JPH0994100A - Bacteria identification and kit therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly and accurately identify bacteria based on, the amplification of a specific DNA, as indicator by applying PCR method to a chromosomal DNA extracted from bacteria to be identified by using a DNA with specific base sequences present in the 16S ribosome RNA of the bacteria as a primer. SOLUTION: This method is to identify bacterial genus, using the amplification of a specific DNA, as indicator, by applying PCR method to a chromosomal DNA extracted from bacteria to be identified as a template and using a DNA having specific base sequences in the 16S ribosome RNA gene of the bacteria as a primer. The base sequences, specific to Brevibacillus, Aneurinobacillus, Paenibacillus and Thermophilic-Bacillus, respectively, are expressed by respective formulas, I, II, III and IV. The chromosomal DNA extracted from bacteria to be identified is amplified by PCR method, and using the resultant DNA amplified, the genus or group (cluster) of the target bacteria is identified quickly and accurately.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a bacterial identification method and kit. In recent years, microorganisms have been widely used in various industrial fields, and for example, they have an important position as production bacteria in the production of antibiotics and physiologically active substances in the pharmaceutical industry and in the production of enzymes and the like in the detergent and food industries. The classification and identification of the producing bacteria is an important condition in developing pharmaceuticals for industrial use. It is also an essential item for obtaining patents and the like, and it is very useful to be able to do this accurately and quickly in the industrial field utilizing microorganisms.

[0002]

2. Description of the Related Art Conventionally, as a method for classifying and identifying bacteria, phenotypic traits (morphology and physiological properties), quinone composition,
Fatty acid composition, cell wall amino acid composition, DNA base composition, D
Methods such as measurement of NA homology and analysis of the nucleotide sequence of 16S ribosomal RNA gene are known.

However, it is very complicated and time-consuming to determine the attribution by combining these methods and comparing the result with a reference strain (type strain), and it is highly skilled in technique and judgment. There were drawbacks such as the need for specialized engineers.

Recently, the genus of bacteria is 16S ribosomal RNA.
A group (cluster) obtained by systematic classification based on the nucleotide sequence of a gene has been used as an index for determination. Ash et al., 16S ribosomal R of Bacillus
As a result of systematic classification based on the nucleotide sequence of NA gene, B. pol
A new genus P for the group consisting of 11 species including ymyxa
We proposed aenibacillus, and as a method for identifying this genus, we prepared a single-stranded probe of 20 bases based on the specific nucleotide sequence present in the 16S ribosomal RNA gene, and slotted this with the 16S ribosomal RNA gene cloned from bacteria.
We reported a method of hybridizing with blot (Ash et al.,
Autonie van Leeuweuhoek, 84 , 253-260 (1993)). This method made it possible to identify the genus Paenibacillus without comparing the entire sequence of 16S ribosomal RNA genes. However, in this method, it takes time to set conditions such as hybridization time, temperature, membrane washing and buffer composition, and
There were drawbacks such as the need to clone the ribosomal RNA gene and the expensive reagents for slot blots.

[0005] Therefore, the development of a new system capable of simply and accurately identifying bacteria, such as determining the genus of bacteria, grouping, for example, determining groups (or clusters) that do not reach the genus of bacteria, has been developed in the industry. Was strongly sought after.

[0006]

SUMMARY OF THE INVENTION The present invention has been made for the purpose of meeting the above strong demands of the industry.

[0007]

Means for Solving the Problems The present invention has been made in order to achieve the above-mentioned object, and as a result of investigations from various directions, a classification method using the nucleotide sequence of 16S ribosomal RNA gene as an index Focusing on its effectiveness as a classification and identification method, the present inventors have conducted diligent research, and as a result, have found a DNA having a nucleotide sequence specific to a bacterial genus or group (cluster) present in the 16S ribosomal RNA gene. By applying the PCR method using the chromosomal DNA obtained from the bacteria to be identified as a template as a primer, it is possible to determine the bacterial genus or group (cluster) depending on whether or not the specific DNA containing the base sequence of the primer used is amplified. Based on the finding that there is, the present invention has been completed.

The present invention is directed to bacterial 16S ribosomal RNA.
DNA having a specific nucleotide sequence for each genus or group (cluster) of bacteria existing in the gene was used as a primer, and chromosomal DNA obtained from the bacteria to be identified was used as a template for P
The present invention relates to a bacterial identification method characterized by applying the CR method and determining the bacterium genus or group (cluster) using the amplification of specific DNA containing the base sequence of the primer used as an index.

The present invention also relates to the genus Brevibacillus, Aneur.
inobacillus, Paenibacillus and Thermophilic B
16S ribosomal RN of bacteria belonging to acillus group
Using the DNA having a specific nucleotide sequence present in the A gene as a primer, the PCR method is applied using the chromosomal DNA obtained from the bacteria to be identified as a template, and the amplification of a specific DNA containing the nucleotide sequence of the primer used as an index The bacteria to be identified
Brevibacillus, Aneurinobacillus, Paenibacillus
The present invention relates to a bacterial identification method for determining whether a genus, Thermophilic Bacillus group, or a bacterial genus or group (cluster) other than them. Furthermore, the present invention relates to a bacterial identification kit which uses as a primer a DNA having a specific base sequence present in the bacterial 16S ribosomal RNA gene.

In the present invention, a DNA having a specific base sequence present in the 16S ribosomal RNA gene of each bacterium or group (cluster) is used as a primer, and chromosomal DNA obtained from the bacterium to be identified is used as a template for PC.
Since it is possible to determine the bacterial genus or group (cluster) by simply applying the R method and determining whether or not a specific DNA containing the base sequence of the primer used is amplified,
The bacteria can be identified very simply, more quickly and more accurately based on the nucleotide sequence of the 16S ribosomal RNA gene.

To carry out the present invention, 16S ribosomal R
Although it is necessary to identify a nucleotide sequence specific to the bacterial genus or group (cluster) existing in the NA gene, a known method may be used. For example, first, several species of the bacterial genus or group (cluster) may be identified. The chromosomal DNA is extracted from the reference strain belonging to, and the 16S ribosomal RNA gene is cloned from the extracted chromosomal DNA, and then the nucleotide sequence of the 16S ribosomal RNA gene is analyzed. Finally, 16S ribosomal RNA obtained from each bacterium
By aligning the nucleotide sequences of the genes, the nucleotide sequences specific to the bacterial genus or group (cluster) are specified.

On the other hand, extraction of chromosomal DNA from bacteria
For example, it can be carried out by culturing the bacterium in a liquid medium for 0.5 to 3 days, centrifuging and collecting the microbial cells, and then lysing the microbial cells. As the lysis method, for example, treatment with a bacterial wall-dissolving enzyme such as lysozyme or β-glucanase, or ultrasonic treatment is used. If necessary, other enzyme agents such as protease and ribonuclease and a surfactant such as sodium lauryl sulfate may be used in combination. In addition, freeze-thaw treatment may be applied. From the extracted chromosomal DNA, the method of Wisotzkey et al. (IJSB, 42 , 263-269
(1992)), the 16S ribosomal RNA gene can be cloned.

Cloned 16S ribosomal RNA
The nucleotide sequence of the gene is 16S ribosomal RNA gene
After ligating to a commercially available E. coli cloning vector such as pUC119 or pGEM-7zf and transforming E. coli,
The insertion of the 6S ribosomal RNA gene can be confirmed by agarose gel electrophoresis or the like, and can be analyzed by an automatic DNA sequencer using this plasmid as a template.

16S ribosomal R obtained from each reference strain
CLUSTAL W verl.4 program package (J. Thompson & T. Gibson, Nucleic aci based on the nucleotide sequence of NA gene)
d Research, 1994) and the like, it is possible to determine whether it is specific to a bacterial genus or group (cluster).
The nucleotide sequence present in the 6S ribosomal RNA gene can be specified.

The bacterium or group (cluster)
Of course, if there is one base sequence specific to the 16S ribosomal RNA gene of the bacterium belonging to the group, the DNA having the base sequence can be used as a primer, but the 16S of the bacterium belonging to the genus or group (cluster) of the bacterium. When the ribosomal RNA gene has a plurality of specific base sequences, DNA having one to all of the base sequences may be used as a primer for PCR. In addition, DNA by PCR
DNA of different base sequences in order to make the amplification of bacterium more specific to the genus or group (cluster)
Can also be used as a primer. Two kinds of primers, a sense primer and an antisense primer, are used for amplification of a gene by the PCR method. One of the primers must have a sequence specific to the genus or group (cluster) of bacteria, but both primers must have a sequence specific to the genus or group of bacteria (cluster) Good.
Only one of them is a bacterium or group (cluster)
When a primer having a sequence specific to is used, the highly conserved region of the 16S ribosomal RNA gene may be used as the other primer.

As described above, a DNA having a nucleotide sequence specific to the genus or group (cluster) of the bacterium existing in the 16S ribosomal RNA gene can be easily synthesized by a commercially available DNA synthesizer, if necessary. The PCR reaction can be performed using a commercially available PCR reaction device. PCR reaction conditions do not have to be set strictly,
It is only necessary that the specific DNA is amplified. For example, a predetermined amount of chromosomal DNA obtained from the bacterium to be identified, sense primer, antisense primer, buffer, DNA polymerase and the like are mixed, and P
CR can be performed.

[0017]

[Table 1]

The confirmation of the DNA amplification after the PCR reaction can be easily confirmed by, for example, agarose electrophoresis. As described above, in the present invention, since it is only necessary to confirm the presence or absence of DNA amplification, unlike the case of examining the degree of DNA amplification, no highly skilled person is required, and the determination can be performed easily and in a short time. Can be done at.

Below, Brevibacillus
The genus, Aneurinobacillus genus, Paenibacillus genus and Thermophilic Bacillus group bacteria are identified, but other genus or group (cluster) bacteria can be identified by similar techniques.

Bacillus agri, B. centrosporus, B. cho
shinensis, B. parabrevis, B. reuszeri, B. formosu
s, B. borstelensis, B. aneurinolyticus, B. migulan
Us reference strain (Shida et al., IJSB, 45 , 93-100 (1995)
& Shida et al., IJSB, 44 , 143-150 (1994)), PCR was performed using 10 ng of chromosomal DNA extracted as a template, and the 16S ribosomal RNA gene of each strain was cloned. The conditions of PCR, the nucleotide sequences of primers, and other experimental methods are described by Wisotzkey et al. (IJSB 42 , 263-2
69 (1992)). The resulting 16S ribosomal RNA gene was cloned into E. coli cloning vector pGEM-7zf
(Promega) and transformed into E. coli JM109. It was confirmed that the desired 16S ribosomal RNA gene was inserted into the plasmid possessed by the obtained transformant by extracting the plasmid with an alkaline extraction method, and
After confirmation by agarose gel electrophoresis, this plasmid was used as a template for sequencing. Fluorescent cycle sequencing method using an automatic DNA sequencer is sequence (Kawamura et al. Japanese Journal of Bacteriology 49, 837-8
38 (1994)). 1 sequence primer
17 kinds of universal primers for 6S ribosomal RNA gene sequence (Koyanazu Journal of Bacteriology, Japan 4
9 , 812-821 (1994)) was used.

The nucleotide sequences of the 16S ribosomal RNA genes of the 10 reference strains obtained above and the nucleotide sequences already registered in the database (EMBL / Gen Bank / DDBJ Data base) 1
Phylogenetic classification was performed using the nucleotide sequences of 16S ribosomal RNA genes of 0 species of Paenibacillus, 43 species of Bacillus, Sporolactobacillus, and Alicyclobacillus. Alignment of base sequences, calculation of base substitution rate,
CLUSTAL W ver
1.4 Program Package (J. Thompson & T. Gibson,
1994).

The phylogenetic tree thus obtained is shown in FIG. Among 9 species of which the nucleotide sequence of 16S ribosomal RNA gene was determined, Bacillus agri, B. centrosporus, B. c
hoshinensis, B. parabrevis, B. reuszeri, B. formos
7 base sequences of us, B. borstelensis, and B. brevis,
The three kinds of nucleotide sequences of B. laterospocus and B. thermoruber each had a homology of 95.9% or more. on the other hand,
The nucleotide sequences of B. aneurinolyticus and B. migulanus are 99.
It had a homology of 3%. In addition, the above 10 species including B. brevis, B. aneurinolyticus and B. migulanus
The homology between the two was less than 91.3%. Furthermore, the homology between these and the other 53 species was 90% or less. From the phylogenetic tree shown in Figure 1, 10 species including B. brevis and B. aneu
Rinolyticus and B. migulanus formed independent clusters, although they were closely related to each other. They were also systematically separated from the other 52 species.

From these results, it was found that it is appropriate to make these two groups (clusters) independent of each other from the genus Bacillus in the classification method based on the nucleotide sequence of the 16S ribosomal RNA gene. And B. b
A new genus B for a group consisting of 10 species including revis
revibacillus gen. nov., B. aneurinolyticus and B. m
A new genera Aneurinobaci to the group composed of igulanus
llus gen. nov. was named respectively and made a new genus.

That is, Brevibacillus is a conventional Bacil
lus bacteria B. agri, B. centrosporus, B. choshinens
is, B. parabrevis, B. reuszeri, B. formosus, B. bo
rstelensis, B. thermoruber, B. brevis, B. laterosp
It is a bacterial group consisting of 10 species, orus. Also An
The genus eurinobacillus is a conventional Bacillus bacterium B. aneurin
It is a bacterial genus composed of two species, olyticus and B. migulanus.

The above 10 species of Brevibacillus bacteria include
It was confirmed that a region having a base sequence specific to the genus was present in the 16S ribosomal RNA gene, and based on this base sequence, as a primer for the genus Brevibacillus, the sequence number in the sequence listing shown in Table 2 below was used. A DNA having the base sequence of 1 was prepared.

[0026]

[Table 2]

Similarly, for the genus Aneurinobacillus, a DNA having the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing shown in Table 3 below was prepared as a primer.

[0028]

[Table 3]

Similarly, for the genus Paenibacillus, a DNA having a nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing shown in Table 4 below was prepared as a primer.

[0030]

[Table 4]

Also, Bacillus stearothermophilus, B.
kaustophilus, B. thermoglucosidasius, “B. thermod
enitrificans ”, B. thermocatenulautus,“ B. caldov
elox ”,“ B. caldotenax ”,“ B. caldolyticus ”, B.
Nine species of thermoleovorans had 95% DNA homology and formed one group (cluster).
Similarly, for this group (cluster), a DNA having the nucleotide sequence represented by SEQ ID NO: 4 in the sequence listing shown in Table 5 below was prepared as a primer.

[0032]

[Table 5]

As the base sequence of the universal antisense primer for identification, the DNA shown in SEQ ID NO: 5 in the sequence listing shown in Table 6 below was used.

[0034]

[Table 6]

[0035]

[Experimental Example 1] Bacillus, Brevibacillus, Aneurinob
genus acillus, Thermophilic Bacillus group, Sporolac
Tobacillus, Alicyclobacillus and Paenibacillus
47 kinds of bacteria belonging to the genus (shown in Table 7 below) were prepared.

[0036]

[Table 7]

Chromosomal DNA was isolated from these 47 strains by Sa
The method of ito & Miura (Saito, H., and Miura, K., Bioch
Em. Biophys. Acta., 72 , 629 (1993)) and the obtained chromosomal DNA and the Brevibacillus genus bacterium A obtained above.
neurinobacillus genus, Paenibacillus genus and Thermoph
PCR was carried out by preparing the samples shown in Table 8 below using DNA having a nucleotide sequence specific to each of the ilic Bacillus group bacteria as a sense primer.

[0038]

[Table 8]

After completion of the PCR reaction, whether or not the specific DNA was amplified was confirmed by electrophoresis using 0.8% agarose gel. As a result, when a sense primer for Brevibacillus was used, amplification of DNA was observed only in 10 species of Brevibacillus and no amplification of DNA was observed in species belonging to other genera. Similarly Aneuri
When a sense primer of the genus nobacillus is used, DNA
Aneurinobacillus genus is the only two species that can be amplified. When a sense primer for Paenibacillus genus is used, the DNA amplification is Paenibacillu.
There were only 10 species of s genus, and when a sense primer of Thermophilic Bacillus group was used, only 9 species of Thermophilic Bacillus group showed DNA amplification.

As is clear from the above-mentioned experimental examples and the examples described below, when the method of the present invention is used, only the primer on the sense side is prepared according to the genus of interest, and then an extremely small amount of chromosomal DNA is used. The genus can be identified simply by applying PCR. The PCR reaction device is currently a general-purpose experimental device, and the device is widely used. In addition, primers can be easily synthesized at low cost and used as templates.
Since the amount of NA is extremely small, its extraction is extremely simple, and further, there are advantages that PCR conditions are constant and various conditions need not be set depending on the bacteria to be identified and the genus of bacteria of the primers.

The present invention is a method for identifying a bacterium that determines a bacterium genus or group (cluster) by using a PCR reaction.
Chromosomal DNA obtained from the bacterium to be identified is used as a primer with a nucleotide sequence specific to the bacterial genus or group (cluster) present in the S ribosomal RNA gene, and the presence or absence of amplification of specific DNA is used as an index. It is a method for easily determining the genus or group (cluster) of bacteria, and it is effective to apply the present invention to primary or secondary screening when identifying unidentified bacteria.

That is, in the present invention, it is first determined which genus or group (cluster) of bacteria is an unidentified bacterium, and if necessary, phenotypic traits (morphology and physiological properties), quinone composition, fatty acid composition. , Subcellular species composition can be determined by analyzing cell wall amino acid composition, DNA base composition, measurement of DNA homology and base sequence of 16S ribosomal RNA gene.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

[0044]

[Example 1: Identification of strains isolated from nature] Unidentified bacteria isolated from soil were treated with T2 medium (glucose 1%, peptone 1%, meat extract 0.5%, yeast extract 0.2%, p.
H7.0). The strain is observed under a microscope for morphology,
From the presence or absence of spore formation, presence or absence of flagella, motility, etc., Bacillacea
It seems to be a bacterium of the e family. Collect the cells by centrifugation and
aito & Miura method (Saito, H., and Miura, K., Bioc
hem. Biophys. Acta., 72 , 629 (1993)) on chromosome DN
A was extracted.

Extracted chromosomal DNA (1 ng / μl) 1 μ
1, antisense primer of SEQ ID NO: 5 (100 pmol
/ μl) 0.25 μl, dNTP (1.25 mM) 4 μl,
2.5-μl 10-fold diluted Taq buffer, Taq polymelase
Brevibacillus of SEQ ID NO: 1 and Aneurinobacil of SEQ ID NO: 2 in 0.1 μl, water 16.9 μl, wax 25 μl
lus genus, Paenibacillus genus of SEQ ID NO: 3 and SEQ ID NO: 4
0.25 μl of the sense primer (100 pmol / μl) for Thermophilic Bacillus group of
℃, 0.5 minutes) × 1 cycle, ((94 ℃, 1 minute) +
The PCR reaction was performed under the conditions of (58 ° C., 1 minute) + (72 ° C., 2.5 minutes) × 25 cycles, (72 ° C., 5 minutes) × 1 cycle. After the PCR reaction, 5 μl of each sample
Was subjected to 0.8% agarose gel electrophoresis, the DNA in the gel was stained with ethidium bromide, and the DNA was colored under UV light of 254 nm to confirm the amplification of the DNA.

As a result, amplification of only DNA was observed in the sample subjected to PCR reaction with the chromosomal DNA of the bacterium to be identified, using the nucleotide sequence specific to the 16S ribosomal RNA gene of Brevibacillus as a sense primer. The strain was found to be a bacterium of the genus Brevibacillus. Next, as a result of examining the DNA homology with 10 reference strains belonging to the genus Brevibacillus, 90% DNA homology with the reference strain of Brevibacillus brevis was confirmed, and this identified bacterium was identified as Brevibacillus brevis.

[0047]

EFFECTS OF THE INVENTION According to the present invention, it is only necessary to confirm the presence or absence of amplification of DNA, and since it is not necessary to clone the 16S ribosomal RNA gene from the bacterium to be identified, no particular skill is required. Bacteria can be identified quickly and accurately.

Further, according to the present invention, a specific nucleotide sequence existing in the bacterial 16S ribosomal RNA gene is used as a primer, each component commonly used in the PCR reaction, and if necessary, electrophoresis and other necessary components. By selecting components and setting them, they can be put on the market as an identification kit.

[Brief description of drawings]

FIG. 1 shows a phylogenetic tree of Bacillaceae bacteria.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12Q 1/68 C12R 1:01) (C12N 15/09 ZNA C12R 1:13) (C12N 15 / 09 ZNA C12R 1:01) (C12Q 1/04 C12R 1:13) (C12Q 1/04 C12R 1:01)

Claims (6)

[Claims] 1. A chromosomal DNA obtained from a bacterium to be identified is used as a template with a DNA having a specific nucleotide sequence present in the bacterial 16S ribosomal RNA gene as a primer.
A bacterial identification method characterized by applying the CR method to determine the genus of bacteria using amplification of specific DNA as an index. 2. The specific base sequence is Brevibacil.
Sequence number 1 in the sequence listing, which is a nucleotide sequence specific to the genus lus
The method for identifying a bacterium according to claim 1, wherein the DNA having the nucleotide sequence shown in (1) is used as a primer. 3. Aneurinoba as the specific base sequence
The method for identifying a bacterium according to claim 1, wherein a DNA having a nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing, which is a nucleotide sequence specific to the genus cillus, is used as a primer. 4. The specific base sequence is Paenibacil.
Sequence number 3 in the sequence listing, which is a nucleotide sequence specific to the genus lus
The method for identifying a bacterium according to claim 1, wherein the DNA having the nucleotide sequence shown in (1) is used as a primer. 5. The specific base sequence is Thermophile.
The method for identifying a bacterium according to claim 1, wherein a DNA having a nucleotide sequence represented by SEQ ID NO: 4 in the sequence listing, which is a nucleotide sequence specific to the ic Bacillus group, is used as a primer. 6. A kit for identifying a bacterium, which uses as a primer a specific nucleotide sequence present in a 16S ribosomal RNA gene of a bacterium.