAA YGT KCC RTC-3’

Using the identification primers shown in Table 2, PCR analysis was performed to obtain a PCR product for specific detection and gene sequencing of Bacillus subtilis and its related species. As an experimental strain, a standard strain included in the 13 kinds of Bacillus genus shown in Table 3 was used.

1, in the primer sequence of the recQ336F primer shown in Table 2, Y represents T or A, and R represents A or G. In the primer sequence of the recQ1458R primer, M represents A, T or C, Y represents C, T, G or A, K represents T, A, G or C, and R represents G or A.

Table 3 shows the standard strains of the genus Bacillus to be used in the experiment.

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(Lane) Species name Strain name PCR reaction One Bacillus subtilis subsp . subtilis KACC 10854 ^T + 2 Bacillus subtilis subsp . stercoris KACC 19726 ^T + 3 Bacillus subtilis subsp . inaquosorum KACC 19623 ^T + 4 Bacillus subtilis subsp . spizizenii KACC 14394 ^T + 5 Bacillus tequilensis KACC 19682 ^T + 6 Bacillus vallismortis KACC 12149 ^T + 7 Bacillus mojavensis KACC 12096 ^T + 8 Bacillus velezensis KACC 19683 ^T + 9 Bacillus siamensis KACC 16244 ^T + 10 Bacillus atrophaeus KACC 12090 ^T -(Other products) 11 Bacillus sonorensis KACC 12102 ^T -(Other products) 12 Bacillus licheniformis KACC 10476 ^T -(Non-generated) 13 Bacillus cereus KACC 11240 ^T -(Non-generated)

For PCR amplification reaction, 10 μL of a PCR reaction solution (EmeraldAmp PCR Master Mix, Takara) containing DNA polymerase and dNTP, 8 μL of distilled water, 1 μL of template DNA (10 ng/μL), and 0.5 μL of each primer pair (100 pmol/μL) were added. Thus, a reaction product was prepared so that a total of 20 μL was obtained. PCR amplification reaction proceeds by repeating the process of initial denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, binding at 52°C for 30 seconds, stretching at 72°C for 1 minute 30 times, and finally stretching at 72°C for 5 minutes. did.

In order to confirm the PCR product, the reaction product was loaded on a 1.5% (w/v) agarose gel, placed in an electrophoresis device filled with 0.5% (v/v) TAE buffer solution, and subjected to electrophoresis at 125V voltage, followed by UV light. It was irradiated with a light-emitting device (UV illuminator) to confirm the formation and size of the PCR product. To confirm the size of the PCR product, a 1kb+ (ladder marker) ladder marker was used. In Table 3, the strains subjected to PCR and electrophoresis and whether the electrophoresis PCR product was generated are indicated, and FIG. 2 shows the electrophoresis results of the standard strains.

As shown in FIG. 2, 4 types of Bacillus subtilis and 5 types of Bacillus subtilis were confirmed to have a band of about 1,100 bp, and other Bacillus except this Bacillus subtilis and its related species were recQ The PCR product in which the gene sequence was amplified was not detected. Therefore, this primer pair was shown to specifically amplify Bacillus subtilis and its related species under the corresponding PCR conditions.

<Experimental Example 3: Comparison of gene sequence using PCR products>

The gene sequence of the obtained PCR product was decoded, and the decoded gene sequence was aligned using ClusterW together with the RecQ gene sequence obtained from the genomes of Bacillus strains obtained from the NCBI database. A system diagram related to the aligned gene sequence was created as shown in FIG. 3, and in order to increase the reliability, three analysis methods (Neighbor-joining, Maximum-parsimony, and maximum-Rye Creep (Maximum-likelihood)) was used, and the topology of the tree was performed 1,000 times bootstrap.

As a result of creating a phylogenetic tree using the RecQ gene sequence obtained from Bacillus subtilis and its related species, it was confirmed that all species including subspecies were systematically branched clearly.

The homology of the recQ gene sequence between species was 66.2-96.6%, indicating that the mutation rate of the gene sequence was higher than that of the 16S ribosomal gene sequence (98.5-99.9%). From these results, it was found that the identification using the recQ gene sequence was more accurate than the identification using the 16S ribosomal nucleotide sequence.

<Experimental Example 4: Analysis of average similarity of genome sequence>

Analyze the homology between the genomes of Bacillus subtilis and its related species through the analysis of the average genome sequence (Average Nucleotide Identity, ANI), and correlate how accurately the RecQ gene homology reflects the lineage relationship. The ANI analysis value, 16S ribosomal sequence homology value, and RecQ gene sequence homology value were compared through univariate linear analysis. 4A and 4B are results of univariate linear analysis between the homology of the genome between Bacillus subtilis and its related species (ANI value) and the homology of the 16S ribosomal sequence and the RecQ gene sequence.

The results of this analysis show that the identification using the recQ gene is almost identical to the identification using the genome (R ² =0.98).

Claims (12)

DNA helicase (helicase) Bacillus subtilis containing the RecQ gene nucleotide sequence and primers for identification of related species thereof. The method of claim 1,
The identification primer is a forward primer, and the gene nucleotide sequence of the identification primer is 5'-TTA CAT YAC ACC RGA GCG-3' Bacillus subtilis and a primer for identification of a related species thereof. The method of claim 1,
The gene base sequence of the identification primer is 5'-MGT CGG AAA YGT KCC RTC-3' Bacillus subtilis and a primer for identification of related species thereof. Preparing a primer pair using a DNA helicase RecQ gene sequence;
Amplifying a gene sequence by performing PCR using the primer pair; And
A method for identifying Bacillus subtilis and related species thereof, comprising the step of identifying a related species of Bacillus subtilis by comparing the amplified gene sequences. The method of claim 4,
The primer is a forward primer, and the gene base sequence of the primer is 5'-TTA CAT YAC ACC RGA GCG-3' Bacillus subtilis and a method for identifying a related species thereof. The method of claim 4,
The primer is a reverse primer, and the gene base sequence of the primer is 5'-MGT CGG AAA YGT KCC RTC-3' Bacillus subtilis and a method for identifying a related species thereof. The method of claim 4,
The Bacillus subtilis and its related species are Bacillus subtilis subtilis subspecies (Bacillus subtilis subsp. subtilis ), Bacillus subtilis stercoris subspecies (Bacillus subtilis subsp. (Bacillus subtilis subsp. inaquosorum), Bacillus subtilis's Fiji Jenny subspecies (Bacillus subtilis subsp. spizizenii), Bacillus Te kwiren system (Bacillus tequilensis), Bacillus Bali's motiseu (Bacillus vallismortis), Bacillus hats Ben sheath (Bacillus mojavensis) , Bacillus velezensis , Bacillus siamensis , Bacillus atrophaeus , Bacillus sonorensis , Bacillus licheniformis and Bacillus licheniformis ( Bacillus cereus ) Bacillus subtilis at least one of and a method for identifying a related species thereof. The method of claim 4,
Bacillus subtilis and its related species identification method for systematically identifying Bacillus subtilis and its related species by constructing the primer pair using a gene sequence. DNA helicase (helicase) Bacillus subtilis provided with a primer pair for identification containing the RecQ gene nucleotide sequence and a kit for identification of related species thereof. The method of claim 11,
The identification primer is a forward primer, and the gene nucleotide sequence of the identification primer is 5'-TTA CAT YAC ACC RGA GCG-3' Bacillus subtilis and a kit for identification of related species thereof. The method of claim 11,
The identification primer is a reverse primer, and the gene base sequence of the identification primer is 5'-MGT CGG AAA YGT KCC RTC-3' Bacillus subtilis and a kit for identification of related species thereof. The method of claim 11, 12 or 13,
The kit for identification of Bacillus subtilis and related species thereof is a kit for identification of Bacillus subtilis and related species thereof further comprising a PCR amplifier for amplifying the RecQ gene sequence.

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