Evaluation of an automated, highly sensitive, real-time PCR-based assay (COBAS Ampliprep/COBAS TaqMan) for quantification of HCV RNA.
BACKGROUND
Diagnosis of hepatitis C virus (HCV) infection and its therapy is based on qualitative and quantitative measurement of HCV RNA.


OBJECTIVE
A new assay that employs automated specimen extraction and real-time RT-PCR (COBAS Ampliprep/COBAS TaqMan, "CAP/CTM", Roche Diagnostics, Pleasanton, USA) was designed for linear quantification and highly sensitive detection of HCV RNA.


METHODS
The performance characteristics of CAP/CTM were compared to standard RT-PCR-based COBAS Amplicor Monitor 2.0 (CAM) assay in a multicenter study.


RESULTS
The limit of detection of CAP/CTM was 7.4 IU/ml (95% CI 6.2-10.6) and clinical specificity was 99%. The linear range of HCV RNA quantification by CAP/CTM was between 28 and 1.4 x 10(7) IU/ml, with a correlation coefficient between expected and observed results of >0.99. A fivefold dilution of serum- or plasma-samples showed a linear correlation of HCV RNA levels in undiluted and diluted samples. Analyses of the mean intra- and inter-assay imprecision within the linear range of quantification showed a coefficient of variation of 3% and 3%, respectively. HCV genotypes 1a/b, 2b, 3a, 4, 5 and 6 were equally quantified by the CAP/CTM and CAM assay with mean deviations ranging from -0.29log(10) to 0.32log(10) IU/ml. HCV RNA quantification by CAP/CTM and CAM was highly concordant (correlation coefficient of 0.96).


CONCLUSIONS
The CAP/CTM assay is a reliable and robust assay for highly sensitive detection and quantification of HCV RNA within a broad linear range.
Sarrazin
Christoph
C
Medizinische Klinik I, Klinikum der J.W. Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. sarrazin@em.uni-frankfurt.de
Dragan
Anette
A
Gärtner
Barbara C
BC
Forman
Michael S
MS
Traver
Stella
S
Zeuzem
Stefan
S
Valsamakis
Alexandra
A
D001331:Automation; D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D019914:Taq Polymerase