This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Papin, J. F.
Right arrow Articles by Dittmer, D. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Papin, J. F.
Right arrow Articles by Dittmer, D. P.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, April 2004, p. 1511-1518, Vol. 42, No. 4
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.4.1511-1518.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

SYBR Green-Based Real-Time Quantitative PCR Assay for Detection of West Nile Virus Circumvents False-Negative Results Due to Strain Variability

James F. Papin, Wolfgang Vahrson, and Dirk P. Dittmer*

Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

Received 11 October 2003/ Returned for modification 26 November 2003/ Accepted 10 January 2004

Real-time quantitative PCR is used routinely for the high-throughput diagnosis of viral pathogens, such as West Nile virus (WNV). Rapidly evolving RNA viruses present a challenge for diagnosis because they accumulate mutations that may render them undetectable. To explore the effect of sequence variations on assay performance, we generated every possible single point mutation within the target region of the widely used TaqMan assay for WNV and found that the TaqMan assay failed to detect 47% of possible single nucleotide variations in the probe-binding site and was unable to detect any targets with more than two mutations. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green. The SYBR green-based assay was as sensitive as the TaqMan assay for WNV. Importantly, it detected 100% of possible WNV target region variants. The assay developed here adds an additional layer of protection to guard against false-negative results that result from natural variations or drug-directed selection and provides a rapid means to identify such variants for subsequent detailed analysis.


* Corresponding author. Mailing address: Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73104. Phone: (405) 271-2133. Fax: (405) 271-3117. E-mail: dirk-dittmer{at}ouhsc.edu.


Journal of Clinical Microbiology, April 2004, p. 1511-1518, Vol. 42, No. 4
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.4.1511-1518.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




This article has been cited by other articles:

  • LaPointe, D. A., Hofmeister, E. K., Atkinson, C. T., Porter, R. E., Dusek, R. J. (2009). EXPERIMENTAL INFECTION OF HAWAI`I `AMAKIHI (HEMIGNATHUS VIRENS) WITH WEST NILE VIRUS AND COMPETENCE OF A CO-OCCURRING VECTOR, CULEX QUINQUEFASCIATUS: POTENTIAL IMPACTS ON ENDEMIC HAWAIIAN AVIFAUNA. J Wildl Dis 45: 257-271 [Abstract] [Full Text]  
  • (2008). Epitope discovery in West Nile virus infection: Identification and immune recognition of viral epitopes. Proc. Natl. Acad. Sci. USA 105: 2981-2986  
  • O'Hara, A. J., Vahrson, W., Dittmer, D. P. (2008). Gene alteration and precursor and mature microRNA transcription changes contribute to the miRNA signature of primary effusion lymphoma. Blood 111: 2347-2353 [Abstract] [Full Text]  
  • Hymas, W. C., Aldous, W. K., Taggart, E. W., Stevenson, J. B., Hillyard, D. R. (2008). Description and Validation of a Novel Real-Time RT-PCR Enterovirus Assay. Clin. Chem. 54: 406-413 [Abstract] [Full Text]  
  • Jimenez-Clavero, M. A., Aguero, M., Rojo, G., Gomez-Tejedor, C. (2006). A New Fluorogenic Real-time Rt-pcr Assay for Detection of Lineage 1 and Lineage 2 West Nile Viruses. jvdi 18: 459-462 [Abstract] [Full Text]  
  • RICHARDSON, J., MOLINA-CRUZ, A., SALAZAR, M. I., BLACK, W. IV (2006). QUANTITATIVE ANALYSIS OF DENGUE-2 VIRUS RNA DURING THE EXTRINSIC INCUBATION PERIOD IN INDIVIDUAL AEDES AEGYPTI. Am J Trop Med Hyg 74: 132-141 [Abstract] [Full Text]  
  • Hilscher, C., Vahrson, W., Dittmer, D. P. (2005). Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability. Nucleic Acids Res 33: e182-e182 [Abstract] [Full Text]  
  • Derbigny, W. A., Kerr, M. S., Johnson, R. M. (2005). Pattern Recognition Molecules Activated by Chlamydia muridarum Infection of Cloned Murine Oviduct Epithelial Cell Lines. J. Immunol. 175: 6065-6075 [Abstract] [Full Text]  
  • Lee, D.-H., Mathew, J., Pfahler, W., Ma, D., Valinsky, J., Prince, A. M., Andrus, L. (2005). Individual Donor Nucleic Acid Amplification Testing for Detection of West Nile Virus. J. Clin. Microbiol. 43: 5111-5116 [Abstract] [Full Text]  
  • Dittmer, D. P., Gonzalez, C. M., Vahrson, W., DeWire, S. M., Hines-Boykin, R., Damania, B. (2005). Whole-Genome Transcription Profiling of Rhesus Monkey Rhadinovirus. J. Virol. 79: 8637-8650 [Abstract] [Full Text]