Multiplexed PCR on Optically Encoded Beads

描述(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法之一，并形成了越来越多的诊断测试和其他分析的基础。非常希望能够通过聚合酶链式反应同时从个人或地点的单个样本中筛选出许多感染性病原体的存在，即执行多重聚合酶链式反应。目前，在增加PCR多重深度时遇到的困难包括：(A)可用于光学多重的可分辨光学标记的数量有限；(B)与最小化可能导致非特异性扩增的引物、模板和扩增之间的复杂和有害的相互作用有关的实质性的生物信息学设计问题。我们建议通过直接在热稳定的多孔玻璃微珠的表面进行固相聚合酶链式反应(SPPCR)来解决这些障碍，这些玻璃微珠已经用Parallume光学编码技术(www.parallume.com)编码，并将两个引物中的一个或两个共价连接到微珠的表面。基于稀土的Parallume编码技术提供了大量的光学编码，再加上通过将其中一个或两个引物隔离在珠面上而减少了引物-引物和引物-扩增子的相互作用，将提供一种比任何现有技术都更具多重深度的聚合酶链式反应方法。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对拟议的多重PCR进行评估。我们将使用鼠疫耶尔森氏菌(鼠疫)的三个签名、苏云金芽孢杆菌以色列杆菌的三个签名和酿酒酵母的四个签名进行基于并行微珠的10-plex聚合酶链式反应。这10个正向引物将共价连接到具有10个不同光学编码的珠子上，并将这些珠子加入含有溶液中的10个反向引物(可标记的)的标准PCR混合物中。PCR产物将通过(A)直接测量由于掺入Cy3(或藻红蛋白)标记的反向引物而在每个珠子上增加的荧光强度，(B)通过变性后通过将互补的Cy3标记的探针杂交到结合的扩增子上而间接地进行定量，或(C)确定SYBR或Pico Green等插层染料的摄取量。我们将通过将我们的微珠结果与LLNL进行的一系列相应的Taqman稀释分析结果进行比较，来评估Parallume编码微珠系统用于PCR的灵敏度和选择性。与公共卫生相关：这项创新寻求通过使用Parallume技术在光学编码的多孔玻璃微珠上安装引物来执行高度多路复用的固相聚合酶链式反应。这一过程的最终结果将是一种化验，在这种化验中，可以根据从这些物种中检测到的已知DNA片段，同时分析未知样本(例如，血液、土壤、组织、空气)中的已知生物制剂或传染病。在多孔珠的大表面积上分离正向或正向和反向引物中的一种或两种，可防止它们与其他珠上的引物相互作用，从而降低了从引物-引物相互作用中产生非特异性扩增产物的可能性。利用这种方法，可以用多重固相聚合酶链式反应代替溶液相聚合酶链式反应，在这种情况下，开发高特异性引物的高成本和繁琐的实验导致了多靶点分析的初始开发和成品之间的较长的滞后时间。除了目前的基于溶液的方法外，目前用于检测扩增产物的荧光探针的成本很高，检测方法的成本也很高。通过使用Parallume编码的珠子和MARS分析系统分别运行聚合酶链式反应和分析聚合酶链式反应后的珠子，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.