Construction and utilization of an internal control (IC) derived from Qb phage in RNA viruses diagnoses.

VILLANOVA, G. VANINA; ADRIANA ANGELICA GIRI; GARDIOL, DANIELA

Bariloche

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2003

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

RT-PCR is an important tool for the detection of RNA viruses in clinical and research laboratories. Due to RNA liability, it is necessary to have a control to verify the efficiency of both, sample processing and amplification reaction. We present the development of an IC derived from Qß phage to be spiked into the specimen, processed and co-amplified with the same target primers. Qß is a coliphage with single strand RNA genome. The primers KY78/KY80, which are commonly used in the detection of HCV, were inserted in the phage genome flanking a phage sequence. They were cloned into the open reading frame of A1 coat protein. The recombinant phage (Qß78-80) was tested in infection experiments and particles viability was demonstrated. To confirm the in vivo results, RT-PCR was performed with the Qß78-80 and HCV RNAs as templates and the KY78 and KY80 primers. Products obtained were the expected ones and they were confirmed by hybridization with specific probes and detection by colorimetric method. Experiments of RNasa-resistance and stability were performed. They showed that this IC is RNasa–resistant and is stable at 4ºC for at least 125 days. It was used in RT-PCR co-amplifications altogether with HCV (+) samples with different viral tittles using the same pair of primers. The results were satisfactory and showed that Qß78-80 was an useful tool. This strategy could be used for the construction of IC for others RNA viruses which molecular diagnosis is important for prevention or therapeutic approaches.