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.
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA. 18298@aruplab.com
Rebecca L
RL
Margraf
Sam
S
Page
Maria
M
Erali
Carl T
CT
Wittwer
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