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Supporting Information
Methods:
Serologic and Molecular Assays
Anti-HCV was tested by second generation enzyme immunoassay (EIA-2; Abbott
Laboratories, Abbott Park, IL). When samples were reactive by EIA-2, a third generation
recombinant immunoblot assay (RIBA-3; Chiron Corp., Emeryville, CA) was performed.
The serum level of HCV RNA was measured using the quantitative (lower limit of
detection (LLD) ≥600 IU/ml) and qualitative (LLD ≥50 IU/ml) COBAS AMPLICOR
assays (Roche Diagnostic Systems, Branchburg, NJ).
Synthesis of positive and negative strand HCV RNA
HCV cDNA encompassing the 5’-UTR and core regions of genotype 1a was obtained by
RT-PCR, and then cloned in a plasmid vector pCR II (Invitrogen, Carlsbad, CA), which
contained T7 and SP6 promoters flanking the multicloning site. To synthesize the
positive and negative strand HCV RNA fragments, the plasmid was linearized by
digestion with Hind III or Xbal I, and then RNA was synthesized by RiboMAX Large
Scale RNA Production System-SP6 or T7 (Promega, Madison, WI). Template linearized
plasmid was removed by two rounds of digestion with RQ1 RNase-free DNase
(Promega) and RNA purification using the RNeasy system (Qiagen, Chatsworth, CA).
The RNA was quantified by spectrophotometry, and the molecular size and purity were
confirmed by electrophoresis through a 1% agarose/formaldehyde gel in 3-(N-
2
morpholino) propanesulfonic acid buffer. Synthesized HCV-RNA copy numbers were
calculated from the quantity and molecular weight of RNA according to conventional
methods (1).
RNA extraction and purification from plasma, uncultured PBMC or tissue samples
RNA was extracted from PBMC or tissue samples with Trizol reagent (Invitrogen,
Carlsbad, CA) or using the RNeasy Plus kit (Qiagen, Valencia, CA) according to the
manufacturer’s instructions. For plasma samples from recovered subjects, MagMAXTM
Viral RNA Isolation Kit (Ambion, Austin, TX) was used. To detect low copy number of
HCV RNA in plasma, a modification to the pre RT-PCR step was performed. Briefly, 1.6
ml of plasma was aliquoted into 4 tubes and RNA extraction was performed with
MagMAXTM Viral RNA Isolation Kit according to the manufacturer’s protocol. 50 µl of
each buffer elution was added to dissolve viral RNA and subsequently four aliquots of
each 50 ul final elution were combined; RNA was further purified and concentrated
using RNeasy MinElute Cleanup Kit (Qiagen, Chatsworth, CA). Total RNA from 1.6 ml
of plasma sample was eluted in 24 µl of buffer and thus, the 10 µl of purified RNA in
each RT-PCR reaction corresponded to 667 μl of original plasma volume.
Purification of HCV RNA from culture supernatants and cultured cells
With the objective of concentrating the virus, 1 ml of culture SN was subjected to
polyethylene glycol 8000 precipitation at final concentration of 8% (W/V) and then
solubilized with buffer RLT Plus (Qiagen , Chatsworth, CA) with 1% 2-mercaptoethanol.
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After adding the RNA carrier, HCV RNA extraction was performed with the QIAmp viral
RNA minikit (Qiagen, Chatsworth, CA) following the manufacturer’s instructions.
Cellular RNA was extracted from cultured cells with RNeasy Plus minikit according to
the manufacturer’s protocol. The purified RNA was quantified and 1µg of total RNA was
used per PCR for every sample.
HCV detection
We followed the RTD-PCR method designed by Takeuchi et al. (1); however, to obtain
better sensitivity, nested-RTD PCR was performed, as reported previously for the
detection of HCV (2, 3). Briefly, the first round RT-PCR was performed using
thermostable rTth polymerase (GeneAmp EZ rTth RNA PCR kit, Applied Biosystems,
Foster City, CA) and primer set KK30 (5’-CTGTCTTCACGCAGAAAGCG-3’) and KM3 (
5’CACTCGCAAGCACCCTATCA-3’ (1). After reverse transcription for 30 minutes at
60ºC, and initial denaturing at 95ºC for 2 minutes, amplification was performed with 15
cycles of denaturing at 95ºC for 20 seconds and annealing and extension at 60ºC for 1
minute, followed by final extension at 72ºC for 7 min. The nested-RTD PCR was
performed using the primer set R6-130-S17 (5’CGGGAGAGCCA TAGGTGG-3’), R6290-R19 5’-AGTACCACAAGGCCTTTCG-3’) and Taqman probe R6-148-S21FT
(6FAM-5’-CTGCGGAACCGGTGAGTACAC-3’-TAMRA) (1). 3 microliter of PCR product
was mixed with the primer sets, the probe, and Taqman Universal PCR Master Mix
(Applied Biosystems). After initial denaturing at 95ºC for 10 minutes, amplification was
performed with 40 cycles of denaturing at 95ºC for 20 seconds and annealing and
extension at 60ºC for 1 minute in Prism 7900HT Sequence Detection System (ABI).
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Quantitative linearity was obtained from 5 to 106 copies per assay. When diluted WHO
HCV standard was used, the detection limit of this nested-RTD PCR with our RNA
extraction-purification method from plasma was 10 IU per ml. For RNA isolated from
PBMC, the assay can detect 2 copies/µg total RNA.
HCV negative strand detection
For negative strand HCV RNA detection, the rTth based method (4, 5) (rTth DNA
polymerase and Buffer kit, Applied Biosystems) was used with minor modification.
Briefly, RNA was denatured at 95ºC for 1 min and then kept at 70ºC followed by the
addition of a reagent mix containing sense primer KK30 and rTth RNA polymerase preheated at 70 ºC. RT was performed at 60ºC for 2 minutes and then 70 ºC for 25
minutes. While the mixture was kept at 70ºC, chelating buffer pre-heated at 70ºC was
added followed by a PCR reagent mix containing the primer KM3 preheated at 70ºC.
After the initial denaturing at 95ºC for 3 minutes, 15 cycles of PCR reaction was
performed with denaturing at 95ºC for 30 seconds, annealing at 60ºC for 30 seconds,
followed by the final extension at 72ºC for 30 seconds. 3 microliter of PCR product was
used for the subsequent nested-RTD PCR. Synthesized negative- strand HCV RNA of
10 to 106 copies per assay was used as the positive quantitative standard and
synthesized positive strand HCV RNA of 10-106 copies per assay was used as the
negative control. Quantitative linearity was obtained from 50 to 106 copies, and the
detection limit was 50 copies per assay. False positive results were observed when
≥106 copies per assay of positive strand HCV RNA were in the reaction.
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References
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Real-time detection system for quantification of hepatitis C virus genome.
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1998:471-481.