Verification of an Assay for Quantification of Hepatitis C Virus RNA by Use of an Analyte-Specific Reagent and Two Different Extraction Methods

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Abstract
Protocols were designed for quantification and detection of hepatitis C virus (HCV) RNA by the use of an analyte-specific reagent (ASR) (Roche COBAS TaqMan48 [CTM48] HCV) after manual and automated RNA extraction. The purposes were to determine (i) assay performance characteristics using manual and automated RNA extraction methods, (ii) whether measurable range and limit of detection (LOD) of the ASR assay were influenced by genotype, and (iii) correlation of quantification by CTM48 HCV ASR and COBAS Monitor HCV v. 2.0. For HCV genotype 1 (Gt1), the lower limits of quantification after manual extraction were slightly lower than those for automated extraction (1.0 versus 1.5 log 10 IU/ml). Results were linear up to the highest concentration tested after extraction by both methods (manual, 6.1 log 10 ; automated, 6.4 log 10 ). Similar results were obtained for Gt2 (1.8 to 6.8 log 10 IU/ml) and Gt3 (1.6 to 6.8 log 10 IU/ml) after automated extraction. The LOD of Gt1 virus was 10 IU/ml after manual extraction and between 25 and 37.5 IU/ml after automated extraction. Results with Gt2 and Gt3 viruses were similar after automated extraction (Gt2, between 25 and 50 IU/ml; Gt3, 25 IU/ml). Variability (intrarun and interrun, at concentrations throughout the range of quantification) was ≤13% for both extraction methods. Clinical specimens tested by Monitor were quantified using the CTM48 HCV ASR assay. Characteristics of the regression line included a slope of 0.98 and y intercept of −0.23. Quantification by the two methods was correlated ( r = 0.97). CTM48 HCV ASR assay values were on average twofold lower than those for Monitor HCV v. 2.0. These data suggest that our assay combines the characteristics of qualitative and quantitative PCR platforms into a single test.