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  Primers for isothermal amplification of hepatitis c virusUSPTO Application #: 20060040261Title: Primers for isothermal amplification of hepatitis c virus Abstract: The present application relates to primers for isothermal amplification of HCV each include at least eighteen consecutive bases corresponding to a 3′ end region of one selected from base sequences of SEQ ID NOs: 1-10, 21 and 22. The primers are specific to HCV subtypes 1a, 1b, 2a, 2b and 3a, respectively and enable genotyping of HCV by isothermal amplification. (end of abstract) Agent: Antonelli, Terry, Stout & Kraus, LLP - Arlington, VA, US Inventors: Maiko Tanabe, Chihiro Uematsu USPTO Applicaton #: 20060040261 - Class: 435005000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Virus Or Bacteriophage The Patent Description & Claims data below is from USPTO Patent Application 20060040261. Brief Patent Description - Full Patent Description - Patent Application Claims
CLAIM OF PRIORITY [0001] The present application claims priority from Japanese application JP 2004-149448 filed on May 19, 2004, the content of which is hereby incorporated by reference into this application. FIELD OF THE INVENTION [0002] The present invention relates to primers for isothermal amplification of subtypes of hepatitis C virus (hereinafter briefly referred to as "HCV"), and methods for detecting or determining HCV using the primers. BACKGROUND OF THE INVENTION [0003] Chiron Corporation, Emeryville, Calif. identified the gene of HCV in 1988. The genome sequence of HCV including about 9,500 bases has been identified, and it has been revealed that HCV has a single strand RNA as its genome. HCV is often accompanied with mutation in its gene sequence, is classified as four to six subgroups based on gene sequences in regions accompanied with frequent mutation, and is further classified as, for example, subtypes 1a, 1b, 2a, 2b and 3a (P. Simmonds, et al., J. Gen Virol., 74, 661-668 (1993)) [0004] Only two- to three-tenths of subjects infected with HCV have subjective symptoms such as malaise, and most of them undergo chronic inflammation without subjective symptoms and may further undergo liver cirrhosis and liver cancer. It is difficult to detect HCV at initial stage of HCV infection by immunoassay using antibodies and to prevent the symptoms from becoming worse and/or the infected blood completely from contaminating into blood for transfusion. [0005] Recent years, however, have seen significant advances as a result of intensive studies and have enabled the detection of HCV infection and genotyping of HCV even at initial stage of HCV infection by a genetic test in which a gene is amplified. The genotyping enables determination of the incidence rate of acute hepatitis, the rate of becoming chronic and the possibility of shifting from hepatitis via liver cirrhosis to liver cancer, enables the prediction of efficacies of administration of an interferon selected depending on the subtype and enables the identification of contagion sources and infection route. Thus, the detection or determination of HCV by such a genetic test becomes superior to immunoassay. [0006] Certain methods for genotyping HCV have been reported. Okamoto et al., for example, have reported a method for genotyping HCV, including the steps of preparing seven different primers using a core region of HCV as a target, and carrying out a reverse transcription polymerase chain reaction (RT-PCR) using a reverse transcriptase in combination with a suitable set of primers in J. Gen. Virol., 73, 673-679 (1992). A certain method for genotyping HCV in its NS5-region has been reported. Japanese Patent Application Laid-Open (JP-A) No. 09-75100 and F. McOmish, et al. in Transfusion, 33, 7-13 (1993) have reported a method including the steps of cleaving an amplified product of the 5'-untranslated region of HCV with a restriction enzyme and then genotyping HCV. Other methods for genotyping have been reported, including the steps of directly sequencing the amplified product just mentioned above, and genotyping HCV based on the resulting base sequence information [Germer J. J., et al., J. Clin. MiCrobiol., 37(8), 2625-2630 (1999); Holland J., et al., Pathology, 30(2), 192-195 (1998); and Doglio A., et al., Res. Virol., 149(4), 219-227 (1998)]. [0007] All the conventional oligonucleotide primers for genotyping HCV are to be amplified by PCR, and no oligonucleotide primer for genotyping HCV by isothermal amplification has been reported. This is probably because the HCV RNA has a complicated secondary structure as a result typically of intramolecular hydrogen bonds, a region that can be amplified by isothermal amplification is restricted, and it is very difficult to design specific primers capable of carrying out genotyping in the region. A method for quantitatively determining HCV by nucleic acid sequence-based amplification (NASBA), a kind of isothermal amplification, has been reported (Guichon A., et al., J. Clin. Virol., 29, 84-91 (2004)). This technique cannot determine the genotype of HCV gene although it can amplify the HCV gene. Specifically, all the conventional HCV genotyping techniques using oligonucleotide primers require complicated temperature control in PCR and invite complicated procedures, long time and high cost for the determination and are not satisfactory. Demands have therefore been made to develop oligonucleotide primers that enable genotyping of HCV by isothermal amplification. SUMMARY OF THE INVENTION [0008] Accordingly, an object of the present invention is to provide a technique for genotyping HCV by isothermal amplification. Another object of the present invention is to provide primers specific to HCV subtypes 1a, 1b, 2a, 2b and 3a for use in the technique. [0009] The present inventors have successfully designed primers that enable genotype-specific isothermal amplification in a core region of gene sequences belonging to HCV subtypes 1a, 1b, 2a, 2b and 3a genes. The isothermal amplification of a HCV sample using these primers results in genotype-specific amplification and thereby enables detection and genotyping of HCV subtypes 1a, 1b, 2a, 2b and 3a genes based on whether or not amplification occurs. [0010] Specifically, the present invention provides, in an aspect, a primer for isothermal amplification of HCV, comprising at least eighteen consecutive bases corresponding to a 3' end region of one base sequence selected from the group consisting of SEQ ID NOs: 1-10, 21 and 22. [0011] The primer can further include a T7 promoter sequence in its 5'-end region typically when used in NASBA. [0012] The present invention further provides, in another aspect, a pair of primers for isothermal amplification of hepatitis C virus, selected from the following pairs of primers (1) to (5): [0013] (1) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3' end region of the base sequences of SEQ ID NOs: 1 and 2, respectively; [0014] (2) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3' end region of the base sequences of SEQ ID NOs: 3 and 4, respectively, or a pair of primers comprising at least eighteen consecutive bases corresponding to a 3' end region of the base sequences of SEQ ID NOs: 3 and 9, respectively; [0015] (3) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3' end region of the base sequences of SEQ ID NOs: 5 and 6, respectively, or a pair of primers comprising at least eighteen consecutive bases corresponding to a 3' end region of the base sequences of SEQ ID NOs: 5 and 10, respectively; [0016] (4) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3' end region of the base sequences of SEQ ID NOs: 7 and 8, respectively; and [0017] (5) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3' end region of the base sequences of SEQ ID NOs: 21 and 22, respectively. [0018] When used typically in NASBA, at least one of the pair of primers can further include a T7 promoter sequence in its 5'-end region. [0019] The present invention further provides, in yet another aspect, a method for detecting HCV, including the steps of subjecting a clinical sample to isothermal amplification with at least one of the pairs of primers according to the present invention, and determining a HCV subtype based on the resulting amplified product. [0020] The method for detecting HCV enables determination of a HCV subtype. When isothermal amplification is carried out by NASBA, at least one of the pair of primers can further include a T7 promoter sequence. Continue reading... Full patent description for Primers for isothermal amplification of hepatitis c virus Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Primers for isothermal amplification of hepatitis c virus patent application. 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