11 A strain switch was classified when two or more distinct viruses were detected during follow-up
but the presence or absence of viremia in the interim period had not been established. In order to detect multiple HCV genotypes in a single sample, a set of four individual nRT-PCR assays were developed using subtype-specific primers that targeted the HCV subtypes 1a, 1b, 2a, and 3a (these are the most prevalent genotypes in Australia, accounting for 94%-99% of all HCV infections).28, 29 Unique sequences (n = 279) of the entire core region were pooled from GenBank and the HCV Los Alamos databases and aligned using Clustal-W. A universal BTK pathway inhibitor JQ1 forward primer (Hep287) was designed for use in the first round amplification (Supporting Information Table 1). The remaining primers (three for each subtype) were designed such that the
last three to five nucleotides of the 3′ end of the primer were specific to the targeted subtype (Supporting Information Table 1). Amplicon lengths for each genotype were 289 bp (1a), 237 bp (1b), 354 bp (2a), and 241 bp (3a). The overlapping 5′-3′ end of the core region for all for subtypes was 228 bp in length. RNA was extracted from sera using the QIAmp Viral RNA extraction kit (QIAGEN, Hilden, Germany). Mixed infection at a single time point was detected 上海皓元 by performing four real-time subtype-specific nRT-PCRs. Reverse-transcription was performed with 5 μL of RNA template added to a 15-μL reaction mix containing 1× VILO reaction mix and 1× Superscript enzyme
mix (Invitrogen, Mount Waverley, Australia). Reverse-transcription was performed at 25°C for 10 minutes, then at 42°C for 60 minutes. Complementary DNA (5 μL) was added to 15 μL of first-round reaction mix containing 1× iQ SYBR green supermix (BioRad, Hercules, CA) and 0.5 μM of each primer. Reactions were performed at 94°C for 2 minutes, then 15 cycles of 95°C, 52°C, and 72°C for 1 minute, respectively. Second-round PCR was performed for 40 cycles using 2 μL of first-round product added to 18 μL of PCR reaction mix. Reaction mix and conditions were as described above, except that the annealing temperature was raised to 56°C. For quantification, standard curves were generated from serial 10-fold dilutions of T7 polymerase-transcribed HCV RNA, and threshold cycle (Ct) values obtained were used to determine the number of viral genome copies/mL of serum, which was subsequently converted into IU/mL.30 The detection limit of the real-time subtype-specific nRT-PCRs was 86 IU/mL.