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Primer and probe design for efficient amplification and detection of HCV 3' non-translating region

a probe design and probe technology, applied in the field of oligonucleotides, can solve the problems of difficult study of rna, difficult identification of this agent, bartenschlager and lohmann

Inactive Publication Date: 2006-01-05
BIOMERIEUX INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] The present invention provides an isolated Hepatitis C virus-derived nucleic acid comprising a nucleotide sequence selected from the group consisting of specific nucleic acid sequences corresponding to a portion of the 3′NTR of HCV. Such oligonucleotides are useful in detecting the presence of HCV nucleic acids. Specifically, t

Problems solved by technology

The identification of this agent turned out to be very difficult (Bartenschlager and Lohmann, J. Gen. Virol. 81: 1631-1648 (2000).
Its RNA has been difficult to study because biological materials are scarce and RNA replication is of low efficiency (Shi and Lai, Cell. Mol. Life Sci. 58: 1276-1295 (2001)).
However, it is often complicated by the low levels of HCV replication or small numbers of infected cells in hepatitis C patients (Shi and Lai (2000)).
However, the sensitivity of this method has been reported to be accompanied by problems of false priming, presumably in areas of RNA secondary structure (Shi and Lai (2000)).
It has not been used in the detection of HCV RNA due, at least in part, to its lack of practical advantages over the well-established tests based on the 5′-UTR sequence (Shi and Lai (2000)).

Method used

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  • Primer and probe design for efficient amplification and detection of HCV 3' non-translating region
  • Primer and probe design for efficient amplification and detection of HCV 3' non-translating region

Examples

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examples

[0041] Within this application and particularly within the Examples, the primers utilized may be referred to as P1 primers or P2 primers. This terminology simply indicates that, within a NASBA or TMA reaction in these examples, the P1 primer is the primer having a T7 promoter sequence attached to its 5′ end (promoter primer); the P2 primer does not. It is not limiting terminology.

TMA Amplification and Detection of 3′ HCV

[0042] To evaluate the usefulness of various primers in HCV amplification, standard VIDAS Probe D2 qHCV assay conditions were used as follows. For lysis, 0.5 ml sample (such as EDTA-plasma, or in vitro RNA transcript diluted in base matrix) was mixed with 0.4 ml urea-based lysis buffer, incubated 67.5° C. (20 min) and cooled to room temp (20 min). During these incubations the released nucleic acid was captured by capB probe (SEQ ID NO: 19), which was linked to magnetic beads by a polyA tail. Complexes were washed and resuspended in RAR buffer (see Table 1). RNA ta...

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Abstract

The present invention provides selected oligonucleotides corresponding to portions of the 3′ non-translating region of Hepatitis C virus. The invention also includes methods of detecting and of quantitating HCV nucleic acids in a sample using these oligonucleotides.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 60 / 538,815 filed on Jan. 23, 2004, U.S. Provisional Patent Application No. 60 / 538,814 filed on Jan. 23, 2004, and U.S. Provisional Patent Application No. 60 / 538,816 filed on Jan. 23, 2004.FIELD OF THE INVENTION [0002] The present invention relates to oligonucleotides that bind to the 3′ non-translating region (3′NTR) of Hepatitis C Virus (HCV), useful for the detection and / or quantification of HCV. Also provided are methods of nucleic acid assays useful in the detection, capture and amplification of HCV utilizing the 3′NTR as the target region. BACKGROUND OF THE INVENTION [0003] After the development of diagnostic tests for hepatitis A and hepatitis B viruses in the 1970s, an additional parenterally transmitted agent responsible for the majority of transfusion-associated non-A, non-B hepatitis cases was recognized. The identification of this agent turned out ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N5/06C12N5/16C12Q1/70
CPCC12Q1/6806C12Q1/6844C12Q1/6846C12Q1/6851C12Q1/686C12Q1/707C12Q2527/137C12Q2527/125C12Q2527/107C12Q2527/101C12Q2527/113
Inventor WANG, HWA-TANGWASHBURN, BRIANPUROHIT, PRAKASHBURG, LAWRENCE
Owner BIOMERIEUX INC
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