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

Myung Jin Mun, Joon Yong Bae, Jin Hyuck Kim, Soo Bok Kim, Ilseob Lee, Jin Il Kim, Mee Sook Park, Man-Seong Park, Yong Suk Nam

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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.

Original languageEnglish
JournalMolecular and Cellular Probes
DOIs
Publication statusAccepted/In press - 2018 Jan 1

Keywords

  • Dengue virus
  • Real-time PCR
  • Serotyping
  • TaqMan probe

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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    Mun, M. J., Bae, J. Y., Kim, J. H., Kim, S. B., Lee, I., Kim, J. I., Park, M. S., Park, M-S., & Nam, Y. S. (Accepted/In press). One-step multiplex real-time RT-PCR for detection and typing of dengue virus. Molecular and Cellular Probes. https://doi.org/10.1016/j.mcp.2018.10.001