( ...)Optimized Dengue RT-PCR

Ravindran Thayran
Institute of Health and Community Medicine, Universiti Malaysia Sarawak

Optimization of annealing temperature for Dengue RT-PCR by using Eppendorf's Mastercycler gradient

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR*) for dengue is often carried out at the Institute of Health and Community Medicine, Universiti Malaysia Sarawak, as a research and diagnostic tool.

The primers used are based on the 5' non-coding region, which enables the detection of all four dengue serotypes, i.e. Den-1, Den-2, Den-3 and Den-4 (Lanciotti et al., 1990).

The experimental parameters are reverse transcriptase reaction at 37C for 1 hour, followed by PCR with denaturation at 94C for 1 minute; annealing at 50C for 1 minute 30 seconds; and extension at 72C for 1 minute 30 seconds for 30 cycles.

Our target PCR product is 511 bp in length. However, the resulting PCR product often includes non-specific bands in addition to the target PCR product.

Objective

Our objective in this evaluation is to determine the best annealing temperature for the RT-PCR which will only result in a single targeted PCR product.

Materials and Methods

RT-PCR was carried out as described below. Briefly, dengue viral RNA was extracted from dengue infected C6/36 cells by using TriReagent (Sigma Bio-sciences). For this experiment, we used a Vietnamese Den-3 isolate (BD98). 5 l of the extracted RNA was mixed with 50 pmol of reverse primer at 70C. Then 4 l of RT mix containin
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