title: A refined long RT-PCR technique to amplify complete viral RNA genome sequences from clinical samples: application to a novel hepatitis C virus variant of genotype 6.
abstract: The goal of this study was to adapt a long RT-PCR technique to amplify large PCR fragments from the genome of hepatitis C virus (HCV) isolates using clinical samples. This was done by using a reverse transcriptase devoid of RNase H activity and a mixture of two antibody-bound thermostable polymerases to combine the high processivity of Taq and the high fidelity of Pwo with its 3'-->5' exonuclease activity. Other modifications included gentle handling during RNA extraction, the absence of tRNA and random primers, a two-step reverse transcription procedure to optimize cDNA synthesis, and increasing the annealing temperature for primers. With this approach, the HCV-1 genome (nucleotides 35-9282) was amplified consistently as two overlapping fragments of 5344 and 4675 bp from a pooled chimpanzee plasma sample containing approximately 10(6) genome copies of HCV RNA/ml. Using the conditions that we identified, 96% of the complete genomic sequence of a distinct HCV genotype 6 variant (km45) was determined from less than 300 microl of serum. This method should prove useful for molecular, epidemiological and clinical studies of hepatitis C where samples are limited but complete virus sequence is required, for example, identifying mutational hot spots of HCV under specific clinical conditions.
authors.affiliation: Division of Gastroenterology/Hepatology, Department of Medicine, University of Kansas Medical Center, 4035 Delp, MS 1023, Kansas City, KA 66160, USA. llu@kumc.edu
authors.forename: Ling
authors.initials: L
authors.lastname: Lu
authors.forename: Tatsunori
authors.initials: T
authors.lastname: Nakano
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Smallwood
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Heffron
authors.forename: Betty H
authors.initials: BH
authors.lastname: Robertson
authors.forename: Curt H
authors.initials: CH
authors.lastname: Hagedorn
mesh_terms: D000818:Animals; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D016679:Genome, Viral; D016174:Hepacivirus / Q000145:classification / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology*; D008969:Molecular Sequence Data; D002679:Pan troglodytes; D010802:Phylogeny; D010949:Plasma / Q000821:virology; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid; D019914:Taq Polymerase / Q000378:metabolism
chemical_list: D012367:RNA, Viral; C118225:Pwo polymerase; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase