Abstract:
Dengue Virus (DENV) is the most common vector-borne viral pathogen and one of the most important disease-causing agents today, affecting millions and causing deaths of thousands of people all over the world, including the Philippines, each year. Fast and accurate diagnostic methods are being developed so that patients are given the quickest and most appropriate treatment for the illness. Of the current protocols developed so far, nucleic acid amplification techniques have proven to be the most sensitive and cost- effective. This study, thus, evaluated a high resolution melt curve analysis protocol to simultaneously detect and serotype DENV infections in Philippine clinical samples. Primers based on Yong et al.’s study (2007) were used in the assay. The 25pL reaction mix consisted of 12.5 pL 2X Master Mix, 0.25 pL reverse transcriptase mix, 150 nM of each of the primers, 5pL template and 4.75 pL PCR water. The optimized reverse transcription temperature was 50°C and the optimum annealing temperature was determined to be 62°C. A standard melt curve analysis was performed thereafter for serotype determination, as well as a high resolution melt curve analysis. Serotypes 1 and 3 were discriminated by standard melt curve analysis while serotypes 2 and 4 yielded similar melting temperatures. With high resolution melt analysis, the four serotypes were discriminated by their unique generated melting temperatures. However, the efficiency of the assay needs to be further improved and optimized because 6 out of the 18 positive control samples were not detected in RT-qPCR but 4 out of these 6 samples were detected in gel electrophoresis. Primer redesign can be performed to further improve the protocol.
