Individual donor nucleic acid amplification testing for detection of West Nile virus.
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.
Lee
Dong-Hun
DH
Laboratory of Virology, The Lindsay F. Kimball Research Institute of the New York Blood Center, 310 East 67th Street, New York, NY 10021, USA.
Mathew
John
J
Pfahler
Wolfram
W
Ma
Dongling
D
Valinsky
Jay
J
Prince
Alfred M
AM
Andrus
Linda
L
D001782:Blood Donors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral / Q000097:blood* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D014901:West Nile Fever / Q000175:diagnosis* / Q000821:virology; D014902:West Nile virus / Q000235:genetics / Q000302:isolation & purification*
D005452:Fluoresceins; D005456:Fluorescent Dyes; D012367:RNA, Viral; C024098:6-carboxyfluorescein; D019914:Taq Polymerase