Rapid, non-radioactive detection of virus infection by polymerase chain reaction.
BACKGROUND
Polymerase chain reaction (PCR) diagnosis of infectious diseases, especially virus diseases, offers a very sensitive and specific technique for clinical diagnosis. However, detection systems for amplified DNA requiring radioactive probe hybridization or signal development using blot transfer or nucleotide capture require overnight incubation or specially labeled probe molecules for analysis of amplified DNA.


OBJECTIVE
To place this technology in the clinical laboratory, rapid and sensitive methods are needed for the detection of amplified DNA which are applicable to the assay of multiple specimens representing many different organisms and requiring a minimum of manipulation.


METHODS
Electrophoretic separation of amplified DNA fragments, stained with the fluorescent dye SYBR Green I, and laser scanning of the gels for detection of virus-specific PCR products was compared with detection of amplified DNA by liquid hybridization with radioactive probes and gel retardation analysis of labeled probe molecules.


RESULTS
Fluorescent scanning methodology was applied to the detection of cytomegalovirus (CMV), herpes simplex virus (HSV) and the human immunodeficiency virus (HIV). This method was at least 10 times more sensitive than 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 scanning of specifically amplified gene fragments. This technology is applicable to the detection of genes from many different organisms, without specially synthesized and/or labeled oligonucleotide primer or probe sequences.
Evans
M J
MJ
Molecular Diagnostics Laboratory, Division of Infectious Diseases. Children's Hospital of Buffalo, 219 Bryant Street, Buffalo, NY 14222, USA.
Edwards-Spring
Y
Y
Povinelli
D
D
Wendt
A
A
Harvey
R
R
Rittenhous-Diakun
K
K
Riepenhoff-Talty
M
M