Optical Encoding Technology for Viral Screening Panels

描述(由申请人提供)：在过去十年中，并行筛查技术的发展取得了实质性进展，旨在检测多种类型的生物威胁。一种类型的筛查技术集中在通过使用多重PCR然后杂交到Luminex的光学编码珠子上来减轻病毒Biothreat试剂造成的威胁。然而，相当少的可用的珠光码(~30-40)和基于流式细胞仪的精密检测器，阻止了在一个面板中检测所有病毒威胁，或者在受控的实验室环境之外使用屏幕。为了解决这些问题，基于Parallume(www.parallume.com)光学编码的系统将被用于对珠子进行编码，这些珠子将被完全便携的电池供电的阅读器读取。最终目标将是开发一个筛查小组，能够通过使用便携式检测系统在任何地点检测、区分和识别所有已知的病毒家族。 与劳伦斯利弗莫尔国家实验室(LLNL)的科学家合作，并行合成技术公司(PSTI)将研制出包含15个光学代码的Parallume编码珠集，用于与LLNL已经使用的基于15珠Luminex的病毒筛查小组(VSP)进行直接比较，以检测人类流感病毒。使用LLNL生物信息学小组设计的生物素化引物，在单一反应中对病毒基因组的15个区域进行了聚合酶链式反应。每个聚合酶链式反应产物的靶区包含与光学编码的珠子上的捕获探针杂交的序列。通过用链霉亲和素-藻红蛋白(SAPE)结合物处理杂交后的PCR产物来标记生物素化的引物，与对照相比，通过在编码的珠子上连续稀释的阈值SAPE水平来检测病毒基因组的存在。在第一阶段的工作中，PARALLEL和LLNL将通过对分离的PCR产物混合物进行相同的反应来比较最先进的Luminex系统和Parallume编码技术。平行样品将使用Parallume珠子和Paralline的稳健多路分析读出系统(MARS)进行表征，该系统用微机械硅珠定位玻片和超亮标志LED取代了脆弱的流式细胞仪和激光，并直接比较了两种系统的测量速度、准确度、精密度和成本。并行技术和LLNL技术的结合可以使所有已知病毒在一次PCR反应中在任何地方被筛选出来。公共卫生相关性这项创新试图创建一个病毒筛查小组(VSP)，在该小组中，对含有DNA的人类、动物或植物样本进行同时筛查，以检测单个瓶子中的多种病原体(本申请中的病毒)。现有的病毒检测技术有限，但受到拟议的VSP不具备的技术限制的阻碍。这种新颖的VSP提出了一种用于扩展目前在单个屏幕中可检测到的病毒数量的方法，并通过能够将其他屏幕(例如细菌和其他非病毒病原体)合并到该单一、多路复用板中来增加面板的潜在功能。这项技术还为这款新产品提供了价格大大降低的阅读器(VSP分析设备)。

DESCRIPTION (provided by applicant): The last decade has witnessed substantial improvement in the development of parallel screening technologies designed to detect many types of biological threats. One type of screening technology has concentrated on mitigating threats caused by viral Biothreat agents by using multiplex PCR followed by hybridization onto optically encoded beads from Luminex. However, the rather small number of usable bead optical codes (~30-40), and the delicate flow cytometer based detector, prevent the detection of all viral threats in one panel or use of the screen outside of a controlled laboratory environment. To address these issues, a system based on Parallume (www.parallume.com) optical encoding, which can support thousands of optical codes, will be used to encode beads that will be read by a completely portable, battery-powered reader. The final goal will be to develop a screening panel capable of detecting, differentiating and identifying all known viral families in any location by using a portable detection system. In collaboration with scientists at Lawrence Livermore National Laboratory (LLNL), Parallel Synthesis Technologies, Inc. (PSTI) will formulate a Parallume-encoded bead set containing 15 optical codes which will be used in a direct comparison against a 15-bead Luminex-based Viral Screening Panel (VSP) already in use at LLNL to detect human influenza viruses. Using biotinylated primers designed by the LLNL Bioinformatics Group, PCR for 15 regions of viral genomes are performed in a single reaction. A target region of each PCR product contains a sequence which is hybridized to a capture probe on the optically encoded bead. By treating the PCR product after hybridization with a streptavidin-phycoerythrin (SAPE) conjugate to label the biotinylated primers, the presence of the viral genome is detected by a serially diluted, threshold SAPE level on an encoded bead as compared to the controls. In the Phase I effort, Parallel and LLNL will compare the state-of-the-art Luminex system with the Parallume encoding technology by characterizing the same reactions on a divided PCR product mixture. The Parallel samples will be characterized using Parallume beads and Parallel's robust Multiplex Assay Reader System (MARS), which has replaced the fragile flow cytometer and lasers with micromachined silicon bead localization slides and super-bright signage LEDs, and the measurement rate, accuracy, precision and cost of the two systems directly compared. The combination of the Parallel and LLNL technologies could allow all known viruses to be screened anywhere in one PCR reaction. PUBLIC HEALTH RELEVANCE This innovation seeks to create a viral screening panel (VSP) whereby a human, animal, or plant sample containing DNA is subjected to a simultaneous screen for multiple pathogens (viruses in the current application) within a single vial. Limited virus detection technologies exist, but are hampered by technical limitations not possessed by the proposed VSP. This novel VSP proposes a method for expanding the number of viruses currently detectable in a single screen and adds to the potential functionality of the panel by being able to incorporate other screens (e.g., bacterial and other non-viral pathogens) into this single, multiplexed panel. This technology also offers a dramatically less-expensive reader (VSP analysis equipment) to accompany this new product.