Method for genotyping and quantifying Hepatitis B Virus
First Claim
1. A pair of primers for amplifying a target nucleic acid of Hepatitis B Virus, comprising, respectively, the sequences of SEQ ID NOs:
- 13 and 14, SEQ ID NOs;
17 and 14, or SEQ ID NOs;
20 and 6, wherein each primer is 8-50 nucleotides in length.
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Abstract
A method for simultaneously genotyping and quantifying Hepatitis B Virus. Also disclosed are (1) a pair of primers containing, respectively, the sequences of SEQ ID NOs: 13 and 14, SEQ ID NOs: 17 and 14, or SEQ ID NOs: 20 and 6, each primer being 8-50 nucleotides in length; (2) a pair of probes, containing, respectively, the sequences of SEQ ID NOs: 18 and 19, SEQ ID NOs: 15 and 16, or SEQ ID NOs: 21 and 22, each probe being 9-50 nucleotides in length; (3) a nucleic acid obtained from amplification of a Hepatitis B Virus nucleic acid template, containing the sequence selected from SEQ ID NOs: 15, 19, or 22, or its complementary sequence, the nucleic acid being 100-1,000 nucleotides in length.
25 Citations
29 Claims
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1. A pair of primers for amplifying a target nucleic acid of Hepatitis B Virus, comprising, respectively, the sequences of SEQ ID NOs:
- 13 and 14, SEQ ID NOs;
17 and 14, or SEQ ID NOs;
20 and 6, wherein each primer is 8-50 nucleotides in length. - View Dependent Claims (2, 3, 4, 12, 13, 14, 15, 16)
- 13 and 14, SEQ ID NOs;
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5. A pair of probes for identifying a single nucleotide polymorphism in a target nucleic acid of Hepatitis B Virus, comprising, respectively, the sequences of SEQ ID NOs:
- 18 and 19, SEQ ID NOs;
15 and 16, or SEQ ID NOs;
21 and 22, wherein each probe is 9-50 nucleotides in length. - View Dependent Claims (6, 7, 8)
- 18 and 19, SEQ ID NOs;
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9. A nucleic acid obtained from amplification of a Hepatitis B Virus nucleic acid template, comprising a sequence selected from the group consisting of SEQ ID NOs:
- 15, 19, and 22, or a sequence complementary thereto, wherein the nucleic acid is 100-1,000 nucleotides in length.
- View Dependent Claims (10, 11)
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17. A method for simultaneously identifying a single nucleotide polymorphism (SNP) in a target nucleic acid from a Hepatitis B Virus (HBV) and quantifying the target nucleic acid, comprising:
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providing a first probe that is identical or complementary to a first sequence of the target nucleic acid that covers a base corresponding to the SNP; and
a second probe that is identical or complementary to a second sequence of the target nucleic acid that does not cover the base corresponding to the SNP;
amplifying, by a polymerase chain reaction with a pair of primers, the target nucleic acid to form a double-stranded nucleic acid product containing the first sequence and the second sequence;
hybridizing to the nucleic acid product in a solution to form a first duplex with the first probe that is covalently bounded to a first fluorophore and to form a second duplex with the second probe that is covalently bounded to a second fluorophore, one of the first and second fluorophores being a donor fluorophore and the other being an acceptor fluorophore, so that, when the first probe and the second probe are hybridized to the nucleic acid product, the donor fluorophore and the acceptor fluorophore are in close proximity to allow fluorescence resonance energy transfer therebetween;
heating the solution to an elevated temperature that is above the melting temperature of the duplex formed by the first probe and its complementary sequence;
identifying the SNP by monitoring fluorescent emission change of the acceptor fluorophore upon irradiation of the donor fluorophore with an excitation light, the change being a function of the elevated temperature; and
quantifying the target nucleic acid by monitoring the fluorescent emission of the acceptor fluorophore. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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Specification