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

RODRIGUEZ LOMBARDI, GONZALO; REYNA, LUCIANA; VITALI, SUSANA; GENTI-RAIMONDI, SUSANA

BIOCELL

Biocell

Lugar: Mendoza; Año: 2015 vol. 39 p. 15 - 15

competitive internal control (IC) adapted to RT-PCR in-house assay was developed for HCVRNA 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 ́non-coding 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