DETECTION OF HEPATITIS-D VIRUS-RNA IN SERUM BY A REVERSE TRANSCRIPTION, POLYMERASE CHAIN REACTION-BASED ASSAY

We designed a reverse transcription, polymerase chain reaction-based a ssay for serum hepatitis D virus RNA. Amplified hepatitis D virus cDNA was revealed by ethidium bromide staining, followed by blotting onto a nylon membrane and hybridization with a (32)phosphorus-labelled olig onucleotide, or by a DNA enzyme immunoassay (DEIA) using a double stra nded DNA-specific monoclonal antibody. The absolute sensitivity was ex pressed as number of hepatitis D virus RNA molecules, using a serum of known viral RNA concentration. Three sets of primers were used, encom passing the base positions 66-686 (variable rod-stabilizing region), 7 01-962 (conserved, viroid-like domain) and 886-1,333 (portion of the o pen reading frame 5 encoding for the carboxyterminus of the hepatitis D antigen) of the viral genome. The lower detection limits, after ampl ification of the three RNA portions, as assessed by ethidium bromide s taining, were 7.5 x 10(6), 7.5 x 10(4) and 7.5 x 10(2) molecules of he patitis D virus RNA per assay, respectively. The region encompassing b ases 886-1,333 was chosen for blotting and hybridization to a radiolab elled oligonucleotide probe or for a capture-based DNA enzyme immunoas say, where the microplate was coated with this same probe. The two pro cedures showed comparable sensitivity, i.e., about 10 molecules of vir al RNA per assay. The specificity of the assay was further on a panel of both anti-hepatitis D-positive and -negative sera. Amplification of serum hepatitis D virus RNA by reverse transcription/polymerase chain reaction followed by detection of the amplified cDNA by DNA enzyme im munoassay is a promising and feasible routine assay for detecting low amounts of circulating virions.