Patent US3715678A successfully refreshed from Google Patents.
EP0409159A2
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EP
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Method for testing causative microorganism of food poisoning and reagents therefor
Assignee: Shimadzu Corp
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Filing: 1990-07-17
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Granted: —
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Expires: 2010-07-17
Abstract
Abstract A method for testing causative bacterial species of food poisoning which is characterized by using two oligonucleotide primers that hybridize to opposite strands of bacterial DNA specifically, and flank a unique region in the target DNA and amplifying the specific fragment of the bacterial DNA, comprising the steps of: (a) hybridizing the primer to specific gene sequence of bacteria in a sample, extending the hybridized primer with deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), and resultantly making the double strand nucleotide; (b) where the primer extension products are cleaved into each single strand of nucleotide by certain external force such as heat, pH and so on, one single strand functioning as a template for nucleotide extension with a primer of the other strand; (c) repeating a series of cycles involving cleavage of primer extension products, primer hybridizing, extension of the hybridized primers to amplify the specific fragment of DNA, and detecting the amplified DNA fragment; and (d) as the result, determining whether or not the specific fragment is present in said sample, thereby to confirm a species of causative bacteria of food poisoning.
Inventors
- 👤 Tetsuo Ohashi
- 👤 Hiroyuki Jikuya
- 👤 Jun Takano
- 👤 Yoshinari Shirasaki
- 👤 Hirohisa Abe
- 👤 Koichi Yamagata
- 👤 Yoshihiro Aoyama
- 👤 Jun Tada
- 👤 Shigeru Fukushima
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-23 19:58
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6888; C12Q1/689; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/832; Y10S435/834; Y10S435/842; Y10S435/879
Claims Analysis
Total Claims: 48
Claim 1
Independent Claim
183 words
A method for testing causative bacterial species of food poisoning which is characterized by using two oligonucleotide primers that hybridize to opposite strands of bacterial DNA specifically, and flank a unique region in the target DNA and amplifying the specific fragment of the bacterial DNA, comprising the steps of: (a) hybridizing the primer to specific gene sequence of bacteria in a sample, extending the hybridized primer with deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), and resultantly making the double strand nucleotide; (b) where the primer extension products are cleaved into each single strand of nucleotide by certain external force such as heat, pH and so on, one single strand functioning as a template for nucleotide extension with a primer of the other strand; (c) repeating a series of cycles involving cleavage of primer extension products, primer hybridizing, extension of the hybridized primers to amplify the specific fragment of DNA, and detecting the amplified DNA fragment; and (d) as the result, determining whether or not the specific fragment is present in said sample, thereby to confirm a species of causative bacteria of food poisoning.
Claim 2
Independent Claim
183 words
A method for testing causative bacterial species of food poisoning which is characterized by using two oligonucleotide primers that hybridize to opposite strands of bacterial DNA specifically, and flank a unique region in the target DNA and amplifying the specific fragment of the bacterial DNA, comprising the steps of: (a) hybridizing the primer to specific gene sequence of bacteria in a sample, extending the hybridized primer with deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), and resultantly making the double strand nucleotide; (b) where the primer extension products are cleaved into each single strand of nucleotide by certain external force such as heat, pH and so on, one single strand functioning as a template for nucleotide extension with a primer of the other strand; (c) repeating a series of cycles involving cleavage of primer extension products, primer hybridizing, extension of the hybridized primers to amplify the specific fragment of DNA, and detecting the amplified DNA fragment; and (d) as the result, determining whether or not the specific fragment is present in said sample, thereby to confirm a species of causative bacteria of food poisoning.
Claim 3
Dependent Claim
30 words
A method as defined in claim 1 wherein said bacterial species of food poisoning is Bacillus cereus and said specific gene of the bacteria is β-lactamase gene of Bacillus cereus.
Claim 4
Dependent Claim
47 words
A method as defined in claim 2 wherein each synthetic oligonucleotide defined below (a)-(e) is used as a primer of extension, and its nucleotide sequence is complementary to the sequence of the gene according to claim 2. (5′)d-GGTTTAAGTATTACAAGCC(3′) (a) (5′)d-GCATATACACCTAATCGAGC(3′) (b) (5′)d-CCACTAAGTCTTCTTTCG(3′) (c) (5′)d-TTCTGTATGCCCTTTCCCTG(3′) (d) (5′)d-ATTTCAGAAGCGCGTAACGG(3′) (e)
Claim 5
Dependent Claim
33 words
A method as defined in claim 3 wherein among the synthetic oligonucleotide described in claim 3, each oligonucleotide of which length is more than 10 bases is used as a primer of extension.
Claim 6
Dependent Claim
34 words
A method as defined in claim 1 wherein said bacterial species of foor poisoning is Vibrio parahaemolyticus and said species gene of the bateria is thermostable directed hemolysin gene (tdh gene) of Vibrio parahaemolyticus.
Claim 7
Dependent Claim
41 words
A method as defined in claim 5 wherein each synthetic oligonucleotide defined below (a)-(b) is used as a primer of extension, and its nucleotide sequence is complementary to the sequence of the gene according to claim 5. (5′)d-GGTAATGTGTATATCCAAC(3′) (a) (5′)d-CTACGTCAAAGTCGCACTAG(3′) (b)
Claim 8
Dependent Claim
33 words
A method as defined in claim 6 wherein among the synthetic oligonucleotide described in claim 6, each oligonucleotide of which length is more than 10 bases is used as a primer of extension.
Claim 9
Dependent Claim
31 words
A method as defined in claim 1 wherein said bacterial species of food poisoning is Salmonella sp. and said specific gene of the bateria is ara C gene of Salmonella sp.
Claim 10
Dependent Claim
43 words
A method as defined in claim 8 wherein each sysnthetic oligonucleotide defined below (a)-(c) is used as a primer of extension, and its nucleotide sequence is complementary to the sequence of the gene according to claim 8. (5′)d-GGCGAGCAGTTTGTCTGTC(3′) (a) (5′)d-TACCGCCATACGTCTGAGC(3′) (b) (5′)d-GTTTCGCCTGGCTGATACG(3′) (c)
Claim 11
Dependent Claim
33 words
A method as defined in claim 9 wherein among the synthetic oligonucleotide defined in claim 9, each oligonucleotide of which length is more than 10 bases is used as a primer of extension.
Claim 12
Dependent Claim
30 words
A method as defined in claim 1 wherein said bacterial species of food poisoning is Clostridium perfringens and said specific gene of the bacteria is enterotoxin gene of Clostridium perfringens.
Claim 13
Dependent Claim
43 words
A method as defined in claim 11 wherein each synthetic oligonucleotide defined below (a)-(c) is used as a primer of extension, and its nucleotide sequence is complementary to the sequence of the gene according to claim 11. (5′)d-AATACATATTGTCCTGCATC(3′) (a) (5′)d-GTAATAGATAAAGGAGATGG(3′) (b) (5′)d-GTAGTAGGATTTATACAAGC(3′) (c)
Claim 14
Dependent Claim
33 words
A method as defined in claim 12 wherein among the synthetic oligonucleotide described in claim 12 each oligonucleotide of which length is more than 10 bases is used as a primer of extension.
Claim 15
Dependent Claim
30 words
A method as defined in claim 1 wherein said bacterial species of food poisoning is Staphylococcus aureus and said specific gene of the bacteria is enterotoxin gene of Staphylococcus aureus.
Claim 16
Dependent Claim
41 words
A method as defined in claim 14 wherein each synthetic oligonucleotide defined below (a)-(b) is used as a primer of extension, and its nucleotide sequence is complementary to the sequence of the gene according to claim 14. (5′)d-CCAGATGAGTTGCACAAATCG(3′) (a) (5′)d-CACCAAATAGTGACGAGTTA(3′) (b)
Claim 17
Dependent Claim
33 words
A method as defined in claim 15 wherein among the sysnthetic oligonucleotide defined in claim 15, each oligonucleotide of which length is more than 10 bases is used as a primer of extension.
Claim 18
Dependent Claim
38 words
A method as defined in claim 1 wherein said bacterial species of food poisoning is enterotoxigenic Escherichia coli capable of producing heat-labile enterotoxin (LT) and said specific gene of the bacteria is enterotoxin gene of enterotoxigenic Escherichia coli.
Claim 19
Dependent Claim
41 words
A method as defined in claim 17 wherein each synthetic oligonucleotide defined below (a)-(b) is used as a primer of extension, and its nucleotide sequence is complementary to the sequence of the gene according to claim 17. (5′)d-TTATCAATTTTGGTCTCGGTC(3′) (a) (5′)d-GAACTATGTTCGGAATATCG(3′) (b)
Claim 20
Dependent Claim
33 words
A method as defined in claim 18 wherein among the synthetic oligonucleotide defined in claim 18, each oligonucleotide of which length is more than 10 base is used as a primer of extension.
Claim 21
Dependent Claim
30 words
A method as defined in claim 1 wherein said bacterial species of food poisoning is Campylobacter jejuni and said specific gene of the bacteria is chromosomal gene of Campylobacter jejuni.
Claim 22
Dependent Claim
41 words
A method as defined in claim 20 wherein each sysnthetic oligonucleotide define below (a)-(b) is used as a primer of extension, and its nucleotide sequence is complementary to the sequence of the gene according to claim 20. (5′)d-AATAATCTGAATCCGATGGT(3′) (a) (5′)d-ATCAGACCATCACCCTTATC(3′) (b)
Claim 23
Dependent Claim
33 words
A method as defined in claim 21 wherein among the synthetic oligonucleotide defined in claim 21, each oligonucleotide of which length is more than 10 bases is used as a primer of extension.
Claim 24
Dependent Claim
41 words
A method as defined in claim 1 wherein said bacterial species of food poisoning is enterotoxigenic Escherichia coli capable of producing heat-stable enterotoxin (ST) and said specific gene of the bacteria is heat-stable enterotoxin gene (ST gene) of enterotoxigenic Escherichia coli.
Claim 25
Dependent Claim
41 words
A method as defined in claim 23 wherein each sysnthetic oligonucleotide defined below (a)-(b) is used as a primer of extension, and its nucleotide sequence is complementary to the sequence of the gene according to claim 23. (5′)d-TAATAGCACCCGGTACAAGC(3′) (a) (5′)d-ATAAAAGTGGTCCTGAAAGC(3′) (b)
Claim 26
Dependent Claim
33 words
A method as defined in claim 24 wherein among the synthetic oligonucleotide defined in claim 24, each oligonucleotide of which length is more than 10 base is used as a primer of extension.
Claim 27
Dependent Claim
30 words
A method as defined in claim 1 wherein said bacterial species of food poisoning is Salmonella typhimurium and said specific gene of the bacteria is flagellin gene of Salmonella typhimurium.
Claim 28
Dependent Claim
41 words
A method as defined in claim 26 wherein each sysnthetic oligonucleotide defined below (a)-(b) is used as a primer of extension, and its nucleotide sequence is complementary to the sequence of the gene according to claim 26. (5′)d-GCGATACTCTTGTCGTCTGG(3′) (a) (5′)d-ATAGCTAATTGCTGCCGAGG(3′) (b)
Claim 29
Dependent Claim
33 words
A method as defined in claim 27 wherein among the synthetic oligonucleotide defined in claim 27, each oligonucleotide of which length is more than 10 bases is used as a primer of extension.
Claim 30
Dependent Claim
28 words
A method as defined in claim 1 wherein detection of said specific fragment is carried out by agarose gel electrophoresis and staining of nucleic acid with ethidium bromide.
Claim 31
Dependent Claim
53 words
A reagent for testing Bacillus cereus which is a synthetic oligonucleotide, targeting β-lactamase gene of Bacillus cereus, and being complementary to the sequence of said -lactamase gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-GGTTTAAGTATTACAAGCC(3′) (a) (5′)d-GCATATACACCTAATCGAGC(3′) (b) (5′)d-CCACTAAGTCTTCTTTCG(3′) (c) (5′)d-TTCTGTATGCCCTTTCCCTG(3′) (d) (5′)d-ATTTCAGAAGCGCGTAACGG(3′) (e) or the corresponding complementary sequence.
Claim 32
Dependent Claim
53 words
A reagent for testing Bacillus cereus which is a synthetic oligonucleotide, targeting β-lactamase gene of Bacillus cereus, and being complementary to the sequence of said -lactamase gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-GGTTTAAGTATTACAAGCC(3′) (a) (5′)d-GCATATACACCTAATCGAGC(3′) (b) (5′)d-CCACTAAGTCTTCTTTCG(3′) (c) (5′)d-TTCTGTATGCCCTTTCCCTG(3′) (d) (5′)d-ATTTCAGAAGCGCGTAACGG(3′) (e) or the corresponding complementary sequence.
Claim 33
Dependent Claim
53 words
A reagent for testing Vibrio parahaemolyticus which is a svnthetic oligonucleotide, targeting thermostable direct remolysin gene (tdri gene) of Vibrio parahemolyticus and being complementary to the sequence of said thermostable direct hemolysin gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-GGTAATGTGTATATCCAAC(3′) (a) (5′)d-CTACGTCAAAGTCGCACTAG(3′) (b) or the corresponding complementary sequence.
Claim 34
Dependent Claim
53 words
A reagent for testing Vibrio parahaemolyticus which is a svnthetic oligonucleotide, targeting thermostable direct remolysin gene (tdri gene) of Vibrio parahemolyticus and being complementary to the sequence of said thermostable direct hemolysin gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-GGTAATGTGTATATCCAAC(3′) (a) (5′)d-CTACGTCAAAGTCGCACTAG(3′) (b) or the corresponding complementary sequence.
Claim 35
Dependent Claim
49 words
A reagent for testing Salmonella sp. which is a synthetic oligonucleotide, targeting ara C gene of Salmonella sp., and being complementary to the sequence of said ara C gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-GCGATACTCTTGTCGTCTGG(3′) (a) (5′)d-ATAGCTAATTGCTGCCGAGG(3′) (b) or the corresponding complementary sequence.
Claim 36
Dependent Claim
49 words
A reagent for testing Salmonella sp. which is a synthetic oligonucleotide, targeting ara C gene of Salmonella sp., and being complementary to the sequence of said ara C gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-GCGATACTCTTGTCGTCTGG(3′) (a) (5′)d-ATAGCTAATTGCTGCCGAGG(3′) (b) or the corresponding complementary sequence.
Claim 37
Dependent Claim
48 words
A reagent for testing Clostridium perfringens which is a synthetic oligonucleotide, targeting enterotoxin gene of Clostridium, and being complementary to the sequence of said enterotoxin gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-AATACATATTGTCCTGCATC(3′) (a) (5′)d-GTAATAGATAAAGGAGATGG(3′) (b) (5′)d-GTAGTAGGATTTATACAAGC(3′) (c) or the corresponding complementary sequence.
Claim 38
Dependent Claim
48 words
A reagent for testing Clostridium perfringens which is a synthetic oligonucleotide, targeting enterotoxin gene of Clostridium, and being complementary to the sequence of said enterotoxin gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-AATACATATTGTCCTGCATC(3′) (a) (5′)d-GTAATAGATAAAGGAGATGG(3′) (b) (5′)d-GTAGTAGGATTTATACAAGC(3′) (c) or the corresponding complementary sequence.
Claim 39
Dependent Claim
45 words
A reagent for testing Staphyrococcus aureus which is a synthetic oligonucleotide, targeting enterotoxin gene of Staphylococcus, and being complementary to the sequence of saidenterotoxin gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-CCAGATGAGTTGCACAAATCG(3′) (a) (5′)d-CACCAAATAGTGACGAGTTA(3′) (b) or the corresponding complementary sequence.
Claim 40
Dependent Claim
45 words
A reagent for testing Staphyrococcus aureus which is a synthetic oligonucleotide, targeting enterotoxin gene of Staphylococcus, and being complementary to the sequence of saidenterotoxin gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-CCAGATGAGTTGCACAAATCG(3′) (a) (5′)d-CACCAAATAGTGACGAGTTA(3′) (b) or the corresponding complementary sequence.
Claim 41
Dependent Claim
53 words
A reagent for testing enterotoxigenic Escherichia coli which is a synthetic oligonucleotide, targeting heat-labile toxin gene (LT gene) of enterotoxigenic Escherichia coli, and being complementary to the sequence of said heat-labile toxin gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-TTATCAATTTTGGTCTCGGTC(3′) (a) (5′)d-GAACTATGTTCGGAATATCG(3′) (b) or the corresponding complementary sequence.
Claim 42
Dependent Claim
53 words
A reagent for testing enterotoxigenic Escherichia coli which is a synthetic oligonucleotide, targeting heat-labile toxin gene (LT gene) of enterotoxigenic Escherichia coli, and being complementary to the sequence of said heat-labile toxin gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-TTATCAATTTTGGTCTCGGTC(3′) (a) (5′)d-GAACTATGTTCGGAATATCG(3′) (b) or the corresponding complementary sequence.
Claim 43
Dependent Claim
47 words
A reagent for testing Campylobacter jejuni which is a synthetic oligonucleotide, targeting chromosomal gene of Campylobacter jejuni and being complementary to the sequence of said chromosomal gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-AATAATCTGAATCCGATGGT(3′) (a) (5′)d-ATCAGACCATCACCCTTATC(3′) (b) or the corresponding complementary sequence.
Claim 44
Dependent Claim
47 words
A reagent for testing Campylobacter jejuni which is a synthetic oligonucleotide, targeting chromosomal gene of Campylobacter jejuni and being complementary to the sequence of said chromosomal gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-AATAATCTGAATCCGATGGT(3′) (a) (5′)d-ATCAGACCATCACCCTTATC(3′) (b) or the corresponding complementary sequence.
Claim 45
Dependent Claim
49 words
A reagent for testing Salmonella typhimurium which is a synthetic oligonucleotide, targeting flagellin gene of Salmonella typhimurium, and being complementary to the sequence of said flagellin gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-GGCGAGCAGTTTGTCTGTC(3′) (a) (5′)d-TACCGCCATACGTCTGAGC(3′) (b) (5′)d-GTTTCGCCTGGCTGATACG(3′) (c) or the corresponding complementary sequence.
Claim 46
Dependent Claim
49 words
A reagent for testing Salmonella typhimurium which is a synthetic oligonucleotide, targeting flagellin gene of Salmonella typhimurium, and being complementary to the sequence of said flagellin gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-GGCGAGCAGTTTGTCTGTC(3′) (a) (5′)d-TACCGCCATACGTCTGAGC(3′) (b) (5′)d-GTTTCGCCTGGCTGATACG(3′) (c) or the corresponding complementary sequence.
Claim 47
Dependent Claim
53 words
A reagent for testing enterotoxigenic Escherichia coli which is a synthetic oligonucleotide, targeting heat-stable enterotoxin gene (ST gene) of enterotoxigenic of Escherichia coli,and being complementary to the sequence of said heat-stable enterotoxin gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-TAATAGCACCCGGTACAAGC(3′) (a) (5′)d-ATAAAAGTGGTCCTGAAAGC(3′) (b) or the corresponding complementary sequence.
Claim 48
Dependent Claim
53 words
A reagent for testing enterotoxigenic Escherichia coli which is a synthetic oligonucleotide, targeting heat-stable enterotoxin gene (ST gene) of enterotoxigenic of Escherichia coli,and being complementary to the sequence of said heat-stable enterotoxin gene, said synthetic oligonucleotide being composed of the following group of sequences; (5′)d-TAATAGCACCCGGTACAAGC(3′) (a) (5′)d-ATAAAAGTGGTCCTGAAAGC(3′) (b) or the corresponding complementary sequence.
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