RAPID, NONRADIOACTIVE DETECTION OF VIRUS-INFECTION BY POLYMERASE CHAIN-REACTION

Background: Polymerase chain reaction (PCR) diagnosis of infectious di seases, especially virus diseases, offers a very sensitive and specifi c technique for clinical diagnosis. However; detection systems for amp lified DNA requiring radioactive probe hybridization or Signal develop ment using blot transfer or nucleotide capture require overnight incub ation or specially labeled probe molecules for analysis of amplified D NA. Objectives: To place this technology in the clinical laboratory, r apid and sensitive methods are needed for the detection of amplified D NA which are applicable to the assay of multiple specimens representin g many different organisms and requiring a minimum of manipulation. St udy Design: Electrophoretic separation of amplified DNA fragments, sta ined with the fluorescent dye SYBR Green I, and laser scanning of the gels for detection of virus-specific PCR products was compared with de tection of amplified DNA by liquid hybridization with radioactive prob es and gel retardation analysis of labeled probe molecules. Results: F luorescent scanning methodology was applied to the detection of cytome galovirus (CMV), herpes simplex virus (HSV) and the human immunodefici ency virus (HIV). This method was at least 10 times more sensitive tha n radioactive probe hybridization in the detection of CMV-specific PCR products. This method also required less time and avoided the use of radioactivity. Conclusions: Clinical diagnosis of virus infections can be conveniently and rapidly accomplished, while avoiding the dangers of radioactive probe handling, by fluorescence staining and laser scan ning of specifically amplified gene fragments. This technology is appl icable to the detection of genes from many different organisms, withou t specially synthesized and/or labeled oligonucleotide primer or probe sequences.