Oligonucleotide primers for efficient detection of hepatitis C virus (HCV) and methods of use thereof
AssigneeOrtho-Clinical Diagnostics, Inc.
InventorsJeffrey M. Linnen, Kevin M. Gorman
Life of this patent
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Abstract
Described herein are methods and kits for the detection of hepatitis C virus RNA is biological samples obtained from human subjects. The invention includes novel amplification primers and probes useful in the amplification of DNA derived from hepatitis C virus RNA, and kits and methods which incorporate the novel primers.
Claims42
A method for detecting the presence of Hepatitis C Virus (HCV) RNA in a biological sample, said method comprising:
- (A) performing a reverse transcription reaction using, as a template, RNA derived from said sample to produce HCV-specific reverse transcription products;
- (B) amplifying said reverse-transcription products using one or more pairs of oligonuclecotide primers specific for HCV to produce HCV-specific amplification products,
- wherein said pairs are selected fom the group consisting of:
- (a) forward primer 5′-CAGAAAGCGTCTAGCCATGGCGTTAGTA-3′ (C69F28) <SEQ ID NO. 1> and reverse primer 5′-CGGTTCCGCAGAGACCACTATGGCTCTC-3′ (C133R26) <SEQ ID NO. 4>;
- and
- (b) forward primer 5′-GGGAGAGCCATAGTGGTCTGCGGAA-3′ (C131F25) <SEQ ID NO. 2> and reverse primer 5′-CGGGGCACTCGCAAGCACCCTATCA-3′ (C294R26) <SEQ ID NO. 7>;
- and
- (C) detecting said amplifcation products, wherein detection of said amplification products indicates the presence of HCV RNA in said sample.
A method for amplifying Hepatitis C Virus (HCV) DNA, which method comprises performing a polymerase chain reaction on a DNA sample containing HCV DNA using one or more pairs of oligonucleotide primers specific for HCV to produce HCV-specific amplification products, wherein said parrs are selected from the group consisting of:
- (a) forward primer 5′-CAGAAAGCGTCTAGCCATGGCGTTAGTA-3′ (C69F28) <SEQ ID NO. 1> and reverse primer 5′-CGGTTCCGCAGACCACTATGGCTCTC-3′ (C133R28) SEQ ID NO. 4>;
- and
- (b) forward primer 5′-GGGAGAGCCATAGTGGTCTGCGGAA-3′ (C131F25) <SEQ ID NO. 2> and reverse primer 5′-CGGGGCACTCGCAAGCACCCTATCA-3′ (C294R25) <SEQ ID NO. 7>.
A method for detecting the presence of Hepatitis C Virus (HCV) RNA in a biological sample, said method comprising:
- (A) performing a reverse transcription reaction using as a template RNA derived from said sample to produce HCV-specific reverse transcription products;
- (B) amplifying said reverse-transcription products using one or more pairs of 5′ NCR oligonucleotide primers specific for HCV and one or more pairs of 3′ NCR oligonucleotide primers to produce HCV-specific amplification products,
- wherein said 5′ NCR primer pairs are selected from the group consisting of:
- (a) forward primer 5′-CAGAAAGCGTCTAGCCATGGCGTTAGTA-3′ (C69F28) <SEQ ID NO. 1> and reverse primer 5′-CGGTTCCGCAGACCACTATGGCTCTC-3′ (C133R26) <SEQ ID NO. 4>;
- and
- (b) forward primer 5′-GGGAGAGCCATAGTGGTCTGCGGAA-3′ (C131 F25) <SEQ ID NO. 2> and reverse primer 5′-CGGGGCACTCGCAAGCACCCTATCA-3′ (C294R25) <SEQ ID NO. 7>;
- and
- wherein each of said pairs of 3′ NCR oligonucleotide primers comprises a forward primer consisting of the oligonucleotide 5′-GGTGGCTCCATCTTAGCCCTAGTCACG-3′ (1F27) <SEQ ID NO. 8> and a reverse primer consisting of the oligonucleotide 5′-AGGCCAGTATCAGCACTCTCTGCAGTC-3′ (57R27) <SEQ ID NO. 9>;
- and
- (C) detecting said amplification products, wherein detection of said amplification products indicates the presence of HCV RNA in said sample.
A method for amplifying Hepatitis C Virus (HCV) DNA, which method comprises performing a polymerase chain reaction on a DNA sample containing HCV DNA using one or more pairs of 5′ NCR oligonucleotide primers specific for HCV and one or more pairs of 3′ NCR oligonucleotide primers to produce HCV-specific amplification products, wherein said 5′ NCR primer pairs are selected from the group consisting of:
- (a) forward primer 5′-CAGAAAGCGTCTAGCCATGGCGTTAGTA-3′ (C69F28) <SEQ ID NO. 1> and reverse primer 5′-CGGTTCCGCAGACCACTATGGCTCTC-3′ (C133R26) <SEQ ID NO. 4>;
- and
- (b) forward primer 5′-GGGAGAGCCATAGTGGTCTGCGGAA-3′ (C131F25) <SEQ ID NO. 2> and reverse primer 5′-CGGGGCACTCGCAAGCACCCTATCA-3′ (C294R25) <SEQ ID NO. 7>;
- and
- wherein each of said pairs of 3′ NCR oligonucleotide primers comprises a forward primer consisting of the oligonucleotide 5′-GGTGGCTCCATCTTAGCCCTAGTCACG-3′ (1F27) <SEQ ID NO. 8> and a reverse primer consisting of the oligonucleotide 5′-AGGCCAGTATCAGCACTCTCTGCAGTC-3′ (57R27) <SEQ ID NO. 9>.
A kit for amplifying HCV DNA derived from HCV RNA, said kit comprising one or more pairs of 5′ NCR oligonucleotide primers, wherein said 5′ NCR primer pairs are selected from the group consisting of:
- (a) forward primer 5′-CAGAAAGCGTCTAGCCATGGCGTTAGTA-3′ (C69F28) <SEQ ID NO. 1> and reverse primer 5′-CGGTTCCGCAGACCACTATGGCTCTC-3′ (C133R26) <SEQ ID NO. 4>;
- and
- (b) forward primer 5′-GGGAGAGCCATAGTGGTCTGCGGAA-3′ (C131F25) <SEQ ID NO. 2> and reverse primer 5′-CGGGGCACTCGCAAGCACCCTATCA-3′ (C294R25) <SEQ ID NO. 7>.
A kit for detecting the presence of HCV DNA, said kit comprising one or more pairs of 5′ NCR oligonucleotide primers, wherein said 5′ NCR primer pairs are selected from the group consisting of:
- (a) forward primer 5′-CAGAAAGCGTCTAGCCATGGCGTTAGTA-3′ (C69F28) <SEQ ID NO. 1> and reverse primer 5′-CGGTTCCGCAGACCACTATGGCTCTC-3′ (C133R26) <SEQ ID NO. 4>;
- and
- (b) forward primer 5′-GGGAGAGCCATAGTGGTCTGCGGAA-3′ (C131F25) <SEQ ID NO. 2> and reverse primer 5′-CGGGGCACTCGCAAGCACCCTATCA-3′ (C294R25) <SEQ ID NO. 7>.
Dependencies are inferred from the claim text.