Molecular diagnosis of dengue by reverse transcriptase-polymerase chain reaction

ABSTRACT

The techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and subsequent PCR were employed in the analysis of serum samples from a range of patients from the Caribbean Epidemiology Centre (CAREC) member countries. Results were compared with those from viral isolation and immunofluorescence. In the second part of the study, ten serum samples were stored for one week under four sets of conditions -20 degrees C, 4 degrees C, 25 degrees C, and thawed (20 degrees C) and frozen (-20 degrees C) daily. After one week of each treatment the samples were analysed by RT-PCR and PCR. 90.4of results from PCR agreed with results from viral isolation (VI) and fluorescent antibody (FA) detection. All PCR positive samples originated from sera collected within five days of the date of onset of fever. Frozen, refrigerated and repeat freeze-thawed samples gave consistent positive results by RT-PCR. After storage at 25 degrees C, however, half the dengue-positive samples were negative by RT-PCR. The results indicate the sensitivity and reliability of this rapid technique and its applicability in the Caribbean. It provides a preliminary assessment of its advantages and limitations under certain conditions of serum collection and storage.