Methods, primers and probes for detection of hepatitis C and novel variants
AssigneeHoffmann-La Roche Inc.
InventorsRobert M. Resnick, Karen K. Y. Young
Life of this patent
Estimating the term…
Abstract
Oligonucleotides primers can be used to amplify and detect Hepatitis C virus nucleic acids in a process that involves reverse transcription of the viral genomic RNA to create cDNA and the subsequent amplification of the cDNA by the polymerase chain reaction. Oligonucleotide probes can be used to detect the presence of amplified DNA by hybridization. The invention provides improved methods, compositions, and kits for amplifying and detecting U.S., Japan, and HCV-C prototype nucleic acids.
Claims23
An oligonucleotide probe for detecting the presence of Hepatitis C virus (HCV) nucleic acids, consisting of a nucleic acid sequence at least 14 nucleotides in length that hybridizes under sequence-specific, stringent hybridization conditions to a nucleotide sequence that distinguishes and is specific to an HCV strain selected from the group consisting of Japan, US, and C9 prototype, and wherein said nucleic acid sequence is contained within a sequence selected from the group consisting of
- KY78 (SEQ ID NO. 18), KY81 (SEQ ID NO. 11), KY86 (SEQ ID NO. 13),
- KY150 (SEQ ID NO. 43), KY145 (SEQ ID NO. 19), KY83 (SEQ ID NO. 7),
- KY87 (SEQ ID NO. 14), KY84 (SEQ ID NO. 8), KY88 CSEO ID NO. 12),
- KY85 (SEQ ID NO. 9), KY100 (SEO ID NO. 21), KY148 (SEQ ID NO. 17),
- KY149 (SEQ ID NO. 16), KY82 (SEQ ID NO. 28), KY109 (SEO ID NO. 24),
- KY111(SEQ ID NO. 25), KY80 (SEQ ID NO. 5), KY144 (SEQ ID NO. 6),
- KY153 (SEQ ID NO. 15), SEQ ID NO. 34. SEQ ID NO. 35,
- SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38,
- SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41,
- SEQ ID NO. 42, and sequences complementary thereto.
An oligonuclcotidc primer for amplifying an HCV nucleic acid, consisting of a nucleic acid sequence at least 14 nucleotides in length that hybridizes under sequence-specific, stringent hybridization conditions to a nucleotide sequence that distinguishes and is specific to an HCV strain or isotype selected from the group consisting of Japan, US and C9 strains, and wherein said nucleic acid sequence is contained within a sequence selected from the group consisting of
- KY78 (SEQ ID NO. 18), KY80 (SEQ ID NO. 5), KY81 (SEQ ID NO. 11),
- KY83 (SEQ ID NO. 7), KY86 (SEQ ID NO. 13), KY88 (SEQ ID NO. 12),
- KY100 (SEQ ID NO. 21), KY144 (SEQ ID NO. 6), KY153 (SEQ ID NO. 15),
- KY145 (SEQ ID NO. 19), KY98 (SEQ ID NO. 3), KY109 (SEQ ID NO. 24),
- KY111 (SEQ ID NO. 25), KY149 (SEQ ID NO. 16), KY87 (SEQ ID NO. 14),
- KY84 (SEQ ID NO. 8), KY148 (SEQ ID NO. 17), KY85 (SEQ ID NO. 9)
- SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40,
- SEQ ID NO. 41, and SEQ ID NO. 42.
A process for detecting the presence of Hepatitis C genomic nucleic acid from a Japan or US prototype strain in a sample, comprising:
- (a) treating said sample under conditions that enable the amplification of a subsequence of HCV genomic nucleic acid, wherein said subsequence is contained in the region of the Hepatitis C genomic nucleic acid corresponding to SEQ ID NO:
- 29;
- (b) mixing the amplified nucleic acid with an oligonucleotide probe consisting of a nucleic acid sequence at least 14 nucleotides in length contained within said amplified region, wherein said nucleic acid sequences distinguishes and is specific to said U.S. and Japan prototype strains and is not specific to a C9 strain, under sequence-specific, stringent hybridization conditions, wherein the probe binds to the subsequence to form a stable hybrid duplex only if said subsequence is from a U.S. or Japan prototype strain;
- and
- (c) detecting hybrids formed between the subsequence and the probe.
A process for detecting a C9 isolate of hepatitis virus, the method comprising the steps of:
- (a) contacting a sample suspected of containing an HCV-C9 nucleic acid sequence with a probe consisting of a nucleic acid sequence at least 14 nucleotides in length contained in a sequence selected from the group consisting of SEQ ID NO;
- 29, SEQ ID NO:
- 30, SEQ ID NO:
- 31, SEQ ID NO:
- 32, SEQ ID NO:
- 33, and the complements thereof, wherein said nucleic acid sequence is complementary to an HCV-C9 nucleic acid subsequence that is specific to the HCV-C9 virus, under sequence-specific, stringent hybridization conditions;
- and
- (b) detecting the hybridization of the probe to said subsequence.
A process for detecting hepatitis C virus (HCV) nucleic acid, comprising the steps of:
- (a) contacting a sample suspected of containing an HCV nucleic acid sequence with a probe consisting of a nucleic acid sequence at least 14 nucleotides in length contained in a sequence selected from the group consisting of SEQ ID NO:
- 29, SEQ ID NO:
- 30, SEQ ID NO:
- 31, SEQ ID NO:
- 32, SEQ ID NO:
- 33, and the complements thereof, wherein said nucleic acid sequence is specific to HCV-C9, US prototype, and Japan prototype virus strains, under sequence-specific, stringent hybridization conditions;
- and
- (b) detecting the hybridization of the probe to said subsequence.
Dependencies are inferred from the claim text.