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. 2017 Jul 24;17(1):164.
doi: 10.1186/s12866-017-1080-9.

Improved detection of genus-specific Alphavirus using a generic TaqMan® assay

Claude Giry  1   2 , Bénédicte Roquebert  3   4   5 , Ghislaine Li-Pat-Yuen  3   4 , Philippe Gasque  6   5 , Marie-Christine Jaffar-Bandjee  3   4   6   5
Affiliations

Improved detection of genus-specific Alphavirus using a generic TaqMan® assay

Claude Giry et al. BMC Microbiol. .

Abstract

Background: Alphaviruses are arthropod borne RNA viruses of medical importance. Geographical expansion of mosquitoes of the Aedes genus in the past decades has been associated with major Alphavirus-associated outbreaks. Climate changes and intensification of air travels have favored vector expansion and virus dissemination in new territories leading to virus emergence not only in tropical areas but also in temperate regions. The detection of emergence is based upon surveillance networks with epidemiological and laboratory investigation.

Method: A specific, sensitive and rapid screening test for genus-specific Alphavirus is critically required. To address this issue, we developed a new molecular assay targeting nsP4 gene and using a TaqMan® real time RT-PCR method for the specific detection of all major Alphavirus genus members.

Results: This assay was tested for specificity using several Alphavirus species. We also tested successfully clinical sensitivity using patient's samples collected during the Chikungunya outbreak of 2005-2006 in the Indian Ocean.

Conclusions: This new pan-Alphavirus molecular diagnostic tool offers great potential for exclusion diagnosis and emergence detection given its broad specificity restricted to Alphavirus genus.

Keywords: Alphavirus; Molecular diagnosis; Virus emergence.

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Conflict of interest statement

Ethics approval and consent to participate

Written informed consent was obtained from healthy subjects or patients undergoing virus screening and attending Reunion Island University Hospital. Our validation study involved healthy controls, patients with confirmed CHIKV and patients with viral infection not related to Alphavirus. The study was approved by the Human Ethics Committee of University of Bordeaux (‘Comité Consultatif de Protection de Personnes se prêtant à des Recherche Biomédicales’, Bordeaux France, ref. 2008-A00151–54).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Design of primers and probes in Alphavirus nsP4 gene. Genome positions refer to CHIKV LR2006_OPY1|DQ443544 sequence. Arrows indicate the 5’➔3′ orientation. Location of forward primers (panel a), probes (panel b) and reverse primers (panel c) used for pan-alphavirus RT-PCR is indicated in boxes. Vertical bars delineate in primers wobble nucleotides that are considered for degeneracy. Use of inosine containing oligonucleotides was an alternative to decrease primers degeneracy and thus improve sensitivity of detection assay
Fig. 2
Fig. 2
Comparison of 3 primers/probes combinations for pan-Alphavirus RT-PCR. Primers containing Inosine were assayed using MIX2 containing probe P1 or MIX5 containing probe P2. Primers without Inosine were tested using MIX3 containing probe P1 or MIX6 containing probe P2. Reference primers from Grywna [31] were used with probe P1 (MIX1) or probe P2 (MIX4)
Fig. 3
Fig. 3
Specific detection of 5 Alphavirus species. Positive controls for CHIKV, ONNV, SINV, SFV or RRV were obtained from reference strains or were isolated from clinical samples in our laboratory. Negative controls included water and negative plasma controls. Fluorescence was read on LightCycler 480® (Roche) using Cyan filter (Excitation at 450 nM/Emission at 500 nM)
Fig. 4
Fig. 4
Clinical sensitivity of pan-Alphavirus assay versus CHIKV specific assay. (ns, no statistical difference observed under 5% risk α using a t-test)
See this image and copyright information in PMC

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