Multiplexed PCR on Optically Encoded Beads

描述（由申请人提供）：聚合酶链式反应（PCR）是分子生物学中最有用和最普遍适用的方案之一，并构成越来越多的诊断检测和其他检测的基础。非常希望能够通过PCR，即进行多重PCR，同时筛选来自个体或位置的单个样品中是否存在许多感染因子。目前，当增加PCR多重深度时遇到的困难包括（a）可用于光学多重的有限数量的可分辨光学特征和（B）与最小化引物、模板和扩增子之间的复杂和有害的相互作用相关的实质性生物信息学设计问题，所述相互作用可导致非特异性扩增。我们建议通过直接在热稳定的多孔玻璃珠表面上进行固相PCR（SPPCR）来解决这些障碍，所述玻璃珠已经用Parallume光学编码技术（www.parallume.com）编码，并且两种引物中的一种或两种共价结合到珠的表面。可从基于稀土的Parallume编码技术获得的大量光学代码，与通过将一种或两种引物螯合到珠表面上而减少引物-引物和引物-扩增子相互作用相结合，将提供具有比任何现有技术更大的多重深度的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，评估拟议的多重PCR的易用性，灵敏度和选择性。我们将使用来自鼠疫耶尔森氏菌（鼠疫）的三个签名、来自苏云金芽孢杆菌以色列亚种的三个签名和来自酿酒酵母的四个签名进行基于Parallume珠的10重PCR。将十种正向引物与具有十种不同光学代码的珠共价连接，并将珠添加到溶液中含有10种反向引物（其可以被标记）的标准PCR混合物中。通过以下方式定量PCR产物：（a）直接测量每个微珠上掺入Cy 3（或藻红蛋白）标记的反向引物后荧光强度的增加，（B）变性后互补Cy 3标记的探针与结合的扩增子杂交间接测定，或（c）测定嵌入染料（如SYBR或皮科绿色）的摄取。通过将我们的珠结果与在LLNL进行的相应Taqman测定的系列稀释系列进行比较，评价Parallume编码珠系统用于PCR的灵敏度和选择性。公共卫生相关性：这项创新旨在利用附着在多孔玻璃珠上的引物进行高度多重的固相PCR，该多孔玻璃珠采用Parallume技术进行光学编码。该过程的最终结果将是一种测定，其中未知样品（例如，血液、土壤、组织、空气）中的已知生物威胁或传染病的同时分析。在多孔珠的大表面积上隔离正向引物或正向引物和反向引物两者阻止它们与其他珠上的引物相互作用，从而降低了引物-引物相互作用产生非特异性扩增产物的可能性。使用这种方法，可以使用多重固相PCR反应代替液相PCR，其中开发高度特异性引物的高成本和繁琐的实验导致多靶测定的初始开发和成品之间的长滞后时间。除了目前的方法是基于溶液的之外，目前用于检测扩增产物的荧光探针的成本高，并且检测方法的成本高。这两种较高的成本都通过使用Parallume编码的珠粒和MARS分析系统来减轻，在所述MARS分析系统上分别运行PCR和分析PCR后珠粒。

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.