Development and Designation of Primers and Probes for Detection and Serotype Identification of Dengue Viruses with One-Step Multiplex Fluorogenic TaqMan Real-Time RT-PCR

Abstract

Dengue fever is caused by dengue viruses. These viruses are transmitted to human via a common house mosquito, Aedes aegypti. Dengue viruses are classified into 4 serotypes, 1-4 by distinct immunogenicity. The aim of this study was to develop an in-house assay one-step multiplex real-time RT-PCR for dengue viruses identifying. Specific primers and probes were designed for each viral serotype and all serotypes. All sets of primers were examined for viral specificity by conventional RT-PCR. The results showed that the designed primers were able to amplify dengue serotype 1, 2, 3 and 4. All primers were then applied for multiplex real-time RT-PCR. The result demonstrated that the multiplex real-time RT-PCR with specific probes were capable of detecting and classifying serotype of dengue virus. Finally, the results from in-house real-time RT-PCR and those from AmpliSens® Dengue virus type-FRT PCR kit were compared. It was demonstrated that the results from real-time RT-PCR were concordant to those from AmpliSens kit. The preliminary result led to the conclusion that the developed in-house real-time RT-PCR assay with specific primers and probes is capable of detecting and classifying dengue virus serotypes. However, in-house real-time RT-PCR should be evaluated further to identify low detection limit and cross reactivity with other viruses.