Bead-array for rapid risk-stratification in leukemia

描述（由申请人提供）：准确区分染色体易位衍生的融合转录物对于优化白血病治疗策略至关重要。然而，随着白血病相关易位的数量稳步增加，单管多重检测多个融合基因转录本变得具有挑战性。该提案的总体目标是开发一种用于白血病染色体易位快速风险分层的测定系统。该系统将多重RT-PCR与96孔板形式的荧光珠上的多重信号检测相结合。临床决策是基于特定融合靶标与内部或外源对照的相对表达水平。该检测方法在速度和成本方面比目前可用的检测方法有显著的改进，并且可以应用于白血病诊断中的高通量风险分层。该测定是灵活的，以容易地适应于可用于癌症/疾病诊断的其他基于核酸的测定。 第一阶段的具体目标： (1)开发一种快速单管检测方法，用于在单管中同时鉴定与白血病相关的常见染色体易位（多达21个易位）。 (2)为该检测试剂盒的临床试验开发一个全面的控制系统。 在第二阶段，我们将把这种检测方法改造成一种强大的、高通量的临床诊断试剂盒，用于白血病的快速风险分层。第二阶段的第二个目标是使用完善的内部系统生产用于外源对照和所有阳性靶对照的装甲RNA，并测试这些装甲RNA对照的扩增和检测效率。装甲RNA的使用确保了我们的阳性对照在测定系统中的稳定性。

DESCRIPTION (provided by applicant): Accurate differentiation of fusion transcripts derived from chromosome translocations is of critical importance for optimizing treatment strategies for leukemia. However, as the number of leukemia associated translocations is steadily increasing, single-tube, multiplex detection of multiple fusion gene transcripts becomes challenging. The overall goal of this proposal is to develop an assay system for rapid risk-stratification of chromosomal translocations in leukemia. The system combines multiplex RT-PCR with multiplex signal detection on fluorescent beads in a 96-well plate format. Clinical decision-making is based on the relative expression level of a specific fusion target to the internal or exogenous controls. The assay offers significant improvements in speed and cost over currently available assays and can be applied for a high-throughput risk-stratification in leukemia diagnostics. This assay is flexible to be easily adapted to other nucleic-based assays useful for cancer/diseases diagnostics. Specific aims for Phase I: (1) Develop a rapid single-tube assay for simultaneous identification of common chromosome translocations related to leukemia in a single tube (up to 21 translocations). (2) Develop a comprehensive control system for the clinical test of this assay. In Phase II we will adapt this assay into a robust, high-throughput clinic diagnostic kit for rapid risk stratification in leukemia. Our second aim for Phase II is to produce armored RNAs for exogenous control and all positive target controls using well-established in-house system and test the amplification and detection efficiency of these armored RNA controls. The use of armored RNA ensures the stability of our positive controls in the assay system.