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 RNAdetection in human pooled plasma. Also, it was applied in a multiplex RT-PCR for the HIV-1 and HCV RNAscreening in human pooled plasma andplasma-derivedproducts.A 258-bp PCR product from the 5´noncoding region of HCV genome was obtained. A competitive ICtemplate was constructed by inserting a 52-bp double strand sequenceinto the NheI site of the 258-bpamplicon. Thissequence 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.ICwas also used to detect all major genotypes of HCV and HIV-1 strainswith 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 satisfactoryresults.This IC is an essential reagent in PCR techniquesto detect and identify HCV and HIV-1 inpooled plasma samplesinvolved in the manufacture of plasma-derived productsas well as in the field of clinical microbiology with limited resources.