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Journal of Clinical Microbiology, October 2005, p. 5111-5116, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5111-5116.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Individual Donor Nucleic Acid Amplification Testing for Detection of West Nile Virus

Dong-Hun Lee,1 John Mathew,1 Wolfram Pfahler,1 Dongling Ma,1 Jay Valinsky,2 Alfred M. Prince,1* and Linda Andrus1

Laboratory of Virology, The Lindsley F. Kimball Research Institute of The New York Blood Center, 310 East 67th St., New York, New York 10021,1 Blood Services, The New York Blood Center, 310 East 67th St., New York, New York 100212

Received 2 May 2005/ Returned for modification 5 July 2005/ Accepted 5 August 2005

We have developed an economical, high-throughput nucleic acid amplification test (NAT) for blood-borne viruses, suitable for use in the screening of plasma samples from individual blood donors. This assay system includes a semiautomated procedure, using 96-well glass fiber plates for the extraction of viral nucleic acids from plasma and "universal beacon" technology which permits the detection of all genotypes of highly variable viruses (e.g., human immunodeficiency virus and hepatitis C virus). In this detection system, two fluorescent- detection technologies were employed successfully in a single tube: molecular beacon for West Nile virus (WNV) detection using a 6-carboxyfluorescein fluorophore and TaqMan for internal control detection using a VIC fluorophore. To establish proof of concept, we focused on the development of a robust individual donor NAT for WNV. The assay showed no reactivity to 15 other viruses tested or to 420 blood donor samples from the WNV pre-epidemic season. No cross-contamination was observed on an alternating positive-/negative-well test. The sensitivity (limit of detection, 95%) of the assay for WNV is between 3.79 and 16.3 RNA copies/ml, depending on which material was used as a standard. The assay detected all positive blood donation samples identified by the Roche WNV NAT. The assay can be performed qualitatively for screening and quantitatively for confirmation.

* Corresponding author. Mailing address: Laboratory of Virology, The New York Blood Center, 310 East 67th Street, New York, NY 10021. Phone: (212) 570-3279. Fax: (212) 570-3180. E-mail: aprince{at}nybloodcenter.org.

Journal of Clinical Microbiology, October 2005, p. 5111-5116, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5111-5116.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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