Oligonucleotides and methods for detecting hepatitis C viral nucleic acids

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.

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.