Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-11-21
2003-10-07
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S091200, C435S091320, C435S091500, C435S091520, C435S235100, C536S024300, C536S024320, C536S024330
Reexamination Certificate
active
06630300
ABSTRACT:
FIELD OF THE INVENTION
SRSV (Small Round Structured Virus) is commonly known as a causative virus of viral food poisoning. The present invention relates to nucleic acid sequences, oligonucleotides and method for detection of SRSV and, in particular, a virus which belongs to Genotype II (GII) in clinical examinations, public health examinations, food evaluations and food poisoning examinations.
PRIOR ART
SRSV belongs to the human Calicivirus group. Human Caliciviruses are classified according to their three genetic types: Genogroup I (GI), Genogroup II (GII) and Genogroup III (GIII). Generally speaking, GI and GII Caliciviruses are generally referred to as SRSV, and GIII Caliciviruses are referred to as human Caliciviruses in the narrow sense.
Approximately 20% of the food poisoning cases reported in Japan are attributed to viral causes. SRSV is detected in over 80% of these viral food poisoning cases. The major source of infection is food, and raw oysters are often implicated. SRSV has also been detected in infant (sporadic) acute enterogastritis, thus suggesting the possibility of propagation from human to human. SRSV detection therefore provides an important contribution to public health and food quality.
To date, SRSV detection has been relied on electron microscope observation. Detection by this method, however, requires the virus to be present in an amount of 10
6
/ml or greater, and thus the detection subject was limited to patient's feces. Further, even though observation of the virus was possible, it could not be identified.
In recent years, it has become possible to produce viroid hollow particles for human caliciviruses, and research is advancing toward a specific antibody-detecting ELISA employing such particles. However, the detection sensitivity is still on the same level as electron microscopy, and the method is therefore far from highly sensitive.
As mentioned above, since a complex procedure and a long time are required for the conventional method and it is difficult to detect trace amounts of SRSV in samples within a short time, it has been desired to provide a detection method satisfying the high-speed and high-sensitivity requirements for food evaluation and the like. There has also been a demand for development of an automated examination device which allows more convenient examination.
Methods of amplifying target nucleic acid can be utilized as highly sensitive detection methods. One known method for amplification of specific sequences of genomic RNA such as that of SRSV is the reverse transcription-polymerase chain reaction (RT-PCR). This method comprises synthesis of a cDNA for the target RNA by a reverse transcription step, and then repeating a cycle of heat denaturation, primer annealing and extension reaction in the presence of a pair of primers which are complementary and homologous to both ends of specific sequences of the cDNA (the antisense primer may be the one used in the reverse transcription step) as well as a thermostable DNA polymerase, thereby amplifying the specific DNA sequence. However, the RT-PCR method requires a two-step procedure (a reverse transcription step and a PCR step), as well as a procedure involving rapidly increasing and decreasing the temperature, which prevent its automation.
Other methods known for amplification of specific RNA sequences include the NASBA and 3SR methods which accomplish amplification of specific RNA sequences by the concerted action of reverse transcriptase and RNA polymerase. In these methods, the target RNA is used as a template in the synthesis of a promoter sequence-containing double-stranded DNA using a promoter sequence-containing primer, reverse transcriptase and Ribonuclease H; this double-stranded DNA provides a template in the synthesis of an RNA containing the specific base sequence of the target RNA using an RNA polymerase; subsequently, this RNA provides a template in a chain reaction for synthesizing a double-stranded DNA containing the promoter sequence.
Thus, the NASBA and 3SR methods allow nucleic acid amplification at a constant temperature and are therefore considered suitable for automation. However, as these amplification methods involve relatively low temperature reactions (41° C., for example), the target RNA forms an intramolecular structure which inhibits binding of the primer and may reduce the reaction efficiency. Therefore, they require subjecting the target RNA to heat denaturation before the amplification reaction so as to destroy the intramolecular structure of the target RNA and thus to improve the primer binding efficiency. Further, even when carrying out the detection of an RNA at a lower temperature, these methods require an oligonucleotide capable of binding to the RNA forming such a molecular structure.
Thus, an object of the present invention is to provide nucleic acid sequences, oligonucleotides or suitable combination thereof, capable of specifically cleaving or amplifying SRSV and, in particular, a virus which belongs to GII type, preferably at a relatively low and constant temperature (between 35° C. and 50° C., preferably 41° C.), useful in detecting and identifying such a virus at high sensitivity.
DETAILED DESCRIPTION OF THE INVENTION
The invention of claim
1
, which has been accomplished to achieve this object, relates to a cDNA as shown in SEQ. ID. No.1, or fragment or derivative thereof having a size sufficient to bind to Genogroup II type Small Round Structured Virus (SRSV).
The invention of claim
2
, which has been accomplished to achieve the aforementioned object, relates to an oligonucleotide for detection of GII type SRSV, which oligonucleotide is capable of binding to said GII type SRSV at specific site, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos.2 to 9.
The invention of claim
3
, which has been accomplished to achieve the aforementioned object, relates to the oligonucleotide according to claim
2
, wherein said oligonucleotide is an oligonucleotide probe for cleaving said RNA at said specific site by binding to said specific site of said RNA.
The invention of claim
4
, which has been accomplished to achieve the aforementioned object, relates to the oligonucleotide according to claim
2
, wherein said oligonucleotide is an oligonucleotide primer for a DNA elongation reaction.
The invention of claim
5
, which has been accomplished to achieve the aforementioned object, relates to the oligonucleotide according to claim
2
, wherein said oligonucleotide is an oligonucleotide probe a portion of which is modified or labeled with a detectable marker.
The invention of claim
6
, which has been accomplished to achieve the aforementioned object, relates to the oligonucleotide according to claim
2
, wherein said oligonucleotide is a synthetic oligonucleotide in which a portion of its base(s) is (are) modified without impairing the function of said oligonucleotide as an oligonucleotide probe.
The oligonucleotides of the present invention, which have been accomplished to achieve the aforementioned object, are oligonucleotides that complementarily bind in a specific manner to intramolecular structure-free regions of the target RNA in the aforementioned RNA amplification, and they are capable of binding specifically to the target RNA without the heat denaturation described above. In this manner, the present invention provides oligonucleotides that bind to intramolecular structure-free regions of the GII type SRSV RNA at a relatively low and constant temperature (35-50° C., and preferably 41° C.), which are useful for specific cleavage, amplification, detection or the like of GII type SRSV RNA. More specifically, the present invention relates to an oligonucleotide primer which cleaves the target RNA mentioned above at specific site, an oligonucleotide primer for amplifying the above target DNA with PCR, an oligonucleotide primer for amplifying the above target DNA with NASBA or the like, and an oligonucleotide probe for detecting the target nucleic acid without or after these amplifications, thereby accompl
Ishiguro Takahiko
Masuda Noriyoshi
Saito Juichi
Taya Toshiki
Yasukawa Kiyoshi
Foley Shanon
Housel James
Tosoh Corporation
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