Development of a universal immunoenzyme quantitative assay for detecting amplified products of nucleic acid and its preliminary application in hepatitis C virus

Objective To develop a universal quantitative immunoenzyme assay (EIA) for detecting amplified products of nucleic acid and its application in hepatit is C virus (HCV). Methods The appropriate cycle number of amplification was selected to stop polymerase chain reaction (PCR) before the "plateau stage". At the same tim e, primers HCV(3) of the second PCR were modified with biotin so that the a mplified products were labeled. The products were diluted and subsequently added to the streptavidin-coated wells, and the biotinylated products were captured, followed by denaturation of NaOH, and non-biotinylated strands we re removed. Hybridization was performed by adding the specific probe labele d with fluorescein. Finally antifluorescein horse radish peroxidase (HRP) c onjugates were added, after washing, 3, 3', 5, 5',- tetramethylbenzidine (T MB) was added to the wells and then measured on a microplate reader. Results EIA detection of amplified products of HCV showed that this assay w as rapid, sensitive, specific and accurate. Correlation between the initial number of viral template and the EIA of amplified products was good. We al so prospectively investigated the! response to interferon in five patients with HCV coinfection. Results showed that this assay could be used as a gui dance to the clinical therapy in directing the use of antiviral drugs. Conclusions This assay could be widely used as a universal technique for th e quantitative detection of amplified products of all nucleic acid (such as virus, bacterium) and other human genes (such as HLA B-27), if has vast vi stas.