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 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.
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 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