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Optimized Dengue RT-PCR

le lane 9 is water (template negative) control. Discussion

Lanciotti et al. (1992) designed the primers using prototype dengue virus strains Den-1 (Hawaii), Den-2 (New Guinea C), Den-3 (H-87) and Den-4 (H-241). In addition, they also included 33 virus isolates from different geographical regions to test the efficiency of their designed primers. According to their paper, only a single PCR product of 511 bp in length was amplified. In our study we also detected, in addition to the target PCR product, the presence of another PCR product measuring at 190 bp. In the beginning, we had assumed that this extra band was a non-specific PCR product.

However, by experimenting at various different annealing temperatures ranging from 56.4C to 72C in our PCR by using Eppendorf's Mastercycler gradient, we found that the 190 bp product persisted in all the different annealing temperatures wheras the target PCR product (511 bp) was not detected at 72C.This could indicate that the primers used by Lanciotti et al. (1992) annealed at least at 2 sites (repeat sequence?) for this particular strain (Vietnamese B98). We hope to sequence this strain to identify the genotype.

Reference

Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV (1992). Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. Journal of Clinical Microbiology, 30(3) 545-551


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