An internal control applied to RT-PCR detection of HCV and HIV-1 in human pooled plasma and plasma-derived medicinal products

Abstract

A competitive internal control (IC) adapted to RT-PCR in-house assay was developed for HCV RNA detection in human pooled plasma. Also, it was applied in a multiplex RT-PCR for the HIV-1 and HCV RNA screening in human pooled plasma and plasma-derived products. A 258-bp PCR product from the 5´noncoding region of HCV genome was obtained. A competitive IC template was constructed by inserting a 52-bp double strand sequence into the NheI site of the 258-bp amplicon. This sequence was cloned and the obtained plasmid was used to generate a synthetic RNA. The IC/RNA was incorporated in in-house HCV and/or HIV PCR technique to monitor the efficiency of extraction, reverse transcription, and PCR amplification steps. IC was also used to detect all major genotypes of HCV and HIV-1 strains with similar sensitivity. The detection limit of the assay for HCV and HIV-1 was 52.7 IU/mL and 164.2 IU/mL, respectively. These techniques have been evaluated in international programs of external quality assurance with highly satisfactory results. This IC is an essential reagent in PCR techniques to detect and identify HCV and HIV-1 in pooled plasma samples involved in the manufacture of plasma-derived products as well as in the field of clinical microbiology with limited resources.