Molecular detection and serotypic characterization of dengue viruses by single-tube multiplex reverse transcriptase–polymerase chain reaction

Abstract

This study was designed to develop a reverse transcriptase–polymerase chain reaction (RT-PCR)–based rapid and cost-effective diagnostic test for detection as well as serotypic characterization of dengue viruses in the acute phase of illness. In this regard, 2 methods of RT-PCR, namely, 2-step nested RT-PCR and 1-tube multiplex RT-PCR, have been evaluated. One-tube multiplex RT-PCR was found superior; thus, its sensitivity and specificity were compared with gold standard “cell culture”. In the present study, serum samples of all individuals were evaluated by cell culture and RT-PCR. Of 200 clinically suspected patients tested, 66 were found to be positive for the dengue virus RNA and could be successfully characterized into the serotypes. Of these 66, 62 patients were found to be serologically positive by Dengue Duo IgM and IgG Rapid Strip test (Pan Bio, Windsor, Australia). The sensitivity of the cell culture procedure was found to be lower; 63% as compared with multiplex RT-PCR, with predictive value of positive test being 100% and predictive value of negative test being 84.8%. Specificity was found to be 100% for both the assays. It is thus proposed that 1-tube multiplex RT-PCR–based assay would prove to be an extremely useful tool for routine laboratory diagnosis.