Development of a immuno-PCR assay for the detection of HCMV

Objective:The objective of this study was to establish a newly immuno-PCR method for the detection of HCMV.Methods:The immuno-PCR approach developed in the study is similar to conventional enzyme-linked immunosorbent assay（ELISA）. We immobilized various amounts of HCMV antigen on the surface of microtiter plate wells. A mouse monoclonal anti-HCMV primary antibody and biotinylated secondary antibody are stepwise immobilized. After that, free streptavidin and a marker biotinylated linear pBV 220 DNA are immunobilized consecutively. A segment of the immunobilized DNA are amplified by PCR with the appropriate primers. Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide. Viral antigen present in the sample is analyse by PCR amplification of the marker DNA. Results:Comparison with ELISA demonstrates that enhancement （approximately 1×10³~10⁵） in detection sensitivity was obtained with the use of immuno-PCR. 5 infected cells of HCMV could be detected by the immuno-PCR. While HSV-1、VZV、BKV antigen could not be detected with the immuno-PCR.Conclusion:This immuno-PCR assay has a extremely high sensitivity, could be applied to measure low level viral antigen in biological samples for diagnostic infection of HCMV.