Oligonucleotides for detection of Vibrio parahaemolyticus and detection method for Vibrio parahaemolyticus using the same oligonucleotides

Abstract

An oligonucleotide for detection or amplification of a gene selected from the group consisting of Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin genes (trh1 and trh2) and Vibrio parahaemolyticus thermostable direct hemolysin gene (tdh2) or RNA derived therefrom is provided. Further, method for detecting trh1, trh2 or tdh2 using said oligonucleotide is provided.

What is claimed is:  
     
         1 . An oligonucleotide for detection or amplification of  Vibrio parahaemolyticus  thermostable direct hemolysin-related hemolysin genes (trh1 and trh2) or RNA derived from these genes, which oligonucleotide is capable of binding specifically to trh1 and trh2 or RNA derived therefrom, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos. 1 to 11, or an oligonucleotide complementary to said oligonucleotide.  
     
     
         2 . An oligonucleotide for detection or amplification of a  Vibrio parahaemolyticus  thermostable direct hemolysin-related hemolysin gene (trh1) or RNA derived from said gene, which oligonucleotide is capable of binding specifically to trh1 or RNA derived therefrom, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos. 12 to 14, or an oligonucleotide complementary to said oligonucleotide.  
     
     
         3 . An oligonucleotide for detection or amplification of a  Vibrio parahaemolyticus  thermostable direct hemolysin-related hemolysin gene (trh2) or RNA derived from said gene, which oligonucleotide is capable of binding specifically to trh2 or RNA derived therefrom, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos. 15 to 17, or an oligonucleotide complementary to said oligonucleotide.  
     
     
         4 . An oligonucleotide primer for DNA extension reaction, comprising an oligonucleotide according to any of  claims 1  to  3 .  
     
     
         5 . An oligonucleotide probe which is an oligonucleotide according to any of  claims 1  to  3  a portion of which is modified or labeled with a detectable marker.  
     
     
         6 . An oligonucleotide for detection or amplification of  Vibrio parahaemolyticus  thermostable direct hemolysin gene (tdh2) or RNA derived from said gene, which oligonucleotide is capable of binding specifically to tdh2 or RNA derived from said gene, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos. 18 to 24, or an oligonucleotide complementary to said oligonucleotide.  
     
     
         7 . An oligonucleotide primer for DNA extension reaction, comprising an oligonucleotide according to  claim 6 .  
     
     
         8 . An oligonucleotide probe which is an oligonucleotide according  claim 6  a portion of which is modified or labeled with a detectable marker.  
     
     
         9 . A detection method employing a RNA amplification process, which comprises the steps of: forming a cDNA with a RNA-dependent DNA polymerase using a specific sequence of a RNA derived from a  Vibrio parahaemolyticus  thermostable direct hemolysin-related hemolysin gene (trh1) present in a sample as a template, with a first primer having a sequence complementary to said specific sequence and a second primer having a sequence homologous to said specific sequence, wherein either the first or second primer has a sequence having the RNA polymerase promoter sequence added at its 5′-region, thereby producing a RNA-DNA double-strand; digesting the RNA of said RNA-DNA double-strand with Ribonuclease H to form a single-stranded DNA; and then forming a double-stranded DNA that includes a promoter sequence allowing transcription of said RNA sequence or a RNA comprising a sequence complementary to said RNA sequence with a DNA-dependent DNA polymerase using said single-stranded DNA as a template, said double-stranded DNA produces a RNA transcription product in the presence of a RNA polymerase, and said RNA transcription product is subsequently used as the template for the single-stranded DNA production with said RNA-dependent DNA polymerase; characterized in that the oligonucleotide of SEQ. ID. No.25 is used as the first primer and the oligonucleotide of SEQ. ID. No.26 is used as the second primer.  
     
     
         10 . The detection method of  claim 9 , characterized in that said first primer is an oligonucleotide comprising at least 10 contiguous bases of the sequence of SEQ. ID. No.25.  
     
     
         11 . The detection method of  claim 9 , characterized in that said second primer is an oligonucleotide comprising at least 10 contiguous bases of the sequence of SEQ. ID. No.26.  
     
     
         12 . A detection method for a  Vibrio parahaemolyticus  thermostable direct hemolysin-related hemolysin gene, which comprises the steps of: conducting the RNA amplification process according to  claim 9  in the presence of an oligonucleotide probe labeled with an intercalator fluorescent dye, wherein the sequence of said probe is complementary to at least a portion of said RNA transcription product, and complementary binding of said probe to said RNA transcription product results in a change of the fluorescent property relative to that of a situation where a complex formation is absent; and then measuring the fluorescence intensity of the reaction solution.  
     
     
         13 . A detection method employing a RNA amplification process, which comprises the steps of: forming a cDNA with a RNA-dependent DNA polymerase using a specific sequence of a RNA derived from a  Vibrio parahaemolyticus  thermostable direct hemolysin-related hemolysin gene (trh2) present in a sample as a template, with a first primer having a sequence complementary to said specific sequence and a second primer having a sequence homologous to said specific sequence, wherein either the first or second primer has a sequence having the RNA polymerase promoter sequence added at its 5′-region, thereby producing a RNA-DNA double-strand; digesting the RNA of said RNA-DNA double-strand with Ribonuclease H to form a single-stranded DNA; and then forming a double-stranded DNA that includes a promoter sequence allowing transcription of said RNA sequence or a RNA comprising a sequence complementary to said RNA sequence with a DNA-dependent DNA polymerase using said single-stranded DNA as a template, said double-stranded DNA produces a RNA transcription product in the presence of a RNA polymerase, and said RNA transcription product is subsequently used as the template for the single-stranded DNA production with said RNA-dependent DNA polymerase; characterized in that the oligonucleotide of SEQ. ID. No.33 is used as the first primer and the oligonucleotide of SEQ. ID. No.34 is used as the second primer.  
     
     
         14 . The detection method of  claim 13 , characterized in that said first primer is an oligonucleotide comprising at least 10 contiguous bases of the sequence of SEQ. ID. No.33.  
     
     
         15 . The detection method of  claim 13 , characterized in that said second primer is an oligonucleotide comprising at least 10 contiguous bases of the sequence of SEQ. ID. No.34.  
     
     
         16 . A detection method for a  Vibrio parahaemolyticus  thermostable direct hemolysin-related hemolysin gene, which comprises the steps of: conducting the RNA amplification process according to  claim 13  in the presence of an oligonucleotide probe labeled with an intercalator fluorescent dye, wherein the sequence of said probe is complementary to at least a portion of said RNA transcription product, and complementary binding of said probe to said RNA transcription product results in a change of the fluorescent property relative to that of a situation where a complex formation is absent; and then measuring the fluorescence intensity of the reaction solution.  
     
     
         17 . A detection method employing a RNA amplification process, which comprises the steps of: forming a cDNA with a RNA-dependent DNA polymerase using a specific sequence of a RNA derived from a  Vibrio parahaemolyticus  thermostable direct hemolysin gene (tdh) present in a sample as a template, with a first primer having a sequence complementary to said specific sequence and a second primer having a sequence homologous to said specific sequence, wherein either the first or second primer has a sequence having the RNA polymerase promoter sequence added at its 5′-region, thereby producing a RNA-DNA double-strand; digesting the RNA of said RNA-DNA double-strand with Ribonuclease H to form a single-stranded DNA; and then forming a double-stranded DNA that includes a promoter sequence allowing transcription of said RNA sequence or a RNA comprising a sequence complementary to said RNA sequence with a DNA-dependent DNA polymerase using said single-stranded DNA as a template, said double-stranded DNA produces a RNA transcription product in the presence of a RNA polymerase, and said RNA transcription product is subsequently used as the template for the single-stranded DNA production with said RNA-dependent DNA polymerase; characterized in that the oligonucleotide of SEQ. ID. No.39 is used as the first primer and the oligonucleotide of SEQ. ID. No.40 is used as the second primer, or the oligonucleotide of SEQ. ID. No.39 is used as the first primer and the oligonucleotide of SEQ. ID. No.41 is used as the second primer, or the oligonucleotide of SEQ. ID. No.42 is used as the first primer and the oligonucleotide of SEQ. ID. No.41 is used as the second primer.  
     
     
         18 . The detection method of  claim 17  characterized in that said first primer is an oligonucleotide comprising at least 10 contiguous bases of the sequence of SEQ. ID. No.39 or of SEQ. ID. No.42.  
     
     
         19 . The detection method of  claim 17 , characterized in that said second primer is an oligonucleotide comprising at least 10 contiguous bases of the sequence of SEQ. ID. No.40 or SEQ. ID. No.41.  
     
     
         20 . A detection method for a  Vibrio parahaemolyticus,  which comprises the steps of: conducting the RNA amplification process according to  claim 17  in the presence of an oligonucleotide probe labeled with an intercalator fluorescent dye, wherein the sequence of said probe is complementary to at least a portion of said RNA transcription product, and complementary binding of said probe with the RNA transcription product results in a change of the fluorescent property relative to that of a situation where a complex formation is absent, and then measuring the fluorescence intensity of the reaction solution.