Multiplexed PCR on Optically Encoded Beads

描述（由申请人提供）：聚合酶链反应（PCR）是分子生物学中最有用和最普遍适用的方案之一，是越来越多的诊断测试和其他分析的基础。如果能够通过聚合酶链反应同时筛选来自个体或地点的单个样本中是否存在多种感染因子，即进行多重聚合酶链反应，那将是非常理想的。目前，增加PCR复用深度时遇到的困难包括(a)可用于光复用的可解析光学特征数量有限，以及(b)与最小化引物、模板和扩增子之间复杂和有害的相互作用有关的大量生物信息学设计问题，这些相互作用可能导致非特异性扩增。我们建议通过直接在热稳定的多孔玻璃珠表面进行固相PCR （SPPCR）来解决这些障碍，这些玻璃珠已经用平行光学编码技术（www.parallume.com）编码，并且两个引物中的一个或两个共价键合在珠的表面。稀土平行编码技术可获得大量光学编码，再加上通过将一个或两个引物分离到头表面来减少引物与引物和引物与扩增子的相互作用，将提供比任何现有技术更具有多路复用深度的PCR方法。在第一阶段，我们将与劳伦斯利弗莫尔国家实验室（LLNL）的化学和生物对策（CB）部门合作，从易用性、灵敏度和选择性方面评估所提出的多重PCR。我们将使用来自鼠疫耶尔森菌（鼠疫）的三个特征，来自苏云金芽孢杆菌（Bacillus thuringiensis Israeliensis）的三个特征和来自酿酒酵母（Saccharomyces cerevisiae）的四个特征进行平行珠状PCR。10个正向引物将共价连接到具有10种不同光学编码的珠子上，并将这些珠子添加到含有10个反向引物（可能被标记）的标准PCR混合物中。PCR产物的定量将通过(a)直接测量每个头上Cy3（或藻红蛋白）标记的反向引物的荧光强度增加，(b)间接通过将互补的Cy3标记探针在变性后杂交到结合的扩增子上，或(c)测定插入染料（如SYBR或Pico Green）的吸收。平行编码的PCR头系统的灵敏度和选择性将通过将我们的头结果与在LLNL进行的一系列相应的Taqman稀释试验进行比较来评估。公共卫生相关性：这项创新旨在执行高复用固相PCR，引物附着在多孔玻璃珠上，用平行技术进行光学编码。该过程的最终结果将是一种分析，其中可以同时分析未知样本（例如血液、土壤、组织、空气），以检测来自这些物种的已知DNA片段，以确定已知的生物威胁或传染病。在多孔珠的大表面积上分离正引物或同时分离正引物和反向引物，可以阻止它们与其他引物的相互作用，从而降低引物与引物相互作用产生非特异性扩增产物的概率。使用这种方法，多重固相PCR反应可以用来代替液相PCR，其中开发高特异性引物的高成本和繁琐的实验导致多目标测定的初始开发和最终产品之间的长滞后时间。除了目前的方法是基于溶液的，目前用于扩增产物检测的荧光探针成本高，检测方法成本高。通过使用平行编码微球和MARS分析系统分别运行PCR和分析PCR后微球，这两种较高的成本都得到了缓解。

DESCRIPTION (provided by applicant): The Polymerase Chain Reaction (PCR) is one of the most useful and generally applicable protocols in molecular biology and forms the basis of an increasing number of diagnostic tests and other assays. It would be highly desirable to be able to screen a single sample from an individual or location for the presence of many infectious agents simultaneously by PCR, i.e. to perform multiplex PCR. Presently, difficulties encountered when increasing the PCR multiplex depth include (a) the limited number of resolvable optical signatures available for optical multiplexing and (b) the substantial bioinformatic design issues related to minimizing the complex and deleterious interactions among primers, templates and amplicons which can lead to nonspecific amplification. We propose to address these impediments by carrying out Solid Phase PCR (SPPCR) directly on the surface of thermally stable, porous glass beads which have been encoded with Parallume optical encoding technology (www.parallume.com) and with one or both of the two primers covalently bonded to the surface of the bead. The large number of optical codes available from the rare earth-based Parallume encoding technology, combined with a reduction in primer-primer and primer-amplicon interactions by sequestering one or both of the primers onto the bead surface, will provide a PCR methodology with a multiplexing depth greater than any current technology. In Phase I we will evaluate the proposed multiplex PCR in terms of ease of use, sensitivity and selectivity in collaboration with the Chemical and Biological Countermeasures (CB) Division at Lawrence Livermore National Laboratory (LLNL). We will perform a Parallume bead-based 10-plex PCR using three signatures from Yersinia pestis (Plague), three from Bacillus thuringiensis Israeliensis and four from Saccharomyces cerevisiae. The ten forward primers will be covalently attached to beads with ten different optical codes and the beads added to a standard PCR mix containing the 10 reverse primers (which may be labeled) in solution. The PCR product will be quantitated by either (a) directly measuring the increasing fluorescent intensity on each bead from incorporation of a Cy3 (or phycoerythrin)-labeled reverse primer, (b) indirectly by hybridization of a complementary Cy3-labeled probe onto the bound amplicon after denaturing or (c) determination of the uptake of an intercalation dye like SYBR or Pico Green. The sensitivity and selectivity of Parallume encoded bead system for PCR will be evaluated by comparison of our bead results to a serial dilution series of corresponding Taqman assays performed at LLNL. PUBLIC HEALTH RELEVANCE: This innovation seeks to perform highly-multiplexed solid phase PCR with primers attached to a porous glass bead optically-encoded with the Parallume technology. The end result of this process will be an assay where an unknown sample (e.g., blood, soil, tissue, air) can be simultaneously analyzed for known biothreats or infectious diseases based on the detection of a known DNA fragment from those species. Segregating either the forward or both the forward and reverse primers on the large surface area of the porous beads prohibits their interaction with primers on other beads thereby diminishing the probability of nonspecific amplification products to occur from primer-primer interactions. Using this methodology, a multiplexed solid phase PCR reaction can be used in place of solution phase PCR where the high cost and tedious experimentation in developing highly specific primers lead to a long lag time between the initial development of a multi-target assay and the finished product. In addition to the current methods being solution-based, there is currently a high cost of the fluorescent probes used in the detection of the amplified product and a high cost to the method of detection. Both of these higher costs are mitigated by the use of Parallume-encoded beads and the MARS analysis system on which to run the PCR and analyze the post-PCR beads, respectively.