EVALUATION OF STRAND-SPECIFIC RNA PROBES VISUALIZED BY COLORIMETRIC AND CHEMILUMINESCENT REACTIONS FOR THE DETECTION OF B19 PARVOVIRUS DNA

A dot-blot hybridization assay was developed to detect B19 DNA using s trand-specific RNA probes labelled with digoxigenin. The sensitivity o f the assays was evaluated either using `plus' and `minus' sense RNA p robes in two different hybridization assays, or in two successive reac tions of the same assay. The hybridized probes were revealed immunoenz ymatically using anti-digoxigenin Fab fragments conjugated with alkali ne phosphatase. The enzyme was visualized by colorimetric reaction. Si nce `minus' sense RNA probe gave the best results in the dot-blot proc edures, we increased the sensitivity of the hybridization assay visual izing the `minus' sense digoxigenin-labelled RNA probe by chemilumines cent reaction. In these experimental conditions up to 20 fg of target B19 DNA could be visualized In the search for B19 DNA, 4656 serum samp les were analyzed by chemiluminescent reaction of `minus' sense digoxi genin-labelled RNA probe and for comparison with the digoxigenin-label led DNA probe. Positive results were confirmed by Southern blotting. O ut of 4656 serum samples analyzed, 4648 gave negative results, 1 resul ted positive to all the hybridization assays, 6 only using RNA probe a nd 1 only by DNA probe.