Detection of herpes simplex virus DNA in cerebrospinal fluid samples using the polymerase chain reaction and microplate hybridization

Abstract

As conventional polymerase chain reaction (PCR) procedures are time-consuming and laborious, we developed and evaluated a rapid semi-automatic microplate method to detect the amplified PCR products. The use of PCR, with subsequent hybridization in microplates, is described for the detection of herpes simplex virus (HSV) DNA in cerebrospinal fluid samples. The principle of the method is based on two phases. Firstly, the amplification of the viral DNA in the sample is undertaken using a pair of primers of which one is biotinylated. Secondly, the amplified viral genomic sequences are bound to the wells of streptavidin-coated microplates and hybridized with digoxigenin-labeled oligonucleotide probes which are then detected using anti-digoxigenin antibody enzyme conjugates and either a photometric, fluorometric or luminometric substrate and microplate reader. The method is highly sensitive allowing the detection of as few as five purified DNA molecules. Compared to conventional gel electrophoresis followed by Southern blotting the established microplate hybridization is also much less time-consuming and involves less manual work. The applicability of the method is described for use as a routine diagnostic procedure for detection of early central nervous system infections caused by HSV-1 and HSV-2.