JPH0789957B2 - Oligonucleotide for detection of Vibrio parahaemolyticus and detection method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to a Vibrio parahaemoly (Vibrio parahaemoly) in clinical tests, particularly tests for food poisoning, or food tests.
ticus) detection.

[Conventional technology and problems] When the test material is vomiting, feces, food or wipes of the patient, the procedure from enrichment culture, separation culture to differential culture is performed until it is identified as Vibrio parahaemolyticus. There must be. The time required for each culture step is 10 ~
It takes 16 hours, 18 to 24 hours in separate culture, and 18 to 24 hours in differential culture, and the total required time is 2 to 4 days, which requires a long time. In the test in the differential culture, it is necessary to perform a growth test on a NaCl-containing agar medium, a Gram stain, an oxidase test, etc., and the operation is complicated and time-consuming and expensive.
As a method for detecting heat-resistant hemolytic toxin produced by Vibrio parahaemolyticus, there is a reverse passive hemagglutination reaction using specific immunoglobulin obtained from antitoxin serum.
It takes ~ 24 hours. On the other hand, recently, a DNA probe method or a hybridization method using an oligonucleotide has been tried. However, it is difficult to obtain sufficient detection sensitivity and selectivity in a bacterial test when performing hybridization on a membrane or on another support with a probe that has been modified by labeling an oligonucleotide and detecting the hybridization.

[Object of the Invention] The present invention detects a tdh gene derived from Vibrio parahaemolyticus by a gene amplification technique in which an oligonucleotide functions as a primer for a nucleic acid synthesis reaction. A simple, rapid and highly sensitive detection method is a food poisoning bacteria. It is to provide in inspection.

[Means and Actions for Solving Problems] The present invention produces an oligonucleotide that selectively hybridizes with the tdh gene of Vibrio parahaemolyticus, and uses this oligonucleotide as a primer for gene amplification,
It is characterized by selectively detecting Vibrio parahaemolyticus. Here, the oligonucleotide of the present invention is a target gene (that is, a target gene (i.e., a detection target gene) that is narrowed down based on the following viewpoints, based on the following wide-ranging experience and comprehensive knowledge accumulated by the present inventor in the field. The gene to be mutated) is a gene of a pathogenic factor peculiar to the bacterial species. When each oligonucleotide is appropriately combined and used as a primer for the gene amplification method, the size of the amplified region is 200 to 500 bp. Being about.

Each oligonucleotide consists of about 19 to 25 bases, of which the composition ratio of G and C is about 50%. The base sequence of each oligonucleotide has no homology with the base sequence of other bacteria. Tm (℃) is above a certain temperature (however, Tm is
At the temperature at which half of the hybrid of the oligonucleotide and the target gene dissociates, it becomes an indicator of the stability of the hybrid.) Next, the selected oligonucleotides are combined and used as a primer for the gene amplification method, and the gene amplification method and agarose gel are used. The selectivity was examined and determined using the electrophoresis method.

Gene amplification is based on Polymerase Chain developed by Saiki et al.
Reaction method (hereinafter abbreviated PCR method; Science.230, 1350 (19
85)). This method involves the use of a specific nucleotide sequence region (in the case of the present invention, a V. parahaemolyticus
A sample prepared by preparing an oligonucleotide that hybridizes by recognizing the + strand on the one side and the strand on the other side when detecting the (tdh gene) and heat-denaturing it. Function as a primer for template-dependent nucleotide polymerization reaction on nucleic acid,
The produced double-stranded nucleic acid is again separated into single strands, and the same reaction is caused again. By repeating this series of operations, the copy number increases until the region sandwiched between the two primers can be detected. The sample may be a clinical test material such as feces, urine, blood, tissue homogenate, or a food material. In order to use these materials as a PCR sample, an operation of releasing the nucleic acid component from the bacterial cells present in the material is required as a pretreatment. But,
PCR will proceed if there are several molecules to several tens of tens of molecules of nucleic acid to which the primer can hybridize, so simply test the test material with lytic enzyme, surfactant, alkali etc. for a short time.
A sample solution having a sufficient amount of nucleic acid for proceeding the PCR reaction can be prepared. The oligonucleotide used as a primer in the present invention is a nucleotide fragment having a length of 10 bases or more, preferably 15 bases or more, in view of selectivity, detection sensitivity and reproducibility, and may be either chemically synthesized or natural. . Further, the primer does not have to be labeled particularly for detection. The amplification region in the nucleotide sequence of the tdh gene of Vibrio parahaemolyticus defined by the primer may be 50 to 2,000 bases, preferably 100 to 1,000 bases. Although a thermostable DNA polymerase is used in the template-dependent nucleotide polymerization reaction, the origin of this enzyme may be derived from any species as long as it retains the activity at a temperature of 90 to 95 ° C. The heat denaturation temperature is 90 to 95 ° C, the annealing temperature for hybridizing the primers is 37 to 65 ° C, and the polymerization reaction is 50 to 75 ° C.
Then, PCR is carried out for 20 to 42 cycles with this as one cycle for amplification. For detection, the presence of the amplified nucleotide fragment and its length can be confirmed by subjecting the enzyme reaction solution to agarose gel electrophoresis as it is. From the result, it can be determined whether or not a nucleotide having a sequence to be recognized by the primer is present in the sample. This determination directly determines the presence or absence of Vibrio parahaemolyticus. Other electrophoresis and chromatography are also effective for detecting the amplified nucleotide fragment.

[Examples] (Example 1) Preparation of Specimens Vibrio parahaemolyticus 5 strains shown in the vertical column of Table 1 were used to inoculate each into an appropriate enrichment medium, and at 37 ° C under aerobic conditions. Culture was carried out overnight, and cells were collected from the medium (1.5 ml) by centrifugation. 10 mM Tris-HCl buffer (pH 7.
After washing once with 5), 0.5 ml of a solution prepared by dissolving lysozyme in the same buffer to a concentration of 1 mg / ml was suspended and lysed at 37 ° C for 10 minutes. The same volume of phenol saturated with the above buffer was added to the lysate, and the mixture was stirred well. After centrifugation, the upper layer liquid was collected and subjected to ethanol precipitation to precipitate the nucleic acid component, and the precipitate was dissolved in 1 ml of the above buffer solution to obtain a sample.

Synthesis of primer Nucleotide sequence of tdh gene of Vibrio parahaemolyticus (Nisibuchi, Ma
nd Kaper, JB; J. Bacteriol. 162, 558-564 (1985)), the sequence shown in claim 1 was selected, and an oligonucleotide having the same sequence was chemically synthesized. Chemical synthesis was carried out by the triester method using a Shimadzu DNA synthesizer NS-1. Purification of the synthesized oligonucleotide fragments
Performed using a C18 reverse phase column.

PCR Using 3 μl of the sample solution, add sterile distilled water 16.05 μ
l, 10 × reaction buffer 3 μl, dNTP solution 4.8 μl,
30 μl of reaction solution was prepared by adding 1.5 μl of primer (a), 1.5 μl of primer (b) and 0.15 μl of thermostable DNA polymerase. To the container containing this reaction solution, 50 μl of mineral oil (manufactured by SIGMA) is added, and the reaction solution is overlaid. The contents of each added liquid are shown below.

10 x reaction buffer: 500 mM KCl, 100 mM Tris-HCl
(PH 8.3), 15 mM MgCl ₂ , 0.1% (w / v) gelatin. dNTP solution: a mixture of dATP, dCTP, dGTP, and dTTP with a final concentration of 1.25 mM. Primers (a) and (b): Each aqueous solution (50 DU / ml) of the above-mentioned chemically synthesized purified product Thermostable DNA polymerase : Teq DNA Polymerase (5unit /
ml; Perkin Elmer Cetus). The reaction conditions are as follows.

Thermal denaturation: 94 ° C for 1 minute Annealing: 37 ° C for 1 minute Polymerization reaction: 60 ° C for 1 minute The process from thermal denaturation to annealing through polymerization 1
A cycle (required time of 5.7 minutes) was used, and this was performed for 42 cycles (total required time of about 4 hours). These operations are
It was performed by programming the above reaction conditions in a DNA Thermal Cycler manufactured by in Elmer Cetus.

Detection To detect the amplified nucleotide fragment from the reaction solution, agarose gel electrophoresis was performed as follows.

The agarose gel had a gel concentration of 2% (w / v) and contained ethidium bromide (0.5 μg / ml). The electrical conditions for electrophoresis were a constant voltage of 100 V and a time of 30 minutes. The operating method and other conditions are described in Maniatis et al., Molecular Cloning (198
The technique described in 2) was used. In addition to the reaction solution, molecular weight markers were also electrophoresed at the same time, and the length of the nucleotide fragment was calculated by comparing the relative mobilities.

Results As described above, the nucleotide sequence of the tdh gene of Vibrio parahaemolyticus has already been determined, and the size of the oligonucleotide of the present invention, that is, the nucleotide amplified by the primer by PCR, can be estimated. According to this, nucleotides with a length of 439 bases should be amplified by the primers (a) and (b). The numerical values shown in Table 1 are the results of measuring the length of nucleotides amplified by the above method, and the unit is kilobase pairs. As can be seen from the table, each primer combination matches the estimated nucleotide length, and these are tdh
It shows that the target region of the gene is being amplified correctly.

(Example 2) In order to confirm whether or not the results obtained in Example 1 are selective for Vibrio parahaemolyticus, a clinical examination was conducted to compare and examine bacterial species other than Vibrio parahaemolyticus that can be tested.

The method is the same as that shown in Example 1, except that (1
For strains 3), 14) and 15) under anaerobic conditions, 37
Cultivation was carried out overnight at 0 ° C to prepare a sample applicable to the PCR method. The bacteria cultured in the preparation of the sample are 16 strains shown in the vertical heading of Table 2. In addition, human placenta-derived DNA
Was prepared at a concentration of 1 μg / ml, and this was also subjected to PCR in the same manner.

The results are shown in Table 2. As in Table 1, the unit of numerical values in the column is kilobase pairs. It appears to be a by-product of the PCR reaction in some bacterial species. Amplified nucleotide fragments were detected, all of which differ from the length of the nucleotide fragment deduced from the tdh gene sequence. If these strains have the same tdh gene as that of Vibrio parahaemolyticus, Example 1
A nucleotide fragment of the same length as the result of 1 should be detected. Therefore, it is clear that the amplified nucleotides derived from these bacterial species were not generated by recognizing the tdh gene of Vibrio parahaemolyticus, and it can be easily distinguished from Vibrio parahaemolyticus and detected. If the agarose electrophoresis used in the examples of the present invention is carried out under the above-mentioned electrophoretic conditions, it will be 5 to 10 within a range of 100 base pairs or less.
It is possible to distinguish between nucleotide length differences of 10 to 20 base pairs within the range of 100 to 500 base pairs. Further, if the accuracy of nucleotide length measurement is improved by using acrylamide or the like for the gel, the reliability in selective detection is considered to be further enhanced.

[Effect of the invention] In the present invention, by using the PCR method, in the detection of Vibrio parahaemolyticus, high detection sensitivity due to the gene amplification action,
High selectivity can be obtained by defining the reaction with two or more primers. Further, because of high detection sensitivity, a large amount of sample is not required, and sample pretreatment is simple. Moreover, the reaction time is short, detection can be done only with simple equipment, and the operation is easy, so the time to identification can be greatly shortened. As shown in the following examples, the reaction time is 4 hours and the detection operation is 30 minutes. In addition, by using agarose gel electrophoresis and nucleic acid staining with ethidium bromide for detection, detection can be performed without labeling primers etc., and the length of the nucleic acid can be confirmed, so the result is highly reliable. Becomes

Vibrio parahaemolyticus is generally known as a causative bacterium of infectious food poisoning, and in fact, most of the health disorders caused by this bacterium are gastroenteritis with diarrhea, abdominal pain and the like as its main features. The most noteworthy factor for this pathogenic factor is thermostable hemolysin (thermostab).
le direct hemolysin (TDH). Therefore, by using the tdh gene in the nucleotide sequence targeted by the primer, it is possible to selectively detect Vibrio parahaemolyticus as a food poisoning-causing bacterium.

Claims (2)

[Claims] 1. Vibrio parahaemolyticus present in a specimen (Vibrio)
parahaemolyticus), it is a chemically synthesized oligonucleotide that targets the nucleotide sequence encoding the heat-resistant hemolytic poison gene (tdh gene) of Vibrio parahaemolyticus and is complementary to the nucleotide sequence. The synthetic nucleotide is selected from the following sequence group, (5 ') d-GGTAATGTGTATATCCAAC (3') ... (a) (5 ') d-CTACGTCAAAGTCGCACTAG (3') ... (b) or the corresponding complementary sequence. An oligonucleotide, characterized in that it consists of 2. A target nucleotide sequence is selected by causing two oligonucleotides, which define both ends of the nucleotide sequence to be amplified, of the oligonucleotide sequences according to claim 1 to function as primers for chain length reaction. A method for detecting Vibrio parahaemolyticus characterized in that the primer is hybridized to a target nucleotide sequence in a single-stranded state in a sample, and the chain length is obtained by a polymerization reaction of four nucleotides. (B) when the obtained double-stranded nucleotide sequence is separated into single strands, the complementary strand functions as a template for further chain length reaction, (c) the chain length reaction by the primer, Amplification of a specific nucleotide sequence by repeating separation of the long product from the template and hybridization with additional primers So, the method of detecting Vibrio parahaemolyticus by determining whether or not there is a nucleic acid having a nucleotide sequence to be recognized during; (d) specimen.