Simultaneous Assay for Multiple Pathogens in Blood (SAMP-B)

描述（由申请人提供）：使用核酸扩增检测技术（NAT）的市售检测试剂盒的最新发展使得血液中心可以考虑将这些检测应用于献血者筛查。但是，NAT测试系统，到目前为止，是时间密集型的，需要有限的实验室空间和训练有素的技术人员，通常不适合大规模筛选供体样本。美国血液中心采用了两种基本的“合并和合并解决”策略。一种是24的直池，其中“反应池”的分辨率需要测试单个样品。在另一种策略中，准备并组合较小的（中间）池以创建最终（主）池。通过首先检测中间池，然后（如有必要）检测单个样本，可解析反应性主池。样本池大小和合并策略平衡了检测灵敏度与确保血液可用性的实际需求。 麦克斯韦传感器公司(MSI)提出将联合收割机解旋酶依赖性扩增（HDA）和数字磁珠（DM-Bead）技术相结合，以高通量96孔微孔板形式产生血液中多种病原体的单样品快速同时测定（SAMP-B）。在单个微孔中，用几滴献血者的血液与DM珠结合，可以同时鉴定HIV 1型和2型、乙型肝炎B（HBV）、丙型肝炎（HCV）、人类嗜T细胞病毒（HTLV I型和II型）、西尼罗河病毒（WNV）和许多其他血液传播疾病。DM微珠通过光刻法用数字条形码批量制造，然后用核酸探针标记，能够在单一均匀介质中准确识别许多靶标。 该组合技术非常强大，并具有以下优点： 样本合并是没有必要的：筛选多种病原体可以在个人的样本进行。 少量样品：微孔中的几滴血就足以鉴定多种目标病原体。它不仅确定了反应样品，而且还确定了“对什么反应”。 窗口期缩短：HDA提供高灵敏度和特异性，而没有与基于抗体的筛查技术相关的“窗口期”。 快速和高通量：简单的等温操作和96个患者样本可以在一个96孔微孔板上进行。 灵活性：可轻松添加新探针，用于在微珠上筛选其他靶标。 低成本，易于使用，高可靠性：DM微珠成本低，操作步骤简单，批次间差异极小。 在该项目中，我们将重点关注四个关键任务：1）数字磁性条形码珠的设计和制造，2）检测系统的设计和构建，3）基于DM-Bead的多重HDA检测方法的测试和4）系统和生物测定性能的评估。公共卫生相关性：麦克斯韦传感器公司(MSI)提出开发一种基于解旋酶依赖性扩增的系统，使用数字磁性条形码珠，提供快速，准确和易于使用的筛选多种血源性病原体的小样本的供体血液。这种血液中多种病原体的同时检测将有助于血库和临床实验室快速检测大量捐献的血液，并有助于防止血液传播感染的传播。将先进的半导体制造工艺与分子特征扩增和探测相结合的拟议技术将允许以相对低的成本对个体进行高通量分子诊断分析。

DESCRIPTION (provided by applicant): The recent development of commercially-available assays using nucleic acid amplification test technologies (NAT) have made it possible for blood centers to consider applying these tests to blood donor screening. But NAT test systems, so far, are time intensive, require restricted laboratory space and highly-trained technical staff, and generally are unsuited to large-scale screening of donor samples. U.S. blood centers have adopted two basic "pooling and pool resolution" strategies. One is a straight pool of 24 in which resolution of a "reactive pool" requires testing individual samples. In the other strategy, smaller (intermediate) pools are prepared and combined to create a final (master) pool. Resolution of reactive master pools occurs by first testing the intermediate pools and then, if necessary, the individual samples. Pool size and pooling strategy balance assay sensitivity with the practical demands of assuring blood availability. Maxwell Sensors, Inc. (MSI) proposes to combine Helicase-Dependent Amplification (HDA) and Digital Magnetic Bead (DM-Bead) technologies to produce a one sample rapid, Simultaneous Assay for Multiple Pathogens in Blood (SAMP-B), in a high throughput 96-well microplate format. With a few drops of a donor's blood combined with DM-beads in a single microwell, it is possible to simultaneously identify HIV type 1 and 2, Hepatitis B (HBV), Hepatitis C (HCV), Human T- Lymphotropic Virus (HTLV type I and II), West Nile Virus (WNV), and many other blood-borne diseases. The DM-Bead, mass fabricated with digital bar codes by photolithography, then tagged with nucleic acid probes, are able to accurately identify many targets in a single homogeneous medium. The combined technology is very powerful and offers the following advantages: Sample pooling is not necessary: screening for multiple pathogens can be performed on an individual person's sample. Small quantity of sample: a few drops of blood in a microwell are all that's needed to identify multiple target pathogens. It not only determines a reactive sample, but also identifies "reactive to what". Reduced window period: HDA offers high sensitivity and specificity without the "window period" associated with antibody based screening technologies. Rapid and high throughput: simple isothermal operation and 96 patient samples can be performed on a single 96-well microplate. Flexibility: easy addition of new probes for additional screening targets on beads. Low cost, easy to use, and high reliability: DM-beads are low cost, simple operation steps, and batch to batch variation is minimal. During this project, we will focus on four key tasks: 1) design and fabrication of digital magnetic barcode beads, 2) design and construction of a detection system, 3) testing the DM-Bead-based multiplex HDA assay and 4) evaluation of system and bioassay performance. PUBLIC HEALTH RELEVANCE: Maxwell Sensors Inc. (MSI) proposes to develop a helicase-dependent amplification-based system, using digital magnetic barcoded beads that provide rapid, accurate, and easy-to-use screening for multiple blood-borne pathogens in small specimens of donor blood. This Simultaneous Assay for Multiple Pathogens in Blood will help blood banks and clinical laboratories to quickly test very large volumes of donated blood, and will help prevent the spread of blood-borne infections. The proposed technology that marries an advanced semiconductor fabrication process with molecular signature amplification and probing will allow high-throughput molecular diagnostic profiling of individuals at relatively low cost.