Oligonucleotide primers for efficient detection of hepatitis...

Details Oligonucleotide primers for efficient detection of hepatitis... Oligonucleotide primers for efficient detection of hepatitis...

2000-01-28

2003-10-28

Benzion, Gary (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120, C435S091200, C536S023100, C536S024300

Reexamination Certificate

active

06638714

ABSTRACT:

FIELD OF THE INVENTION
The present invention pertains to improved methods for detecting nucleic acid sequences in biological samples, particularly sequences derived from infectious microorganisms.
BACKGROUND OF THE INVENTION
Hepatitis C Virus (HCV) is a parenterally transmitted virus responsible for the majority of cases of post-transfusion hepatitis and a substantial portion of sporadic (or community acquired) hepatitis cases worldwide. It is estimated that more than 1% of the world's population is infected with HCV. HCV infection is associated with acute hepatitis, chronic hepatitis, cirrhosis, and hepatocellular carcinoma.
HCV is currently classified as a separate genus, Hepacivirus, in the family Flaviviridae. Its genome consists of a positive-stranded RNA molecule of about 9,500 nucleotides with a single, large open reading frame (ORF) which encodes a polyprotein precursor of about 3,000 amino acids. The large ORF is preceded by a 5′ non-coding region (NCR) of about 340 nucleotides, which is the most highly conserved region of the genome. The 5′ region of the ORF encodes (in a 5′-to-3′ direction) a capsid protein, two envelope glycoproteins (E1 and E2), and a small protein of unknown function (P7). The 3′ portion of the ORF encodes nonstructural proteins which include a protease, protease/helicase bi-functional protein, RNA polymerase, and regulatory peptides. The 3′ portion also includes an NCR.
Analysis of HCV coding sequences from around the world has revealed considerable sequence variation among individual viral isolates. Furthermore, analyses of HCV sequences from individual patients have shown that the virus circulates as so-called “quasi-species,” which contain related but not identical sequences. The variation that exists among isolates and within individual patients is believed to be the result of the low fidelity of the virally-encoded RNA-dependent RNA polymerase. The degree of genetic variability of HCV has important implications for prevention, diagnosis, and control of infection.
Serodiagnosis of HCV infection is typically determined by commercially available enzyme immuno-assays (EIA) which detect antibodies that bind recombinant HCV proteins or peptides. Positive EIA results can be confirmed by a recombinant immunoblot assay (RIBA), but neither EIA nor RIBA assays distinguish past from present infections. Because of the typically low titer of circulating virus, a direct assay for viral proteins has not been successfully developed. Furthermore, antibody-based assays fail to detect HCV infection for usually 2 to 3 months after exposure
Thus, there is a need in the art for improved assays for HCV that are sensitive enough to detect HCV viremia within a few days after initial exposure of a patient to HCV.
SUMMARY OF THE INVENTION
Thus, in a first aspect, the invention is directed to a method for detecting the presence of Hepatitis C Virus (HCV) RNA in a biological sample. The method comprises:
(A) performing a reverse transcription reaction using as a template RNA derived from the sample to produce HCV-specific reverse transcription products;
(B) amplifying the reverse-transcription products using one or more pairs of oligonucleotide primers specific for HCV to produce HCV-specific amplification products,
 where each of the pairs comprises:
(a) a forward primer selected from the group consisting of:
(i) 5′-CAGAAAGCGTCTAGCCATGGCGTTAGTA-3′
(C69F28) <SEQ ID NO. 1>,

(ii) 5′-GGGAGAGCCATAGTGGTCTGCGGAA-3′
(C131F25) <SEQ ID NO. 2>, and

(iii) 5′-GTGGTCTGCGGAACCGGTGAGTACAC-3
(C143F26) <SEQ ID NO. 3>; and
(b) a reverse primer selected from the group consisting of:
(i) 5′-CGGTTCCGCAGACCACTATGGCTCTC-3′
(C133R26) <SEQ ID NO. 4>,

(ii) 5′-GCAAGCACCCTATCAGGCAGTACCACA-3′
(C282R27) <SEQ ID NO. 5>,

(iii) 5′-CACTCGCAAGCACCCTATCAGGCAGTA-3′
(C287R27) <SEQ ID NO. 6>, and

(iv) 5′-CGGGGCACTCGCAAGCACCCTATCA-3′
(C294R25) <SEQ ID NO. 7>; and
(C) detecting the amplification products,
 where detection of the amplification products indicates the presence of HCV RNA in the sample.
In another aspect, the invention is directed to a method for amplifying Hepatitis C Virus (HCV) DNA. The method comprises:
(A) 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,
 where each of the pairs comprises:
(a) a forward primer selected from the group consisting of:
(i) 5′-CAGAAAGCGTCTAGCCATGGCGTTAGTA-3′
(C69F28) <SEQ ID NO. 1>,

(ii) 5′GGGAGAGCCATAGTGGTCTGCGGAA-3′
(C131F25) <SEQ ID NO. 2>, and

(iii) 5′-GTGGTCTGCGGAACCGGTGAGTACAC-3
(C143F26) <SEQ ID NO. 3>; and
(b) a reverse primer selected from the group consisting of:
(i) 5′-CGGTTCCGCAGACCACTATGGCTCTC-3′
(C133R26) <SEQ ID NO. 4>,

(ii) 5′-GCAAGCACCCTATCAGGCAGTACCACA-3′
(C282R27) <SEQ ID NO. 5>,

(iii) 5′-CACTCGCAAGCACCCTATCAGGCAGTA-3′
(C287R27) <SEQ ID NO. 6>, and

(iv) 5′-CGGGGCACTCGCAAGCACCCTATCA-3′
(C294R25) <SEQ ID NO. 7>.
In a third aspect, the invention is directed to a method for detecting the presence of Hepatitis C Virus (HCV) RNA in a biological sample. The method comprises:
(A) performing a reverse transcription reaction using as a template RNA derived from the sample to produce HCV-specific reverse transcription products;
(B) amplifying the reverse-transcription products using a forward primer and a reverse primer to produce HCV-specific amplification products,
 where the forward primer consists of the oligonucleotide 5′-GGTGGCTCCATCTTAGCCCTAGTCACG-3′ (1F27) <SEQ ID NO. 8> and the reverse primer consists of the oligonucleotide 5′-AGGCCAGTATCAGCACTCTCTGCAGTC-3′ (57R27) <SEQ ID NO. 9>; and
(C) detecting the amplification products,
 where detection of the amplification products indicates the presence of HCV RNA in the sample.
In a fourth aspect, the invention is directed to a method for amplifying Hepatitis C Virus (HCV) DNA. The method comprises:
(A) performing a polymerase chain reaction on a DNA sample containing HCV DNA using a forward primer and a reverse primer to produce HCV-specific amplification products,
 where the forward primer consists of the oligonucleotide 5′-GGTGGCTCCATCTTAGCCCTAGTCACG-3′ (1F27) <SEQ ID NO. 8> and the reverse primer consists of the oligonucleotide 5′-AGGCCAGTATCAGCACTCTCTGCAGTC-3′ (57R27) <SEQ ID NO. 9>.
In a fifth aspect, the invention is directed to a method for detecting the presence of Hepatitis C Virus (HCV) RNA in a biological sample. The method comprises:
(A) performing a reverse transcription reaction using as a template RNA derived from the sample to produce HCV-specific reverse transcription products;
(B) amplifying the 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,
 where each of the pairs of 5′ NCR oligonucleotide primers comprises:
(a) a forward primer selected from the group consisting of:
(i) 5′-CAGAAAGCGTCTAGCCATGGCGTTAGTA-3′
(C69F28) <SEQ ID NO. 1>,

(ii) 5′-GGGAGAGCCATAGTGGTCTGCGGAA-3′
(C131F25) <SEQ ID NO. 2>, and

(iii) 5′-GTGGTCTGCGGAACCGGTGAGTACAC-3
(C143F26) <SEQ ID NO. 3>; and
(b) a reverse primer selected from the group consisting of:
(i) 5′-CGGTTCCGCAGACCACTATGGCTCTC-3′
(C133R26) <SEQ ID NO. 4>,

(ii) 5′-GCAAGCACCCTATCAGGCAGTACCACA-3′
(C282R27) <SEQ ID NO. 5>,

(iii) 5′-CACTCGCAAGCACCCTATCAGGCAGTA-3′
(C287R27) <SEQ ID NO. 6>, and

(iv) 5′-CGGGGCACTCGCAAGCACCCTATCA-3′
(C294R25) <SEQ ID NO. 7>; and
 where each of the pairs of 3′ NCR oligonucleoti

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