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
| Autor: | Carmina Gallardo, Javier del Amo, Jovita Fernández-Pinero, Elena Sotelo, Francisco Llorente, Miguel Ángel Jiménez-Clavero, Montserrat Agüero |
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| Rok vydání: | 2013 |
| Předmět: |
Lineage (genetic)
West Nile virus viruses RT-PCR Biology Real-Time Polymerase Chain Reaction medicine.disease_cause Sensitivity and Specificity Fluorescence Virus Virology Diagnosis medicine Animals Multiplex Usutu virus DNA Primers Genetics Bird Diseases Flavivirus virus diseases biology.organism_classification Europe Real time Real-time polymerase chain reaction Molecular Diagnostic Techniques Lineage 2 Oligonucleotide Probes Multiplex Polymerase Chain Reaction West Nile Fever Lineage 1 |
| Zdroj: | Repositorio de Resultados de Investigación del INIA INIA: Repositorio de Resultados de Investigación del INIA Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria INIA |
| Popis: | 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. © 2013 Elsevier B.V. |
| Databáze: | OpenAIRE |
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