A LightCycler Real Time PCR protocol for accurate quantification of HCV viraemia followed by sequencing of different genotypes

Abstract number: 1133_38

Tagliaferro L., Menegazzi P., De Donno M.A., McDermott J.L., Varnier O.E.

Objectives:  

The main objectives of this project were the development of a rapid LightCycler Real Time PCR (RTiPCR) protocol for accurate quantification of HCV viraemia, its use in the testing of a large number of clinical samples and its cost-effective integration in the genotyping protocol.

Methods:  

Serial ten-fold dilutions of two HCV RNA positive controls (Accurun, BBI), 170,000 and 910,000 IU/ml, respectively, were amplified and the data from the resulting calibration curve were stored in the LightCycler database for the quantification of plasma HCV RNA. Clinical samples were tested in a rapid single-tube RTiPCR reaction and cost-effective HCV RNA quantification was determined using one reference standard during each assay. The quantification obtained for the single reference standard was validated in a comparative analysis with the stored calibration curve followed by interpolation of the amount of HCV RNA in the clinical samples. HCV sequencing was directly performed using 10 ml of purified real time PCR amplicons.

Results:  

A total of 2,470 plasma samples were tested from Oct 2002 to Sept 2004. HCV RNA was quantified in 1,226 samples with a dynamic range of 57 to 2.5 × 10.9 IU/ml and the theoretical threshold was 53 IU/ml. The standard deviation (SD) and the coefficient of variation (CV) of the HCV RNA levels were 1.08 and 3.55% for the 170,000 IU/ml standard and 1.34 and 4.78% for the 910,000 IU/ml standard. In 266 samples the amplicons were purified and sequenced for HCV genotyping (Bayer Diagnostics). Thirty five different HCV strains were typed: the majority were genotype 1b (36%) and 2 (30%), but genotypes 4 and 5 were also identified.

Conclusions:  

Our study shows that this HCV RTiPCR protocol, which has been used for 2 years to test over 2,000 samples with minimal SD and CV values, allows accurate quantitation of HCV RNA in clinical samples. The characteristics of rapidity and accuracy have been extended to include cost-effectiveness due to subsequent use of the amplicons for sequencing.