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 am...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 s top polymerase chain reaction (PCR) before the “plateau stage”. At the same ti me, primers HCV(3) of the second PCR were modified with biotin so that the ampli fied products were labeled. The products were diluted and subsequently added t o the streptavidin coated wells, and the biotinylated products were captured, f ollowed by denaturation of NaOH, and non biotinylated strands were removed. Hy bridization was performed by adding the specific probe labeled with fluorescein. Finally anti fluorescein horse radish peroxidase (HRP) conjugates were added, after washing, 3,3',5,5', tetramethylbenzidine (TMB) was added to the well s and then measured on a microplate reader. Results EIA detection of amplified products of HCV showed that this ass ay was rapid, sensitive, specific and accurate. Correlation between the initial number of viral template and the EIA of amplified products was good. We also pro spectively investigated the response to interferon in five patients with HCV coi nfection. Results showed that this assay could be used as a guidance to the clin ical therapy in directing the use of antiviral drugs. Conclusions This assay could be widely used as a universal technique fo r the quantitative detection of amplified products of all nucleic acid (such as virus, bacterium) and other human genes (such as HLA B 27 ), it has vast vistas.展开更多
文摘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 s top polymerase chain reaction (PCR) before the “plateau stage”. At the same ti me, primers HCV(3) of the second PCR were modified with biotin so that the ampli fied products were labeled. The products were diluted and subsequently added t o the streptavidin coated wells, and the biotinylated products were captured, f ollowed by denaturation of NaOH, and non biotinylated strands were removed. Hy bridization was performed by adding the specific probe labeled with fluorescein. Finally anti fluorescein horse radish peroxidase (HRP) conjugates were added, after washing, 3,3',5,5', tetramethylbenzidine (TMB) was added to the well s and then measured on a microplate reader. Results EIA detection of amplified products of HCV showed that this ass ay was rapid, sensitive, specific and accurate. Correlation between the initial number of viral template and the EIA of amplified products was good. We also pro spectively investigated the response to interferon in five patients with HCV coi nfection. Results showed that this assay could be used as a guidance to the clin ical therapy in directing the use of antiviral drugs. Conclusions This assay could be widely used as a universal technique fo r the quantitative detection of amplified products of all nucleic acid (such as virus, bacterium) and other human genes (such as HLA B 27 ), it has vast vistas.
