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Optimized Dengue RT-PCR

g 2 l of 5X RT buffer, 0.5 l of 10mM dNTP, 1.0 l of sterile UHQ water and 100 U of MMLV reverse transcriptase (PROMEGA, USA) were added. The mixture was incubated at 37C for 1 hour for the RT reaction and then heated to 65C to inactivate the RT.

PCR was subsequently carried out by adding 20 l of PCR mix containing 3 l of 10X Taq Polymerase buffer (PROMEGA, USA), 1.8 l of 25mM Mg Cl2, 50 pmol each of sense and reverse primers, 0.9 l of 10mM dNTP, 4 U of Taq DNA Polymerase (PROMEGA, USA) and 7.55 l of sterile UHQ water to the 1st strand synthesis tube containing cDNA. The PCR parameters were maintained as above. For the annealing temperature a temperature gradient was introduced by using Eppendorf's Mastercycler gradient. This thermal cycler has the ability to run various different annealing temperatures within a single cycle.

The mean temperature was set at 64C with a variation of 8C, which gave the following annealing temperatures: 56.4C, 58.6C, 60.5C, 65.0C, 67.2C and 72C. Negative cell control and water negative (absence of template) controls were also used as negative controls.

Results

The results indicated the presence of two distinct PCR products, measuring at 511 bp (target PCR product) and 190 bp (unknown PCR product), as shown in Figure 1. This pattern was seen up to an annealing temperature of 68C. At 72C only the 190 bp PCR product was seen.

Figure 1: Agarose gel (1 %) electrophoresis of PCR products. Lane 1 and 10: 501000 DNA Marker (FMC). Lanes 27: PCR products at the following temperatures: 56.4C, 58.6C, 60.5C, 65.0C, 67.2C and 72C. Lane 8 is negative cell control whi
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