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

摘要 快速、高通量和高准确性地识别潜在的血液传播疾病将有助于血库快速检测大量捐献的血液，并有助于防止进一步的感染。目前血液检测存在以下问题： 1.耗时的检测和样本操作，必须对所有疾病“逐一”进行。 2.需要大量的样品，这取决于测试的数量。 3.随着测试次数的增加，测试程序变得复杂。 4.复杂的操作程序，必须由分子实验室中训练有素的人员进行。 最近，使用核酸扩增检测技术（NAT）的商业化检测的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，而不是在“多路复用（> 2）的方式”，因此不适合于各种病原体的供体样品的大规模筛选。 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和条形码磁珠（BMB）技术相结合，以产生血液中多种病原体的快速同时测定（SAMP-B）。只需几滴献血者的血液与数字磁珠结合在一个微孔中，就可以同时鉴定HIV 1型和2型、B肝炎（HBV）、梅毒、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。在这个I期项目中，我们已经完成了所有的任务，并成功地证明了通过制造128重BMB，集成分析仪，并进行HIV，HBV和Syphilis多重检测来同时检测多种病原体的技术可行性。由此产生的组合技术非常强大，并将提供以下优势： (1)少量样品：微孔中的几滴血就足以鉴定多种目标病原体。它 不仅确定了反应性样品，而且还确定了“对什么反应” (2)缩短窗口期：提供高灵敏度和特异性，而无需基于抗体的筛查技术的“窗口期” (3)快速和高通量：该系统每小时可自动处理多达100个样本--最多可报告12，800个96微孔格式的结果 (4)灵活性：可轻松添加新探针，用于在微珠上筛选其他靶标 (5)低成本、易于使用和高可靠性：BMB成本低，操作步骤简单，批次间差异最小。 在这个II期项目中，我们将重点关注：（1）将血液筛查的血源性病原体面板从（3-plex到8-plex）扩展，（2）将样本处理与SAMP-B分析仪集成，以及（3）开发和评估用于血液筛查的多重病原体。

ABSTRACT Rapid, high-throughput, and highly accurate identification of potential blood borne diseases will help blood banks to quickly test a very large volume of donated blood, and will help to prevent further infections. Current blood testing has the following issues: 1. Time consuming testing and sample operation that must be performed for all diseases "one-by-one". 2. A large sample volume is required that depends on the number of tests. 3. Testing procedure is complicated as the number of tests increase. 4. Complicated operational procedures that must be run by highly trained personnel in molecular laboratories. 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, not in "multiplexed (> 2) fashion", thus are unsuited to large-scale screening of donor samples for various pathogens. Maxwell Sensors, Inc. (MSI) proposes to combine Helicase-Dependent Amplification and Barcode Magnetic Bead (BMB) technologies to produce a rapid, Simultaneous Assay for Multiple Pathogens in Blood (SAMP-B). With a few drops of a donor's blood combined with digital magnetic beads in a single microwell, it is possible to simultaneously identify HIV type 1 and 2, Hepatitis B (HBV), Syphilis, Hepatitis C (HCV), Human T-Lymphotropic Virus (HTLV type I and II), West Nile Virus (WNV), and many other blood-borne diseases. During this Phase I project, we have completed all tasks and successfully demonstrated the technical feasibility of simultaneous assay for multiple pathogens by fabricating 128-plex BMBs, integrating the analyzer, and performing HIV, HBV, and Syphilis multiplexed assays. The resulting combined technology is very powerful and will offer the following advantages: (1) 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" (2) Reduced window period: offers high sensitivity and specificity without the "window period" associated with antibody based screening technologies (3) Rapid and high throughput: the system can automatically process up to 100 samples per hour - for a maximum of 12,800 reportable results in a 96-microwell format (4) Flexibility: easy addition of new probes for additional screening targets on beads (5) Low cost, easy to use, and high reliability: BMB are low cost, simple operation steps, and batch to batch variation is minimal. In this Phase II project, we will focus on: (1) expand blood-borne pathogens panels from (3-plex to 8-plex) for Blood screening, (2) integrate the sample process with SAMP-B analyzer, and (3) develop and evaluate multiplex pathogens for blood screening.