Lee Dong-Hun, Mathew John, Pfahler Wolfram, Ma Dongling, Valinsky Jay, Prince Alfred M, Andrus Linda
Laboratory of Virology, The Lindsay F. Kimball Research Institute of the New York Blood Center, 310 East 67th Street, New York, NY 10021, USA.
J Clin Microbiol. 2005 Oct;43(10):5111-6. doi: 10.1128/JCM.43.10.5111-5116.2005.
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.
我们开发了一种经济、高通量的血源病毒核酸扩增检测（NAT）方法，适用于对个体献血者血浆样本进行筛查。该检测系统包括一个半自动程序，使用96孔玻璃纤维板从血浆中提取病毒核酸，并采用“通用信标”技术，可检测高变异性病毒（如人类免疫缺陷病毒和丙型肝炎病毒）的所有基因型。在这个检测系统中，两种荧光检测技术成功地应用于同一试管中：使用6-羧基荧光素荧光团的分子信标用于检测西尼罗河病毒（WNV），使用VIC荧光团的TaqMan用于检测内部对照。为了建立概念验证，我们专注于开发一种针对WNV的稳健的个体献血者NAT检测方法。该检测方法对其他15种测试病毒或来自WNV流行前季节的420份献血者样本均无反应。在交替进行的阳性/阴性孔测试中未观察到交叉污染。根据用作标准的材料不同，该检测方法对WNV的灵敏度（检测限，95%）在3.79至16.3个RNA拷贝/毫升之间。该检测方法检测出了罗氏WNV NAT鉴定出的所有阳性献血样本。该检测方法可进行定性筛查和定量确认。
