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Journal of virological methods2009; 157(2); 155-160; doi: 10.1016/j.jviromet.2008.12.014

A highly sensitive method for the detection and genotyping of West Nile virus by real-time PCR.

Abstract: In recent years, West Nile virus has been responsible for outbreaks in regions where it has not previously been found. Five genetic lineages with specific geographic distributions exist. Recent outbreaks of WNV associated with the introduction of lineage 1 strains into the western hemisphere, together with the emergence of lineage 2 WNV in Central Europe, has highlighted the potential for spread of pathogenic WNV strains beyond their expected geographical boundaries. Therefore, genotyping of WNV strains may have important applications in surveillance and epidemiology. We report here the development of a nested real-time PCR for the detection and genotyping of WNV strains by means of dissociation-curve analysis, using fluorescence resonance energy transfer (FRET) probe technology. Eight WNV strains, representing three lineages were tested and correctly genotyped at a detection limit of 0.07 viral genome copies/ml in one-step real-time RT-PCR or 7x10(-16) viral genome copies/ml in a nested real-time PCR. WNV could be identified and typed in serum and brain specimens from a human and horse with neurological disease. To our knowledge, this is the first assay designed for the simultaneous detection and genotyping of WNV by rapid, sensitive real-time PCR which may be implemented in diagnostic and epidemiology laboratories.
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Publication Date: 2009-01-10 PubMed ID: 19138708DOI: 10.1016/j.jviromet.2008.12.014Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The researchers developed a highly sensitive method thatuses real-time PCR to detect and genotype West Nile virus (WNV). This method can efficiently identify the specific geographic strains of WNV and could prove valuable for surveillance and epidemiology purposes.

Understanding West Nile Virus and Its Importance

  • West Nile Virus (WNV) is responsible for causing disease outbreaks in areas where it was not previously observed. It is primarily known for causing severe and sometimes fatal neurological illness in humans.
  • The virus has five genetic lineages, each prevalent in a specific geographical area. In recent years, outbreaks associated with lineage 1 strains have been observed in the western hemisphere. Equally concerning is the emergence of lineage 2 WNV in Central Europe, despite its expected geographical limitation.
  • This geographical expansion of the virus points to its growing potential of causing disease in new territories and highlights the need for advanced detection methods to control its spread.

The Designer Detection Method: Nested Real-Time PCR

  • The researchers designed a nested real-time PCR that can serve as a highly sensitive method to detect and genotype WNV.
  • PCR (Polymerase Chain Reaction) is a widely-used laboratory method in genetics that allows for copying and amplifying specific DNA sequences. Nested real-time PCR enhances the sensitivity of the method by performing two rounds of PCR with two sets of primers.
  • This specific PCR protocol was developed using Fluorescence Resonance Energy Transfer (FRET) probe technology, which aids in dissociation-curve analysis for genotyping.

Testing and Results

  • Eight WNV strains, which represented three distinct lineages, were tested using this method.
  • The detection limit (smallest amount the test can identify) was found to be incredibly low, making this method highly sensitive.
  • Moreover, the method could successfully identify and type the WNV in serum and brain specimens from a human patient and a horse showing neurological symptoms.

Significance

  • This is the first known assay designed for simultaneous detection and genotyping of WNV using rapid and sensitive real-time PCR.
  • This method can be employed in diagnostic and epidemiology labs, potentially improving surveillance and response measures to outbreaks.

Cite This Article

APA
Zaayman D, Human S, Venter M. (2009). A highly sensitive method for the detection and genotyping of West Nile virus by real-time PCR. J Virol Methods, 157(2), 155-160. https://doi.org/10.1016/j.jviromet.2008.12.014

Publication

Journal of virological methods
ISSN: 1879-0984
NlmUniqueID: 8005839
Country: Netherlands
Language: English
Volume: 157
Issue: 2
Pages: 155-160

Researcher Affiliations

Zaayman, Dewald
  • Department of Medical Virology, University of Pretoria, South Africa.
Human, Stacey
    Venter, Marietjie

      MeSH Terms

      • Animals
      • Base Sequence
      • Brain / virology
      • Fluorescence Resonance Energy Transfer
      • Genotype
      • Horses
      • Humans
      • Molecular Sequence Data
      • Polymerase Chain Reaction / methods
      • RNA, Viral / genetics
      • Sensitivity and Specificity
      • Serum / virology
      • West Nile virus / classification
      • West Nile virus / genetics
      • West Nile virus / isolation & purification

      Citations

      This article has been cited 25 times.
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