M2000rt instrument

The Plus assay (Abbott Molecular Inc., Des Plaines, IL, USA) uses a multiplex RT-PCR to generate HCV RNA amplicons from clinical specimens. An unrelated RNA sequence serves as an internal control (IC) and is simultaneously extracted and amplified to verify the correct processing of each individual sample. The assay selectively detects HCV subtypes 1a and 1b and genotype 6 in a single reaction, using specific fluorescently labeled oligonucleotide probes targeting the HCV core region. The assay is fully automated; the Abbott m2000sp platform performs HCV RNA extraction, mastermix preparation, and PCR plate setup, while the Abbott m2000rt instrument is used for amplification and detection. The Abbott m2000rt instrument automatically reports, on the Abbott m2000rt workstation, the genotype call results as gt 1a, gt 1b, gt 6, or “not detected,” which could correspond to a gt 1 subtype other than 1a or 1b, as only samples with gt 1 results from the GT II test were subjected to the Plus test in the present study.

HBV DNA was extracted from 0.5 ml of plasma on the Abbott m2000sp instrument using the HBV DNA protocol (Abbott Molecular, Des Plaines, IL) and eluted in 50 µl. Three sets of real-time PCR primers and probes were designed to target the conserved regions within the 3’ end of the core gene and the 5’ splice site (the reverse primer RPwt crossing the 5’ splice site), the 3’ end of the core gene and the SP1 splice junction (the reverse primer RPsp crossing the SP1 splice junction), and the 5’ end of the X gene, respectively (Fig. 1B,C). Real-time PCRs were performed by using the TaqMan Universal PCR master mix II (Applied Biosystems, Foster City, CA). Purified HBV DNA (10 µl) was added to a 40 µl PCR mixture containing 25 µl of TaqMan Universal PCR master mix II, 0.3 µM of each primer, and 0.3 µM of fluorogenic probe (the FAM probe for the core gene and the CY5 probe for the X gene). The reaction consisted of one initiating step of 10 min at 95 C, followed by 40 cycles of amplification and reading including 15 s at 95 C, 30 s at 62 C and 90 s at 56 C. The real-time PCR reactions were performed on the Abbott m2000rt instrument. Data acquisition and analyses were done with Abbott MultiAnalyze v5.04 software. The standard curve for quantification was calculated using serial tenfold dilutions (10¹–10⁶ copies per PCR reaction) of wtHBV or spHBV plasmids.

All samples were processed using the Abbott-Real Time-HCV genotype II (Abbott Laboratories, Illinois, USA) according to manufacturer instructions. First, viral RNA was extracted from 200µl of plasma sample using the QIAsymphony instrument, the commercial mini kit DSP Virus/Pathogen, and the protocol Cellfree 200 v.7. (Qiagen, Hilden, Germany). Internal, positive and negative controls from Abbott-RT-HCV II kit were incorporated into the extraction process. Then, multiplexed RT-PCR reactions were performed using the kit amplification reagent packs and the Abbott m2000rt instrument.