Qualitative measurement of HCV-RNA by home-made PCR in chronic liver diseases compared with commercial kits

ABSTRACT

Hepatitis C is a major health problem in Egypt. As global population estimates reach 170 million infected with the hepatitis C virus [HCV], there has never been a more pressing need for sensitive, precise tests for active infection. This study aimed to develop a sensitive, precise, cost saving protocol for measuring qualitative HCV RNA by PCR. To accomplish this aim, we optimized an-already published-direct RT-PCR protocol without extracting viral nucleic acid. Neat serum [3UL] was used, denatured with phosphate-buffered saline, then nested RT-PCR was performed. Two pairs of primers [inner and outer] were used, which had been selected from the most conservative 5'- untranslated region of the HCV genome. Designing of the inner primers based on nucleotide sequence of the genotype IV which is the more prevalent among Egyptian HCV patients. Positive result showed an 237 bp-sizing band by gel electrophoresis. Sensitivity of the optimized method was estimated also, and revealed a detection limit of 460 copies/ml. This emphasizes that the optimized protocol is a good screening test in comparison with other commercially used ones. Negative cases should be confirmed by extraction method to avoid any loss of false negatives