Oligonucleotides for detecting Vibrio parahaemolyticus

Abstract: Oligonucleotides (SEQ ID NOs 1-8) selectively hybridizable with a specific gene of Vibro parahaemolyticus , oligonucleotides (SEQ ID NOs 9-13) selectively hybridizable with the LT gene of toxigenic Escherchia coil , oligonucleotides (SEQ ID NOs 14-21) selectively hybrizable with the STh or STp gene of toxigenic Escherchia coil , oligonucleotides (SEQ ID NOs 22-47) selectively hybridizable with the ent A, B, C, or D gene of Staphylococcus aureus , or oligonucleotides (SEQ ID NOs 48-53) selectively hybridizable with the ent E gene of Staplyloccus aureus are prepared and used as primers for gene amplification to thereby selectively detect only respective microorganisms causing food poisoning.

Description: This invention relates to means for detecting Vibrio parahaemolyticus, in clinical examination, in particular testing in case of food poisoning, or in food inspection. In traditional techniques for detection of bacteria, when feces, food, or wipe, a series of operations, namely enrichment culture, isolation culture and differential culture, are required for final identification of the pathogen or contaminant as Vibrio parahaemolyticus, if present. The time periods required for the respective culture steps are 10-16 hours for enrichment culture, 18-24 hours for isolation culture, and 18-24 hours for differential culture, the total time being as long as 2-4 days. Tests to be included in differential culture include growth test in agar medium supplemented with NaCl, gram staining, oxydase test and so forth. They involve complicated and troublesome procedures and are time-consuming and expensive. For detecting the pathogenic factor of Vibrio parahaemolyticus , the so-called reverse passive hemagglutination reaction is available which uses a specific immunoglobulin obtained from an antiserum to thermostable (thermostable) direct hemolysin (TDH) produced by Vibrio parahaemolyticus. However, this reaction needs 20-24 hours until a result is obtained. As mentioned above, the prior art methods invariably need a very complicated procedure and a long period of time for identification as vibrio parahaemolyticus, and are not suited for use in clinical laboratory testing, among others, which demands speediness. Recently, the DNA probe or hybridization techniques, which use oligonucleotides, have been attempted. However, these techniques, which comprise hybridization with oligonucleotide label-modified probes on a membrane or some other support, followed by detection, scarcely have a satisfactory detection sensitivity and selectivity. Moreover, strains of Vibrio parahaemolyticus that have a novel pathogenic factor, namely TDH-related hemolysin (TRH), which is different from those so far reported, has been discovered recently and, further, it has become clear that the gene coding for TRH includes two types, namely trh 1 and trh 2, which differ in base sequence from each other. However, no method has been established as yet for directly testing for Vibrio parahaemolyticus strains having this new pathogenic factor. JP-A-4020299 (Derwent Publications Ltd., London, GB AN 92-076290 Week 9210) describes oligonucleotides for the detection of V. parahaemolyticus. Similarly Nishibuchi M., et al in J. Clinical Microbiology June 1986 Vol. 23 No.6 pages 1091-1095 describes synthetic oligodeoxyribonucleotide probes to examine the association between the Kanagawa phenomenon and the thermostable direct haemolysin gene (tdh) of V. parahaemolyticus. The present invention provides a simple, speedy and highly sensitive method useful in testing for microorganisms causative of food poisoning, which method comprises detecting a Vibrio parahaemolyticus -derived specific gene by the gene amplification technique using oligonucleotides as primers in the nucleic acid synthesis reaction. The invention further provides primers as defined in claims 1 to 3 which can be used in accordance with the first aspect of the invention. The gene amplification can be effected by the polymerase chain reaction method developed by Saiki et al. [hereinafter, PCR method for short; Science, 230, 1350 (1985)]. This method comprises preparing two oligonucleotides, one recognizing and hybridizing with the + chain and the other recognizing and hybridizing with the - chain at the ends of a specific nucleotide sequence region to be detected (in the present case, the trh gene or tdh gene, or trhl gene of Vibrio parahaemolyticus ). In this method, these function as primers for the template-dependent nucleotide polymerization reaction against the sample nucleic acid, in the single stranded form as a result of heat denaturation. The resulting double-stranded nucleic acid is separated into single strands allowing the hybridizing of primers followed by primer extension to be repeated. By repeating this serial procedure, the number of copies of the region between the two primers is increased so that said region can be detected. The sample to be tested may be a laboratory test sample such as feces, urine, blood or tissue homogenate, or a food sample. For testing such samples using PCR, pretreatment is necessary by which the nucleic acid components are released from microbial cells occurring in the sample. Since, however, the PCR can proceed if only a small number of molecules of the nucleic acid hybridizable with the primers are present, a short period of treatment of the laboratory sample with a lytic enzyme, a surfactant, an alkali or the like can give a sample solution containing the nucleic acid in a sufficient amount to enable the PCR to proceed. The primers of the invention may be chemically synthesized ones or natural ones. The primers need not be particularly labeled for detection purposes. The primer-specified amplification region of the nucleotide sequence of a particular gene of Vibrio parahaemolyticus may comprise 50 bases to 2,000 bases, desirably 100 bases to 1,000 bases. In carrying out the template-dependent nucleotide polymerization reaction, a thermostable DNA polymerase may be used. This enzyme may be of any origin provided that it can retain its activity at temperatures of 90-95°C. The heat denaturation is usually carried out at 90-95°C, the annealing for primer hybridization at 37-65°C, and the polymerization reaction at 50-75°C, and these constitute one cycle of PCR. Amplification is effected by repeating PCR cycles, perhaps 20-42 cycles. For detection, the reaction mixture after completion of the PCR may be subjected, as such, to agarose gel electrophoresis, whereby the presence or absence of the amplified nucleotide fragment and, if present, the length thereof can be confirmed. Based on the results, judgment can be made as to whether the nucleotide having the sequence to be recognized by the primers is present in the sample or not. This judgment directly serves as a judgment as to whether Vibrio parahaemolyticus carrying the trh gene or the like is present or not. Other electrophoretic techniques and chromatographic techniques are also effective in detecting the amplified nucleotide fragment. As mentioned above, the present invention makes it possible to detect Vibrio parahaemolyticus. Detection can be made in a simple and speedy manner and with high sensitivity by detecting a Vibrio parahaemolyticus -derived specific gene by the gene amplification technique using certain oligonucleotides as primers in the nucleic acid synthesis reaction. The present invention will now be illustrated by exemplification. EXAMPLE 1 Detection of the trh gene of Vibrio parahaemolyticus (Experiment 1) Sample preparation A total of 326 strains of Vibrio parahaemolyticus and other Vibrio species as specified in Tables 1 to 15 under the columns "Strain" and "Strain No." were used. Each was inoculated into an appropriate enrichment medium and incubated overnight at 37°C under aerobic conditions. Cells were recovered from the medium (1.5 ml) by centrifugation. They were washed once with 10 mM Tris-hydrochloride buffer (pH 7.5) and then subjected to lysis by suspending them in 0.5 ml of a 1 mg/ml lysozyme solution in the same buffer at 37°C for 10 minutes. A phenol-chloroform mixture (mixing ratio 1:1) saturated with the above-mentioned buffer was added to the lysate solution, followed by thorough stirring. After centrifugation, the upper layer was recovered and subjected to ethanol treatment for precipitating nucleic acid components. The precipitate was dissolved in 1 ml of the above-mentioned buffer and the solution was used as a sample. Primer synthesis Based on the base sequence of the trh gene of Vibrio parahaemolyticus [Nishibuchi, M. et al.: Infect. Immun., 57 , 2691-2697 (1989) and Kishishita et al.: Japan. J. Bacteriol., 45 , 340 (1990)], the sequences specifically shown in Claim 1 were selected and oligonucleotides identical in sequence therewith were chemically synthesized. The chemical synthesis was carried out by the β-cyanoethylphosphamidite method using a Cyclone Plus DNA synthesizer (Milligen/BioResearch). The oligonucleotides synthesized were purified by high-performance liquid chromatography using a C18 reversed phase column. PCR A reaction mixture (30 µl) was prepared by adding, to 3 µl of the sample solution mentioned above, 17.55 µl of sterile distilled water, 3 µl of 10 x reaction buffer, 4,8 µl of dNTP solution, 0.75 µl of primer (a) (SEQ.ID NO.1) 0.75 µl of primer (b) (SEQ.ID. NO.2) and 0.15 µl of thermostable DNA polymerase. Mineral oil (50 µl; Sigma) was layered on said reaction mixture placed in a container. The solutions used in preparing the reaction mixture were as 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: containing dATP, dCTP, dGTP and dTTP each at a final concentration of 1.25 mM. Primer (a) (SEQ.ID NO.1) and primer (b) (SEQ.ID NO.2): Each was an aqueous solution of each product chemically synthesized and purified as mentioned above (concentration 5 ODU/ml). Thermostable DNA polymerase: Taq DNA polymerase (5 units/ml; Perkin Elmer Cetus) The reaction conditions were as follows. Heat denaturation: 94°C, 1 minute. Annealing: 55°C, 1 minute. Polymerization reaction: 72°C, 1 minute. The time require for each cycle covering the process from heat denaturation, through annealing, to polymerization reaction was 5.7 minutes. This cycle was repeated 35 times (total time required about 3 hours). For carrying out these operations, the above reaction conditions were programmed into a DNA Thermal Cycler apparatus (Perkin Elmer Cetus). Detection For detecting the amplified nucleotide fragment in the reaction mixture, agarose gel electrophoresis was performed in the following manner. The agarose gel used had a gel concentration of 2% (w/v) and contained ethidium bromide (0.5 µl/ml). The electrophoresis was conducted at a constant voltage of 100 V for 30 minutes. The procedure and other conditions were as described in Maniatis et al.: Molecular Cloning (1982). In addition to the reaction mixture, molecular weight markers were simultaneously electrophoresed and, based on the comparison of relative mobilities, the length of the nucleotide fragment was estimated. Results As mentioned hereinabove, the base sequence of the trh gene of Vibrio parahaemolyticus has already been determined and two types differing in base sequence, namely trh1 and trh2 have been reported. Therefore, the size of the nucleotide amplified by the PCR using the oligonucleotides, namely primers, of the present invention can be estimated. Thus, with primers (a) (SEQ.ID NO.1) and (b) (SEQ.ID NO.2), the nucleotide amplified is expected to be 251 bases long. When the size of the nucleotide amplified was in agreement with such estimated value, then it was judged that each primer contributed to correct amplification of the target region in the trh gene. The results obtained with 264 strains of Vibrio parahaemolyticus and 98 strains of other Vibrio species are shown in Table 1. As can be seen from Table 1, the primers could correctly detect both the trh1 and trh2 genes of Vibrio parahaemolyticus. Experiment 2 To ascertain whether the results obtained in Experiment 1 were selective for Vibrio parahaemolyticus strains having the trh1 or trh2 gene, diarrhea-causing microorganisms other than Vibrio haemolyticus but generally included as targets in clinical laboratory testing were further tested for comparison. The method was the same as shown in Experiment 1 except that the strains listed in Tables 16 and 17 under Nos. 12, 13, 19 and 20 were cultured overnight at 37°C under anaerobic conditions for preparing samples submissible to the PCR method. A total of 39 strains listed in Table 16 and 17 were cultured for preparing samples. The human placenta-derived DNA was diluted to a concentration of 1 µg/ml and subjected to PCR in the same manner. The results thus obtained are shown in Tables 16 and 17. Some microorganisms gave amplified nucleotide fragments, which were presumably byproducts of the PCR. All of said fragments differed in size from the nucleotide fragment anticipated from the sequence of the trh1 or trh2 gene. If these diarrhea-causing microorganisms had the same trh gene as that of Vibrio parahaemolyticus, the same 251-base-long nucleotide fragment as found in Experiment 1 would have been detected. It is therefore clear that the amplified nucleotides detected for some diarrhea-causing microorganisms were not the products resulting from recognition of the trh gene of Vibrio parahaemolyticus but that trh gene-carrying strains of Vibrio parahaemolyticus and other Vibrio species can be readily detected in distinction from other diarrhea-causing microorganisms. The agarose gel electrophoresis employed in the experiment described herein, when conducted under the conditions mentioned above, can distinguish nucleotides of 100 base pairs or less in size from each other when they differ by 5 to 10 base pairs, and uncleotides of 100 to 500 base pairs in size from each other when they differ by 10 to 20 base pairs. When the precision of nucleotide size determination is improved by using an acrylamide gel, for instance, the reliability in the selective detection will presumably be increased further. EXAMPLE 2 Detection of the tdh gene of vibrio parahaemolyticus Experiment 1 Sample preparation Samples were prepared in the same manner as in Example 1. Primer synthesis Based on the base sequence of the rdh gene of Vibrio parahaemolyticus [Nishibuchi, M. and kaper, J. B.: Mol. Microbiol., 4 , 87-99 (1990)], the sequences shown in Claim 2 were selected, and oligonucleotides having the same sequences as those were chemically synthesized. The chemical synthesis and purification of the oligonucleotides synthesized were carried out in the same manner as in Example 1. PCR The same procedure as used in Example 1 was followed except that the following primer combinations were used. Primer (1) Primer (2) (c) (SEQ. ID NO. 3) (d) (SEQ. ID NO. 4) (e) (SEQ. ID NO. 5) (d) (SEQ. ID NO. 4) (e) (SEQ. ID NO. 5) (f) (SEQ. ID NO. 6) Detection The same procedure as used in Example 1 was followed. Results As mentioned above, the base sequence of the tdh gene of Vibrio parahaemolyticus has already been determined. Therefore, the size of the uncleotide amplified by the PCR using the oligonucleotides, or primers, of the present invention can be estimated. Thus, when primers (c) and (d), (e) and (d), or (e) and (f) are used (SEQ.ID NOS.3 and 4, 5 and 4, or 5 and 6), the oligonucleotide amplified is expected to be 373, 199, or 251 bases long, respectively. When the size of the amplified uncleotide was in agreement with such estimated value, it was judged that each primer contributed to correct amplification of the target region in the tdh gene. The results thus obtained with 264 strains of Vibrio parahaemolyticus and 98 strains of other Vibrio species are shown in Tables 1-15. As can be seen from Tables 1-15, the three primer combinations each could correctly detect tdh gene-containing strains alone among the strains of Vibrio parahaemolyticus and Vibrio species tested. Experiment 2 To ascertain whether the results obtained in Experiment 1 were selective for tdh gene-carrying strains of Vibrio parahaemolyticus and Vibrio species, diarrhea-causing microorganisms other than Vibrio parahaemolyticus but generally included as targets in clinical laboratory testing were tested for comparison in the same manner as in Example 1. The results obtained are shown in Tables 16 and 17. In some microorganisms, amplified nucleotide fragments, which were presumably byproducts of the PCR, were detected. However, they all differed in size from the nucleotide fragment anticipated from the sequence of the tdh gene. If these diarrhea-causing microorganisms had the same tdh gene as that of Vibrio parahaemolyticus, a nucleotide fragment identical in size as that detected in Experiment 1, namely 373, 199 or 251 bases long, would have been detected. It is therefore evident that the amplified nucleotides observed for said certain diarrhea-causing microorganisms were not the products resulting from recognition of the tdh gene of Vibrio parahaemolyticus but that those stains of Vibrio parahaemolyticus and Vibrio species that have the tdh gene can be readily detected in distinction from other diarrhea-causing microorganisms. EXAMPLE 3 Detection of the trh 1 gene of Vibrio parahaemolyticus Experiment 1 Sample preparation, primer synthesis, PCR and detection were carried out in the same manner as in Example 1 except that the primers (g) (SEQ.ID NO.7) and (h) (SEQ.ID. NO.8) defined in Claim 3 were used. When the primers (g) (SEQ.ID NO.7) an (h) (SEQ.ID NO.8) of the invention are used, it is expected that a nucleotide 211 bases long be amplified. When the size of the nucleotide amplified was in agreement with said estimated value, it was judged that each primer contributed to correct amplification of the target region in the trh1 gene. The results obtained with 264 strains of Vibrio parahaemolyticus and 98 strains of Vibrio species are shown in Tables 1-15. As can be seen in Tables 1-15, the primer combination could correctly detect only those strains having the trhl gene among the strains of Vibrio parahaemolyticus and Vibrio species. Experiment 2 To ascertain whether the results obtained in Experiment 1 were selective for the trhl gene-containing strains of Vibrio parahaemolyticus and Vibrio species, diarrhea-causing microorganisms other than Vibrio parahaemolyticus but generally involved as targets in clinical laboratory testing were tested for comparison by the same method as used in Example 1. The results obtained are shown in Tables 16 and 17. With some microorganisms, amplified nucleotide fragments, which were presumably byproducts of the PCR, were detected. However, none of them was identical in size with the nucleotide fragment anticipated from the sequence of the trh1 gene. If these diarrhea-causing microorganisms had the same trh1 gene as that of Vibrio parahaemolyticus, the same, 211-base-long nucleotide fragment as detected in Experiment 1 would have been detected. Therefore, it is evident that the amplified nucleotides found in said certain diarrhea-causing microorganisms were by no means the products of recognition of the trh1 gene of Vibrio parahaemolyticus but that trh1 gene-containing strains of Vibrio parahaemolyticus can be readily detected in distinction from other diarrhea-causing microorganisms. Tables 1-17 Notes to the above Tables: (1) in column "trh': strain having the type 1 trh gene. (2) in column "trh': strain having the type 2 trh gene. (-) in column "trh': strain having no trh gene. (+) in column "tdh': strain having the tdh gene. (-) in column "tdh': strain having no tdh gene. (O) in column "Primer combination": The amplified nucleotide had the same size as the expected value. (o ○) in column "Primer combination": The amplified nucleotide, though detected, had a size different from the expected value. (-) in column "Primer combination": No amplified nucleotide was detected. The strains shown in Tables 1-15 were obtained from: Department of Microbiology, Faculty of Medicine, Kyoto University. The strains shown in Tables 16 an 17 were obtained from: ATCC (American Type Culture collection), JCM (Japanese Collection of Microorganisms, RIKEN Institute of Physical and Chemical Research), and IFO (Institute for Fermentation, Osaka). By using, in accordance with the invention the PCR method and the primers the targets of which are genes most closely associated with pathogenicity, it is possible, in detecting bacteria having such genes, namely Vibrio parahaemolyticus . Since the detection sensitivity is high, samples are needed only in small amounts, and this makes sample pretreatment simple and easy. In the examples described herein, the reaction time was 3 hours and the time for detection procedure was as short as 30 minutes. The detection may be made easily by using a simple apparatus or instrument. Moreover, when agarose gel electrophoresis and nucleic acid staining with ethidium bromide are used for detection purposes, the detection can be carried out without labeling primers or others and, in addition, nucleic acid size determination in possible. The test results thus become highly reliable. Vibrio parahaemolyticus is a pathogen ranking highest among food poisoning-causing bacteria in Japan. The health disturbance caused by this pathogen mostly lies in gastroenteritis, with diarrhea and abdominal pain as main symptoms. Among pathogenic factors of such gastroenteritis, thermostable direct hemolysin (TDH) is currently at the center of concern. Vibrio parahaemolyticus strains having the tdh gene, which are causative of food poisoning, can be detected selectively by taking the tdh gene coding for thermostable direct hemolysin as the target nucleotide for the primers. Furthermore, thermostable direct hemolysin-related hemolysin (TRH), a pathogenic factor similar to TDH but essentially different therefrom, has recently been discovered and, currently, importance has been attached to the pathogenicity of TRH-producing Vibrio parahaemolyticus in food poisoning cases. In accordance with the invention, trh gene-containing Vibrio parahaemolyticus strains as food poisoning-causing organisms can be selectively detected by taking the trh gene coding for TRH as the target nucleotide to the primers. "Sequence Listing" SEQ ID NO: 1 SEQUENCE LENGTH 19 bases SEQUENCE TYPE nucleic acid STRANDEDNESS single TOPOLOGY linear MOLECULE TYPE genomic DNA HYPOTHETICAL NO ANTISENSE NO ORIGINAL SOURCE Vibrio parahaemolyticus FEATURE IDENTIFICATION METHOD S SEQUENCE GGCTCAAAATGGTTAAGCG SEQ ID NO: 2 SEQUENCE LENGTH 19 bases SEQUENCE TYPE nucleic acid STRANDEDNESS single TOPOLOGY linear MOLECULE TYPE genomic DNA HYPOTHETICAL NO ANTISENSE NO ORIGINAL SOURCE Vibrio parahaemolyticus FEATURE IDENTIFICATION METHOD S SEQUENCE CATTTCCGCTCTCATATGC SEQ ID NO: 3 SEQUENCE LENGTH 20 bases SEQUENCE TYPE nucleic acid STRANDEDNESS single TOPOLOGY linear MOLECULE TYPE genomic DNA HYPOTHETICAL NO ANTISENSE NO ORIGINAL SOURCE Vibrio parahaemolyticus FEATURE IDENTIFICATION METHOD S SEQUENCE CCATCTGTCCCTTTTCCTGC SEQ ID NO: 4 SEQUENCE LENGTH 19 bases SEQUENCE TYPE nucleic acid STRANDEDNESS single TOPOLOGY linear MOLECULE TYPE genomic DNA HYPOTHETICAL NO ANTISENSE NO ORIGINAL SOURCE Vibrio parahaemolyticus FEATURE IDENTIFICATION METHOD S SEQUENCE CCAAATACATTTTACTTGG SEQ ID NO: 5 SEQUENCE LENGTH 20 bases SEQUENCE TYPE nucleic acid STRANDEDNESS single TOPOLOGY linear MOLECULE TYPE genomic DNA HYPOTHETICAL NO ANTISENSE NO ORIGINAL SOURCE Vibrio parahaemolyticus FEATURE IDENTIFICATION METHOD S SEQUENCE GGTACTAAATGGCTGACATC SEQ ID NO: 6 SEQUENCE LENGTH 20 bases SEQUENCE TYPE nucleic acid STRANDEDNESS single TOPOLOGY linear MOLECULE TYPE genomic DNA HYPOTHETICAL NO ANTISENSE NO ORIGINAL SOURCE Vibrio parahaemolyticus FEATURE IDENTIFICATION METHOD S SEQUENCE CCACTACCACTCTCATATGC SEQ ID NO: 7 SEQUENCE LENGTH 20 bases SEQUENCE TYPE nucleic acid STRANDEDNESS single TOPOLOGY linear MOLECULE TYPE genomic DNA HYPOTHETICAL NO ANTISENSE NO ORIGINAL SOURCE Vibrio parahaemolyticus FEATURE IDENTIFICATION METHOD S SEQUENCE GGCTCAAAATGGTTAAGCGC SEQ ID NO: 8 SEQUENCE LENGTH 20 bases SEQUENCE TYPE nucleic acid STRANDEDNESS single TOPOLOGY linear MOLECULE TYPE genomic DNA HYPOTHETICAL NO ANTISENSE NO ORIGINAL SOURCE Vibrio parahaemolyticus FEATURE IDENTIFICATION METHOD S SEQUENCE TGGCGTTTCATCCAAATACG

Claims: An oligonucleotide which is complementary to nucleic acid which comprises a target sequence encoding the thermostable direct hemolysin-related hemolysin gene type 1 or type 2 ( trh 1 trh 2 gene) of Vibrio parahaemolyticus, said oligonucleotide consisting of a sequence selected from among the sequences: and or from among the corresponding complementary sequences. An oligonucleotide which is complementary to nucleic acid which comprises a target sequence encoding the thermostable direct hemolysin gene (tdh gene) of Vibrio parahaemolyticus , said oligonucleotide consisting of a sequence selected from among the sequences: and or from among the corresponding complementary sequences. An oligonucleotide which is complementary to nucleic acid which comprises a target sequence encoding the thermostable direct hemolysin gene type 1 ( trh1 gene) of Vibrio parahaemolyticus , said oligonucleotide consisting of a sequence selected from among the sequences: and or from among the corresponding complementary sequences. A method of detecting Vibrio parahaemolyticus in a test sample by selectively amplifying a target nucleotide sequence using, as primers for chain extension reaction, at least one pair of oligonucleotides each defined in claim 1, 2 or 3, said method comprising (a) hybridizing the primers to the target sequence occurring in the single-stranded form in the sample and causing chain extension by the polymerization reaction involving four nucleotide triphosphates (dNTPs), (b) separating the thus-obtained double-stranded target nucleotide sequence into single strands, each complementary strand being capable of functioning as a template for chain extension reaction involving a complementary primer, (c) repeating the chain extension reaction involving the primers, separating primer chain extension products from the templates and hybridizing with primer molecules thereby to amplify the target sequence, and detecting the thus-amplified nucleotide fragment, and (d) judging, based on the detection result, whether the sequence to be recognized is present in said sample.

Inventors: FUKUSHIMA SHIGERU, NISHIMURA NAOYUKI, OHASHI TETSUO, OZAKI HIROKO, SHIRASAKI YOSHINARI, TADA JUN, YAMAGATA KOICHI, FUKUSHIMA, SHIGERU, NISHIMURA, NAOYUKI, OHASHI, TETSUO, OZAKI, HIROKO, SHIRASAKI, YOSHINARI, TADA, JUN, YAMAGATA, KOICHI, NISHIMURA, NAOYUKI, KASUTORUMU-RAKUHOKU 713, OZAKI, HIROKO, GRACE COURT HORIKAWA 201
Applicants: SHIMADZU CORP, SHIMADZU CORPORATION
Application year: 1992
Main classification: C12Q1
IPC codes: C12Q1