Development of a SYBR Green I Real Time polymerase Chain Reaction (qRT-PCR) for the Detection and Serotyping of Dengue Virus Infections

Abstract

Dengue has long been an epidemic in the Philippines and nowadays it is still a major cause of hospitalization and death among children. Dengue infections occur in different levels of severity which could be from mild illnesses to haemorrhagic fevers and shock syndromes. The early diagnosis and treatment of the patients is at stake since most of the laboratories today employ cell cultures and serology techniques which require at least a week to complete. Recently, Taiwanese researchers made use of SYBR Green I for real-time RT-PCR which provided significant results in the molecular diagnosis of dengue fever infections. In light of that success, this study aims to develop the optimized conditions for SYBR Green I-based one-step qRT-PCR in the detection and serotyping of Philippine dengue infections. The 3'NC amplimer set using DC 10418 in combination with CDC10564 was applied to detect and identify the specific serotypes of dengue viral RNA. SYBR Green I-based qRT-PCR was performed through the utilization of the Superscript III Platinum SYBR Green One-Step qRT-PCR Kit. Melting curve analysis was clone for the detection and serotyping of the samples. A bioinformatic approach was performed for the analysis of current primers and the design of new ones. The optimized primer and template concentrations and temperature conditions for the SYBR Green I qRT-PCR profile was able to detect DENV-2 and DENV-3 strains from the extracted RNA samples. The study showed that SYBR Green I R.T-PCR. protocol is able to detect and serotype dengue strains 2 and 3; however, as the other serotypes were not provided in the samples, the ability of the suggested SYBR Green protocol detect and serotype all dengue serotypes found in the Philippines has not yet been proven.