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

• Published in: Molecular and cellular probes 2019-02, Vol.43, p.86-91
• Main Authors: Mun, Myung-Jin, Bae, Joon-Yong, Kim, Jin Hyuck, Kim, Soo Bok, Lee, Ilseob, Kim, Jin Il, Park, Mee Sook, Park, Man-Seong, Nam, Yong Suk
• Format: Article
• Language: English
• Subjects: Dengue virus; Real-time PCR; Serotyping; TaqMan probe
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1016/j.mcp.2018.10.001

Previous studies reported that severity of dengue is associated with multiple factors, including secondary infection, age, viral load and infecting serotype and genotype. In addition, other studies have reported that a dengue virus-2 (DENV-2) infection is associated with a prognosis of more severe clinical manifestations than DENV-1 and DENV-4 infections. For these reasons, the ability to identify the DENV serotypes is critical for optimal patient diagnosis and epidemiological studies. 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. •We developed a TaqMan probe-based real-time reverse transcriptase-polymerase chain reaction (RT-PCR) system for DENV serotyping.•Detection limits of DENV-1, 2, 3 and 4 were 10, 100, 10 and 100 copies/reactions, respectively.•Comparison test of different real-time PCR instruments revealed that our system was consistent in five different instruments.