Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
  • C12Q1/706—Specific hybridization probes for hepatitis
  • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the invention relates to the field of sequence variations within the HCV genome. More specifically, the invention relates to methods for amplification of an NS5B nucleic acid of any one of HCV genotypes 1 to 6 as a tool for analysis of sequence variations within the amplified NS5B fragment that may be correlated with HCV drug resistance.
• the methods comprise the design of sense and antisense primers according to genotype within NS5B genomic regions.
• Further subject of the invention are sets of primers designed according to genotype and capable of annealing to said NS5B genomic regions.
• the current invention further relates to kits based on said methods and primers.
• the about 9.6 kb single-stranded RNA genome of the HCV virus comprises a 5'- and 3 '-non-coding region (NCRs) and, in between these NCRs, a single long open reading frame of about 9 kb encoding an HCV polyprotein of about 3000 amino acids.
• NCRs 5'- and 3 '-non-coding region
• HCV polypeptides are produced by translation from the open reading frame and cotranslational proteolytic processing.
• Structural proteins are derived from the amino- terminal one-fourth of the coding region and include the capsid or Core protein (about 21 kDa), the El envelope glycoprotein (about 35 kDa) and the E2 envelope glycoprotein (about 70 kDa, previously called NSl), and p7 (about 7kDa).
• the E2 protein can occur with or without a C-terminal fusion of the p7 protein (Shimotohno et al. 1995).
• the non-structural HCV proteins are derived which include NS2 (about 23 kDa), NS3 (about 70 kDa), NS4A (about 8 kDa), NS4B (about 27 kDa), NS5A (about 58 kDa) and NS5B (about 68 kDa) (Grakoui et al. 1993).
• HCV is the major cause of non-A, non-B hepatitis worldwide. Acute infection with HCV (20% of all acute hepatitis infections) frequently leads to chronic hepatitis (70% of all chronic hepatitis cases) and end-stage cirrhosis. It is estimated that up to 20% of HCV chronic carriers may develop cirrhosis over a time period of about 20 years and that of those with cirrhosis between 1 to 4%/year is at risk to develop liver carcinoma (Shiffman 1999; Lauer and Walker 2001). An option to increase the life-span of HCV-caused end- stage liver disease is liver transplantation (30% of all liver transplantations world- wide are due to HCV-infection).
• HCV variants are primarily classified into 6 genotypes, representing the 6 genetic groups defined by phylogenetic analysis of core/El and NS5B subgenomic sequences as well as of complete genome sequences. Within each genotype, HCV variants can be further divided into subtypes (Simmonds et al. 1994). An overview of the current unified system of nomenclature of HCV genotypes is given by Simmonds et al. (2005).
• HCV has a high mutation rate, which is thought to help the virus in eluding its host's immune system.
• the high mutation rate is reflected in the presence of many distinct HCV genome sequences, known as quasispecies, within infected individuals (Bukh et al. 1995; Farci et al. 1997).
• Quasispecies result from the activity of the virally- encoded NS5B RNA-dependent RNA polymerase, which, because of its lack of proofreading function, is inherently a low-fidelity enzyme.
• the compounds under clinical evaluation comprise polymerase inhibitors (ViroPharma/Wyeth; Idenix Pharmaceuticals; Roche; XTL) and protease inhibitors (Schering-Plough; Vertex Pharmaceuticals; Gilead/Achillion; Intermune).
• HCV sub-genomic replicons have been used to study viral resistance to both nucleoside and non-nucleoside NS5B inhibitors as well as to NS3/4A protease inhibitors (Kukolj et al. 2005; Mo et al. 2005; reviewed in: Tomei et al. 2005). An understanding of HCV resistance mutants would further progress towards effective HCV treatments.
• the present invention relates to a method for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6 comprising the step of designing a set of primers per genotype in the NS5B genomic region, wherein said set of primers for amplifying said HCV NS5B nucleic acid is characterized in that at least one of the primers is an antisense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 9276 to 9298, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention further envisages the above described method for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6 wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7500 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the above described method for amplification of an HCV NS5B nucleic acid from HCV genotype 1 wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7536 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the above described method for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 2 to 6 wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the above described method for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6 wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 8258 to 8278, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention relates to a method for amplification of an HCV NS5B nucleic acid of HCV genotype 1 comprising the step of designing a set of primers in the NS5B genomic region, wherein said set of primers for amplifying said
• HCV NS5B nucleic acid is characterized in that at least one of the primers is an antisense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 8529 to 8546, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the above method for amplification of an HCV NS5B nucleic acid of HCV genotype 1 wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7536 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention relates to a method for amplification of an HCV NS5B nucleic acid of HCV genotype 2 comprising the step of designing a set of primers in the NS5B genomic region, wherein said set of primers for amplifying said HCV NS5B nucleic acid is characterized in that at least one of the primers is an antisense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 8620 to 8638, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the above method for amplification of an HCV NS5B nucleic acid of HCV genotype 2 wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention relates to a method for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 3 to 5 comprising the step of designing a set of primers per genotype in the NS5B genomic region, wherein said set of primers for amplifying said HCV NS5B nucleic acid is characterized in that at least one of the primers is an antisense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 8470 to 8491, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the abovem method for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 3 to 5, wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention relates to a method for amplification of an HCV NS5B nucleic acid of HCV genotype 6 comprising the step of designing a set of primers in the NS5B genomic region, wherein said set of primers for amplifying said HCV NS5B nucleic acid is characterized in that at least one of the primers is an antisense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 8529 to 8554, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the above method for amplification of an HCV NS5B nucleic acid of HCV genotype 6, wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• a method for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6 comprising the step of designing a set of primers per genotype in the NS5B genomic region, wherein said set of primers for amplifying said HCV NS5B nucleic acid is characterized in that one of the primers is an antisense primer chosen from the group consisting of
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 9276 to 9298
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8529 to 8546 - an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8620 to 8638,
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8470 to 8491 - an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8529 to 8554, and that the other primer is a sense primer chosen from the group consisting of
• a sense primer capable of annealing to the NS5B genomic region spanning nucleotides 7500 to 7555 - a sense primer capable of annealing to the NS5B genomic region spanning nucleotides 7536 to 7555,
• a sense primer capable of annealing to the NS5B genomic region spanning nucleotides 8258 to 8278, and wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• a further step in any of the above methods is the amplification of said NS5B nucleic acid with the set of primers as designed in any of these methods.
• said amplification can also be part of a multiplex amplification method.
• the invention in another aspect, relates to a method for determining the sequence of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6 wherein said NS5B nucleic acid is amplified according to any of the above described methods prior to or concurrent with determining said sequence.
• the invention relates to a method for evaluating the sequence variations in an HCV NS5B nucleic acid of any of HCV genotypes 1 to 6 wherein the sequence of said NS5B nucleic acid is determined according to the above described method prior to evaluating the sequence variations.
• Said sequence variations can be mutations that are induced by HCV antiviral agents.
• Said sequence variations can be mutations causing HCV drug resistance.
• Said sequence variations can also be genotype-specific nucleotide variations.
• said amplification can be a one-step RT-PCR and said HCV NS5B nucleic acid can be present in a biological sample.
• the invention also relates to a recombinant vector comprising an NS5B fragment amplified according to any of the above described methods.
• the invention in another aspect, relates to a set of primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6, said set of primers characterized in that at least one of the primers is an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 9276 to 9298, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention further relates to the above set of primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• the invention also relates to the above set of primers for amplification of an HCV NS5B nucleic acid from HCV genotype 1, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7536 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also relates to the above set of primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 2 to 6, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• the invention also relates to the above set of primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8258 to 8278, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• the invention relates to a set of primers for amplification of an HCV NS5B nucleic acid of HCV genotype 1, said set of primers characterized in that at least one of the primers is an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8529 to 8546, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• the invention further relates to the above set of primers for amplification of an HCV NS5B nucleic acid of HCV genotype 1, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7536 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention relates to a set of primers for amplification of an HCV NS5B nucleic acid of HCV genotype 2, said set of primers characterized in that at least one of the primers is an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8620 to 8638, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• the invention also relates to the above set of primers for amplification of an HCV NS5B nucleic acid of HCV genotype 2, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention relates to a set of primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 3 to 5, said set of primers characterized in that at least one of the primers is an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8470 to 8491, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also relates to the above set of primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 3 to 5, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• the invention relates to a set of primers for amplification of an HCV NS5B nucleic acid of HCV genotype 6, said set of primers characterized in that at least one of the primers is an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8529 to 8554, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• the invention also relates to the above set of primers for amplification of an HCV NS5B nucleic acid of HCV genotype 6, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention relates to a set of primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6, said set of primers characterized in that one of the primers is an antisense primer chosen from the group consisting of - an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 9276 to 9298,
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8529 to 8546 - an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8620 to 8638,
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8470 to 8491
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8529 to 8554, and that the other primer is a sense primer chosen from the group consisting of
• a sense primer capable of annealing to the NS5B genomic region spanning nucleotides 8258 to 8278, and wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention relates to the use of any of the above sets of primers for the manufacture of a kit for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6.
• the invention relates to a kit for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6, said kit comprising any of the above sets of primers.
• the above described kit can be used for multiplex amplification.
• the invention also relates to a kit for assaying sequence variations in an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6, said kit comprising any of the above set of primers, wherein said sequence variations can be mutations that are induced by HCV antiviral agents, mutations causing HCV drug resistance, or genotype-specific nucleotide variations.
• a robust and consistent amplification and sequencing methodology for analyzing sequence variations in potential HCV anti- viral genomic targets is a primary tool for drug resistance profiling. Such a methodology is useful for monitoring patients under treatment with novel antiviral drugs and therapeutic regimens, such as those targeting the HCV NS3 protease and the HCV NS5B RNA polymerase.
• the current invention provides the basis for such methodologies and tools relating to the HCV NS5B polymerase.
• the current invention provides diagnostic tools and methodologies for analyzing sequence variations in potential HCV anti-viral genomic targets. More specifically, the present invention provides for a robust and consistent amplification and sequencing methodology for analyzing drug resistance mutants in the NS5B RNA polymerase region of genotypes 1 to 6 of the hepatitis C virus. This methodology is useful for the sequential analysis and follow-up of patients under treatment with novel antiviral drugs targeting HCV NS5B.
• the primer sets were further optimized to allow their use for amplification of each region and genotype under one set of conditions, i.e. under one specific amplification protocol. This was done for "ease of use” purposes, especially in view of time managment and reduction of reagent costs associated with the procedure.
• a single extraction method and one-step PCT protocol was optimized for all genotypes. The final protocol resulted in the amplification of one full length NS5B fragment or, in the alternative, two overlapping NS5B fragments covering the complete NS5B gene.
• the present invention relates to a method for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6 comprising the step of designing a set of primers per genotype in the NS5B genomic region, wherein said set of primers for amplifying said HCV NS5B nucleic acid is characterized in that at least one of the primers is an antisense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 9276 to 9298, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention further envisages the above described methods for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6 wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7500 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the above described methods for amplification of an HCV NS5B nucleic acid from HCV genotype 1 wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7536 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the above described methods for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 2 to 6 wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the above described methods for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6 wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 8258 to 8278, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention relates to a method for amplification of an HCV NS5B nucleic acid of HCV genotype 1 comprising the step of designing a set of primers in the NS5B genomic region, wherein said set of primers for amplifying said HCV NS5B nucleic acid is characterized in that at least one of the primers is an antisense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 8529 to 8546, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the above method for amplification of an HCV NS5B nucleic acid of HCV genotype 1 wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7536 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention relates to a method for amplification of an HCV NS5B nucleic acid of HCV genotype 2 comprising the step of designing a set of primers in the NS5B genomic region, wherein said set of primers for amplifying said HCV NS5B nucleic acid is characterized in that at least one of the primers is an antisense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 8620 to 8638, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the above method for amplification of an HCV NS5B nucleic acid of HCV genotype 2 wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention relates to a method for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 3 to 5 comprising the step of designing a set of primers per genotype in the NS5B genomic region, wherein said set of primers for amplifying said HCV NS5B nucleic acid is characterized in that at least one of the primers is an antisense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 8470 to 8491, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the abovem method for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 3 to 5, wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention relates to a method for amplification of an HCV NS5B nucleic acid of HCV genotype 6 comprising the step of designing a set of primers in the NS5B genomic region, wherein said set of primers for amplifying said HCV NS5B nucleic acid is characterized in that at least one of the primers is an antisense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 8529 to 8554, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention also envisages the above method for amplification of an HCV NS5B nucleic acid of HCV genotype 6, wherein said set of primers is further characterized in that it comprises a sense primer that is capable of annealing to the NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• a method for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6 comprising the step of designing a set of primers per genotype in the NS5B genomic region, wherein said set of primers for amplifying said HCV NS5B nucleic acid is characterized in that one of the primers is an antisense primer chosen from the group consisting of
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 9276 to 9298 - an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8529 to 8546,
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8620 to 8638
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8470 to 8491 ,
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8529 to 8554, and that the other primer is a sense primer chosen from the group consisting of
• nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the method for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6 can comprise the steps of
• Consistency in numbering of nucleotides in the current invention is based on taking HCV strain HCV-H as a reference strain (Inchauspe et al. 1991).
• the complete genome sequence of HCV-H which is a type Ia isolate, is available at GenBank (Accession Number M67463).
• the HCV-H sequence consists of 9416 nucleotides including the 5' and 3' untranslated or non-coding regions (UTR; NCR). Nucleotide numbering starts at the first nucleotide with position 1.
• the adenine residue at position 342 is the first residue of the ATG start codon that initiates the long HCV polyprotein of 3011 amino acids.
• type Ia isolates contain 1 extra amino acid in the NS5A region as compared to type Ib isolates, coding sequences of type Ia and Ib have identical numbering in the Core, El, E2, NS2, NS3 and NS4 region, but a slightly different numbering in the NS5 region.
• Type 2 isolates have 4 extra amino acids in the E2 region, and 17 or 18 extra amino acids in the NS5 region compared to type 1 isolates, and will differ in numbering from type 1 isolates in the NS2, NS3, NS4 and NS5 regions. Similar insertions compared with type 1 (but of a different size) can also be observed in genotype 3 to 6 sequences which affect the numbering of the respective types accordingly.
• insertions or deletions may be readily observed in genotype 1 to 6 sequences after alignment with the reference strain HCV-H.
• a given genomic region of an HCV genotype or isolate of interest can always and easily be placed at the same relative position of the corresponding region of HCV-H.
• an insertion or deletion of one or more nucleotides in the Core region of a given HCV isolate changes the nucleotide numbering in the NS5 region relative to HCV-H.
• the NS5 region of the HCV isolate can be integrally aligned with the NS5 region of HCV-H.
• Consistency in nucleotide numbering means that the nucleotide numbering of the NS5 region of HCV-H is taken as the standard numbering when refering to nucleotides of the NS5 region of another HCV isolate.
• HCV genotypes 1 to 6 refer to all HCV variants characterized since the initial molecular characterization of HCV in 1989.
• HCV genotype 1 refers to all HCV variants characterized since the initial molecular characterization of HCV in 1989.
• HCV genotype 1 refers to all HCV variants characterized since the initial molecular characterization of HCV in 1989.
• HCV genotype 1 refers to all HCV variants characterized since the initial molecular characterization of HCV in 1989.
• HCV genotype 1 refers subgroups of said HCV variants.
• HCV variants are classified into 6 HCV major genotypes and a series of subtypes (up to 80) by phylogenetic analysis of Core/El and NS5B subgenomic sequences as well as of complete genome sequences. A comprehensive listing of all currently classified HCV variants and provisionally assigned HCV genotypes/subtypes is provided in Table 1 and 2 of Simmonds et al. (2005).
• HCV variants may also
• HCV NS5B nucleic acid or NS5B genomic region any HCV NS5B genomic sequence.
• Said HCV NS5B genomic sequence can be part of a larger HCV genomic sequence.
• Amplification of said HCV NS5B genomic sequence according to the invention results in an amplified HCV NS5B nucleic acid fragment starting at around nucleotide position 7500 and ending at around nucleotide position 9298, or fragments thereof, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H as outlined above.
• HCV NS5B nucleic acid fragments starting at nucleotides 7500 to 7555 and ending at nucleotides 9276 to 9298 are meant, for example starting at nucleotides 7500 to 7517, 7500 to 7518, 7500 to 7521, 7536 to 7555, and ending at nucleotides 9276 to 9296, 9276 to 9297, 9276 to 9298.
• HCV NS5B nucleic acid fragments starting at around nucleotide position 8258 to around nucleotide position 9298 are also included.
• HCV NS5B nucleic acid fragments starting at nucleotides 8258 to 8278 and ending at nucleotides 9276 to 9298 are meant, for example starting at nucleotides 8259 to 8278 and ending at nucleotides 9276 to 9296, 9276 to 9297, 9276 to 9298.
• Amplification of said HCV NS5B genomic sequence according to the present invention also results in an amplified HCV NS5B nucleic acid fragment starting at around nucleotide position 7500 and ending at around nucleotide position 8546, or fragments thereof, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H as outlined above.
• HCV NS5B nucleic acid fragments starting at nucleotides 7536 to 7555 and ending at nucleotides 8529 to 8546 are meant.
• Amplification of said HCV NS5B genomic sequence according to the present invention further results in an amplified HCV NS5B nucleic acid fragment starting at around nucleotide position 7500 and ending at around nucleotide position 8638, or fragments thereof, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H as outlined above.
• HCV NS5B nucleic acid fragments starting at nucleotides 7500 to 7521 and ending at nucleotides 8620 to 8638 are meant.
• Amplification of said HCV NS5B genomic sequence according to the present invention further results in an amplified HCV NS5B nucleic acid fragment starting at around nucleotide position 7500 and ending at around nucleotide position 8491, or fragments thereof, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H as outlined above.
• HCV NS5B nucleic acid fragments starting at nucleotides 7500 to 7521 and ending at nucleotides 8470 to 8491 are meant, for example starting at nucleotides 7500 to 7517, 7500 to 7518, 7500 to 7521 and ending at nucleotides 8470 to 8488, 8470 to 8491, 8473 to 8491.
• Amplification of said HCV NS5B genomic sequence according to the present invention further results in an amplified HCV NS5B nucleic acid fragment starting at around nucleotide position 7500 and ending at around nucleotide position 8554, or fragments thereof, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H as outlined above.
• HCV NS5B nucleic acid fragments starting at nucleotides 7500 to 7521 and ending at nucleotides 8529 to 8554 are meant, for example starting at nucleotides 7500 to 7517, 7500 to 7521 and ending at nucleotides 8529 to 8554, 8534 to 8554.
• the length of the resulting amplified HCV NS5B nucleic acid fragments is not restricted to the above described examples. In fact, it should be clear by the skilled man that the exact start and endpoints of the HCV NS5B nucleic acid fragments are determined by the primers used for amplifying said fragments (see infra).
• a "primer” is the at least one oligonucleotide in a nucleic acid amplification reaction mixture that is required to obtain specific amplification of a target nucleic acid.
• Nucleic acid amplification can be linear or exponential and can result in an amplified single stranded nucleic acid derived from a single- or double-stranded nucleic acid or can result in both strands of a double-stranded nucleic acid.
• Specificity of a primer in directing amplification of a nucleic acid can be improved by introducing modified nucleotides in said primer or by adapting hybridization or annealing conditions (see e.g. Example 1).
• oligonucleotide referred to herein is meant to include all nucleic acid molecules comprising or consisting of a part of the HCV nucleic acids according to the invention.
• the lower size limit of an oligonucleotide is a nucleic acid comprising or consisting of at least 8 contiguous nucleotides of HCV nucleic acids.
• An oligonucleotide thus can comprise or consist of at least and/or comprise or consist of up to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more contiguous nucleotides of HCV nucleic acids.
• Primers as short as 8 nucleotides in length have been applied successfully in directing specific amplification of a target nucleic acid molecule (for instance: Majzoub et al. 1983).
• a "sense primer" or a forward primer is a primer that will direct amplification by annealing to the antisense strand of a target nucleic acid, and will generate the sense strand of an amplification product.
• an "antisense primer” or a reverse primer is a primer that will direct amplification by annealing to the sense strand of a target nucleic acid, and will generate the antisense strand of an amplification product.
• a target nucleic acid refers to the nucleic acid that is to be amplified.
• a target region refers to a region of the nucleic acid that is to be amplified.
• a polynucleic acid or a nucleic acid or an oligonucleotide of the present invention may be of genomic origin, RNA, DNA, cDNA or a synthetic polynucleic acid and may comprise modified nucleotides, wherein said modification can occur in the nucleotide base or the nucleotide sugar. Any of the modifications can be introduced in a nucleic acid or oligonucleotide to increase/decrease stability and/or reactivity of the nucleic acid or oligonucleotide and/or for other purposes such as labeling of the nucleic acid or oligonucleotide.
• Modified nucleotides include phophorothioates, alkylphophorothioates, methylphosphonate, phosphoramidate, peptide nucleic acid monomers and locked nucleic acid monomers.
• a polynucleic acid or a nucleic acid can be single- or double-stranded or may be a triplex- forming nucleic acid.
• the terms nucleic acid and polynucleic acid are used interchangeably herein.
• Nucleotide sequence amplification methods may include but are not limited to the polymerase chain reaction (PCR; DNA amplification), strand displacement amplification (SDA; DNA amplification), ligase chain reaction (LCR; DNA amplification), transcription-based amplification system (TAS; RNA amplification), self-sustained sequence replication (3SR; RNA amplification), nucleic acid sequence-based amplification (NASBA; RNA amplification), transcription-mediated amplification (TMA; RNA amplification), Qbeta-replicase-mediated amplification and run-off transcription.
• PCR polymerase chain reaction
• SDA DNA amplification
• LCR DNA amplification
• TAS transcription-based amplification system
• NASBA nucleic acid sequence-based amplification
• TMA transcription-mediated amplification
• Qbeta-replicase-mediated amplification and run-off transcription PCR; DNA amplification), strand displacement amplification (SDA; DNA amplification),
• DNA sequencing (classical, such as based on Sanger reaction, or via PCR) is herewith also included as an amplification method (see further).
• the amplified products can be conveniently labeled either using labeled primers or by incorporating labeled nucleotides. Labels may be isotopic ( 32 P, 35 S, etc.) or non-isotopic (biotin, digoxigenin, etc.).
• the most widely spread nucleotide sequence amplification technique is PCR. Basically, two primers, a sense and an antisense primer are annealed to a denatured DNA substrate and extended by a thermostable DNA polymerase.
• PCR rapid and repeated thermal cycling (denaturing/annealing/extension in three-step PCR; denaturing/annealing + extension in two-step PCR).
• the target DNA is exponentially amplified.
• the amplification reaction is repeated between 20 and 70 times, advantageously between 25 and 45 times.
• Many methods rely on PCR including AFLP (amplified fragment length polymorphism), IRS-PCR (interspersed repetitive sequence PCR), iPCR (inverse PCR), RAPD (rapid amplification of polymorphic DNA), RT-PCR (reverse transcription PCR) and real-time PCR.
• RT- PCR can be performed with a single thermostable enzyme having both reverse transcriptase and DNA polymerase activity (Myers and Gelfand 1991).
• RT-PCR single-tube RT-PCR
• one-step RT-PCR RT-PCR in which all components of the RT and PCR are mixed in one tube prior to starting the reactions and in which RT and PCR are thus carried out sequentially in one tube.
• two-step RT-PCR is meant RT-PCR in which the components of the RT and PCR are respectively separated in two tubes prior to starting the reaction and in which RT and PCR are thus carried out sequentially in two tubes.
• Multiplex PCR another variant of PCR which enables simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers, is herewith also included as an amplification method.
• multiplex PCR include but are not limited to simultaneous amplification in one reaction of a single HCV NS5B nucleic acid fragment for multiple HCV genotypes, or of multiple NS5B fragments for a single HCV genotype, or of multiple NS5B fragments for multiple HCV genotypes according to the current invention.
• said multiplex PCR may further include primers for simultaneous amplification of any other HCV or non- HCV nucleic acid target sequence, such as a HCV NS3 nucleic acid sequence, a HBV nucleic acid sequence, a HIV nucleic acid sequence etc.
• the invention further relates to any of the methods as described above wherein said amplification can be part of a multiplex amplification method.
• the invention in another aspect, relates to a method for determining the sequence of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6 wherein said NS5B nucleic acid is amplified according to any of the above described methods prior to or concurrent with determining said sequence. Further, the invention relates to a method for evaluating the sequence variations in an HCV NS5B nucleic acid of any of HCV genotypes 1 to 6 wherein the sequence of said NS5B nucleic acid is determined according to the above described method prior to evaluating the sequence variations. More specifically, said sequence variations can be mutations that are induced by HCV antiviral agents. More specifically, said sequence variations can be mutations causing HCV drug resistance. Said sequence variations can also be genotype-specific nucleotide variations. The evaluation of sequence variations, more specifically the discrimination between either HCV drug resistance mutations or genotype-specific nucleotide variations, can occur simultaneously.
• sequence variations or “nucleotide variations” is meant at least one nucleotide of an HCV nucleic acid or genomic region according to the invention that is different from the corresponding nucleotide in another HCV nucleic acid sequence to which the comparison is made.
• Corresponding nucleotide in this context is referring to a nucleotide at the same relative position in the HCV genome (whether noted as RNA or DNA) wherein the relative position takes into account the possible occuring genotype-, subtype-, or iso late-specific insertions and/or deletions (see for instance section on Genotype-specific insertions and deletions on page 198 of Maertens and Stuyver 1997); a corresponding nucleotide can also be present in a nucleic acid that is the complement of the nucleic acid to which the comparison is made. Differences between nucleotide sequences can easily be determined by the skilled person, for instance after aligning said sequences.
• genotype-specific nucleotide variations is meant at least one nucleotide of an HCV nucleic acid or genomic region according to the invention that is unique to or specific to any genotype, subtype or isolate of an HCV classifying in any of the known genotype classes 1 to 6.
• “Genotype-specific” is referring to the specificity or uniqueness of a nucleotide to an HCV nucleotide sequence belonging to any of HCV genotypes 1 to 6.
• the term “genotype-specific” is generally accepted as can for instance be derived from EP 0 637 342 Bl or any of US 6,548,244, US 5,882,852 or US 5,514,539.
• Said unique or specific nucleotides can occur as a single nucleotide or a set of nucleotides specific to a nucleic acid sequence of any of the HCV types identified to date.
• the specific or unique nucleotides of said set can form a contiguous sequence or can be dispersed in a larger region of a nucleic acid sequence of any of the HCV types identified to date.
• HCV antiviral agents are meant any nucleoside or nucleotide analogs as well as immunological reagents and chemical, proteinaceous and nucleic acid agents which inhibit or otherwise interfere with HCV viral replication, maintenance, infection, assembly or release.
• HCV antiviral agents or inhibitors may include but are not limited to HCV NS5B polymerase inhibitors that currently can be broadly divided into three categories: (i) nucleoside analogues, (ii) pyrophosphate mimics, and (iii) non-nucleoside inhibitors (reviewed in: Beaulieu and Tsantrizos 2004).
• nucleoside analogues such as those having a 2'-methylribose structure, act as chain terminators of the polymerase reaction (Carroll et al. 2003).
• the pyrophosphate mimics exemplified by a series of diketo acids and hydroxypyrimidine carboxylic acids that selectively bind the NS5B active site divalent cations, act as product-like inhibitors of the polymerase reaction (Summa et al. 2004).
• the non-nucleoside inhibitors are by far the most diverse class of inhibitors and represent compounds that bind to distinct pockets on the HCV polymerase: the thiophene 2-carboxylic acids, phenylalanine derivatives, and cyclopentyl dihydropyran-2-ones classes of compounds all bind to a pocket with a central Met 423 residue (Love et al. 2003; Wang et al. 2003).
• Another class of compounds is exemplified by benzothiadiazines that give rise to resistant replicon mutants at position Met 414 (Nguyen et al. 2003; Tomei et al. 2004).
• HCV antiviral agents are also meant combinations of compounds binding to different sites of one HCV target or to other HCV targets, such as HCV protease inhibitors.
• mutants that are induced by HCV antiviral agents relates to a nucleotide variation in an HCV gene encoding an HCV structural or non- structural protein caused by said HCV antiviral agents as described above. More specifically, the mutation may or may not result in an altered amino acid sequence (amino acid addition, substitution and/or deletion) in the targeted region. Viral variants carrying said mutations may or may not exhibit reduced sensitivity to one or more of said HCV antiviral agents as described above.
• the term "mutation" is to be read in its broadest context and may include one or more mutations.
• mutations causing HCV drug resistance relates to acquired mutations induced by said HCV antiviral agents as described above or to mutations that spontaneously occurred, causing HCV drug resistance.
• viral variants carrying HCV drug resistance mutations exhibit reduced sensitivity to one or more of said HCV antiviral agents as described above.
• HCV drug resistant mutations that emerged with the benzimidazole 5- carboxamide class of NS5B inhibitors were found at three amino acid positions, each conferring different levels of resistance to this drug class: Pro 495 with substitutions to Ser, Leu, Ala or Thr; Pro 496 with substitutions to Ser or Ala; and VaI 499 with substitutions to Ala (Tomei et al. 2003; Kukolj et al. 2005).
• the occurrence of certain HCV drug resistance mutations may be correlated with a certain HCV genotype.
• a large number of assays capable of detecting nucleotide sequences and nucleotide sequence variations is currently available. These assays can identify specific mutations, single nucleotide polymorphisms (SNPs), genotype-specific nucleotides or the like. Some of these assays are based on physical methods whereas others use enzymatic approaches.
• nucleotide sequence variation detection with one or more physical processes for detection although not excluding the enzymatic process of prior amplification of the target DNA sequence comprising one or more nucleotide sequence polymorphisms.
• Said physical processes include electrophoresis, chromatography, spectrometry, optical signal sensing and spectroscopy.
• the nucleotide sequence variations to be detected more specifically are the polymorphisms occurring in the nucleic acids of different HCV genotypes, -subtypes, or -isolates.
• Physical nucleotide sequence variation detection assays include electrophoretic methods such as SSCP (single stranded conformation polymorphism), CDCE (constant denaturant capillary electrophoresis), CDGE (constant denaturant gel electrophoresis), DGGE (denaturing gradient gel electrophoresis), TGCE (thermal gradient capillary electrophoresis), DGCE (double gradient capillary electrophoresis), nonisocratic CZE (capillary zone electrophoresis), TDGS (two-dimensional gene scanning), CSGE (conformation sensitive gel electrophoresis) , MADGE (microplate array diagonal gele electrophoresis), DSCA (double stand conformation analysis), HMA (heteroduplex mobility assay), HTA (heteroduplex tracking assay); chromatographic methods include DHPLC (denaturing high performance liquid chromatography).
• SSCP single stranded conformation polymorphism
• CDCE constant denaturant capillar
• Physical nucleotide sequence variation detection assays may be effective for identification of known or new mutations and may require confirmation by direct DNA sequencing. Probes or primers utilized in any of these physical nucleotide sequence variation detection assays may be modified to increase their discriminatory power, such modifications including the addition of a GC-clamp (i.e., an artificial high-melting domain) to a probe or primer.
• a GC-clamp i.e., an artificial high-melting domain
• MALDI-TOF MS matrix-assisted laser desorption-ionization time-of- flight mass spectrometry
• Enzymatic approaches for the generation of products signaling nucleotide sequence variations is meant in the present context approaches relying on the activity of one or more enzymes for generation of said signaling products.
• Enzymes include DNA restriction endonucleases, DNA polymerases, DNA ligases, DNA/RNA structure-specific endonucleases, DNA/RNA flap endonucleases, DNA exonucleases and reverse transcriptases (RTs).
• Enzymatic approaches usually require a physical process (e.g. as described supra) for detection of the enzymatically produced signal.
• Said enzymatic approaches include RFLP (restriction fragment length polymorphism), AFLP (amplified fragment length polymorphism), ASO-PCR (allele-specific oligonucleotide PCR), real-time PCR, LCR (ligase chain reaction) and any variation thereof, LDR (ligase detection reaction), CFLP (Cleavase fragment length polymorphism), EMD (enyzmatic mutation detection), NIRCA (non-isotropic RNase cleavage assay), InvaderTM assay and its modifications, MIDAS (mutation identification DNA analysis system), ddF (dideoxy fingerprinting), Bi-ddF 21
• Probes or primers utilized in any of these enzymatically-based nucleotide sequence polymorphism detection assays may carry specific modifications in order to detect the target nucleotide sequence polymorphism.
• Such modifications include labeling with a single label, with two different labels (for instance two fluorophores or one fluorophore and one quencher), the attachment of a different 'universal' tail to two probes or primers hybridizing adjacent or in close proximity to each other with the target nucleotide sequence, the incorporation of a target-specific sequence in a hairpin probe or primer (for instance Molecular Beacon-type primer), the tailing of such a hairpin probe or primer with a 'universal' tail (for instance Sunrise-type probe and AmplifluorTM-type primer).
• a special type of hairpin probe/primer incorporates in the hairpin a sequence capable of hybridizing to part of the newly amplified target DNA.
• Amplification of the hairpin is prevented by the incorporation of a blocking nonamplifiable monomer (such as hexethylene glycol).
• a fluorescent signal is generated after opening of the hairpin due to hybridization of the hairpin loop with the amplified target DNA.
• This type of hairpin probe/primer is know as scorpion primers (Whitcombe et al. 1999).
• Another special type of probe is a padlock probe (or circularizable probe or open circle probe or C-probe) that are used in RCA (rolling circle amplification).
• the technique of CFLP fingerprinting has already been applied to perform HCV genotyping (Sreevatsan et al. 1998).
• a chemical alternative for CFLP or NIRCA is the CCM assay (chemical cleavage of mismatch).
• DNA sequencing methods include the Maxam and Gilbert protocol, the Sanger reaction (dideoxynucleotide chain termination reaction) and modifications thereof, pyrosequencing, cycle sequencing, SBH (sequencing by hybridization).
• GBA Genetic Bit Analysis
• DNA sequencing methods are based on (partial) amplification of the target nucleic acid (cfr. supra).
• DNA sequencing methods include molecular resonance sequencing which uses electrospray ionization (ESI) combined with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (Smith et al. 1994) and, for smaller DNA fragments, MALDI-TOF MS). Diagnostic sequencing by combining specific cleavage of DNA followed by mass spectrometric analysis of the fragments has also been described (see e.g. Stanssens and Zabeau 2000 - WO00/66771).
• ESI electrospray ionization
• FTICR Fourier transform ion cyclotron resonance
• both ribozymes hammerhead-, hairpin-, group I intron-, ribonuclease P- or hepatitis delta viral-type ribozymes
• deoxyribozymes 'DNAzymes'
• the DNA sequencing methodology known as SBH or sequencing-by-hybridization uses an array of all possible n-nucleotide oligomers (n-mers) to identify said n-mers comprised in an unknown DNA sample (Drmanac et al. 1993).
• n-mers n-mers
• VDA variable detector array
• Microscope slides can be replaced by optical fibers as solid support for the oligonucleotides (Healey et al. 1997).
• a variation of the above- described SBH is based on solution hybridization of probes with a known information region or information tags with the target DNA fragments to be sequenced.
• the information tag can be a DNA bar code (eventually comprising modified bases), a molecular bar code or a nanoparticle bar code and forms the basis for identification and characterization of the hybridized target DNA (Drmanac 2000 - WO/0056937).
• Said high-density oligonucleotide arrays or DNA chips abolish the need to design a set of oligonucleotides specifically hybridizing under the same conditions to a set of polymorphic nucleotide sequences.
• Single-stranded target DNA targets are electrophoresed through said gel and, depending on electrophoresis conditions (temperature and/or denaturant), captured by the oligonucleotides immobilized in a capture gel layer.
• This method is also applicable for detecting nucleotide sequence polymorphisms (Kenney et al. 1998).
• hybridization-based methods for detecting nucleotide sequence variations include the solution phase sandwich hybridization assay in which the target DNA is captured by a target-specific immobilized capture probe and detected via an amplifier or linker probe.
• Two methods of signal generation have been described. A first one utilizes a branched oligonucleotide hybridizing to the flap of the linker probe not binding to the target DNA. Subsequently a labeled probe is hybridized to the branches of the amplifier probe and the amount of bound label is quantified. In a second method, a (partially) double stranded amplifier probe is hybridized to the flap of the linker probe not binding to the target DNA.
• the double stranded (part of) said amplifier probe comprises a promoter recognized by a DNA-dependent RNA polymerase.
• the signal generated is formed by newly transcribed RNA from the amplifier probe, the amount of which is quantified, (see e.g. Urdea 1991 -
• the invention further relates to any of the methods as described above wherein said amplification is a one-step RT-PCR.
• the invention further relates to any of the methods as described above wherein the HCV NS5B nucleic acid is present in a biological sample.
• biological sample generally refers to any biological sample (tissue or fluid) containing traces of HCV such as nucleic acid sequences. More particularly, blood serum or plasma samples are meant.
• the invention also relates to a recombinant vector comprising an NS5B fragment amplified according to any of the above described methods.
• Said vector may be a cloning tool or may additionally comprise regulatory sequences such as promoters, enhancers and terminators or polyadenylation signals. It may also comprise any other HCV or non-HCV nucleic acid sequence, different from the amplified NS5B nucleic acid according to the invention, for example the 5' end of an HCV NS5B nucleic acid, the HCV NS3 nucleic acid, the HCV NS4 nucleic acid, an indicator gene, a reporter gene, etc.
• Said vector can be a "resistance test vector" as defined in e.g.
• Said vector can be an expression vector capable of directing expression of the HCV NS5B protein or any other protein encoded by the HCV nucleic acid sequence comprised in said vector. More specifically, said vector can be used for determining susceptibility or resistance of the comprised nucleic acid to an antiviral drug (e.g. WO 99/06597).
• Host cells transformed with a vector according to the invention are also part of the present invention and are known in the art.
• the present invention relates to a set of primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6, said set of primers characterized in that at least one of the primers is an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 9276 to 9298, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• SEQ ID Nos: 2, 4, 6, 8, 10, 12 are oligonucleotide sequences representing the above described antisense primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6. More specifically,
• SEQ ID NO 2 is the oligonucleotide sequence of an antisense primer designed for HCV genotype 1, capable of annealing to the HCV NS5B genomic region from nucleotide position 9276 to 9297
• SEQ ID NO 4 is the oligonucleotide sequence of an antisense primer designed for HCV genotype 2, capable of annealing to the HCV NS5B genomic region from nucleotide position 9276 to 9298 -
• SEQ ID NO 6 is the oligonucleotide sequence of an antisense primer designed for HCV genotype 3
• SEQ ID NO 8 is is the oligonucleotide sequence of an antisense primer designed for HCV genotype 4, capable of annealing to the HCV NS5B genomic region from nucleotide position 9276 to 9297
• SEQ ID NO 10 is the oligonucleotide sequence of an antisense primer designed for HCV genotype 5, capable of annealing to the HCV NS5B genomic region from nucleotide position 9276 to 9298
• SEQ ID NO 12 is the oligonucleotide sequence of an antisense primer designed for HCV genotype 6, capable of annealing to the HCV NS5B genomic region from nucleotide position 9276 to 9296
• the invention further relates to the set of primers as described above for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention further relates to the set of primers as described above for amplification of an HCV NS5B nucleic acid from HCV genotype 1 , said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7536 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• the invention further relates to the set of primers as described above for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 2 to 6, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• SEQ ID Nos: 13, 14, 15, 16, 17, 18 are oligonucleotide sequences representing the above described sense primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6. More specifically, - SEQ ID NO 13 is the oligonucleotide sequence of a sense primer designed for HCV genotype 1, capable of annealing to the HCV NS5B genomic region from nucleotide position 7536 to 7555 - SEQ ID NO 14 is the oligonucleotide sequence of a sense primer designed for HCV genotype 2, capable of annealing to the HCV NS5B genomic region from nucleotide position 7500 to 7521
• SEQ ID NO 15 is the oligonucleotide sequence of a sense primer designed for HCV genotype 3, capable of annealing to the HCV NS5B genomic region from nucleotide position 7500 to 7517
• SEQ ID NO 16 is the oligonucleotide sequence of a sense primer designed for HCV genotype 4, capable of annealing to the HCV NS5B genomic region from nucleotide position 7500 to 7518
• SEQ ID NO 17 is the oligonucleotide sequence of a sense primer designed for HCV genotype 5, capable of annealing to the HCV NS5B genomic region from nucleotide position 7500 to 7517
• SEQ ID NO 18 is the oligonucleotide sequence of a sense primer designed for HCV genotype 6, capable of annealing to the HCV NS5B genomic region from nucleotide position 7500 to 7517
• the invention further relates to the set of primers as described above for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8258 to 8278, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• SEQ ID Nos: 1, 3, 5, 7, 9, 11 are oligonucleotide sequences representing the above described sense primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6. More specifically,
• SEQ ID NO 1 is the oligonucleotide sequence of a sense primer designed for HCV genotype 1, capable of annealing to the HCV NS5B genomic region from nucleotide position 8258 to 8278
• SEQ ID NO 3 is the oligonucleotide sequence of a sense primer designed for HCV genotype 2, capable of annealing to the HCV NS5B genomic region from nucleotide position 8258 to 8278
• SEQ ID NO 5 is the oligonucleotide sequence of a sense primer designed for HCV genotype 3, capable of annealing to the HCV NS5B genomic region from nucleotide position 8259 to 8278
• SEQ ID NO 7 is is the oligonucleotide sequence of a sense primer designed for HCV genotype 4, capable of annealing to the HCV NS5B genomic region from nucleotide position 8258 to 8278 - SEQ ID NO 9 is the oligonucleotide sequence of a sense primer designed for HCV genotype 5, capable of annealing to the HCV NS5B genomic region from nucleotide position 8258 to 8278
• SEQ ID NO 11 is the oligonucleotide sequence of a sense primer designed for HCV genotype 6, capable of annealing to the HCV NS5B genomic region from nucleotide position 8259 to 8278
• the present invention relates to a set of primers for amplification of an HCV NS5B nucleic acid of HCV genotype 1, said set of primers characterized in that at least one of the primers is an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8529 to 8546, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• SEQ ID No: 19 is an oligonucleotide sequence representing the above described antisense primers for amplification of an HCV NS5B nucleic acid of HCV genotype 1. More specifically,
• SEQ ID NO: 19 is the oligonucleotide sequence of an antisense primer designed for HCV genotype 1, capable of annealing to the HCV NS5B genomic region from nucleotide position 8529 to 8546
• the invention further relates to the set of primers as described above for amplification of an HCV NS5B nucleic acid of HCV genotype 1, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7536 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• SEQ ID NO: 13 is an oligonucleotide sequence representing the above described sense primers for amplification of an HCV NS5B nucleic acid of HCV genotype 1. More specifically,
• SEQ ID NO 13 is the oligonucleotide sequence of a sense primer designed for HCV genotype 1, capable of annealing to the HCV NS5B genomic region from nucleotide position 7536 to 7555
• the present invention also relates to a set of primers for amplification of an HCV NS5B nucleic acid of HCV genotype 1, said set of primers characterized in that at least one of the primers is a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7536 to 7555, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention further relates to the set of primers as described above for amplification of an HCV NS5B nucleic acid of HCV genotype 1, said set of primers further characterized in that it comprises an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8529 to 8546, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• the present invention relates to a set of primers for amplification of an HCV NS5B nucleic acid of HCV genotype 2, said set of primers characterized in that at least one of the primers is an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8620 to 8638, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• SEQ ID NO: 20 is an oligonucleotide sequence representing the above described antisense primers for amplification of an HCV NS5B nucleic acid of HCV genotype 2. More specifically,
• SEQ ID NO: 20 is the oligonucleotide sequence of an antisense primer designed for HCV genotype 2, capable of annealing to the HCV NS5B genomic region from nucleotide position 8620 to 8638
• the invention further relates to the set of primers as described above for amplification of an HCV NS5B nucleic acid of HCV genotype 2, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• SEQ ID NO: 14 is an oligonucleotide sequence representing the above described sense primers for amplification of an HCV NS5B nucleic acid of HCV genotype 2. More specifically,
• SEQ ID NO: 14 is the oligonucleotide sequence of a sense primer designed for HCV genotype 2, capable of annealing to the HCV NS5B genomic region from nucleotide position 7500 to 7521
• the present invention also relates to a set of primers for amplification of an HCV NS5B nucleic acid of HCV genotype 2, said set of primers characterized in that at least one of the primers is a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention further relates to the set of primers as described above for amplification of an HCV NS5B nucleic acid of HCV genotype 2, said set of primers further characterized in that it comprises an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8620 to 8638, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• the present invention relates to a set of primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 3 to 5, said set of primers characterized in that at least one of the primers is an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8470 to 8491, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• SEQ ID Nos: 21, 22, 23 are oligonucleotide sequences representing the above described antisense primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 3 to 5. More specifically,
• SEQ ID NO 21 is the oligonucleotide sequence of an antisense primer designed for HCV genotype 3, capable of annealing to the HCV NS5B genomic region from nucleotide position 8470 to 8491
• SEQ ID NO 22 is the oligonucleotide sequence of an antisense primer designed for HCV genotype 4, capable of annealing to the HCV NS5B genomic region from nucleotide position 8473 to 8491
• SEQ ID NO 23 is the oligonucleotide sequence of an antisense primer designed for HCV genotype 5, capable of annealing to the HCV NS5B genomic region from nucleotide position 8470 to 8488
• the invention further relates to the set of primers as described above for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 3 to 5, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• SEQ ID Nos: 15, 16, 17 are oligonucleotide sequences representing the above described sense primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 3 to 5. More specifically, - SEQ ID NO 15 is the oligonucleotide sequence of a sense primer designed for HCV genotype 3, capable of annealing to the HCV NS5B genomic region from nucleotide position 7500 to 7517 - SEQ ID NO 16 is the oligonucleotide sequence of a sense primer designed for HCV genotype 4, capable of annealing to the HCV NS5B genomic region from nucleotide position 7500 to 7518
• SEQ ID NO 17 is the oligonucleotide sequence of a sense primer designed for HCV genotype 5, capable of annealing to the HCV NS5B genomic region from nucleotide position 7500 to 7517
• the present invention also relates to a set of primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 3 to 5, said set of primers characterized in that at least one of the primers is a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention further relates to the set of primers as described above for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 3 to 5, said set of primers further characterized in that it comprises an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8470 to 8491, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the present invention relates to a set of primers for amplification of an HCV NS5B nucleic acid of HCV genotype 6, said set of primers characterized in that at least one of the primers is an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8529 to 8554, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• SEQ ID NO: 24 is an oligonucleotide sequence representing the above described antisense primers for amplification of an HCV NS5B nucleic acid of HCV genotype 6. More specifically,
• SEQ ID NO: 24 is the oligonucleotide sequence of an antisense primer designed for HCV genotype 6, capable of annealing to the HCV NS5B genomic region from nucleotide position 8534 to 8554
• the invention further relates to the set of primers as described above for amplification of an HCV NS5B nucleic acid of HCV genotype 6, said set of primers further characterized in that it comprises a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• SEQ ID NO: 18 is an oligonucleotide sequence representing the above described sense primers for amplification of an HCV NS5B nucleic acid of HCV genotype 6. More specifically,
• SEQ ID NO: 18 is the oligonucleotide sequence of a sense primer designed for HCV genotype 6, capable of annealing to the HCV NS5B genomic region from nucleotide position 7500 to 7517
• the present invention also relates to a set of primers for amplification of an HCV NS5B nucleic acid of HCV genotype 6, said set of primers characterized in that at least one of the primers is a sense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 7500 to 7521, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the invention further relates to the set of primers as described above for amplification of an HCV NS5B nucleic acid of HCV genotype 6, said set of primers further characterized in that it comprises an antisense primer that is capable of annealing to the HCV NS5B genomic region spanning nucleotides 8529 to 8554, wherein the nucleotide positions correspond with the nucleotide numbering for HCV strain HCV- H.
• the invention relates to a set of primers for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6, said set of primers characterized in that one of the primers is an antisense primer chosen from the group consisting of
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 9276 to 9298 - an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8529 to 8546,
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8620 to 8638
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8470 to 8491
• an antisense primer capable of annealing to the NS5B genomic region spanning nucleotides 8529 to 8554, and that the other primer is a sense primer chosen from the group consisting of
• nucleotide positions correspond with the nucleotide numbering for HCV strain HCV-H.
• the antisense and sense primers according to the invention can also be used in nucleic acid sequencing reactions or as genotype-specific probes.
• the invention relates to the use of any of the sets of primers as described above for the manufacture of a kit for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6.
• the invention relates to a kit for amplification of an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6, said kit comprising any of the sets of primers as described above.
• Said kit can be used for multiplex amplification.
• a further embodiment relates to a kit for assaying sequence variations in an HCV NS5B nucleic acid of any one of HCV genotypes 1 to 6, said kit comprising any of the sets of primers as described above.
• Said sequence variations can be mutations that are induced by HCV antiviral agents, mutations causing HCV drug resistance, or genotype-specific nucleotide variations.
• kits may further comprise:
• At least one probe containing a sequence complementary to and capable of hybridizing to said amplified HCV NS5B nucleic acid according to the invention, said at least one probe serving to detect the amplified product and/or to detect sequence variations; - a means for denaturing nucleic acids;
• - a wash solution, or components necessary for producing said solution; - a means for detecting partially or completely denatured nucleic acids and/or a means for detecting hybrids formed in the preceding hybridization and/or a means for detecting enzymatic modifications of nucleic acids, wherein said means is producing a discriminatory signal;
• NS5B nucleic acid according to the invention, the presence of said HCV NS5B nucleic acid, or the presence of at least one unique or genotype-specific or specific nucleotide therein, and, therefrom, the presence of said HCV virus in said biological sample and/or the genotype/subtype of said HCV virus in said biological sample; - a means for infering from a detected discriminatory signal, and/or from the obtained NS5B nucleic acid according to the invention, the presence of said HCV NS5B nucleic acid, or the presence of at least one mutation therein, or the presence of at least one mutation therein which is a drug resistance mutation, in said HCV virus in said biological sample.
• a means for infering the presence of at least one unique or genotype-specific or specific nucleotide is meant any technique or method to localize and identify in a target nucleic acid sequence said genotype-specific or specific nucleotide.
• Said means can include a method performed manually, or performed computationally, or performed manually and/or computationally.
• Said means may include aligning and/or comparing an obtained nucleic acid sequence with a set of nucleic acid sequences contained within a database.
• Said means may furthermore include the result of the method being presented in the form of a report wherein said report can be in paper form, in electronic form or on a computer readable carrier or medium.
• Said means may furthermore include the searching of (nucleic acid and/or amino acid) sequence databases and/or the creation of (nucleic acid and/or amino acid) sequence alignments, the results of which may or may not be included in said report.
• Said means may furthermore include a device for detecting a discriminatory signal, or a kit insert or kit chart indicating how to interpret a detected discriminatory signal, or indicating where a specific discriminatory signal should appear, e.g. on a solid carrier carrying multiple oligonucleotides which can be arranged as spots, lines, dots, etc and possibly interpreting said discriminatory signal occurring on a specific location. Further examples of genotype-specific nucleotides and how to discriminate within a target HCV nucleic acid sequence are described for instance in WO 94/25601, WO 96/13590 and WO 03/020970.
• a means for infering the presence of at least one mutation is meant any technique or method to localize and identify in a target nucleic acid sequence said mutation. It will be clear by the man skilled in the art that said means can include a similar method as for infering the presence of at least one unique or genotype-specific or specific nucleotide (cfr. supra). It should also be clear by the skilled person that infering the presence of at least one genotype-specific nucleotide and infering the presence of at least one mutation and discriminating between them can occur simultaneously.
• the examples of the present invention presented below are provided only for illustrative purposes and not to limit the scope of the invention.
• Sense and antisense primers were designed for amplification of the 3' end of an NS5B nucleic acid of HCV genotypes 1 to 6 within the HCV NS5B region spanning nucleotides 8258 to 8278 and nucleotides 9276 to 9298 respectively.
• the nucleotide positions are according to the nucleotide numbering for HCV strain HCV-H (GenBank accession no. M67463).
• different primers were designed for each genotype. Due to the variability of the genome within a certain genotype, wobbles were sometimes included in the primer sequence. This was only done for frequently common polymorphisms that gave rise to a too low Tm when screened by the Metcalc software (Gottingen, Germany).
• genotype-specific primers were screened for their capability of amplifying the 3' end of an NS5B nucleic acid present in HCV-positive serum samples.
• Table 1 gives examples of sense and anti-sense primers per genotype that are capable of amplifying the NS5B fragment, according to the further described methodology.
• the efficiency of the amplification protocols was evaluated using HCV-positive samples from all major HCV genotypes covering a wide range of viral loads. This will be further illustrated by taking genotype 1 (subtype Ia or Ib) as an Example.
• RNA from all serum samples was performed with the Qiagen QiAamp MiniElute Virus Spin kit (Venlo, The Netherlands) according to the manufacturar's instructions.
• RT-PCR was performed to amplify the targeted 3' end NS5B fragment using the Invitrogen Superscript III One Step RT-PCR System with Platinum Taq DNA polymerase (Merelbeke, Belgium), as further described below.
• the PCR protocol was optimized by evaluating different temperatures of the RT step (50 0 C; 55°C; 60 0 C) and the annealing step (52°C; 54°C; 56°C), by testing different MgCl 2 concentrations (l,6mM; 2,OmM; 2,4mM), different primer concentrations (0,4 ⁇ M-0,6 ⁇ M-0,8 ⁇ M) and different annealing times (30s and 60s), by testing different additives (DMSO and glycerine) at several concentrations (5%; 7,5%; 10%) and addition of wheat germ RNA.
• Optimal conditions in terms of sensitivity/specificity were as follows: an RT step of 30 minutes at 55°C, annealing temperature of 54°C and 1,6 mM MgCl 2 . Both DMSO and Glycerine at a concentration of 7.5% can intensify the amplification signal without altering the specificity. Addition of wheat germ RNA increases the aspecif ⁇ city of the PCR reaction.
• PCR was performed from 15 uL of RNA, starting with an RT step consisting of 30 min at 55 0 C, followed by 2 min at 94 0 C. Next, 40 cycles were performed with each consisting of 30 s at 94 0 C, 30 s at 54 0 C and 2 min at 68 0 C . The extension time for the last cycle was increased to 5 min.
• the reactions were carried out with 2 ⁇ L Superscript III RT / Platinum Taq polymerase mix in 25 uL 2X reaction mix (containing 200 ⁇ M dNTP and 1.6 mM MgCl 2 ) and 5.5 ⁇ L RNAse free water, in the presence of 2.5 ⁇ l primer mix (0.5 ⁇ M, containing 10 pmol/ ⁇ L of each primer).
• the PCR products were electrophoresed in 2% agarose gel and fragments were detected by ethidium bromide staining and UV illumination.
• the amplification efficiency of the developed one-step RT-PCR protocol was confirmed by evaluating 18 HCV genotype 1 positive serum samples with the genotype 1 -specific set of sense and antisense primers (SEQ ID NO 1 and 2, respectively). The results are summarized in Table 2. The expected sizes of NS5B cDNA bands of about 1000 bp were detected in all serum samples. As such, the selected pairs of primers with SEQ ID NO 1 and SEQ ID NO 2 designed for genotype 1 within the above described regions are widely effective for amplification of the targeted NS5B region.
• the described methodology provides a standardized and rapid amplification system for the targeted 3' end of an HCV NS5B nucleic acid fragment of any of HCV genotypes 1 to 6.
• Table 1 it can moreover be derived that all genotype-specific sense primers locate in the same HCV NS5B region spanning nucleotides 8258 to 8278 and similarly, that all genotype-specific antisense primers locate in the same HCV NS5B region spanning nucleotides 9276 to 9298.
• these two defined HCV NS5B regions are universal regions wherein any primer according to genotype can be designed capable of amplification of the NS5B fragment. Table 1.
• a dilution pool was made of 2 serum samples 242-029 (genotype lb-positive) and 248- 085 (genotype la-positive). Each diluted sample was evaluated as a template for amplification of the targeted 3' end NS5B fragment using the genotype 1 specific set of sense and antisense primers in a RT-PCR reaction as described in Example 1. The results are shown in Table 8.
• the methodology outlined in Example 1 thus appears to be robust and works at HCV viral loads as low as 3500 to 4500 cp/mL. As such, the described methodology thus provides a highly sensitive and specific amplification system for the targeted 3' end HCV NS5B region.
• the designed sense and antisense primers according to HCV genotypes 1 to 6 as described in Example 1 were successivefully used as sequencing primers to obtain complete coverage of both strands of the resulting amplified NS5B fragment of each genotype.
• the nucleic acid sequence of the amplified NS5B fragments derived from the above-mentioned HCV-positive serum samples about 1000 bp was confirmed.
• the present invention thus provides a uniform and rapid RT- PCR based tool for amplification and sequencing of the 3' end of an NS5B nucleic acid fragment of any one of HCV genotypes 1 to 6.
• Example 2 The same methodology as described in Example 1 was shown to be applicable for amplification of the entire NS5B nucleic acid of HCV genotypes 1 to 6, except that sense and antisense primers were now designed for amplification of the full length of the NS5B nucleic acid of HCV genotypes 1 to 6 within the HCV NS5B region spanning nucleotides 7500 to 7555 and nucleotides 9276 to 9298 respectively.
• the nucleotide positions are according to the nucleotide numbering for HCV strain HCV- H (GenBank accession no. M67463).
• genotype-specific primers were screened for their capability of amplifying the full length of an NS5B nucleic acid present in HCV-positive serum samples.
• Table 9 gives examples of sense and anti-sense primers per genotype that are capable of amplifying the entire NS5B nucleic acid, according to the methodology described in Example 1.
• nucleotide positions correspond with the nucleotide numbering HCV strain HCV-H (GenBank accession no. M67463).
• Sense and antisense primers were designed for amplification of the 5' end of an NS5B nucleic acid of HCV genotype 1 within the HCV NS5B region spanning nucleotides
• nucleotides 8529 to 8546 respectively.
• the nucleotide positions are according to the nucleotide numbering for HCV strain HCV-H (GenBank accession no. M67463). Due to the variability of the genome within a genotype, wobbles were sometimes included in the primer sequence. This was only done for frequently common polymorphisms that gave rise to a too low Tm when screened by the Metcalc software (G ⁇ ttingen, Germany).
• genotype-specific primers were screened for their capability of amplifying the 5' end of an NS5B nucleic acid present in HCV-positive serum samples.
• Table 11 gives examples of sense and anti-sense primers for genotype 1 that are capable of amplifying the NS5B fragment, according to the further described methodology.
• the efficiency of the amplification protocols was evaluated using HCV-positive samples of HCV genotype 1 covering a wide range of viral loads.
• RNA from all serum samples was performed with the Qiagen QiAamp MiniElute Virus Spin kit (Venlo, The Netherlands) according to the manufacturar's instructions.
• RT-PCR was performed to amplify the targeted 5' end NS5B fragment using the Invitrogen Superscript III One Step RT-PCR System with Platinum Taq DNA polymerase (Merelbeke, Belgium), as further described below.
• the PCR protocol was optimized by evaluating different temperatures of the RT step (50 0 C; 55°C; 60 0 C) and the annealing step (52°C; 54°C; 56°C), by testing different MgCl 2 concentrations (l,6mM; 2,OmM; 2,4mM), different primer concentrations (0,4 ⁇ M-0,6 ⁇ M-0,8 ⁇ M) and different annealing times (30s and 60s), by testing different additives (DMSO and glycerine) at several concentrations (5%; 7,5%; 10%) and addition of wheat germ RNA.
• Optimal conditions in terms of sensitivity/specificity were as follows: an RT step of 30 minutes at 55°C, annealing temperature of 54°C and 1,6 mM MgCl 2 . Both DMSO and Glycerine at a concentration of 7.5% can intensify the amplification signal without altering the specificity. Addition of wheat germ RNA increases the aspecificity of the PCR reaction.
• PCR was performed from 15 uL of RNA, starting with an RT step consisting of 30 min at 55 0 C, followed by 2 min at 94 0 C. Next, 40 cycles were performed with each consisting of 30 s at 94 0 C, 30 s at 54 0 C and 2 min at 68 0 C . The extension time for the last cycle was increased to 5 min.
• the reactions were carried out with 2 ⁇ L Superscript III RT / Platinum Taq polymerase mix in 25 uL 2X reaction mix (containing 200 ⁇ M dNTP and 1.6 mM MgCl 2 ) and 5.5 ⁇ L RNAse free water, in the presence of 2.5 ⁇ l primer mix (0.5 ⁇ M, containing 10 pmol/ ⁇ L of each primer).
• the PCR products were electrophoresed in 2% agarose gel and fragments were detected by ethidium bromide staining and UV illumination.
• the amplification efficiency of the developed one-step RT-PCR protocol was confirmed by evaluating 21 HCV genotype 1 positive serum samples with the genotype 1 -specific set of sense and antisense primers (SEQ ID NO 13 and
• NS5B cDNA bands of about 1050 bp were detected in all serum samples.
• the selected pairs of primers with SEQ ID NO 13 and SEQ ID NO 19 designed for genotype 1 within the above described regions are widely effective for amplification of the targeted NS5B region.
• the described methodology provides a standardized and rapid amplification system for the targeted 5' end of an HCV NS5B nucleic acid fragment of HCV genotype 1.
• the two defined HCV NS5B regions are universal regions wherein any primer according to genotype 1 can be designed capable of amplification of the NS5B fragment. Table 11.
• a dilution pool was made of 2 serum samples 242-029 (genotype lb-positive) and 248- 085 (genotype la-positive). Each diluted sample was evaluated as a template for amplification of the targeted 5' end NS5B fragment using the genotype 1 specific set of sense and antisense primers in a RT-PCR reaction as described in Example 5. The results are shown in Table 13. The methodology outlined in Example 5 thus appears to be robust and works at HCV viral loads as low as 1000 cp/mL. As such, the described methodology thus provides a highly sensitive and specific amplification system for the targeted 5' end HCV NS5B region.
• EXAMPLE 7 Sequencing of the 5' end NS5B amplified product derived from HCV genotype 1 positive serum samples
• the designed sense and antisense primers according to HCV genotype 1 as described in Example 5 were successivefully used as sequencing primers to obtain complete coverage of both strands of the resulting amplified NS5B fragment of genotype 1.
• the nucleic acid sequence of the amplified NS5B fragments derived from the above-mentioned HCV-positive serum samples of about 1000 bp was confirmed.
• the present invention thus provides a uniform and rapid RT-PCR based tool for amplification and sequencing of the 5' end of an NS5B nucleic acid fragment of HCV genotype 1.
• Sense and antisense primers were designed for amplification of the 5' end of an NS5B nucleic acid of HCV genotype 2 within the HCV NS5B region spanning nucleotides
• nucleotide positions are according to the nucleotide numbering for HCV strain HCV-H (GenBank accession no. M67463). Due to the variability of the genome within a genotype, wobbles were sometimes included in the primer sequence. This was only done for frequently common polymorphisms that gave rise to a too low Tm when screened by the Metcalc software (G ⁇ ttingen, Germany).
• genotype-specific primers were screened for their capability of amplifying the 5' end of an NS5B nucleic acid present in HCV-positive serum samples.
• Table 14 gives examples of sense and anti-sense primers for genotype 2 that are capable of amplifying the NS5B fragment, according to the further described methodology.
• the efficiency of the amplification protocols was evaluated using HCV-positive samples of HCV genotype 2 covering a wide range of viral loads.
• RNA from all serum samples was performed with the Qiagen QiAamp MiniElute Virus Spin kit (Venlo, The Netherlands) according to the manufacturar's instructions.
• RT-PCR was performed to amplify the targeted 5' end NS5B fragment using the Invitrogen Superscript III One Step RT- PCR System with Platinum Taq DNA polymerase (Merelbeke, Belgium), as further described below. This approach minimizes the possibility for introduction of contaminants into reactions between the RT and PCR steps, since the RT and PCR reactions are carried out sequentially, without opening of reaction tubes between steps.
• PCR was performed from 15 uL of RNA, starting with an RT step consisting of 30 min at 55 0 C, followed by 2 min at 94 0 C.
• 40 cycles were performed with each consisting of 30 s at 94 0 C, 30 s at 54 0 C and 2 min at 68 0 C .
• the extension time for the last cycle was increased to 5 min.
• the reactions were carried out with 2 ⁇ L Superscript III RT / Platinum Taq polymerase mix in 25 uL 2X reaction mix (containing 200 ⁇ M dNTP and 1.6 mM MgCl 2 ) and 5.5 ⁇ L RNAse free water, in the presence of 2.5 ⁇ l primer mix (0.5 ⁇ M, containing 10 pmol/ ⁇ L of each primer).
• the PCR products were electrophoresed in 2% agarose gel and fragments were detected by ethidium bromide staining and UV illumination.
• the described methodology provides a standardized and rapid amplification system for the targeted 5' end of an HCV NS5B nucleic acid fragment of HCV genotype 2.
• the two defined HCV NS5B regions are universal regions wherein any primer according to genotype 2 can be designed capable of amplification of the NS5B fragment.
• the designed sense and antisense primers according to HCV genotype 2 as described in Example 8 were successivefully used as sequencing primers to obtain complete coverage of both strands of the resulting amplified NS5B fragment of genotype 2.
• the nucleic acid sequence of the amplified NS5B fragments derived from the above-mentioned HCV-positive serum samples of about 1000 bp was confirmed.
• the present invention thus provides a uniform and rapid RT-PCR based tool for amplification and sequencing of the 5' end of an NS5B nucleic acid fragment of HCV genotype 2. Table 14.
• Sense and antisense primers were designed for amplification of the 5' end of an NS5B nucleic acid of HCV genotypes 3 to 5 within the HCV NS5B region spanning nucleotides 7500 to 7521 and nucleotides 8470 to 8491 respectively.
• the nucleotide positions are according to the nucleotide numbering for HCV strain HCV-H
• genotype-specific primers were screened for their capability of amplifying the 5' end of an NS5B nucleic acid present in HCV-positive serum samples.
• Table 16 gives examples of sense and anti-sense primers per genotype that are capable of amplifying the NS5B fragment, according to the further described methodology. The efficiency of the amplification protocols was evaluated using
• RNA from all serum samples was performed with the Qiagen QiAamp MiniElute Virus Spin kit (Venlo, The Netherlands) according to the manufacturar's instructions.
• RT-PCR was performed to amplify the targeted 5' end NS5B fragment using the Invitrogen Superscript III One Step RT- PCR System with Platinum Taq DNA polymerase (Merelbeke, Belgium), as further described below. This approach minimizes the possibility for introduction of contaminants into reactions between the RT and PCR steps, since the RT and PCR reactions are carried out sequentially, without opening of reaction tubes between steps.
• PCR was performed from 15 uL of RNA, starting with an RT step consisting of 30 min at 55 0 C, followed by 2 min at 94 0 C.
• 40 cycles were performed with each consisting of 30 s at 94 0 C, 30 s at 54 0 C and 2 min at 68 0 C .
• the extension time for the last cycle was increased to 5 min.
• the reactions were carried out with 2 ⁇ L Superscript III RT / Platinum Taq polymerase mix in 25 uL 2X reaction mix (containing 200 ⁇ M dNTP and 1.6 mM MgCl 2 ) and 5.5 ⁇ L RNAse free water, in the presence of 2.5 ⁇ l primer mix (0.5 ⁇ M, containing 10 pmol/ ⁇ L of each primer).
• the PCR products were electrophoresed in 2% agarose gel and fragments were detected by ethidium bromide staining and UV illumination.
• the described methodology provides a standardized and rapid amplification system for the targeted 5' end of an HCV NS5B nucleic acid fragment of any of HCV genotypes 3 to 5.
• Table 16 it can moreover be derived that all genotype-specific sense primers locate in the same HCV NS5B region spanning nucleotides 7500 to 7521 and similarly, that all genotype-specific antisense primers locate in the same HCV NS5B region spanning nucleotides 8470 to 8491.
• these two defined HCV NS5B regions are universal regions wherein any primer according to genotype can be designed capable of amplification of the NS5B fragment.
• the designed sense and antisense primers according to HCV genotypes 3 to 5 as described in Example 10 were successivefully used as sequencing primers to obtain complete coverage of both strands of the resulting amplified NS5B fragment of each genotype.
• the nucleic acid sequence of the amplified NS5B fragments derived from the above-mentioned HCV-positive serum samples about 1000 bp was confirmed.
• the present invention thus provides a uniform and rapid RT- PCR based tool for amplification and sequencing of the 3' end of an NS5B nucleic acid fragment of any one of HCV genotypes 1 to 6.
• Y is T or C ; K is G or T; R is G or A
• Sense and antisense primers were designed for amplification of the 5' end of an NS5B nucleic acid of HCV genotype 6 within the HCV NS5B region spanning nucleotides
• nucleotide positions are according to the nucleotide numbering for HCV strain HCV-H (GenBank accession no. M67463). Due to the variability of the genome within a genotype, wobbles were sometimes included in the primer sequence. This was only done for frequently common polymorphisms that gave rise to a too low Tm when screened by the Metcalc software (G ⁇ ttingen, Germany).
• genotype-specific primers were screened for their capability of amplifying the 5' end of an NS5B nucleic acid present in HCV-positive serum samples.
• Table 20 gives examples of sense and anti-sense primers for genotype 6 that are capable of amplifying the NS5B fragment, according to the further described methodology.
• the efficiency of the amplification protocols was evaluated using HCV-positive samples of HCV genotype 6 covering a wide range of viral loads.
• RNA from all serum samples was performed with the Qiagen QiAamp MiniElute Virus Spin kit (Venlo, The Netherlands) according to the manufacturar's instructions.
• RT-PCR was performed to amplify the targeted 5' end NS5B fragment using the Invitrogen Superscript III One Step RT- PCR System with Platinum Taq DNA polymerase (Merelbeke, Belgium), as further described below. This approach minimizes the possibility for introduction of contaminants into reactions between the RT and PCR steps, since the RT and PCR reactions are carried out sequentially, without opening of reaction tubes between steps.
• PCR was performed from 15 uL of RNA, starting with an RT step consisting of 30 min at 55 0 C, followed by 2 min at 94 0 C.
• 40 cycles were performed with each consisting of 30 s at 94 0 C, 30 s at 54 0 C and 2 min at 68 0 C .
• the extension time for the last cycle was increased to 5 min.
• the reactions were carried out with 2 ⁇ L Superscript III RT / Platinum Taq polymerase mix in 25 uL 2X reaction mix (containing 200 ⁇ M dNTP and 1.6 mM MgCl 2 ) and 5.5 ⁇ L RNAse free water, in the presence of 2.5 ⁇ l primer mix (0.5 ⁇ M, containing 10 pmol/ ⁇ L of each primer).
• the PCR products were electrophoresed in 2% agarose gel and fragments were detected by ethidium bromide staining and UV illumination.
• the described methodology provides a standardized and rapid amplification system for the targeted 5' end of an HCV NS5B nucleic acid fragment of HCV genotype 6.
• the two defined HCV NS5B regions are universal regions wherein any primer according to genotype 6 can be designed capable of amplification of the NS5B fragment.
• the designed sense and antisense primers according to HCV genotype 6 as described in Example 12 were successivefully used as sequencing primers to obtain complete coverage of both strands of the resulting amplified NS5B fragment of genotype 6.
• the nucleic acid sequence of the amplified NS5B fragments derived from the above-mentioned HCV-positive serum samples of about 1000 bp was confirmed.
• the present invention thus provides a uniform and rapid RT-PCR based tool for amplification and sequencing of the 5' end of an NS5B nucleic acid fragment of HCV genotype 6. Table 20.
• nucleotide positions correspond with the nucleotide numbering of HCV strain HCV-H (GenBank accession no. M67463).
• Y is T or C ;
• R is G or A
• HCV antiviral resistance the impact of in vitro studies on the development of antiviral agents targeting the viral NS5B polymerase. Antivir.Chem Chemother. 16, 225-245.
• Non- nucleoside analogue inhibitors bind to an allosteric site on HCV NS5B polymerase. Crystal structures and mechanism of inhibition. J Biol Chem 278, 9489-9495.

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