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

ABSTRACT

OBJECTIVE: To develop a universal quantitative immunoenzyme assay (EIA) for detecting amplified products of nucleic acid and its application in hepatitis 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 time, primers HCV (3) of the second PCR were modified with biotin so that the amplified 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 were removed. Hybridization was performed by adding the specific probe labeled with fluorescein. Finally antifluorescein horse radish peroxidase (HRP) conjugates were added, after washing, 3, 3', 5, 5',-tetramethylbenzidine (TMB) was added to the wells and then measured on a microplate reader. RESULTS: EIA detection of amplified products of HCV showed that this assay was rapid, sensitive, specific and accurate. Correlation between the initial number of viral template and the EIA of amplified products was good. We also prospectively investigated the response to interferon in five patients with HCV coinfection. Results showed that this assay could be used as a guidance to the clinical therapy in directing the use of antiviral drugs. CONCLUSIONS: This assay could be widely used as a universal technique for the quantitative detection of amplified products of all nucleic acid (such as virus, bacterium) and other human genes (such as HLA B27), it has vast vistas.