Normalization of RT-PCR protocol for detecting dengue viral RNA in infected clinical residual serum samples

Abstract

Among mosquito-borne viral infections, dengue tops the list with a high prevalence of all four serotypes, especially in tropical regions. The disease has disseminated in over 100 countries and has become a global threat, mainly affecting young individuals. Detecting the dengue virus in clinical samples has a significant role in diagnosing, treating, epidemiology, surveillance, predicting outbreaks etc. To accomplish this, RT-PCR technology has become a practical way out. Although PCR has reached its cutting edge with its improved versions being available, the conventional form of it has still retained the standard for generating more accurate and reliable results cost-effectively. In the present study, the conventional RT-PCR protocol was normalized for detecting dengue viral RNA in infected clinical residual serum samples by adopting a few previous protocols with required modifications. This procedure, with necessary precautions considered particularly for residual samples, resulted in obtaining expected amplicon bands in three samples. Additionally, Sanger sequencing of the amplicons and NCBI nucleotide BLAST analysis of obtained sequences assured the RT-PCR outcomes. The benefits of using clinical residual samples in dengue research encouraged us to make the PCR protocol suitable for such samples by tackling their limitations.