POLYMERASE CHAIN-REACTION IN THE DIAGNOSIS OF VIRUS DISEASES

The detection of viruses in tissues is often difficult and time-consuming. The amplification of virus-specific DNA-regions by the polymerase chain reaction (PCR) leads to an increase of the quantity of the specific DNA which can be analysed by simple molecular biological methods. The DNA was isolated by proteinase K digestion from human renal cryostat sections. For the detection of the human cytomegalovirus (HCMV) a 147 bp fragment of the 4th exon of the immediate early gene of HCMV, between the nucleotides 1767 and 1913 was amplified in 32 cycles and used for slot-blot hybridizations. A 40-mer detection oligonucleotide, having an identical sequence with the amplified DNA-fragment, marked at the 3'-terminus with a digoxigenin-labelled oligonucleotide was used for hybridization. The detection was performed using an alkaline phosphatase bound digoxigenin-antibody. The method was sensitive till 0,1 fg.