Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-03-15
2003-05-13
Horlick, Kenneth R. (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024100, C536S024200, C536S024300, C536S024320, C536S024330, C536S025320, C435S091100, C435S091500, C435S091510, C435S006120
Reexamination Certificate
active
06562955
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to oligonucleotides for detection of
Vibrio parahaemolyticus
for clinical examination, public hygiene, food evaluation or food poisoning evaluation, and to detection methods for
Vibrio parahaemolyticus.
PRIOR ART
Vibrio parahaemolyticus
is known as a common infectious food poisoning bacteria. Over 95% of
Vibrio parahaemolyticus
isolated from gastroenteritis patients are Kanagawa phenomenon-positive bacteria exhibiting hemolytic activity in Wagatsuma agar medium, whereas 99% of these bacteria isolated from fish and water are Kanagawa phenomenon-negative bacteria. This had suggested a strong relationship between pathogenic
Vibrio parahaemolyticus
and the Kanagawa phenomenon, and later investigation revealed that the Kanagawa phenomenon is a phenomenon that occurs due to extracellular release of
Vibrio parahaemolyticus
thermostable direct hemolysin (TDH). As a result, TDH has come to notice as a pathogenic factor of
Vibrio parahaemolyticus.
More recently, certain pathogenic strains even among the Kanagawa phenomenon-negative strains have been confirmed to have a base sequence similar to that of TDH, and produce a hemolysin (TDH-related hemolysin: TRH) with partially common antigenicity.
Detection and identification of
Vibrio parahaemolyticus
has hitherto been complicated and time-consuming as it involves enrichment culturing and isolation culturing followed by determination of the Kanagawa phenomenon. Recently, detection and identification of
Vibrio parahaemolyticus
has been accomplished by the hybridization method using genetic probes specific to sequences in the TDH or TRH genes, but it has been difficult to obtain sufficient detection sensitivity for food evaluation and the like.
Thus, since identification of
Vibrio parahaemolyticus
requires complex procedures and prolonged periods, and a rapid detection of trace amounts of
Vibrio parahaemolyticus
in a sample was difficult to accomplish, a rapid and high sensitive detection method has been desired in fields such as food evaluation. In addition, in order to simplify the examination of interest, development of an automatic examination device has also been desired.
For highly sensitive detection, it is preferable to perform the detection after amplifying a specific sequence in the gene to be detected or identified, or in RNA derived from the gene (hereafter, these genes will collectively be referred to as “target nucleic acid”).
When the target nucleic acid is DNA, Polymerase chain reaction (PCR) is known as an amplification method thereof. This method accomplishes amplification of a specific sequence in the target DNA by repetition of a cycle of heat denaturation, primer annealing and extension reaction in the presence of a pair of primers homologous and complementary to both ends of the specific sequence, as well as a thermostable DNA polymerase. However, the PCR method requires a complicated procedure involving repetition of rapidly increasing and decreasing the temperature, which prevents its automatization. In addition, amplification of a specific sequence by the PCR method requires oligonucleotides highly specific to the specific sequence, and oligonucleotides with high specificity to the target DNA are also required for highly sensitive detection and identification.
As amplification methods in cases where the target nucleic acid is RNA, there are known the NASBA method and 3SR method, whereby the specific sequence is amplified by the concerted action of reverse transcriptase and RNA polymerase. These methods involve a chain reaction, wherein a promoter sequence-containing primer for a specific sequence in the target RNA, reverse transcriptase, and Ribonuclease H are used to synthesize double-stranded DNA containing the promoter sequence, and this double-stranded DNA is used as a template for RNA polymerase-catalyzed synthesis of RNA containing the specific sequence, while the RNA in turn becomes a template for synthesis of double-stranded DNA containing the promoter sequence. The NASBA method and 3SR method can accomplish nucleic acid amplification at a constant temperature, and are therefore considered to be methods suitable for automation. However, since these amplification methods involve reaction at relatively low temperature (for example, 41° C.), the target RNA forms an intramolecular structure which inhibits binding of the primer, and may reduce the reaction efficiency. Consequently, a procedure of heat denaturation of the target RNA prior to the amplification reaction was required to break down the intramolecular structure of the target RNA, thereby to improve the primer binding efficiency. In addition, amplification of the specific sequence by the NASBA method requires an oligonucleotide with high specificity for the specific sequence, and an oligonucleotide with high specificity to the target RNA is also required for highly sensitive detection and identification. Even for RNA detection at low temperature, it is necessary to use an oligonucleotide that can bind to RNA that has formed the aforementioned intramolecular structure.
Therefore, the first object of the present invention is to provide an oligonucleotide that is useful for specific amplification of
Vibrio parahaemolyticus
thermostable direct hemolysin-related hemolysin genes (trh1 and trh2) or RNA derived from the genes, as well as for their highly sensitive detection and identification.
The second object of the present invention is to provide an oligonucleotide that is useful for specific amplification of
Vibrio parahaemolyticus
thermostable direct hemolysin gene (tdh2) or RNA derived from this gene, as well as for its highly sensitive detection and identification.
The third object of the present invention to provide a suitable combination of oligonucleotides useful for specific amplification of RNA derived from a
Vibrio parahaemolyticus
thermostable direct hemolysin-related hemolysin gene (trh1) at relatively low temperature (for example, 41° C.), as well as for highly sensitive detection and identification thereof.
The fourth object of the present invention to provide a suitable combination of oligonucleotides useful for specific amplification of RNA derived from a
Vibrio parahaemolyticus
thermostable direct hemolysin-related hemolysin gene (trh2) at relatively low temperature (for example, 41° C.), as well as for highly sensitive detection and identification thereof.
The fifth object of the present invention to provide a suitable combination of oligonucleotides useful for specific amplification of RNA derived from a
Vibrio parahaemolyticus
thermostable direct hemolysin gene (tdh2) at relatively low temperature (for example, 41° C.), as well as for highly sensitive detection and identification thereof.
DETAILED DESCRIPTION OF THE INVENTION
The invention of 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, which has been accomplished to achieve the first object, relates to 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.
The invention of 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
Ishiguro Takahiko
Ishizuka Tetsuya
Saitoh Juichi
Auerbach Jeffrey I.
Horlick Kenneth R.
Liniak Berenato & White
Spiegler Alexander H.
Tosoh Corporation
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