HIGHLY SENSITIVE NONRADIOACTIVE DETECTION OF TICK-BORNE ENCEPHALITIS-VIRUS

The non-radioactive reverse dot-blot method was used for the detection of tick-borne encephalitis virus (TBEV) in clinical specimens. The me thod involves reverse transcription (RT) and polymerase chain reaction (PCR) using a pair of biotin-labelled oligonucleotide primers. These primers flank a region in the gene of the envelope protein E, which is more conserved than other regions, and initiate the polymerisation wi th RNAs of all the investigated strains. The amplified cDNA was captur ed from solution on a solid support using complementary oligonucleotid es covalently bound to a polyamide membrane. The biotin labels of the resulting hybrids were visualized by means of the streptavidin-horsera dish peroxidase conjugate. The detection limit of the test was about 1 0(3)-10(4) molecules of target RNA. The sensitivity was comparable to that obtained by dot-hybridization of PCR-product with P-32-labelled D NA probe. The method was used for the detection of RNA in specimens of tick and blood.