Bead-array for rapid risk-stratification in leukemia

描述(由申请人提供)：准确区分来自染色体易位的融合转录本对于优化白血病的治疗策略至关重要。然而，随着白血病相关易位数量的稳步增加，单管、多重检测多个融合基因转录本变得具有挑战性。这项建议的总体目标是开发一种检测系统，用于白血病染色体易位的快速风险分层。该系统将多重RT-PCR与对96孔板格式的荧光珠进行多重信号检测相结合。临床决策是基于特定融合靶点相对于内部或外源对照的相对表达水平。该分析在速度和成本方面比目前可用的分析方法有了显著的改进，可以应用于白血病诊断中的高通量风险分层。这种分析方法很灵活，很容易适用于其他用于癌症/疾病诊断的基于核酸的分析方法。 第一阶段的具体目标： (1)建立单管快速鉴定白血病相关常见染色体易位的方法(最多21个易位)。 (2)建立该方法临床试验的综合控制系统。 在第二阶段，我们将把这项试验改造成一种强大的、高通量的临床诊断试剂盒，用于白血病的快速风险分层。我们第二阶段的第二个目标是使用成熟的内部系统生产用于外源对照和所有阳性靶标对照的装甲RNA，并测试这些装甲RNA对照的扩增和检测效率。装甲RNA的使用确保了我们在检测系统中阳性对照的稳定性。

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.