One-step multiplex real-time RT-PCR for detection and typing of dengue virus

In this study, we developed a TaqMan probe-based, one-step real-time reverse transcriptase-polymerase chain reaction (RT-PCR) system for detection and serotyping DENV. Our linear dynamic range (101 to 107 copies/reaction) showed the R2 values of DENV-1, 2, 3 and 4 as 0.998, 0.998, 0.994, and 0.998, respectively. The detection limits of DENV-1, 2, 3, and 4, were 10 copies/reaction, 100 copies/reaction, 10 copies/reaction, and 100 copies/reaction, respectively. Specificity test results indicated that this system is specific for DENV-1, 2, 3, and 4 and does not react with other viruses. Finally, we validated our results with five different real-time PCR instruments. Our results showed that the Ct values of the four serotype templates were similar in five real-time PCR instruments. Thus, this system provides an accurate method for detection and serotyping of DENV, which can be applied in diagnostics, surveillance, and epidemiology. Dengue can be found in many nations with varying socioeconomic and monetary resources. The results of our validation analyses using five different real-time PCR instruments suggest that this method can easily and confidently be used world-wide.
Source: Molecular and Cellular Probes - Category: Molecular Biology Source Type: research