Development and evaluation of laboratory diagnosis and genotyping for dengue virus by flurogenic reverss transcript ase PCR (hybridization probe)

Abstract

Rapid determination of dengue virus has become increasing important for clinician in order to provide proper care. Therefore, numerous different methods have been developed to enable dengue diagnosis. In this study, the LightCycler instrument combined with fluorescence resonance energy transfer (FRET) hybridization probe was developed and evaluated for rapid dengue diagnosis and genotyping simultaneously. Two colors multiplexing of hybridization probes are designed that recognize adjacent internal sequence within target sequences of dengue virus to identified dengue genotype. Sera from 100 dengue infected patients were typing with two set of hybridization probes by melting curve analysis. The DEN-1 and DEN-3 were detected by the PDEN2 LC-Red 640 detection probe and identified based on melting temperature. The DEN-1 has a melting temperature about 47.12°C while the DEN-3 has 48.59°C. On the other hand, the DEN-2 and DEN-4 were detected by the PDEN4 LC-Red 705 detection probe. The melting temperature of the DEN-2 appeared to be approximately 54.21°C while the DEN-4 produces 54.80°C. In addition, the development of in-house master mix are used instead the commercial kit, which is decrease cost expenditure. The application of this study is useful in rapid diagnosis and epidemiology of dengue infection.