Probes and Methods for Hepatitis C Virus Typing Using Multidimensional Probe Analysis

Abstract

This invention provides compositions and methods for HCV typing, e.g., genotyping and/or subtyping. The compositions and methods of the invention can be used to assign an HCV isolate to one of at least five HCV types (for example, selected from types 1, 2, 3, 4, 5 or 6), or to one of at least five subtypes (for example, subtypes 1a/b/c, 2a/c, 2b, 3a, 4a, 5a or 6a). These methods integrate the hybridization data from a plurality of HCV typing probes in a multidimensional analysis to make an HCV type assignment for an HCV in an experimental sample. The invention also provides related compositions, including, for example, the HCV typing probes and HCV typing diagnostic kits.

1 - 34 . (canceled) 
     
     
         35 . A composition comprising a plurality of probes, wherein:
 a) each probe comprises a nucleotide sequence that targets the probe to a same target region within a hepatitis C virus (HCV) genome and is complementary or partially complementary to a nucleotide sequence within the HCV genome;   b) the regions of complementarity or partial complementarity show sequence heterogeneity among at least two HCV types;   c) hybridization complexes comprising (i) any one probe from the plurality of probes, and (ii) nucleotide sequences from at least two HCV types have a distinguishing range of melting temperature (T m ) that permits differentiation of more than two HCV types;   d) the more than two HCV types differentiated by any one probe from the plurality of probes are different than the more than two HCV types differentiated by a second different probe from the plurality of probes; and,   e) the distinguishing range of melting temperature (T m ) of each of the hybridization complexes comprising at least two probes from the plurality of probes correlates with one of at least five HCV types, wherein an assignment of an HCV type is made by considering the distinguishing range of melting temperature (T m ) of the hybridization complexes.   
     
     
         36 . The composition of  claim 35 , wherein the plurality of probes are independently selected from the nucleotide sequences provided in SEQ ID NOs: 9 through 57. 
     
     
         37 . The composition of  claim 35 , wherein the plurality of probes comprises at least a first probe and a second probe, wherein the first and second probes comprise nucleotide sequences selected from SEQ ID NOs: 26 and 54, SEQ ID NOs: 10 and 53, and SEQ ID NOs: 57 and 52. 
     
     
         38 . (canceled) 
     
     
         39 . The composition of  claim 35 , comprising a reverse transcriptase and a primer suitable for the initiation of reverse transcription of the nucleotide sequence within the HCV genome. 
     
     
         40 . The composition of  claim 35 , comprising a nucleic acid that is either:
 a) an HCV amplicon comprising a nucleotide sequence that is complementary or partially complementary to a nucleotide sequence in at least two probes in the plurality of probes;   b) an amplification primer capable of generating one strand of the HCV amplicon; or   c) an amplification primer pair capable of generating the HCV amplicon;   
       wherein the primer and the primer pair are admixed with a thermostable DNA-dependent DNA polymerase, free deoxyribonucleotide triphosphates and a suitable DNA polymerase reaction buffer. 
     
     
         41 . The composition of  claim 35 , wherein at least one probe from the plurality of probes comprises a FRET donor moiety, and wherein the composition comprises a soluble FRET quencher that is capable of quenching the FRET donor moiety. 
     
     
         42 . A diagnostic kit for determining the type of a hepatitis C virus (HCV) in a sample, the kit comprising:
 a) a plurality of HCV typing probes, wherein:
 i) each typing probe comprises a nucleotide sequence that targets the probe to a same target region within an HCV genome and is complementary or partially complementary to a nucleotide sequence within the HCV genome; 
 ii) the regions of complementarity or partial complementarity show sequence heterogeneity among at least two HCV types; 
 iii) hybridization complexes comprising (i) any one probe from the plurality of probes and (ii) nucleotide sequences from at least two HCV types have a distinguishing range of melting temperature (T m ) that permits differentiation of more than two virus types; 
 iv) the more than two HCV types differentiated by one probe from the plurality of probes are different than the more than two HCV types differentiated by a second different probe from the plurality of probes; and, 
 v) the distinguishing range of melting temperature (T m ) of each of the hybridization complexes comprising at least two probes from the plurality of probes correlates with one of at least five HCV types, wherein an assignment of an HCV type is made by considering the distinguishing range of melting temperature (T m ) of the hybridization complexes; and, 
   b) instructions for measuring the distinguishing range of melting temperature (T m ) of hybridization complexes comprising a target probe from the plurality of target probes.   
     
     
         43 . The diagnostic kit of  claim 42 , wherein the plurality of typing probes are independently selected from the nucleotide sequences provided in SEQ ID NOs: 9 through 57. 
     
     
         44 . The diagnostic kit of  claim 42 , wherein the plurality of typing probes comprises at least two typing probes, wherein the two typing probes comprise nucleotide sequences selected from SEQ ID NOs: 26 and 54, SEQ ID NOs: 10 and 53, and SEQ ID NOs: 57 and 52. 
     
     
         45 . The diagnostic kit of  claim 42 , comprising an amplification primer pair for the amplification of an HCV nucleotide sequence, the primer pair comprising the nucleotide sequences of SEQ ID NOS: 58 and 59. 
     
     
         46 . The diagnostic kit of  claim 42 , further wherein the kit is for determining a viral load of the HCV in the sample, the kit comprising, c) an amplification primer pair capable of generating an HCV amplicon, wherein the amplicon comprises nucleotide sequences that are complementary or partially complementary to nucleotide sequences in at least two typing probes from the plurality of typing probes; and, d) an amplicon quantitation probe for real-time detection of amplicon accumulation, wherein the amplicon quantitation probe forms a quantitation hybridization complex with the amplicon under conditions wherein base-pairing occurs. 
     
     
         47 . The diagnostic kit of  claim 42 , wherein one or more components of the kit is packaged in one or more containers. 
     
     
         48 . The diagnostic kit of  claim 42 , wherein at least one typing probe comprises a FRET donor moiety, and further wherein the kit comprises at least one soluble FRET quencher comprising a thiazine dye that is capable of quenching the FRET donor moiety. 
     
     
         49 . The diagnostic kit of  claim 42 , comprising one or more additional components selected from a reverse transcriptase, a thermostable DNA-dependent DNA polymerase, free deoxyribonucleotide triphosphates, standardization samples, positive control samples, negative control samples, buffers suitable for enzymatic reactions, sample collection tubes, amplification reaction tubes and multi-well plates. 
     
     
         50 . A system that correlates detection of a plurality of signals with a hepatitis C virus (HCV) type, the system comprising:
 a) a detector for detecting the signals, wherein the signals correlate with a distinguishing range of melting temperature (T m ) of a plurality of hybridization complexes; and,   b) a correlation module operably coupled to the detector, wherein the correlation module receives the signals detected by the detector and correlates the signals with an HCV type by collating the detected signals with predicted or predetermined signals observed when detecting the distinguishing range of melting temperature (T m ) of hybridization complexes comprising a plurality of known HCV types, wherein during operation of the system:
 i) the signals are generated from a plurality of hybridization complexes comprising an amplicon comprising an HCV nucleotide sequence and at least a first and second probe; 
 the first and second probe nucleotide sequences comprise a nucleotide sequence that targets the probe to a same target region within an HCV genome and are complementary or partially complementary to nucleotide sequences within the HCV genome; 
 iii) the regions of hybridization complex complementarity or partial complementarity show sequence heterogeneity among at least two HCV types; 
 iv) hybridization complexes comprising the first probe have a distinguishing range of melting temperature (T m ) that differentiates more than two HCV types; 
 v) hybridization complexes comprising the second probe have a distinguishing range of melting temperature (T m ) that differentiates more than two HCV types, wherein the more than two virus types differentiated by the first probe are different than the more than two virus types differentiated by the second probe; and, 
 vi) the distinguishing range of melting temperature (T m ) of each of the hybridization complexes comprising each of the at least two probes correlates with one of at least five HCV types. 
   
     
     
         51 . The system of  claim 50 , wherein the correlation module comprises a dataset of predicted or predetermined T m  values for hybridization complexes comprising the at least first and second probes and each HCV type in the plurality of known HCV types, wherein the dataset is in a computer readable format.