Multiplexed Diagnosis of Four Serotypes of Dengue Virus by Real-time RT-PCR

Abstract

The number of people at risk for dengue viral infection is about 50% of the worlds population. Dengue viral infection is classified as dengue fever and severe dengue depending on the severity. Severe dengue may be associated with infection by the different serotype from the primary infected serotype. The circulating epidemic serotypes have changed continuously and periodically. It is very important to diagnose dengue viral infection as early as possible to be treated adequately. The purpose of this study was the development of a simultaneous detection method for all the serotypes of dengue virus in a single sample. The consensus primers and four different fluorescein-tagged probes that specifically binds to each serotype of dengue virus were designed from the multiple alignment data of full-length dengue viral sequences, which were retrieved from GenBank. They were located between the 5’ untranslated region and the capsid gene of the dengue viruses. Each serotype of dengue viruses isolated in Korea was detected correctly with the established multiplex real time RT-PCR at the level of 5-10 copies/reaction. Other RNA viruses such as the Japanese encephalitis virus, Zika virus, yellow fever virus, and Chikungunya virus were not amplified with this method. The results of the inter-assays and intra-assays were within the acceptable variation ranges. Conclusively, the developed method can detect each serotype of dengue virus specifically and quantitatively even when different serotypes of dengue virus are present in a sample.