JPH0789959B2 - Oligonucleotide for detecting C. perfringens and detection method using the same - Google Patents

Description

Description: [Industrial field of application] The present invention is applicable to Clostridium perfringens in clinical tests, particularly food poisoning tests or food tests.
To detect.

[Prior Art and Problems] When the test material is a patient's vomiting substance, feces, food or wiping material, operations such as enrichment culture and confirmation culture must be performed before identification as C. perfringens. The time required for each culture step is 18 to 24 hours, and the total time required is about 2 days, which requires a long time. In the confirmation culture, it is necessary to perform an aerobic culture test, a motility test, a gelatin liquefaction test, a carbohydrate decomposition test, a lecithinase reaction inhibition test and a milk coagulation digestion test, which is a heavy burden in terms of time and cost. Furthermore, it is necessary to investigate whether enterotoxin, which causes Clostridium perfringens enteropathogenicity, that is, food poisoning, is produced. As a test method for enterotoxin, rabbit ligation intestinal reaction, mouse lethal reaction,
Biological methods such as guinea pig or rabbit skin reaction, cultured cell killing activity, and gel precipitation, counter immunoelectrophoresis, latex agglutination, ELISA,
There are immunological methods such as radioimmunoassay. One of the latex agglutination reactions, which is a method of directly detecting the reverse passive latex agglutination reaction, has been the most effective method until now, but it takes 20 to 24 hours to obtain the result.

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 hybridization is carried out on a membrane or on another support by using a probe which is labeled with an oligonucleotide and modified.

[Object of the Invention] The present invention detects the enterotoxin gene of C. perfringens by a gene amplification technique in which an oligonucleotide functions as a primer for a nucleic acid synthesis reaction. To provide a detection method.

[Means and Actions for Solving Problems] The present invention is characterized by selectively detecting C. perfringens by a gene amplification method using an oligonucleotide as a primer. 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. The gene amplification technology was developed by Saiki et al.
olymerase chain reaction method (hereinafter abbreviated PCR method; Scie
nce.230,1350 (1985)). This method is used for detecting a specific nucleotide sequence region (enterotoxin gene of C. perfringens in the case of the present invention).
An oligonucleotide is prepared so that one of both ends of the region recognizes the + strand and the other of the − strand and hybridizes, and is heat-denatured into a single-stranded state. It functions as a primer for the reaction, separates the generated double-stranded nucleic acid into single strands again, and causes the same reaction 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. However, if there are several molecules to several tens of tens of molecules of nucleic acid to which the primer can hybridize, the PCR reaction will proceed, so simply treating the test material with lytic enzyme, surfactant, alkali etc. for a short time is sufficient for the PCR to proceed. A sample solution having a large amount of nucleic acid can be prepared. The oligonucleotide used as a primer in the present invention has 10 bases or more, preferably 15 bases or more in view of selectivity, detection sensitivity and reproducibility.
A nucleic acid fragment having a length of at least the base, which may be chemically synthesized or natural. In addition, the primer is
In particular, it does not have to be labeled for detection. The amplification region of the enterotoxin gene of C. perfringens specified 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
Amplify by performing 20 to 42 cycles of PCR at ˜75 ° C. as one cycle. For detection, the enzyme reaction solution as it is,
The presence and length of the amplified nucleic acid fragment can be confirmed by subjecting it to agarose gel electrophoresis. From the result, it can be judged whether or not the nucleic acid having the sequence to be recognized by the primer is present in the sample. This determination directly determines the presence or absence of C. perfringens. Other electrophoresis and chromatography are also effective for detecting the amplified nucleic acid fragment.

[Examples] (Example 1) Preparation of Specimens The 11 strains of C. perfringens shown in the vertical column of Table 1 were used to inoculate each into an appropriate enrichment medium, and the mixture was incubated overnight at 40 ° C under anaerobic conditions. After culturing, the cells were collected from 1.5 ml of the culture medium 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.

Primer Synthesis Information on the nucleotide sequence of the enterotoxin gene of C. perfringens can be found in the amino acid sequence of enterotoxin (Richard
son M. and Granum PE; FEBS Lett−ers182: 479−484
(1985)), the sequence shown in claim 1 was selected using the information obtained by reverse translation, and an oligonucleotide having the same sequence as that was chemically synthesized. Shimadzu
It was performed by the triester method using a DNA synthesizer NS-1. Purification of the synthesized oligonucleotide fragments was 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 (1), 0.5 μl of primer (2) 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, dTTP with a final concentration of 1.25 mM. Primers (1) and (2): aqueous solutions of the above-mentioned chemically synthesized purified products (50 DU / ml). As the primer combination, the following combinations were used from the sequences ((a) to (c)) shown in claim 2. Primer (1) + Primer (2) (a) + (b) (a) + (c) Thermostable DNA polymerase: Taq DNA polymerase (5 unit /
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 (5.7 minutes) was used, and this was performed for 42 cycles (total required time: about 4 hours). These operations are perkin Elmer C
It was carried out by programming the above reaction conditions in a DNA Thermal Cycler manufactured by etus.

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 reverse-translated C. perfringens enterotoxi
From the nucleotide sequence of the n gene, the size of the oligonucleotide of the present invention, that is, the size of the nucleotide that is amplified by the primer by PCR can be estimated. According to it, the primers (a) and (b) have 728 bases,
In (a) and (c), a nucleotide having a length of 602 bases should be amplified. 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. In the table, (5),
Except for strains (7) and (11), the combinations of each primer matched the length of the deduced nucleotide, and the enterotoxin gene is present in these strains, and the target region has been amplified correctly. It indicates that

(Example 2) In order to confirm whether or not the results obtained in Example 1 are selective for Clostridium perfringens, comparative examinations were carried out with respect to bacterial species other than Clostridium perfringens that can be tested.

The method is the same as that shown in Example 1, but
(6), (7) and (9) were cultured overnight at 37 ° C under anaerobic conditions at 40 ° C and under other aerobic conditions at 37 ° C. The bacteria cultured in the preparation of the sample are 10 strains shown in the vertical heading of Table 2. Human placenta-derived DNA is 1 μg / ml
Was prepared and the PCR was performed 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. Amplified nucleotide fragments, which are considered as a by-product of the PCR reaction, were detected in some bacterial species, but they are all different in nucleotide length from the nucleotide sequence of the enterotoxin gene. If these strains have the same enterotoxin gene as that of C. perfringens, then the nucleotides should have the same length as the results of Example 1. Therefore, it is clear that the amplified nucleotides derived from these bacterial species were not generated by recognizing the enterotoxin gene, and it can be easily distinguished and detected from C. perfringens. The agarose electrophoresis used in the examples of the present invention is 10
5 to 10 base pairs, 100 to 50 in the range of 0 base pairs or less
Within the 0 base pair range, it is possible to distinguish between nucleotide length differences of 10 to 20 base pairs. Furthermore, if the accuracy of measurement of nucleotide length is improved by using acrylamide or the like for the gel, the reliability in selective detection is considered to be further enhanced.

(Example 3) Clostridium perfring used in Example 1
ens) 11 strains were examined for enterotoxin productivity.

Method Using a commercially available enterotoxin detection kit (PET-RPLA: Denka Seiken) that uses the latex agglutination method, the enterotoxin activity for each C. perfringens was examined according to its manual.

Results Table 3 shows the results. The judgment of + and-was performed according to the judgment standard of the same manual. The result of PCR is
The same method as in Example 1 is used.

As is clear from the results shown in the table, for the strains marked with + by the latex agglutination method using a commercial kit, nucleotide fragments of the correct length were also generated by the PCR method.
Negative-negative strains do not produce amplified nucleotide fragments by the PCR reaction, or the lengths differ greatly even if they are produced. Thus, the results of the latex agglutination method using a commercial kit and the method of the present invention are in agreement, which indicates that the enterotoxin-producing ability of C. perfringens can be tested for the presence or absence of the enterotoxin gene. Therefore, directly detecting the presence of the enterotoxin gene has the same meaning as detecting C. perfringens capable of producing enterotoxin. The present invention detects C. perfringens as a food poisoning bacterium by directly detecting the enterotoxin gene of C. perfringens. The above results are in line with the object of the present invention.

[Effect of the Invention] In the present invention, by using the PCR method, it is possible to obtain high detection sensitivity due to the gene amplification action and high selectivity due to the reaction being defined by 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, the equipment is simple to detect, and the operation is easy, so that the time until identification can be greatly shortened, and the efficiency of inspection can be improved. As shown in the following examples, the reaction time is 4 hours and the detection operation is 30 minutes. In addition, by using the nucleic acid staining method with agarose gel electrophoresis and ethidium bromide for detection, it is possible to detect without labeling the primer etc., and moreover, since the length of the nucleic acid can be confirmed, the result is highly reliable. Become.

The effects of using the enterotoxin gene of C. perfringens for the nucleotide sequence targeted by the primer are as follows. There are strains of C. perfringens that produce enterotoxin and strains that do not, and the former strain producing enterotoxin is the causative agent of food poisoning. In addition, there are indigenous Clostridium perfringens that does not produce enterotoxin in human feces. Especially when performing food poisoning tests using feces as a sample, enteroto
Whether or not xin is produced is important. Therefore, enter
By targeting the otoxin gene, it is possible to selectively detect C. perfringens as a causative bacterium of food poisoning.

Claims (2)

[Claims] 1. An enterotoxin of Clostridium perfringens for selective detection of Clostridium perfringens, which is a causative agent of food poisoning, in a sample.
na) target the nucleotide sequence encoding the gene,
An oligonucleotide chemically synthesized so as to be complementary to the nucleotide sequence, wherein the synthetic nucleotide is the following sequence group: (5 ') d-AATACATATTGTCCTGCATC (3') ... (a) (5 ') d- GTAATAGATAAAGGAGATGG (3 ') ... (b) (5') d-GTAGTAGGATTTATACAAGC (3 ') ... (c) or a sequence selected from the corresponding complementary sequences. 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 Clostridium perfringens, characterized in that the primer is hybridized with a target nucleotide sequence in a single-stranded state in a sample, and a chain length reaction is carried out by a polymerization reaction of four kinds of 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, and (c) the chain length reaction by the primer, the chain length A specific nucleotide sequence is amplified by repeating the separation of the product from the template and hybridization with additional primers. The method for detecting C. perfringens by determining whether or not there is a nucleic acid having a nucleotide sequence to be recognized during; (d) specimen.