Universal primers that amplify RNA from all three flavivirus subgroups

Abstract

Background: Species within the Flavivirus genus pose public health problems around the world. Increasing cases of Dengue and Japanese encephalitis virus in Asia, frequent outbreaks of Yellow fever virus in Africa and South America, and the ongoing spread of West Nile virus throughout the Americas, show the geographical burden of flavivirus diseases. Flavivirus infections are often indistinct from and confused with other febrile illnesses. Here we review the specificity of published primers, and describe a new universal primer pair that can detect a wide range of flaviviruses, including viruses from each of the recognised subgroups.

Results: Bioinformatic analysis of 257 published full-length Flavivirus genomes revealed conserved regions not previously targeted by primers. Two degenerate primers, Flav100F and Flav200R were designed from these regions and used to generate an 800 base pair cDNA product. The region amplified encoded part of the methyltransferase and most of the RNA-dependent-RNA-polymerase (NS5) coding sequence. One-step RT-PCR testing was successful using standard conditions with RNA from over 60 different flavivirus strains representing about 50 species. The cDNA from each virus isolate was sequenced then used in phylogenetic analyses and database searches to confirm the identity of the template RNA.

Conclusion: Comprehensive testing has revealed the broad specificity of these primers. We briefly discuss the advantages and uses of these universal primers.

Figure 1

a, b, c) Representative PCR results showing the ~800 bp fragment that was amplified. d) A range of template concentrations was tested for TAMV and YOKV. Reactions marked 1 and 2 have 2 μL template RNA, reactions 3 and 4 have 6 μL RNA; reaction 5 has 10 μL template, reactions 2, 4 and 5 have 4 μL of MgSO₄. All reactions were performed under identical conditions. (NTC- no template control, L- ladder).

Figure 2

An alignment of the regions targeted by the Flav100F/Flav200R primers. Identities are marked by a dot, gaps are marked by a dash, and nucleotide variants are shown.

Figure 3

A maximum likelihood tree of the cDNA and references sequence found using an alignment of the 800 base long region. The mosquito-borne (m), tick-borne (t) and no known-vector (n) groups were partitioned as marked.