JPH07213299A - Method for identifying and detecting bacterium with dna gyrase gene - Google Patents

Abstract

PURPOSE:To accurately detect and identify a bacterium playing an important role in a medical field, various industrial fields, and environmental maintenance in the level of genes by identifying the bacterium with the base sequence of the DNA gyrase gene of the bacterium. CONSTITUTION:When a bacterium is identified with the base sequence of the DNA gyrase gene of the bacterium, at least the partial base sequence of a gyrase B sub-unit coding an amino acid sequence nipped with an amino acid sequence of formula I and an amino acid sequence of formula II on the DNA gyrase B sub-unit is multiplied by a PCR method using a DNA coding the amino acid sequences of formulas I and II. Thus, the kind and strain of the bacterium can more accurately defined than by conventional methods, and the bacterium can be detected and identified, thereby enabling to utilize this method for the diagnosis of infectious diseases, the developments of microorganism environmental clarification systems, the control of food production processes, etc.

Description

Detailed Description of the Invention

[0001]

[Field of Industrial Application] The present invention identifies and detects, at the gene level, bacteria that play an important role in the fields of medicine, various industries (food, chemistry, etc.) and environmental protection (water treatment, biodegradation of pollutants). On how to do.

[0002]

2. Description of the Related Art Conventionally, biochemical tests such as sugar assimilation have been used to identify bacteria. However, the inspection using these is very complicated and time-consuming,
Nevertheless, it was difficult to obtain accurate results.
In recent years, gene level analysis of microbial species has been conducted using the nucleotide sequence of 16S rRNA. However, 16Sr
RNA has a slow molecular evolution rate, and its difference is insignificant among closely related bacterial species. Therefore, it was difficult to determine the species and strain of bacteria with the identification system using the 16S rRNA sequence.

[0003]

[Problems to be Solved by the Invention] In order to classify, identify or monitor bacteria with high accuracy, comparison at the gene level
It is desirable to perform detection, but for this it is essential that the gene sequence information be readily available for any bacterium. The above-mentioned rRNA gene has a DNA base sequence that is highly conserved among species, and by using this sequence as a so-called universal primer, it is relatively easy to PC in most bacterial species.
R amplification can be performed, and sequencing can be performed from the amplified fragment. However, in other structural genes, since there is no DNA base sequence conserved among such species, it has not been possible to obtain sequence information by the same method until now. For this reason, conventionally, it has been practically impossible to apply these genes for the purpose of classification and identification in order to carry out sequencing for each species.

An object of the present invention is to establish a method for easily obtaining gene sequence information from an arbitrary bacterium and to provide a simple and highly accurate method for identifying and detecting a bacterium.

[0005]

Means for Solving the Problems As a result of repeated studies to achieve the above object, the present inventors have found that a highly conserved amino acid sequence across species is present on a DNA gyrase protein ubiquitously present in bacteria. The present invention was found, and the present invention was completed based on this finding. That is, the first aspect of the present invention is a method for identifying a bacterium, which comprises identifying the bacterium by using a nucleotide sequence of a DNA gyrase gene of the bacterium. Here, as the base sequence, the amino acid sequence represented by His-Ala-Gly-Gly-Lys-Phe-Asp on the DNA gyrase B subunit and Met-Thr-Asp-Ala-Asp-Val-Asp-
A partial base sequence of the gyrase B subunit encoding an amino acid sequence sandwiched by the amino acid sequences represented by Gly can be used.

A second aspect of the present invention is a method for detecting a bacterium, which comprises detecting the bacterium by using the nucleotide sequence of the DNA gyrase gene of the bacterium. The base sequence here is on the DNA gyrase B subunit.
Jai encoding an amino acid sequence sandwiched between the amino acid sequence represented by His-Ala-Gly-Gly-Lys-Phe-Asp and Met-Thr-Asp-Ala-Asp-Val-Asp-Gly A partial base sequence of Race B subunit can be used.

The present invention will be described in detail below. The present invention identifies and detects the bacterium by using the nucleotide sequence of the DNA gyrase gene of the bacterium. Specifically, a DNA fragment containing a specific base sequence on the DNA gyrase gene is amplified by the PCR method, and then the base sequence of the DNA fragment is determined by the dideoxy method or the like, and based on this base sequence, identification and detection of bacteria is carried out. Is to do.

As a primer used in the PCR method, Hi
Sense primer containing a nucleotide sequence encoding the amino acid sequence represented by s-Ala-Gly-Gly-Lys-Phe-Asp and Met-Thr-
Two types of primers, an antisense primer containing a nucleotide sequence encoding the amino acid sequence represented by Asp-Ala-Asp-Val-Asp-Gly, are used. The template DNA binding site of each primer is pos.97 of the E. coli K12 strain gyrase B subunit amino acid sequence (GYRB ECOLI [SWISS-PROT]).
Equivalent to -104, pos.495-501. By using these primers, the amino acid sequence represented by His-Ala-Gly-Gly-Lys-Phe-Asp on the DNA gyrase B subunit and Met-Thr-Asp-Ala-Asp-Val-Asp- A DNA fragment containing a partial base sequence of the gyrase B subunit encoding an amino acid sequence sandwiched by the amino acid sequences represented by Gly can be amplified. The sense primer and antisense primer are designed based on the 7 and 8 amino acid sequences as shown in FIGS. As shown in the figure, in the sense primer, delete the third base of the codon corresponding to aspartic acid on the C-terminal side, and in the antisense primer, fix the third base of the codon corresponding to glycine on the N-terminal side. Therefore, 512 types of each primer are mixed.

The amplified fragment with such a mixed primer cannot be directly sequenced because the entire base sequence is unknown and a primer sequence cannot be obtained.
Therefore, in the present invention, a known sequence is added to the 5'end of the primer in advance as shown in FIG. 3, and this base sequence is used as a sequence primer to directly determine the base sequence from the amplified fragment. It should be noted that this method can sequence about 350 bp from each primer.

The base sequence of the amplified DNA fragment can be obtained by a known base sequence determination method such as the dideoxy method. As shown in the examples, this nucleotide sequence differs between the same species or strains, and the degree of difference is significantly higher than that of the 16S rRNA nucleotide sequence when compared between the same species. It is not necessary to determine a long nucleotide sequence using multiple primers as in 16S rRNA. D obtained by utilizing the specificity of such a base sequence
AN gyrase gene nucleotide sequence information is called signa
It can be used as a ture base sequence, which enables simple and highly accurate identification of bacteria without complicated biochemical tests. In addition, the obtained nucleotide sequence can be used as a probe as it is. Furthermore, by similarly obtaining the nucleotide sequences of closely related bacterial species and performing multiple comparison, it has a very high specificity at the species or strain level. It is possible to create a probe. With these probes, a target bacterium can be specifically detected even in a natural flora containing a large number of unknown bacteria. In addition, since the bacterium can be easily expressed at the gene level, it is possible to describe an unidentified new species.

The bacteria which can be identified and detected by the present invention are most of Gram-negative bacteria and some of Gram-positive bacteria. Specific examples of the genera include, but are not limited to, Escherichia, Pseudomonas, Acinetobacter, Salmonella, Bacillus, and the like.

[0012]

EXAMPLES Example: Application to Gram-negative bacterium identification Using the primers shown in FIG. 3, Escherichia sp.
Strain ( E.coli K12), Pseudomonas 4 strains ( Pseudomonas
putida , Pseudomonas alkanolytica , Pseudomonas stu
tzeri , Pseudomonas aeruginosa ), 14 strains of Acinetobacter ( A. calcoaceticus CIP81.08, A. baumannii CI)
P70.34, Acinetobacter sp. 3 CIP70.29, A. haemolyticus
CIP64.3, A .juni CIP64.5, Acinetobacter sp. 6 CIPA
165, A.jhonsonii CIP64.6, A.lwoffii CIP64.10, Acin
etobacter sp. 9 CIP70.31, Acinetobacter sp. 10 CIP7
0.12, Acinetobacter sp. 11 CIP63.46, Acinetobacter
sp.12 SEIP12.81: CIP and SEIP are conservation numbers of Pasteur Institute) and PCR amplification of DNA gyrase genes of unidentified novel hydrocarbon-degrading bacteria T4 strain and SM-8 4L strain were attempted.

As a result, an almost single amplification product could be obtained in all strains. The nucleotide sequence of the amplified product was determined by the dideoxy method using the sequence primer shown in FIG. The amino acid sequence data of E. coli K12 and Pseudomonas putida matched the data registered in the database. Next, the nucleotide sequence of the T4 strain and the nucleotide sequence of the 3 strains of Acinetobacter were obtained by the same method as above, and the nucleotide sequences of both were compared. The result is shown in FIG. As shown in the figure, the T4 strain belongs to the genus Acinetobacter and has ATCC3101
It was confirmed that it is the newest species closest to the two strains. Also, Ac
inetobacter is different from the base sequence of DNA gyrase gene even within the genus, the difference was observed by strains within a species of Acinetobacter calacoacetius. That is, the strain could be identified by comparing the base sequences of the DNA gyrase gene. In addition, from the obtained nucleotide sequence, T
It was possible to discriminate the ATCC31012 strain by producing PCR primers specific to the 4 strains and determining the presence or absence of amplification.

[0014]

Industrial Applicability According to the present invention, it becomes possible to more accurately define the species and strains of bacteria than ever before. This is, for example,
It can be used for diagnosis of infectious diseases, etc., and it becomes possible to follow the prevalence of specific bacteria from the complicated flora, and it can also be used for development of a microbial environment purification system, management of food manufacturing processes, etc. it can.

[Brief description of drawings]

FIG. 1 shows a sense primer used in the present invention.

FIG. 2 shows an antisense primer used in the present invention.

FIG. 3 shows an example of a sequence primer used in the present invention.

FIG. 4 shows the nucleotide sequences of T4 strain and known Acinetobacter strains.

Claims (4)

[Claims] 1. A method for identifying a bacterium, which comprises identifying the bacterium using a nucleotide sequence of a DNA gyrase gene of the bacterium. 2. The base sequence is at least His-Ala-Gly-Gly-Lys- on the DNA gyrase B subunit.
Amino acid sequence represented by Phe-Asp and Met-Thr-Asp-Ala-As
The method for identifying a bacterium according to claim 1, which is a partial base sequence of a gyrase B subunit encoding an amino acid sequence sandwiched between the amino acid sequences represented by p-Val-Asp-Gly. 3. A method for detecting a bacterium, which comprises detecting the bacterium using a nucleotide sequence of a DNA gyrase gene of the bacterium. 4. The base sequence is at least His-Ala-Gly-Gly-Lys- on the DNA gyrase B subunit.
Amino acid sequence represented by Phe-Asp and Met-Thr-Asp-Ala-As
The method for detecting bacteria according to claim 3, which is a partial base sequence of a gyrase B subunit encoding an amino acid sequence sandwiched between the amino acid sequences represented by p-Val-Asp-Gly.