The PROCLEIX West Nile virus assay (WNV assay) is a qualitative nucleic acid test based on transcription-mediated amplification (TMA). The assay was used under an investigational protocol in the United States to screen blood donations for West Nile virus (WNV) RNA starting in the summer of 2003, and was licensed by the FDA in December 2005 for use on the PROCLEIX System, also known as the enhanced semi-automated system (eSAS). Performance characteristics for the assay were determined on both eSAS and the fully automated PROCLEIX TIGRIS (TIGRIS) System. Detection of both lineage 1 and lineage 2 WNV was demonstrated on both systems. For lineage 1, the 95% detection limit was 8.2 copies/ml for eSAS and 9.8 copies/ml for the TIGRIS system. For lineage 2, > or =95% detection was seen at > or =30 copies/ml on both systems. The overall specificity of the assay was >99.9% in fresh and frozen plasma specimens. Reproducibility studies on the TIGRIS system yielded > or =99.1% agreement with expected results for the 3-member panel tested (0, 30, and 100 copies/ml). The WNV assay exhibited robust performance in cadaveric specimens and specimens representing various donor and donation conditions, including specimens from different plasma collection tubes that were subjected to multiple freeze/thaw cycles; specimens with elevated levels of endogenous substances; specimens containing other viruses and microorganisms; and specimens from patients with autoimmune and other diseases. Overall, these studies demonstrate high sensitivity, specificity, and reproducibility of the WNV assay on both the semi-automated and automated systems.
Detection of infectious pathogens such as HIV-1, HPV and SARS-CoV-2 from biospecimens is critical to healthcare. Particularly sensitive and specific diagnostic techniques to accomplish this include molecular amplification and detection tests of nucleic acids from pathogens. Such tests are comprised of reagent compositions to facilitate hybridization of primers and probes that are complementary to specifically amplified sequences of the analyte target. One of these reagents from an isothermal molecular assay occasionally changed its physical appearance over time, generating interest into the cause of the transformation and suitability of the reagent in diagnostic testing. A preliminary hypothesis was that the 2,2’-dithiodipyridine component was the pre-chromophoric compound of its distinctly yellow reduced form, 2-thiopyridine. However, under oxidizing conditions, 2-thiopyridine is a minor constituent of hybridization reagents and not a major contributor to the yellow color. Instead, a new yellow compound was isolated from colored hybridization reagent, identified as 1-(2’-pyridyl)-2-thiopyridone and determined to be the result of an intramolecular cyclic rearrangement and sulfur extrusion from 2,2’-dithiodipyridine under acidic and oxidizing conditions. Neither the appearance of 1-(2’-pyridyl)-2-thiopyridone nor the concomitant depletion of 2,2’-dithiodipyridine reduced the sensitivity or specificity of in vitro diagnostic screening assay results for detecting amplified nucleic acids from viral pathogens, ensuring the safety of tested blood transfusion products.
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