One-tube semi-nested PCR-ELISA for the detection of human cytomegalovirus DNA sequences; comparison with hybridization-based and semi-nested-based PCR-ELISA procedures

Abstract

Amplification of DNA targets by polymerase chain reaction (PCR) followed by their colorimetric detection in an enzyme-linked immunosorbent assay (ELISA) is increasingly used in both immunological research and clinical practice. Several methods for the labeling and detection of amplified DNA sequences have been previously described. In this study, we compared the conventional hybridization-based PCR-ELISA with a modified semi-nested and one-tube semi-nested PCR-ELISA for the detection of human cytomegalovirus (HCMV) DNA. Amplified DNA sequences were labeled with biotin and 2,4-dinitrophenyl (DNP), and detected by DNP-specific monoclonal antibody conjugated to alkaline phosphatase. Using a cloned HCMV DNA as a template, we found that the one-tube semi-nested PCR-ELISA gave a strong positive response when 20 copies of the template were used, whereas both the hybridization-based and the semi-nested-based PCR-ELISA required at least 200 template copies. The claim of higher sensitivity and robustness of the one-tube semi-nested assay was also supported by the analysis of plasma samples from patients treated for HCMV infection. Since the modified one-tube semi-nested PCR-ELISA is quick, sensitive and easy-to-perform, it can be used with advantage in routine identification of DNA targets in clinical and other specimens.