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Open Access Highly Accessed Research

Development of a SYBR green I based RT-PCR assay for yellow fever virus: application in assessment of YFV infection in Aedes aegypti

Paban Kumar Dash1, Alain Boutonnier1, Eric Prina2, Shashi Sharma3 and Paul Reiter1*

Author Affiliations

1 Institut Pasteur, Insects and Infectious Disease Unit, CNRS URA 3012, 25 rue du Docteur Roux, 75724 Paris, France

2 Institut Pasteur, Immunophysiology and Intracellular Parasitism Unit, 25 rue du Docteur Roux, 75724 Paris, France

3 Division of Virology, Defence R & D Establishment (DRDE), Jhansi Road, Gwalior, India

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Virology Journal 2012, 9:27  doi:10.1186/1743-422X-9-27

Published: 22 January 2012

Abstract

Background

Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. The incidence of human infections in endemic areas has risen in recent years. Prompt and dependable identification of YFV is a critical component of response to suspect cases.

Results

We developed a one-step SYBR Green I-based real-time quantitative RT-PCR (qRT-PCR) assay targeting the 5'NTR and capsid-gene junction--for rapid detection and quantification of YFV. The detection limit was 1 PFU/mL, 10-fold more sensitive than conventional RT-PCR, and there was no cross-reactivity with closely related flaviviruses or with alphaviruses. Viral load in samples was determined by standard curve plotted from cycle threshold (Ct) values and virus concentration. The efficacy of the assay in mosquitoes was assessed with spiked samples. The utility of the assay for screening of pooled mosquitoes was also confirmed. Replication of a Cameroon isolate of YFV in Ae. aegypti revealed a marked variation in susceptibility among different colonies at different days post infection (pi).

Conclusions

The SYBR Green-1 based qRT-PCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of YFV than other currently used methods.