Current commercially available laboratory methods for the detection and quantification of hepatitis C virus (HCV) RNA in clinical specimens include transcription-mediated amplification, branched-DNA, and real-time PCR (1, 2, 4-6, 9, 10, 13, 18). All of these methods target the highly conserved HCV 5Ј untranslated region (UTR) rather than the similarly conserved 3Ј UTR, which was not recognized during initial characterization of the HCV genome (3, 7) but was subsequently identified (8). Recently, a prototype real-time PCR assay based on MultiCode-RTx PCR technology (i.e., the RTx assay; EraGen Biosciences, Inc., Madison, WI) (11,(15)(16)(17) and targeting the HCV 3Ј UTR was developed for HCV detection and quantification. Herein, we report the results of the initial analytical evaluation of this novel RTx assay along with preliminary correlation studies comparing the RTx assay to the Versant HCV RNA 3.0 assay (the bDNA assay; Siemens Healthcare Diagnostics, Inc., Tarrytown, NY) and a laboratory-developed assay based on the TaqMan HCV analyte-specific reagent (the TaqMan assay; Roche Molecular Systems, Inc.) (1, 2, 4, 6, 9).For the initial analytical studies, individual RTx assay reaction mixtures contained 0.5 U/l Moloney murine leukemia virus reverse transcriptase and 1ϫ Titanium Taq DNA polymerase (Clontech, Mountain View, CA) in 1ϫ ISOlution 1482 buffer (EraGen Biosciences, Inc.) along with primer sets to specifically amplify two targets. The first primer set, labeled with 6-carboxyfluorescein, targeted a 48-bp region of the HCV 3Ј UTR, while the second primer pair, labeled with hexachlorofluorescein, targeted a 72-bp segment of an RNA internal control sequence added at 250 copies/reaction. Thermal cycling was performed with an ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA), with the following parameters: 15 min at 50°C, 2 min at 95°C, followed by 50 cycles of 95°C for 5 s, 58°C for 10 s, and 72°C for 20 s. Following PCR, a 5-min thermal ramp (60°C to 95°C) was performed to determine melting point (T m ) values of the amplified products.An in vitro RNA transcript containing both the HCV 5Ј UTR and the 3Ј UTR was used to prepare a series of RTx assay reaction mixtures containing 10-fold dilutions of this transcript ranging from 10 8 to 10 1 copies/reaction. Data obtained from RTx assay testing of these analytical samples in quadruplicate were exported to MultiCode-RTx analysis software (EraGen Biosciences, Inc.) for further analysis. The presence of HCV RNA was confirmed by a 6-carboxyfluorescein amplification curve with a T m of 80.4 Ϯ 1.0°C occurring within 50 cycles and crossing a cycle threshold (C T ) set at 10 times the standard deviation (SD) of the baseline noise. Results showed that the RTx assay was linear over 7 logs (Fig. 1) and sensitive enough to detect two of four replicates at 10 copies/reaction.For all subsequent studies, the RTx assay was modified to include two manual nucleic acid extraction procedures, both of which incorporate the use of an unrelated extracta...