Applications of Polymerase Chain Reaction for Identification of Dengue Viruses Isolated from Patient Sera

A simple and sensitive procedure of reverse transcriptase polymerase chain reaction (RT‐PCR) was developed previously such that all 4 serotypes of dengue viruses could be detected and their serotypes identified simultaneously in a single‐step procedure. In this study we compared the RT‐PCR with a conventional immunoperoxidase (PAP) staining method for the identification of dengue viruses currently isolated from patient sera. Sixty‐six sera taken from dengue hemorrhagic fever (DHF) patients were subjected to virus isolation by inoculating onto C6/36 cell cultures. Screening for the presence of dengue viruses in culture fluids was done after 7 days of incubation by PAP staining using hyperimmune rabbit anti‐dengue virus antibody as the primary reagent. Dengue viruses in positive cultures were further identified for their serotypes by PAP using type‐specific monoclonal antibodies (MAb) and by RT‐PCR. Thirty‐two out of the 66 serum specimens tested (48.5%) were positive for dengue viruses. Of these, 5 were type 1 (DEN‐1), 25 were type 2 (DEN‐2) and 2 contained both DEN‐1 and DEN‐2. All cultures that were positive by PAP method were also positive by RT‐PCR and vice versa. Thus, the results obtained by RT‐PCR were in good agreement with those by PAP. It is important to point out that while all 5 DEN‐1 isolates reacted readily with the MAb 1F1, only 2 of them could be identified by the MAb 15F3. Our data suggest that antigenic variation among DEN‐1 isolates occur frequently and this should be taken into consideration in the selection of appropriate type‐specific MAb for serotyping of dengue viruses. We also demonstrated dual infection of DEN‐1 and DEN‐2 in two patients' sera by RT‐PCR.