Development and validation of an RT-qPCR assay for rapid detection and quantification of hepatitis C virus RNA for routine testing in Moroccan clinical specimens

A one-step reverse transcription quantitative PCR (RT-qPCR) assay in combination with rapid RNA extraction was evaluated for routine testing of hepatitis C virus (HCV) RNA. Specific primers and probes were designed for the detection of a 150 bp sequence located in the 5 ' untranslated region (5 ' UTR) of HCV RNA. The target sequence was selected as the most conserved region between the six known HCV subtype sequences following an alignment. The assay was able to quantify a dynamic linear range of 10(8) to 10(1) plasmid copies/reaction (r(2) = 0.98) containing the target sequence. Two copies of this HCV plasmid corresponds to one international unit (IU) measured using a standard obtained by serial dilutions of the World Health Organization (WHO) standard. The detection limit of the assay was about 10 IU/mL of HCV RNA (20 copies/mL) in plasma samples. The assay was comparable to Cobas AmpliPrep/Cobas TaqMan (R) HCV Test, v2.0 Quantitative assay (Roche Molecular Systems, Inc., Branchburg, NJ) with correlation coefficient r(2) = 0.98. The present assay could be completed within 3 hours from RNA extraction to data analysis of at least 30 plasma samples. Our test provides sufficient sensitivity, specificity, and reproducibility and proved to be fast, labor-saving, and cost-effective. Indeed, our system will definitely allow low-income countries to monitor accurately this viral infection and to efficiently treat their infected patients.