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Development of Field-Based Real-Time Reverse Transcription–Polymerase Chain Reaction Assays for Detection of Chikungunya and O’nyong-nyong Viruses in Mosquitoes

Chikungunya (CHIK) and O’nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription–polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

Received March 16, 2009. Accepted for publication June 3, 2009.

Acknowledgments: We thank David Dohm for technical assistance and Diana Fisher for statistical analysis support.

Financial support: This study was supported by the Military Infectious Disease Research Program, Step U, project # U0176_09_RD.

Disclaimer: The mention of trade names or commercial products does not constitute endorsement or recommendation for use by the Department of Defense. The views of the authors do not necessarily reflect the position of the Department of Defense or the Department of the Army.

^* Address correspondence to Monica L. O’Guinn, Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702. E-mail: monica.oguinn{at}us.army.mil

Authors’ addresses: Darci R. Smith, John S. Lee, Michael J. Turell, Jennifer L. Groebner, and Monica L. O’Guinn, Virology Division, Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, E-mails: darci.smith1{at}us.army.mil, john.s.lee{at}us.army.mil, michael.turell{at}amedd.army.mil, jennifer.groebner{at}amedd.army.mil, and monica.oguinn{at}us.army.mil. Jordan Jahrling and David A. Kulesh, Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, E-mails: jbjahrli{at}utmb.edu and david.kulesh{at}amedd.army.mil.

Reprint requests: Monica L. O’Guinn, Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, E-mail: monica.oguinn{at}us.army.mil.