HCV RNA purification optimisation results in higher analytical sensitivity for the artus HCV RG RT-PCR assay Micalessi MI., Cuypers A., Peeters B., Smismans A., Van Meensel B., Frans J. Laboratory of Clinical Microbiology, Imelda Hospital, Bonheiden, Belgium. 8th EMMD 2013 Scheveningen BACKGROUND The artus HCV RG RT-PCR kit (Qiagen) combined with the QIAamp MinElute Virus Spin kit (Qiagen) for viral RNA purification is used in our daily routine for the detection and quantification of HCV RNA in plasma. The assay can quantify HCV RNA over the range of 400 to 1 x 106 IU/ml and is also able to pick up 50 IU/ml with 95% probability. Moreover, this approach is accredited under the ISO 15189 standard. However, the current guidelines for therapy of chronic hepatitis C genotype 1 with protease inhibitors require the use of quantitative HCV RNA assays with a lower limit of quantification (LLOQ) of less than or equal to 25 IU/ml and a lower limit of detection (LLOD) of approximately 10-15 IU/ml. AIM The HCV RNA purification protocol was adapted to increase the analytical sensitivity of the artus HCV RG RT-PCR assay. MATERIAL AND METHODS • Dilution series of the 4th WHO international standard (NIBSC): 200 IU/ml to 6.25 IU/ml. HCV RNA - 1000 µl instead of 400 µl HCV RNA sample volume - 30 µl instead of 60 µl detection elution volume purification QIAamp MinElute Virus Spin Vacuum protocol artus HCV RG RT-PCR assay RESULTS Figure 1 Determination of the LLOD for the artus HCV RT-PCR assay with the adapted purification protocol. Minimum 4 replicates at each concentration (200, 50, 25, 12.5, 6.25 IU/ml) were tested on 2 different days. Probit analysis was performed using xlstat (version 2013.1). Probability 0.05 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 0.95 0.99 IU/ml 2.270 4.176 6.483 8.147 9.569 10.898 12.226 13.648 15.312 17.619 19.525 23.099 Table 2 Probit analysis showed a detection rate of 95% and 80% at 19.5 IU/ml and 15.3 IU/ml, respectively. Table 1 Results of the artus HCV RT-PCR assay for the HCV RNA extracts of several dilutions. Pos, positive; Neg, negative. CONCLUSION • The optimised QIAamp MinElute Virus Vacuum protocol combined with the artus HCV RG RT-PCR kit resulted in a 95% LLOD of 19.5 IU/ml, which is slightly higher than the targeted LLOD. • In future experiments, the sample volume will be increased to obtain an LLOD of 10-15 IU/ml and the lower limit of linearity will be assessed to determine the LLOQ. ACKNOWLEDGEMENT We would like to thank Qiagen for providing the QIAvac 24 Plus vacuum manifold and all reagents to conduct this study.