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Open Access Highly Accessed Methodology

Development of one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses

Pranav Patel1*, Olfert Landt2, Marco Kaiser13, Oumar Faye4, Tanja Koppe2, Ulrich Lass2, Amadou A Sall4 and Matthias Niedrig1

Author Affiliations

1 Robert Koch Institute, Center for Biological Security 1 (ZBS1), Nordufer 20, Berlin, 13353, Germany

2 TIB MOLBIOL Syntheselabor, Berlin, Germany

3 GenExpress, Berlin, Germany

4 Institut Pasteur de Dakar, Dakar, Senegal

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Virology Journal 2013, 10:58  doi:10.1186/1743-422X-10-58

Published: 14 February 2013

Abstract

Background

The genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses.

Methods

A Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively.

Results

Two degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10–100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays.

Conclusion

The assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis.

Keywords:
Pan-Flavi assay; Locked-nucleic acid; Flaviviruses; qRT-PCR