Detection of West Nile Viral RNA from Field-Collected Mosquitoes in Tropical Regions by Conventional and Real-Time RT-PCR

  • Ana Silvia González-Reiche
  • María de Lourdes Monzón-Pineda
  • Barbara W. Johnson
  • María Eugenia Morales-Betoulle
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 630)

Abstract

West Nile virus (WNV) is an emerging mosquito-borne flavivirus, which has rapidly spread and is currently widely distributed. Therefore, efforts for WNV early detection and ecological surveillance of this disease agent have been increased around the world. Although virus isolation is known to be the standard method for detection and identification of viruses, the use of RT-PCR assays as routine laboratory tests provides a rapid alterative suitable for the detection of viral RNA on field-collected samples. A method for WNV RNA genome detection in field-collected mosquitoes is presented in this chapter. This method has been designed for virus surveillance in tropical regions endemic for other flaviviruses. Reverse Transcriptase-PCR (RT-PCR) assays, both standard and real time, to detect WNV and other flaviviruses are described. A first screening for flavivirus RNA detection is performed using a conventional RT-PCR with two different sets of flavivirus consensus primers. Mosquito samples are then tested for WNV RNA by a real-time (TaqMan) RT-PCR assay. Sample preparation and RNA extraction procedures are also described.

Key words

RT-PCR Real-time RT-PCR Viral RNA Flavivirus West Nile virus Field-collected mosquitoes 

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Ana Silvia González-Reiche
    • 1
  • María de Lourdes Monzón-Pineda
    • 1
  • Barbara W. Johnson
    • 2
  • María Eugenia Morales-Betoulle
    • 1
  1. 1.Centro de Estudios en Salud, Centers for Disease Control and Prevention, Universidad del Valle de Guatemala, Regional Office for Central America and PanamaGuatemala CityGuatemala
  2. 2.Diagnostic & Reference Laboratory, Arbovirus Diseases Branch, Division of Vector-Borne Infectious Diseases (DVBID)Centers for Disease Control and Prevention (CDC)Fort CollinsUSA

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