Optimization of real time PCR for checking the activity of siRNA in Dengue Serotypes

Dengue, the most prevalent infectious disease among the arbovirus community is emerging as a first order health complication, especially, in the tropics and in subtropical regions. It is untreatable, since, there is no advanced diagnostic method with which tendency of the infection can be traced with great precision in its acute phase. In our previous studies we showed that, siRNA targeting the 5NTR among all the 4 serotypes of dengue successfully inhibited the replication of respective serotypes to the greatest extent as confirmed by the realtime RT-PCR. In the present study, we monitored the relative expression of dengue genomic RNA in treated (rAdsh-5b) and non treated cells and control (rAdsh-N) to reveal the efficiency of the real-time RT-PCR in detecting the viral load in the presence of antiviral agent (rAdsh-5b) to discriminate the disease severity in the presence and absence of antiviral therapeutics. The cDNA for all the samples were prepared and performed real-time RT-PCR with the aid of SYBR green 1 and the post PCR amplicons were analyzed with melting curve analysis to check the specificity and efficiency of the process. The results showed that, the relevant viral RNA expression was found to be less in rAdsh-5b treated sample than the non treated sample as marked by the Ct-values and the melting curve peaks. This indicates that real-time RT-PCR can serve as a gold standard for diagnosing the dengue infection in the acute phase and in distinguishing the relative viral content in the sample treated with antiviral agent.