title: A novel quantitative multiplex real-time RT-PCR for the simultaneous detection and differentiation of West Nile virus lineages 1 and 2, and of Usutu virus.
abstract: An increase in activity of two mosquito-borne flaviviruses, West Nile virus (WNV) and Usutu virus (USUV), has been reported in Europe in recent years. The current epidemiological situation calls for RT-PCR methods that are able to detect not only the widespread lineage 1 (L1) WNV, but also lineage 2 (L2) WNV. In addition, the presence in Europe of the closely related USUV requires methods that can identify these three flaviviruses and permit an efficient and accurate differential diagnosis. Here we describe a new one-step real-time multiplex RT-PCR that detects and differentiates efficiently WNV-L1, WNV-L2 and USUV in a single reaction. The assay is based on different sets of primers and fluorogenic probes specific to each virus that are labelled with selective, non-overlapping fluorogen-quencher pairs. This enables the fluorescence emitted by each probe, characterized by distinct wavelengths, to be differentiated. This multiplex assay was very sensitive to all of the target viruses; in addition, there were no cross-reactions between the viruses and the assay did not react to any other phylogenetically or symptomatically related viruses. Quantitation was enabled through the use of in vitro-transcribed RNAs developed specifically for each virus as copy number standards. This new assay was validated using different types of experimental and field samples.
authors.affiliation: Centro de Investigación en Sanidad Animal, CISA-INIA, Valdeolmos, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Del Amo
authors.forename: Elena
authors.initials: E
authors.lastname: Sotelo
authors.forename: Jovita
authors.initials: J
authors.lastname: Fernández-Pinero
authors.forename: Carmina
authors.initials: C
authors.lastname: Gallardo
authors.forename: Francisco
authors.initials: F
authors.lastname: Llorente
authors.forename: Montserrat
authors.initials: M
authors.lastname: Agüero
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Jiménez-Clavero
mesh_terms: D000818:Animals; D001715:Bird Diseases / Q000175:diagnosis / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D005060:Europe; D005453:Fluorescence; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*; D014901:West Nile Fever / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D014902:West Nile virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes