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

Authors:
Address: Institut Pasteur, Insects and Infectious Disease Unit, CNRS URA 3012, 25 rue du Docteur Roux, 75724 Paris, France.
Journal:

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

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.

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