Toro GG, Valdés RYC , Rosa GMC, Falcón CV, Cabal MCA

Language: Spanish
References: 24
Page: 5-10
PDF size: 118.92 Kb.

ABSTRACT

Molecular assays for diagnosis tests are widespread and essential in these days. Cloning of the representative sequence of the hepatitis C virus (HCV) 5’ untranslated region (5’-UTR) a highly conserved region, was carried out to be used as a positive control in the HCV detection by reverse transcriptase-polimerase chain reaction (RT-PCR). We selected a patient with anti-HCV antibodies by HCV EIA 3.0 and RIBA HCV 3.0 and a viral load > 90,000 copies of viral genome/mL. Standard recombinant DNA technologies were used for all cloning procedures. First, we obtained viral RNA (RNAv) from the patient infected plasma. The RNAv was used to obtain a DNA product (DNAi) of 214 pb by a reverse transcriptase-PCR (RT-PCR) and nested PCR reactions, these assays were development using conserved primers (KY-80 and KY-78/FIP and RIP) deduced from the 5´-UTR region of the HCV. DNAi was used for the plasmid construction pGem-T-5´-UTR which was transformed into E. coli DH5α cells to obtain the E. coli DH5α-pGem-T-5´-UTR cellular lines. 5´-UTR fragment was amplified by PCR using the initiators FIP and RIP from stable cell clones to obtain the 5´-UTR product of 214 pb. This product appears to be a good positive control in the RT-PCR detection assay for HCV.

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