Rapid and sensitive method for the detection of B19 virus DNA using the polymerase chain reaction with nested primers

Abstract

The sensitivity of detection of B19 virus DNA in clinical specimens was evaluated by comparing the results of single PCR and nested PCR assays, with or without subsequent Southern blot hybridization to a radiolabelled B19 DNA probe. The inhibitory activity of human serum components on polymerase reaction was also determined. The sensitivity of B19 virus DNA detection decreased by a factor of 10(7) in the presence of 10% serum in the single PCR reaction mixture, and of 10(3) for nested PCR. When nested PCR products were analysed by Southern blot hybridization to a B19 radioactive DNA probe, the sensitivity of the assay increased to such a level of B19 DNA detection that the reaction was no longer influenced by the presence of serum inhibitors in the original sample. Less than ten B19 genome copies could thus be detected in a 10-microliters sample. A panel of 38 clinical samples, originating from patients with possibility of B19 virus infection, were assayed by this method. Only one sample was found to be positive after single PCR, whereas seven samples (including the former) gave a positive signal after nested PCR. The specificity of the nested PCR products was controlled by hybridization to the B19 DNA probe and DNA sequencing. No discrepancy in the results was observed between nested PCR alone and nested PCR followed by Southern blot analysis.