PO.PR02.02 · 预防研究
采用基于PCR的单分子计数技术，在简化的单管流程中多重检测BCR-ABL1融合转录本
Multiplex detection of BCR-ABL1 fusion transcripts in a streamlined single-tube workflow using PCR-based single molecule counting
该海报暂无可下载的资料
AACR 官方页面
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
准确定量BCR-ABL1融合转录本对于监测慢性髓系白血病（CML）的微小残留病（MRD）至关重要，其中区分不同亚型（p210、p230、p190）在诊断、预后和治疗监测方面具有重要临床意义。传统检测方法依赖两步流程，即先进行逆转录（RT），再通过qPCR或数字PCR（dPCR）进行PCR扩增。虽然dPCR无需标准曲线即可实现绝对定量，但其有限的动态范围和多步骤流程给简化的亚型检测带来了挑战。我们开发了一种一步法多重RNA检测方法，可在单个管内直接检测并进行分子计数，识别p210、p230和p190亚型，即使在大量野生型ABL1分子背景下也能实现。整个流程，包括RT-PCR和计数，均无需样本转移，从而减少了手动操作时间和污染风险。采用3D光片显微镜技术，通过对完整的50 µL反应体积进行成像来直接对分子计数，消除了死体积和分区统计的影响。当含各亚型的BCR-ABL1 RNA以不同比例连续稀释至野生型ABL1背景中时，野生型ABL1的计数保持稳定，接近一百万个分子，而BCR-ABL1的计数随稀释成比例下降。这凸显了该方法在6个数量级动态范围内对RNA亚型的高灵敏度和高特异性检测，且变异度低（CV<10%），能够在单个管内监测丰度高和稀有的转录本。这种单管方法为在一次多重反应中灵敏检测所有BCR-ABL1亚型提供了一种简化的替代方案，在CML的临床评估和治疗决策方面具有潜在应用价值。
查看英文原文 English abstract
Accurate quantification of BCR-ABL1 fusion transcripts is essential for monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML), where distinguishing between isoforms (p210, p230, p190) is clinically important for diagnosis, prognosis, and treatment monitoring. Traditional detection methods rely on two-step workflows involving reverse transcription (RT) followed by PCR amplification, either via qPCR or digital PCR (dPCR). While dPCR enables absolute quantification without standard curves, its limited dynamic range and multi-step workflow present challenges for streamlined isoform detection. We developed a one-step multiplex RNA assay for the direct detection and molecular counting of p210, p230, and p190 isoforms, even amid abundant wild-type ABL1 molecules, within a single tube. The entire workflow, including RT-PCR and counting, occurs without sample transfers, reducing hands-on time and contamination risk. Using 3D light-sheet microscopy, molecules are directly counted by imaging the complete 50 µL reaction volume, eliminating dead volume and partitioning statistics. When BCR-ABL1 RNA containing each isoform was serially diluted into wild-type ABL1 background at varying ratios, wild-type ABL1 counts remained stable, near one million molecules, while BCR-ABL1 counts decreased proportionally with dilution.This highlights the highly sensitive and specific detection of RNA isoforms across a 6-log dynamic range with low variance (<10% CV), enabling monitoring of both abundant and rare transcripts within a single tube. This single-tube approach provides a streamlined alternative for sensitive detection of all BCR-ABL1 isoforms in one multiplexed reaction, with potential applications in clinical assessment and therapeutic decision-making in CML.
利益披露 Disclosure
V. Ratushna, None..
J. Keum, None..
C. Fan, None.