Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-01-28
2003-09-23
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S091100, C435S091200, C536S023100, C536S024300, C536S024320, C536S024310
Reexamination Certificate
active
06623919
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
Human Immunodeficiency Virus (HIV) infects millions of individuals world-wide and consequently represents a serious public health concern. Spread of HIV infection via contaminated blood products means that there is a need for screening methods that can detect small amounts of HIV RNA in patient samples. Furthermore, the increasing availability of ameliorative treatments for HIV infection means that early detection of infection in a patient is vital in order to initiate appropriate therapeutic interventions.
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 subsequent 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 (NC) region 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.
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 usually fail to detect HCV infection for 2 to 3 months after exposure.
Thus, there is a need in the art for improved assays that allow for the simultaneous screening of patient samples for both HIV and HCV.
SUMMARY OF THE INVENTION
The present invention provides methods for the simultaneous detection of the presence of Hepatitis C Virus (HCV) RNA and Human Immunodeficiency Virus (HIV) RNA in a biological sample using a multiplex assay.
Thus, in one aspect, the invention is directed to a method for co-detecting Hepatitis C Virus (HCV) RNA and Human Immunodeficiency Virus (HIV) RNA in a biological sample. The method comprises:
(A) performing a reverse transcription reaction using RNA derived from the sample as a template and at least one reverse transcription primer that will prime reverse transcription of DNA from HCV RNA and at least one reverse transcription primer that will prime reverse transcription of DNA from HIV RNA to produce reverse transcription products comprising (a) HCV-specific reverse transcription products, (b) HIV-specific reverse transcription products, or (c) a combination of (a) and (b);
(B) amplifying the reverse-transcription products using one or more pairs of oligonucleotide primers specific for the 5′ noncoding region of HCV and one or more pairs of oligonucleotide primers specific for HIV to produce amplification products comprising (a) HCV-specific amplification products, (b) HIV-specific amplification products, or (c) a combination of (a) and (b);
where each of the pairs of oligonucleotide primers specific for HCV comprises:
(i) forward primer
5′-GGGAGAGCCATAGTGGTCTGCGGAA-3′ (C131F25) <SEQ ID NO. 1>, and
(ii) reverse primer
5′-CGGGGCACTCGCAAGCACCCTATCA-3′ (C294R25) <SEQ ID NO. 2>;
where each of the pairs of oligonucleotide primers specific for HIV-1 comprises a forward primer with the sequence:
5′-CTGCTTAAGCCTCAATAAAGCTTGCCTTGA-3′ (JBLTR4) <SEQ ID NO. 3>, and a reverse primer specific for HIV-1 selected from the group consisting of
(1) 5′-GGGTCTGAGGGATCTCTAGTTACC AGAGT-3′ (JBLTR6) <SEQ ID NO. 4>, and
(2) 5′-TGTTCGGGCGCCACTGCTAGAGA-3′ (JBLTR8) <SEQ ID NO. 5>, and where each of the pairs of oligonucleotide primers specific for HIV-2 comprises a forward primer with the sequence 5′-GGGAGGTTCTCTCCAGCACTAGCA-3′ (2LTRe) <SEQ ID NO. 6>, and a reverse primer specific for HIV-2 with the sequence:
5′-GCGACTAGGAGAGATGGGAACACACA-3′ (2LTR-R1)
<SEQ ID NO. 7>; and
(C) detecting the amplification products, where detection of HCV-specific amplification products indicates the presence of HCV RNA in the sample, detection of HIV-specific amplification products indicates the presence of HIV RNA in the sample, and the detection of HCV-specific amplification products and HIV-specific amplification products indicates the presence of HCV RNA and HIV RNA in the sample.
In a second aspect, the invention is directed to a method for co-amplifying Hepatitis C Virus (HCV) DNA and Human Immunodeficiency Virus (HIV) DNA. The method comprises:
(A) performing a polymerase chain reaction on a DNA sample suspected to contain HCV DNA, HIV DNA, or a combination of HCV DNA and HIV DNA, using one or more pairs of oligonucleotide primers specific for the 5′ noncoding region of HCV and one or more pairs of oligonucleotide primers specific for HIV to produce amplification products comprising (a) HCV-specific amplification products, (b) HIV-specific amplification products, or (c) a combination of (a) and (b);
where each of the pairs of oligonucleotide primers specific for HCV comprises:
(i) forward primer 5′-GGGAGAGCCATAGTGGTCTGCGGAA-3′ (C131F25) <SEQ ID NO. 1>, and
(ii) reverse primer 5′-CGGGGCACTCGCAAGCACCCTATCA-3′ (C294R25) <SEQ ID NO. 2>;
where each of the pairs of oligonucleotide primers specific for HIV-1 comprises a forward primer with the sequence:
5′-CTGCTTAAGCCTCAATAAAGCTTGCCTTGA-3′ (JBLTR4) <SEQ ID NO. 3>, and a reverse primer specific for HIV-1 selected from the group consisting of:
(1) 5′-GGGTCTGAGGGATCTCTAGTTACC AGAGT-3′ (JBLTR6) <SEQ ID NO. 4>, and
(2) 5′-TGTTCGGGCGCCACTGCTAGAGA-3′ (JBLTR8) <SEQ ID NO. 5>, and where each of the pairs of oligonucleotide primers specific for HIV-2 comprises a forward primer with the sequence 5′-GGGAGGTTCTCTCCAGCACTAGCA-3′ (2LTRe) <SEQ ID NO. 6>, and a reverse primer specific for HIV-2 with the sequence:
5′-GCGACTAGGAGAGATGGGAACACACA-3′ (2LTR-R1) <SEQ ID NO. 7>.
In a third aspect, the invention
Gorman Kevin M.
Linnen Jeffrey M.
Patterson David R.
Song Keming
Darby & Darby
Fredman Jeffrey
Goldberg Jeanine
Ortho-Clinical Diagnostics, INC
LandOfFree
Oligonucleotide primers for efficient multiplex detection of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Oligonucleotide primers for efficient multiplex detection of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Oligonucleotide primers for efficient multiplex detection of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3016589