Oligonucleotides and methods for detecting hepatitis C viral nucleic acids

• US 20050100889A1
• Filed: 10/13/2004
• Published: 05/12/2005
• Est. Priority Date: 12/04/2001
• Status: Abandoned Application

• Current Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Original Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Inventors: Baumann, Russell, Hamdan, Hasnah, Lewinski, Michael
• Application Number:
  US10/964,302
• Publication Number:
  US 20050100889A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/707 non-A, non-B Hepatitis, excluding hepatitis D

Abstract

The present invention provides methods and compositions for determining the presence and/or amount of HCV nucleic acids in a test sample. In particular, substantially purified oligonucleotide primers and probes are described that can be used for qualitatively and quantitatively detecting HCV nucleic acid in a test sample by amplification methods. The present invention also provides primers and probes for generating and detecting control nucleic acid sequences that provide a convenient method for assessing internal quality control of the HCV assay.

Claims

1. A method for detecting the presence or amount of HCV nucleic acids in a test sample, comprising:

(a) introducing lambda phage-HCV nucleic acid hybrids into said test sample;

(b) reverse transcribing and amplifying;

i) HCV nucleic acid if present in said sample and using a pair of oligonucleotide primers having the sequences set forth in SEQ ID NO;

1 and SEQ ID NO;

2, to generate HCV amplicons; and

ii) lambda phage-HCV hybrid nucleic acid using a pair of oligonucleotide primers having the sequences set forth in SEQ ID NO;

1 and SEQ ID NO;

2, to generate a lambda phage-HCV hybrid amplicons;

(c) hybridizing said HCV amplicons with an oligonucleotide probe comprising the sequence set forth in SEQ ID NO;

3 in the presence of an enzyme that cleaves said probe when said probe hybridizes to said HCV nucleic acids, wherein said probe is conjugated to a first detectable label that generates a detectable signal upon said cleavage;

(d) hybridizing said lambda phage-HCV hybrid amplicons to a control oligonucleotide probe in the presence of an enzyme that cleaves said control oligonucleotide probe when said control probe hybridizes to said lambda phage-HCV hybrid amplicons, wherein said control probe is conjugated to a second detectable label that generates a detectable signal upon said cleavage; and

(e) detecting a signal from said first and second detectable labels, wherein said signal from said first detectable label indicates the presence or amount of HCV nucleic acids in said test sample.

2. The method of claim 1 wherein said control probe has the sequence set forth in SEQ ID NO:

6.

3. The method of claim 1, wherein said first and second detectable labels are 2′

-chloro-7′

-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) and 6-carboxytetramethylrhodamine (TAMRA), or 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA).

4. The method of claim 1, wherein said lambda phage-HCV nucleic acid hybrids have the sequence set forth in SEQ ID NO:

5.

5. The method of claim 1, wherein said lambda phage-HCV nucleic acid hybrids further comprises a T7 RNA polymerase promoter nucleic acid sequence.

6. The method of claim 5, wherein said T7 RNA polymerase promoter-lambda phage-HCV nucleic acid hybrids have the sequence set forth in SEQ ID NO:

4.

7. The method of claim 1, wherein said test sample is selected from the group consisting of serum, blood, plasma, cerebral spinal fluid, synovial fluid, and urine.

8. The method of claim 1, wherein nucleic acids are purified from said sample prior to said reverse transcription and amplification step (a).

9. The method of claim 1, wherein lambda phage-HCV ribonucleic acid hybrids are introduced into said test sample prior to isolating nucleic acids from said sample.