Reverse transcriptase -polymerase chain reaction and DNA enzyme immunoassay for diagnosis of dengue fever and for dengue virus typing

ABSTRACT

In this study the use of commercially prepared reagents for detection and typing of dengue viruses from clinical samples using reverse transcriptase- polymerase chain reaction [RT- PCR] was evaluated on local dengue strains isolated in Jeddah Saudi Arabia over the period from February 1994 to July 1999. Total of 80 dengue culture confirmed cases, and 30 culture negative and IgM negative non dengue cases, and additionaly dengue virus infected, 20, and non infected, 20, tissue culture supernatants from C6/36 cell line, were used. All dengue culture positive cases were detected by RT- PCR followed by gel electrophoresis and or DNA enzyme immunoassay [DEIA], except 1 sample, which was negative by RT-PCR but was positive in culture in 1994 epidemic, with a sensitivity of 98.7% and specificity of 100%. All positive cases were correctly typed using gel electrophoresis following nested PCR and also by DEIA from first amplification product, without the need of nested PCR. This study showed that the commercially availabe reagents for extraction, RT- PCR, and detection are effective methods for the rapid and sensitive as well as specific diagnosis of dengue virus infection and are also effective in rapid typing of the infecting serotype, and that the use of DEIA in detection and typing from first amplification products is as effective as nested PCR using specific primers followed by gel electrophoresis or culture followed by antigen detection using polyclonal and monoclonal antibodies in an indirect immunofluorescent assay