JPH08266286A - Determination gene of aeromonas salmonicida - Google Patents

Abstract

PURPOSE: To obtain a new determination gene having a specific base sequence, capable of identifying only Aeromonas Salmonicida which is a pathogenic fungus infected into salmons and trouts and capable of simply and rapidly identifying causative fungus of an infectious disease of fishes in a nursery for fishes. CONSTITUTION: This determination gene of Aeromonas Salmonicida ATCC14174 has a base sequence expressed by formula I. The determination gene is capable of identifying only Aeromonas Salmonicida which is a pathogenic fungus infected into salmons and trouts and useful for simply and rapidly identifying causative fungus of infectious diseases of fishes in a nursery for fishes. The determination gene is obtained by extracting chromosome DNA by ordinary method from Aeromonas Salmonicida ATCC14174, carrying out polymerase chain reaction method by using the DNA as a template and using a primer being a part of chromosome DNA of Aeromonas Salmonicida and having a base sequence expressed by formula II and formula III which are >=300 bases distant to each other and cloning the DNA.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a determinant gene capable of identifying the pathogen Aeromonas salmonicida that infects salmon and trout, etc., its probe and its primer, a reagent for identifying Aeromonas salmonicida containing the probe, and The present invention relates to an identification method thereof, a reagent for amplifying the above gene containing the primer, and a method for amplifying the same.

[0002]

2. Description of the Related Art When an infection caused by a fish pathogen occurs in a farm, identifying the causative pathogen is extremely important in the prevention and treatment of the infection. Conventionally, in classifying and identifying general bacteria including fish pathogens, their morphology, biochemical properties, or biological properties using immunological methods have been observed. Further, with the development of biochemistry and molecular genetics, classification based on chromosomal DNA and RNA of the bacterium, bacterial substance and microbial-produced substance has become possible.

[0003] However, these methods have a problem that they have to go through many complicated steps and consume a great deal of time, and there is a novel method capable of easily and quickly identifying the causative bacterium of a fish infectious disease. Was wanted.

In view of such circumstances, recently, a method of identifying a gene that determines a species possessed by each pathogen and using it to identify a target pathogen has become widespread. This method is based on the determination of the determinant gene of the target pathogen.
An NA probe (single-stranded DNA of this determinant gene is labeled with a label) is prepared, and this DNA probe hybridizes only with the chromosomal DNA of the target pathogen. Furthermore, a polymerase capable of amplifying a target DNA in a large amount in an extremely short time
When the chain reaction (PCR) method was developed,
By using a part of both ends of the nucleotide sequence of the determined gene as a primer, it has become possible to rapidly identify the type of pathogenic bacterium using a test sample (biological tissue containing pathogenic bacterium).

However, in the above-mentioned method, it is an essential condition that the nucleotide sequence of the determinant gene capable of identifying the type of the target pathogen is determined. The present inventors have been studying various pathogenic bacteria that infect fishes in particular, in order to determine the nucleotide sequence of the determinant gene, and have already determined that they are the causative bacteria of Aeromonas disease (hemorrhea and hemorrhagic haemorrhage). It provides a common determinant gene to a certain Aeromonas salmonicida and Aeromonas hydrophila (JP-A-3-280882). These pathogens are particularly susceptible to infecting salmon and trout, which adversely affects fish farming. Of these, Aeromonas salmonicida causes a large amount of loss economically, such as causing a mass infection due to a subclinical infection and the like, and therefore it is earnestly desired to provide a determinant gene that can identify only Eromonas salmonicida. It was

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and an object thereof is to provide a determinant gene capable of identifying only Aeromonas salmonicida, a probe thereof, and a primer thereof. It is in. Another object of the present invention is to provide a reagent for identifying Aeromonas salmonicida containing the above probe and a method for identifying the same, and a reagent for amplifying the above gene containing the above primer and a method for amplifying the same.

[0007]

The determination gene of Aeromonas salmonicida of the present invention, which has been able to solve the above-mentioned problems, has a gist in that it has the nucleotide sequence shown in SEQ ID NO: 1 of the sequence listing described later. is there.

Further, the probe of the determination gene of Aeromonas salmonicida of the present invention has a gist in that the single-stranded DNA of the above determination gene is labeled with a labeling substance. And, a reagent for identifying Aeromonas salmonicida containing this probe, and a method for identifying Aeromonas salmonicida which identifies Aeromonas salmonicida by hybridizing this probe with single-stranded DNA in a sample bacterium. It is included in the range.

Furthermore, the primer of the determination gene of Aeromonas salmonicida of the present invention is 1 at an arbitrary position separated by 300 bases or more in the base sequence of the above probe.
The gist is that it contains a base sequence portion of 0 to 90. And, a reagent for amplifying a determinant gene of Aeromonas salmonicida containing this primer, and a method for amplifying the determinant gene by carrying out a polymerase chain reaction method using the above-mentioned primer or the above-mentioned reagent for amplification is also the present invention. It is included in the range.

[0010]

As described above, the present inventors have already disclosed a determinant gene capable of identifying either Aeromonas salmonicida or Aeromonas hydrophila. Its molecular chain is 352 bp, and The gene used to identify the species was extremely short. The constitution of the determinant gene that can identify only Aeromonas salmonicida, which is the subject of this invention, is
As a matter of course, it is necessary that the above-mentioned known determinant gene base sequence is not included, and the larger the molecular weight thereof, the less likely it is that homology of chromosomal genes between closely related bacterial species will occur. As a result of conducting research from the viewpoint, this invention was completed.

The structure of the present invention will be described below by describing a general method for producing the determinant gene of Aeromonas salmonicida. In the following description including examples, the case where Aeromonas salmonicida ATCC14174 is used as the representative strain will be described, but the present invention is not limited thereto. Here, the Aeromonas salmonicida ATCC14174 is referred to by the American Type Culture Collection as the accession number AT.
It means the strain deposited under CC14174. First, according to the process chart shown in FIG. 1, the chromosomal DNA of Aeromonas salmonicida ATCC14174 was
rmur) method.

The DNA extracted as described above was used as a template, and DNA oligomer A05 (AGCAGCGCCTCA) manufactured by Nippon Gene Co., Ltd. was used as a primer.
These are amplified by applying an automatic temperature controller. A recombinant plasmid is obtained by cloning a DNA fragment of about 0.7 kbp among the amplified DNA fragments using a known plasmid pMosBlue (manufactured by Amersham).

Escherichia coli K-12 MosBlue was subjected to light conversion using this recombinant plasmid, and
A recombinant plasmid DNA containing a chromosomal DNA fragment of Salmonicida ATCC14174 is prepared. After extracting this with alkaline SDS method, the obtained DNA was EcoR
It is cut with I and Hind III, and subjected to agarose gel electrophoresis to select and isolate a clone having a molecular weight of about 0.7 kbp.

The thus obtained DNA fragment having a molecular weight of about 0.7 kbp is used as a probe to carry out colony hybridization with a colony in which Aeromonas salmonicida is grown to form a hybrid only with Aeromonas salmonicida (ie, , Which do not hybridize with other bacterial species), the target determinant gene of Aeromonas salmonicida can be obtained.

The nucleotide sequence of the determination gene of Aeromonas salmonicida thus obtained is as shown in SEQ ID NO: 1 in the sequence listing described later.

The determinant gene of Aeromonas salmonicida is useful for identifying Aeromonas salmonicida. That is, if a DNA probe obtained by labeling the single-stranded DNA of this determinant gene with a labeling substance can be hybridized with the single-stranded DNA in the sample bacterium, it is possible that Aeromonas salmonicida is present in this sample. I understand.

Here, as the label used for preparing the above-mentioned probe, one commonly used in this field can be used. Such examples include radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, biological chromophoric compounds and the like.

Using the above-mentioned probe, a reagent or kit for identifying Aeromonas salmonicida in a sample bacterium can be prepared. These identifying reagents and kits may contain various carriers such as alkaline phosphatase, biotin and avidin, and diluents such as sodium chloride, sodium citrate, formamide, sodium hydrogen phosphate and dodecyl sulfate.

To amplify the above gene, DN
In the base sequence of the A probe, it is effective to apply to the PCR method a portion containing a base sequence of 10 to 90 at an arbitrary position 300 bases or more apart as a primer. Among these, those containing 10 to 90 base sequences counted from the 5'end side and 3'end side thereof are preferable, and those having 20 to 25 base sequences are more preferable. By using such an amplification method, it becomes possible to detect a small amount of Eromonas salmonicida in the living body, and it is possible to easily confirm the presence of this bacterium in the early stage of infection, and thus to prevent infection. Is also very useful.

This primer can be used in the reagent for amplifying the determinant gene, the kit, etc. of the present invention together with the above-mentioned various carriers, diluents and the like. The apparatus used in this PCR method is not particularly limited as long as it can set a predetermined temperature for each step.

For detection of Aeromonas salmonicida, the above DNA probe may be used, or the DNA amplified by the above PCR method or the like may be directly measured by the electrophoresis method.

[0022]

EXAMPLES In the following examples, the following abbreviations are used, and the experimental methods are otherwise described as follows.
Molecular Cloning 2nd Edition (Cold Spring Harbar Labor
atory)). LB: Luria Bertani medium (adjusted to pH 7.5) Bactotryptone 10 g Yeast extract 5 g Salt 10 g in 1 L 1NB: Polypeptone 10 g Meat extract 10 g Salt 10 g in 1 L of 1% salted agar medium

HIA: Heart infusion agar medium with 0.5% salt 2HIA: Heart infusion agar medium with 1.5% sodium chloride and 0.3% yeast extract LA: Luria Bertani medium (adjusted to pH 7.5) 1 L, Bactotryptone 10 g Yeast extract 5 g Salt 5 g Agar 15 g contained dDW: Purified water Tris: Hydroxymethylaminomethane DTT: Dithiothreitol ATP: Adenosine-5'-triphosphate BSA: Bovine serum albumin

X-gal: 5-bromo-4-chloro-3
-Indolyl-β-D-galactopyranoside TBE buffer: solution containing trisborate (0.089M), boric acid (0.089M) and EDTA (0.002M) 2ME: 2-mercaptoethanol EDTA: Ethylenediaminetetraacetic acid STEP solution: 0.5% sodium lauryl sulfate, 50
Solution containing mM Tris-hydrochloric acid (pH 7.5), 0.4 M EDTA and 1 mg / ml proteinase K (manufactured by Merck) TE: 10 mM Tris-hydrochloric acid (pH 8.0) and 1 mM E
Mixture of DTA TE phenol: saturated TE phenol 250 mL chloroform 240 mL isoamyl alcohol 10 mL quinolinol 70 mL metacresol 7 mL dATP: deoxyadenosine triphosphate dCTP: deoxycytosine triphosphate dGTP: deoxyguanine triphosphate dTTP: deoxythymine triphosphate

Example 1: Determination of Aeromonas salmonicida
Preparation of constant gene (1) Extraction of chromosomal DNA of Aeromonas salmonicida Aeromonas salmonicida ATCC14174 1NB
Cultivated overnight in medium (100 mL), 5 × 10 ⁹ cells / m
A bacterial culture of L was obtained. After centrifuging the bacterial culture at 6000 rpm for 10 minutes, Tris-hydrochloric acid (pH 8.0) and EDTA were added to the remaining precipitate after removing the supernatant by 5 times, respectively.
A solution (5 mL) containing 0 mM each was added. After freezing this solution at -20 ° C, 0.25 M Tris-HCl (pH
A solution (0.5 mL) in which 10 mg / mL of lysozyme (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 8.0) was added. This was left to stand at room temperature and thawed, and then reacted under ice-cooling for 45 minutes. Next, add 1 mL of the STEP solution and stir 50 times with occasional stirring.
The reaction was carried out at 60 ° C for 60 minutes. After the reaction was completed, TE phenol (6 mL) was added and the mixture was centrifuged at 6000 rpm for 15 minutes,
The supernatant was collected.

To this supernatant, 1/10 volume of 3M sodium acetate was added to the supernatant, and then 2 volumes of ethanol was added to the supernatant to precipitate nucleic acids. It was This precipitate was collected while winding it around a thin glass rod, and was mixed with 50 mM Tris-HCl (pH 7.5) and 1 mM.
After dissolving in a solution (5 mL) containing mM EDTA, RNase (manufactured by Sigma) 10 mg / mL solution (1
(00 μL) was added and the mixture was allowed to stand at 4 ° C. overnight.

Next, an equal volume of chloroform was added to this solution and mixed thoroughly, followed by centrifugation at 6000 rpm for 15 minutes to collect the supernatant. To this supernatant, 1/10 volume of 3M sodium acetate and 2 volumes of ethanol were added to the supernatant and mixed to precipitate the desired chromosomal DNA. The precipitated chromosomal DNA was collected while winding it around a thin glass rod, and then a solution containing 50 mM Tris-HCl (pH 7.5) and 1 mM EDTA (2
mL) was added and stored at 4 ° C.

(2) RAPD (Random Amplified Polymorphic DNA: Nucletic Acids Res)
earch, 18: 6531-6535) Amplification of Eromonas salmonicida chromosomal DNA by the method of Eromonas salmonicida chromosomal DNA (20n) obtained as a template in distilled water (20 μL).
g) and 0.4 μM of the above DNA oligomer A05 as a primer were added and mixed. Add dAT to this mixture.
P, dCTP, dGTP, dTTP (all 0.2 m
M), 10 mM Tris-HCl (pH 8.9), 1.5 m
M magnesium chloride solution, 80 mM potassium chloride solution, 0.1% sodium cholate solution, 0.1% Tr
iton X-100, 50 μg / mL BSA solution and 0.4
Unit of Taq DNA Polymerase [Nippon Gene Co., Ltd.
Manufactured] was added and mixed. Put this on an automatic temperature controller,
94 ℃ 30 seconds, 30 ℃ 30 seconds and 72 ℃ 1
The step of reacting for 1 minute was defined as one cycle, and after this step was repeated 40 times in total, the reaction was further performed at 72 ° C. for 5 minutes.

After completion of the reaction, the molecular weight of the amplified DNA fragment was measured by performing electrophoresis for 1 hour at 80 mA in TBE buffer using 0.8% agarose gel. The molecular weight of this amplified DNA fragment is λ phage DN
Mobility of Hind III digestion fragment of A (LH Robinson and
A. Landy, Gene 2 (1977)). The DNA fragment was detected by staining the agarose gel in TBE buffer containing ethidium bromide (1 μg / mL) and then observing fluorescence under UV irradiation.

(3) Aeromonas from agarose gel
Extraction of Salmonicida chromosome (about 0.7 kbp) DNA fragment About 0.7 k after completion of electrophoresis on the agarose gel
The agarose gel containing the bp DNA fragment was cut out and placed in a dialysis tube. After closing one end of the dialysis tube, TBE buffer (300 μL) was added and the other end was closed. Electrophoresis was performed in a TBE buffer solution using the tube thus treated (80 mA for 1 hour) to elute the DNA fragment from the gel. After recovering the solution in the tube, an equal volume of TE phenol was added and mixed well, followed by centrifugation at 13000 rpm for 5 minutes.

The obtained supernatant was transferred to another tube, 1/10 volume of 3M sodium acetate was added to the solution, and then 2.5 times volume of 99% ethanol was added. It was left for 10 minutes at ° C. Then 13000r
After centrifuging at pm for 10 minutes, the supernatant was removed and the resulting precipitate was vacuum-dried to obtain D of about 0.7 kbp.
An NA fragment was obtained.

(4) Aeromonas salmonicida (about 0.
7 kbp) DNA fragment vector plasmid pMosBlue
Cloning into Eromonas salmonicida (about 0.7 kbp) DNA fragment (100 ng) obtained as described above was dissolved in distilled water (7.5 μL), and then the vector plasmid pM
Mixed with osBlue (50 ng). Next, a 10-fold concentration of ligation buffer (1 μL, manufactured by Amersham), 100 mM DTT (0.5 μL) and 10 mM ATP (0.5 μL) were added to this solution, and then T _{4 was} further added.
Ligase (Escherichia coli C75 T ₄ ligase)
A circular recombinant plasmid DNA was obtained by adding 0.5 μL and reacting at 16 ° C. for 2 hours.

(5) Replication of recombinant plasmid DNA by transformation of Escherichia coli Recombinant plasmid D obtained as described above
The NA replication strain was extracted by the alkaline SDS method, EcoR I and
After digestion with HindIII, electrophoresis was performed using 0.8% agarose gel in TBE buffer at 80 mA for 1 hour. The detection of the DNA band and the measurement of the molecular weight on the agarose gel were carried out in the same manner as in (2) above, and the DNA band was measured at about 0.
A 7 kbp DNA fragment was obtained.

(6) Confirmation by hybridization method The DNA fragment of about 0.7 kbp obtained as described above was labeled with ³² P by the random primer method, and then the target bacterium was obtained by the colony hybridization method. It was confirmed that it hybridizes only with the DNA of Aeromonas salmonicida. The results are shown in Table 1.

[0035]

[Table 1]

(7) A DNA clone fragment (about 0.
The nucleotide sequence of 7 kbp) was determined by the cycle sequence method using a Sequother Long Read Cycle Sequence Kit (manufactured by Boxis Brown).
The results are as shown in SEQ ID NO: 1 in the sequence listing (molecular weight: 680 bp).

Example 2 Of the determinant gene fragment of Aeromonas salmonicida obtained in Example 1, the 52nd to 71st portions counted from the 5'end side thereof were used as an upstream primer (primer 1),
A complementary strand from the 544th position to the 563th position was prepared as a downstream primer (primer 2). The base sequence is as shown in FIG. With these primers,
An attempt was made to detect Aeromonas salmonicida by the PCR method.

First, various fish pathogens such as Aeromonas salmonicida were subjected to liquid culture, and chromosomal DNA of each pathogen was extracted by the method of (1) of Example 1 using these bacterial solutions. The types of fish pathogens used in this example are as shown in FIGS. 3 and 4.

Further, the above-mentioned primer portion used in this Example is a DNA synthesizer "Gene Assembler.
Plus "(Pharmacia)
Triester method (Gait, MJ Oligonucleotide synthe
sis, a practical approach, IRL Press Limited, Oxfor
d, Vol. 19). After synthesis, NAP-1
The solvent for the primer (ammonia water) was replaced with distilled water using a 0 column (Pharmacia).

Chromosomal DNA (100 ng) in the sample and the primer (0.4 μM) were added to and mixed with this distilled water (50 μL). DAT in this mixture
P, dCTP, dGTP, dTTP (all 0.2 m
M), 10 mM Tris-HCl (pH 8.9), 1.5 m
M magnesium chloride solution, 80 mM potassium chloride solution,
0.1% sodium cholate solution, 0.1% Triton X-1
00, 50 μg / mL BSA solution and 0.4 units Taq
DNA Polymeraze was added and mixed. Applying this solution to an automatic temperature controller, reacting at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 1 minute constitutes one cycle,
This was repeated for a total of 30 cycles.

After the reaction was completed, the molecular weight of the amplified DNA fragment was measured by performing electrophoresis in 0.8% agarose gel in TBE buffer at 80 mA for 1 hour. The detection of this amplified DNA fragment and the measurement of its molecular weight were carried out in the same manner as described in Example 1 (2).
The obtained results are shown in FIGS. 3 and 4. As is clear from the figure, a clear DNA band was detected near the molecular weight of about 0.7 kbp only for Aeromonas salmonicida.

Example 3 Using the primer described in Example 2, from a culture solution containing a plurality of fish pathogens mixed, Aeromonas
We tried to detect Salmonicida. First, a liquid culture of 5 × 10 ⁹ cells / mL was prepared by liquid-culturing each of the main pathogens that infect the cultured eggs such as Aeromonas salmonicida, Aeromonas hydrophila, and β-streptococcus. These bacterial solutions were added to sterilized physiological saline to prepare various combinations of bacterial solutions so that the concentration of each bacterial solution was about 10 ⁶ cells / mL. The details are as described in FIG.
Chromosomal DNA of each pathogenic bacterium was extracted by the method of (1) of Example 1 using these bacterial solutions.

Using the chromosomal DNA thus extracted, amplification of a DNA fragment and measurement of the molecular weight of the amplified DNA fragment were carried out in the same manner as in Example 2. The result is shown in FIG. As is clear from the figure, a clear DNA band was detected near the molecular weight of about 0.7 kbp only in the sample containing Aeromonas salmonicida.

Example 4 Using the primers described in Example 2, a method for directly detecting Aeromonas salmonicida from the kidney of an egg was attempted by the PCR method. First of all, 15 eggs with a weight of about 4.5g.
The tail is divided into 2 wards, 8 and 7, and each has a separate tank (1
00L). After acclimatization in the water tank for a while, about 8 fish were intraperitoneally infected with Aeromonas salmonicida (2 × 10 ⁷ cells / mL per fish a bacterial solution of 0.
05 mL was administered, this is called the infected section), and the remaining 7 fish were left as they were (non-infected section).

On the 4th day after infection, all the eggs in each section were sacrificed, the kidneys were aseptically removed, and then homogenized by a conventional method. DNA contained in each obtained homogenate solution
Was extracted in the same manner as in the method (1) of Example 1. Using the extracted DNA as a template, amplification by the PCR method was performed to observe the DNA band on the agarose gel. The result is shown in FIG.

As shown in FIG. 6, the DNA in the kidney homogenate in the infected area had a molecular weight of about 0.7, unlike the DNA in the uninfected area.
Since a clear DNA band was detected near kbp, it was suggested that the infection was caused by Aeromonas salmonicida. From this result, it was found that it is possible to identify Aeromonas salmonicida by the PCR method using the primer of the present invention.

[0047]

The determinant gene of the present invention can identify only Aeromonas salmonicida in a sample. That is, if a probe is prepared using the gene of the present invention, only Aeromonas salmonicida in the test sample can be easily identified by a hybridization method or the like. Furthermore, by applying a part of both ends of the base sequence of this probe to the PCR method as a primer, the gene of the present invention can be easily amplified. It has been confirmed that it is very useful in the detection of Aeromonas salmonicida, such as the fact that the pathogenic bacteria can be detected even when a living tissue that does not exist is used. In identifying or amplifying the above-mentioned determinant gene, it is useful to use an identifying reagent or an amplifying reagent containing the probe or primer of the present invention.

[0048]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 680 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Aeromonas salmonicida Strain name: ATCC14174 Sequence characteristics symbol: None present position: 1..680 method to determine the characteristics: E SEQ AGCAGCGCCT CATGGGGAGG CAACCATCGG GCAGCGCTGC ATCATACGTG AAGCCTCCAC 60 GCGCTCACAG CCCCCCAATA CACCGTGGCG ACGCACACCG CCGTGTCTGT GTCACGGAAA 120 CCGCGCTCTT CCGCTCCCGC CTGGGCGCCG CATCAATAAA ATTTTTGGCA AAAGTGGATT 180 ACCGCAAAAC CATAGGCCTA GGCCCCGCCA GATAAAGGTA CTCGGGCGTG GTTAGGATGT 240 GAAAAGAAGG TTCCTTTTTG CGGTTTTTTA CAGTTTGGAG GGGCTCTGGA TTGCTGCACT 300 GAAAACATAA CTCATTGATT ATGCAGGGAG AGTCGTACTT TCCGTGAGAA TTTGAATGTT 360 CAGGAAGGAT CTCATTTGCT GCGGGGGAAT AACGTGAAGG CGTTTTGTTT CAACTGGTTA 420 GGTGTGATGA GGGCTGATCT GATAACTGAA ATTTTGAAAA AGCATGAGCT GTGATGGTTA 480 ACCGAGGTGG TCATAGCAAA TGAGGTTCAT CTTGGCGTTC TCCGCACTCA AAAGCAGAAG 540 AGGCCCACAC TATGGGGCCT C TTCTCGATC TATCGTCTCA GTCTCACATT AGTGAGCAGC 600 TAGCTCACTG TAGAGATCTG GCTTTTCTTG CATAAGCGTC AAGACCTTCG CTGCCAATCC 660 AGTCGGGTTG AGGCGCTGCT 680

[Brief description of drawings]

Figure 1: Aeromonas salmonicida ATCC 14174
It is a figure which shows the extraction process of the chromosomal DNA of.

FIG. 2 is a diagram showing the base sequences of primers used in Examples.

FIG. 3 is a photograph showing the result of electrophoresis in Example 2.

FIG. 4 is a photograph showing the result of electrophoresis in Example 2.

5 is a photograph showing the result of electrophoresis in Example 3. FIG.

FIG. 6 is a photograph showing the results of electrophoresis in Example 4.

Claims (7)

[Claims] 1. A determinant gene of Aeromonas salmonicida having the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing. 2. The single-chain D of the determinant gene according to claim 1.
Aeromonas characterized by labeling NA with a marker
Salmonicida determinant gene probe. 3. A reagent for identifying Aeromonas salmonicida, which comprises the probe according to claim 2. 4. The probe according to claim 2 or 3,
A method for identifying Aeromonas salmonicida, which comprises identifying Aeromonas salmonicida by hybridizing with single-stranded DNA in a sample bacterium. 5. The base sequence of the probe according to claim 2, which has 10 to 10 bases at arbitrary positions separated by 300 bases or more.
A primer for the determinant gene of Aeromonas salmonicida, which contains 90 nucleotide sequence portions. 6. A reagent for amplifying a determinant gene of Aeromonas salmonicida, which comprises the primer according to claim 5. 7. A method for amplifying a determinant gene of Aeromonas salmonicida, which comprises performing a polymerase chain reaction method using the primer according to claim 5 or 6.