کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
3406695 | 1223585 | 2013 | 7 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: 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 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](/preview/png/3406695.png)
• A real-time RT-PCR to detect and identify WNV-L1, WNV-L2 and USUV is described.
• The test is fast, efficient, specific & sensitive for a wide range of applications.
• This technique is suitable for high-throughput screenings, apt for surveillance.
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.
Journal: Journal of Virological Methods - Volume 189, Issue 2, May 2013, Pages 321–327