Studies included in these analyses were conducted in full compliance with the guidelines of Good Clinical Practice and of the World Medical Assembly Declaration of Helsinki. Prior to study initiation, protocols and informed consent forms were reviewed and approved by institutional review boards at each study site. All patients provided written informed consent before participating in any study-related activity. ClinicalTrials Identifiers for studies included in the manuscript are as follows: NCT00336479, NCT00372385, NCT00420784, NCT00535847, NCT00627926, NCT00983853, NCT00758043, NCT00916474, NCT00528528, NCT00703118, NCT00911963, NCT01080222.
This method is based on the amplification protocol originally described in Kwong et al. .
HCV RNA extraction
Viral RNA was extracted from plasma in a 96-well format according to the manufacturer’s instruction with several modifications. Plasma (220 ~ 660 μL, the volume was adjusted based on viral load, i.e., 220 μL for HCV RNA greater than or equal to 50,000 IU/mL, while 660 μL for HCV RNA less than 50,000 IU/mL) was mixed with QIAGEN Protease K (40 ~ 120 μL) and QIAamp AL buffer (240 ~ 720 μL) supplemented with 20 ~ 60 μg of carrier RNA (tRNA) per column. The mixture was incubated for 15 min at 56°C, and absolute ethanol (293 ~ 875 μL) was added to each well and mixed well. The mixture was loaded into QIAamp column and passed through the column by suction with a peristaltic micropump (IPS-16; Ismatec, Zurich, Switzerland). Subsequently, the column was washed with AW1 buffer (1000 μL) and AW2 buffer (1000 μL) (provided in QIAGEN kit), respectively, by applying vacuum. A second wash with AW2 buffer (1000 μL) was spun at 6000 × g for 10 min and an additional spin at 6000 × g for 15 min was applied to remove residual ethanol. 40 μL of RNA storage solution (Ambion) was loaded into each column and incubated at room temperature for 5 min. The RNA was eluted by centrifugation at 6000 × g for 10 min. The elution was repeated once to increase the yield. A total of 80 μL viral RNA was isolated from 220 to 660 μL of plasma.
A complementary DNA (cDNA) fragment was synthesized from HCV RNA that was diluted (1:2 or 1:4, the dilution factor was based on viral load, i.e., 1:2 and 1:4 for HCV RNA less than 50,000 IU/mL, while 1:4 for HCV RNA greater than or equal to 50,000 IU/mL) into a total RT reaction volume of 20 μL composed of 2.5 μM of an oligo-dA20 primer (Table 1), 400 units of Superscript™ III Reverse Transcriptase (Life Technologies Corporation, Carlsbad, CA), 40 units of RNAseOUT (Life Technologies Corporation, Carlsbad, CA), PC2 reaction buffer (50 mM Tris–HCl pH 9.1, 16 mM ammonium sulfate, 3.5 mM magnesium chloride, and 150 μg/mL BSA) (AB Peptides, St. Louis, MO), 500 μM dNTPs (Clontech, Mountain View, CA), and 5 mM DTT (Life Technologies Corporation, Carlsbad, CA). The RNA was denatured at 65°C for 5 min, followed by ramping extension temperatures at 25°C for 10 min, 42°C for 60 min, 50°C for 30 min, 55°C for 30 min, and 70°C for 15 min.
Amplification of HCV via nested PCR
The cDNA from the RT reaction was diluted 1:1 into a 40 μL PCR1 reaction mixture, containing PC2 reaction buffer (AB Peptides, St. Louis, MO), 200 μM dNTPs (Clontech, Mountain View, CA), 1.5 M betaine (Sigma Aldrich, St. Louis, MO), 2.56 units Klentaq DNA polymerase (AB Peptides, St. Louis, MO), 1.28 units pfu DNA polymerase (Stratagene, La Jolla, CA), and 400 μM each subtype-specific primer (Table 1). The PCR1 reaction was incubated at 94°C for 2 min, followed by 30 cycles at 94°C for 15 sec, 68°C −0.5°C/cycle (“touchdown” PCR) for 20 sec, followed by an incubation at 68°C for 12 min. The completed PCR1 reaction was diluted 1:10 into a second PCR mixture (PCR2, 50 μL). The reaction composition and PCR cycling parameters were identical to the PCR1 reaction, with the following changes; 3.2 units Klentaq DNA polymerase, 1.6 units of Pfu DNA polymerase, and nested subtype-specific primers were utilized (Table 1).
Preparation of sample for sequencing
The PCR2 product (8991 bp) was analyzed and verified by agarose gel electrophoresis, and purified using the QIAquick 96 PCR Purification kit (QIAGEN, Valencia, CA). The purified PCR2 product was subsequently quantified using a NanoDrop 8000 Spectrophotometer (Thermo Scientific, Hudson, NH) prior to sequence analysis.