Single-tube method for nucleic acid extraction, amplification, purification, and sequencing.
BACKGROUND
The hepatitis C virus (HCV) genotype determines patient prognosis and duration of treatment, but sequencing of the gene is lengthy and labor-intensive. We used a commercially available nucleic acid extraction system to develop a single-tube extraction-to-sequencing (STETS) method for HCV genotyping.


METHODS
HCV RNA was purified and amplified in tubes coated with a solid-phase matrix that irreversibly bound nucleic acid during the extraction step. After reverse transcription-PCR, the amplicon was adsorbed to the original extraction matrix for purification and use in the subsequent sequencing reactions.


RESULTS
The STETS method generated genotyping-quality sequence for a range of HCV titers from 500 to 6,000,000 IU/mL. If a viral sample was detected during real-time reverse transcription-PCR, it could be sequenced and genotyped. Read lengths >600 bases were observed with the STETS method. Mixed infections were detected and genotyped if at least 15% of the minor species was present. Combining the STETS method with consecutive sequencing provided a means of performing both forward and reverse sequencing in a single tube.


CONCLUSIONS
A single-tube nucleic acid extraction-to-sequencing method, which requires less time and labor than conventional methods, generates HCV sequence data that are equivalent to conventional methods and can be used to genotype HCV.
Margraf
Rebecca L
RL
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA. 18298@aruplab.com
Page
Sam
S
Erali
Maria
M
Wittwer
Carl T
CT
D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA
D012367:RNA, Viral