Methods and compositions for identifying and characterizing hepatitis C

Abstract

The invention provides novel methods and compositions for amplifying portions of the HCV genome. The nucleic acid sequences set forth as SEQ ID NOS:1-64 derived from HCV cDNA and functional equivalents thereof, kits containing same, and methods employing same, are useful for the identification and characterization of HCV in biological samples.

1 . A nucleic acid molecule selected from the group consisting of SEQ ID NOS:1-64.  
     
     
         2 . A nucleic acid molecule having a nucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOS:1-64, wherein the oligonucleotide is capable of annealing to the NS3 or NS4 gene of HCV, or a portion thereof.  
     
     
         3 . A nucleic acid molecule comprising a nucleotide sequence which is at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS:1-64.  
     
     
         4 . A nucleic acid molecule comprising a fragment of at least 8 nucleotides of a nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NOS:1-64.  
     
     
         5 . A set of primers selected from the group consisting of two or more oligonucleotides of SEQ ID NOS:1-64.  
     
     
         6 . A vector comprising a nucleic acid molecule of claims  1 - 4 .  
     
     
         7 . The nucleic acid molecule of claims  1 - 4 , further comprising a label.  
     
     
         8 . The oligonucleotides of  claim 5 , further comprising a label.  
     
     
         9 . The nucleic acid molecule of  claim 7 , wherein the label is selected from the group consisting of a fluorescent group, digoxigenin, biotin, radioactive labels, chemiluminescent groups, enzymes, antibodies, luminescent agents, precipitating agents, and dyes.  
     
     
         10 . The oligonucleotides of  claim 8 , wherein the label is selected from the group consisting of a fluorescent group, digoxigenin biotin, radioactive labels, chemiluminescent groups, enzymes, antibodies, luminescent agents, precipitating agents, and dyes.  
     
     
         11 . A nucleic acid molecule encoding a promoter-primer comprising a sequence selected from the group consisting of SEQ ID NOS:1-64, and a promoter sequence at the 5′ end.  
     
     
         12 . An oligonucleotide for amplifying a nucleic acid sequence in a sample, wherein the sample contains a nucleotide sequence selected from the group consisting of SEQ ID NOS:1-64, or complement thereof.  
     
     
         13 . The oligonucleotide of  claim 12 , wherein the sample is from a patient infected with HCV.  
     
     
         14 . A kit for the detection of HCV, comprising an oligonucleotide, wherein the oligonucleotide comprises a nucleotide sequence which is at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS:1-64.  
     
     
         15 . A kit for the detection of HCV, comprising a set of oligonucleotides, wherein the oligonucleotides comprise a nucleotide sequence which is at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS:1-64.  
     
     
         16 . The kit of  claim 14  or  15 , further comprising at least one labeled oligonucleotide for detecting amplified HCV nucleic acid.  
     
     
         17 . The kit of  claim 14  or  15 , wherein said kit further comprises a component selected from the group consisting of a plurality of nucleotides and a nucleic acid polymerase.  
     
     
         18 . The kit of  claim 17 , wherein said kit further comprises at least one other component for conducting a polymerase amplification reaction.  
     
     
         19 . The kit of  claim 18 , wherein said kit comprises a thermostable DNA polymerase.  
     
     
         20 . The kit of  claim 14  or  15 , further comprising instructions for use.  
     
     
         21 . A method for amplifying a target nucleic acid, the method comprising: 
 combining a target nucleic acid under conditions which allow for an amplification reaction to occur with: 
 a) one or more nucleic acid primer sequences which are at least 80% identical to the sequences set forth as SEQ ID NOS:1-64;  
 b) a nucleic acid polymerase; and  
 c) a plurality of nucleotides,  
   thereby resulting in an amplified target nucleic acid.    
     
     
         22 . The method of  claim 21 , wherein the target nucleic acid is from the HCV genome.  
     
     
         23 . The method of  claim 22 , wherein the target nucleic acid is from the NS3 or NS4 gene.  
     
     
         24 . The method of  claim 22 , wherein the target nucleic acid is from the serine protease portion of the NS3 gene.  
     
     
         25 . The method of  claim 22 , wherein the target nucleic acid is from the helicase portion of the NS3 gene.  
     
     
         26 . The method of  claim 21 , wherein said polymerase is selected from the group consisting of reverse transcriptase and thermostable DNA polymerase.  
     
     
         27 . The method of  claim 21 , wherein the amplified HCV nucleic acid is sequenced.  
     
     
         28 . The method of  claim 27 , wherein the sequence is evaluated for mutations.  
     
     
         29 . The method of  claim 21 , wherein the amplified HCV nucleic acid is cloned into a vector.  
     
     
         30 . The vector of  claim 29 .  
     
     
         31 . A method for specifically detecting HCV nucleic acids in a sample comprising: 
 a) contacting said sample with one or more nucleic acid sequences which are at least 80% identical to the sequences set forth as SEQ ID NOS:1-64, under conditions such that said HCV nucleic acids can hybridize with said primers;    b) reverse transcribing and amplifying said nucleic acids to obtain amplified HCV nucleic acids; and    c) detecting the presence of said amplified HCV nucleic acids.    
     
     
         32 . The method of  claim 31 , wherein detecting the presence of said amplified HCV nucleic acids comprises: 
 a) contacting said amplified HCV nucleic acids with a labeled oligonucleotide to obtain labeled HCV nucleic acids; and    b) identifying said labeled nucleic acids.    
     
     
         33 . The method of  claim 31 , wherein amplifying said nucleic acids is accomplished by nucleic acid sequence based amplification (NASBA), a polymerase chain reaction (PCR), Transcription Mediated Amplification (TMA) or Ligase chain reaction.  
     
     
         34 . The method of  claim 31 , further comprising the step of sequencing the amplified HCV nucleic acids.  
     
     
         35 . The method of  claim 34 , further comprising evaluating the sequence for mutations.  
     
     
         36 . A method for determining whether a patient is resistant to an agent, the method comprising: 
 a) obtaining a sample from a patient comprising DNA;    b) performing amplification PCR on the DNA from the patient sample using a nucleic acid primer sequence which is at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS:1-64; and    c) analyzing the amplified product, thereby determining whether a patient is resistant to an agent.    
     
     
         37 . The method of  claim 36 , wherein the agent is an antiviral agent.  
     
     
         38 . The method of  claim 36 , wherein the agent is a protease inhibitor.  
     
     
         39 . The method of  claim 36 , wherein the agent is an inhibitor of the serine protease portion of the NS3 gene.  
     
     
         40 . The method of  claim 36 , wherein the agent is an inhibitor of the helicase portion of the NS3 gene.  
     
     
         41 . The method of  claim 36 , wherein the agent is alpha-interferon.  
     
     
         42 . The method of  claim 36 , wherein the agent is pegalated interferon.  
     
     
         43 . The method of  claim 36 , wherein the agent is a combination of agents.  
     
     
         44 . A method for determining whether an agent can or can not be used to treat an HCV patient, the method comprising the steps of: 
 a) obtaining a sample from a patient infected with HCV;    b) amplifying the patient sample using a nucleic acid primer sequence which is at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS:1-64;    c) sequencing the resulting amplified HCV nucleic acid sequences; and    d) identifying mutations in the amplified HCV nucleic acid sequence that correlate with resistance or sensitivity to an anti-viral agent, thereby    determining whether an agent can or can not be used to treat an HCV patient.    
     
     
         45 . The method of  claim 44 , wherein the nucleic acid is a set of primers.  
     
     
         46 . A method for determining whether treatment with an agent should be continued in a patient infected with HCV, the method comprising: 
 (a) obtaining two or more samples comprising DNA from a patient during the course of treatment;    (b) performing the method of  claim 21  on the samples;    (c) sequencing the resulting amplified target nucleic acid product of  claim 21;     (d) identifying mutations present in the amplified product that correlate with resistance or sensitivity to an agent; and    (e) continuing treatment when the mutations identified in the amplified product do not change during the course of treatment.    
     
     
         47 . A method for determining whether treatment with an agent should not be continued in a patient infected with HCV, the method comprising: 
 (a) obtaining two or more samples comprising DNA from a patient during the course of treatment;    (b) performing the method of  claim 21  on the samples;    (b) sequencing the resulting amplified target nucleic acid product of  claim 21;     (c) identifying mutations present in the amplified product that correlate with resistance or sensitivity to an agent; and    (d) discontinuing treatment when the mutations identified in the amplified product change during the course of treatment.    
     
     
         48 . A method for evaluating a patient diagnosed as or suspected of having HCV to assess whether anti-viral therapy is likely to be successfuil, the method comprising: 
 (a) obtaining a sample from the patient containing DNA, and    (b) comparing the serine protease portions of the NS3 gene in the sample with a consensus subtype sequence, wherein the presence of one or more mutations is indicative of resistance to the anti-viral agent.    
     
     
         49 . A method for evaluating a patient diagnosed as or suspected of having HCV to assess whether anti-viral therapy is likely to be successful, the method comprising: 
 (a) obtaining a sample from the patient containing DNA, and    (b) comparing the helicase portions of NS3 gene to a consensus subtype sequence, wherein the presence of one or more mutations is indicative of resistance to the anti-viral agent.    
     
     
         50 . A method of assessing the efficacy of an agent for treating HCV, the method comprising comparing: 
 a) nucleic acid sequences in a first sample obtained from a patient exposed to an agent, wherein the nucleic acid sequences are amplified products from one or more nucleic acid primer sequences which are at least 80% identical to the sequences set forth as SEQ ID NOS:1-64, and    b) nucleic acid sequences in a second sample obtained from a patient who has not been exposed to an agent, wherein the nucleic acid sequences are amplified products from one or more nucleic acid primer sequences which are at least 80% identical to the sequences set forth as SEQ ID NOS:1-64,    wherein an increased number of mutations in the nucleic acid sequences from the first sample, relative to the second sample, is an indication that the agent is not efficacious in treating HCV.