BLINK AG, Jena, Germany.
Universitätsklinikum Jena, Klinik für Innere Medizin II, Abteilung Hämatologie und Internistische Onkologie, Jena, Germany.
PLoS One. 2021 Mar 18;16(3):e0242529. doi: 10.1371/journal.pone.0242529. eCollection 2021.
Precise quantification of molecular targets in a biological sample across a wide dynamic range is a key requirement in many diagnostic procedures, such as monitoring response to therapy or detection of measurable residual disease. State of the art digital PCR assays provide for a dynamic range of four orders of magnitude. However digital assays are complex and require sophisticated microfluidic tools. Here we present an assay format that enables ultra-precise quantification of RNA targets in a single measurement across a dynamic range of more than six orders of magnitude. The approach is based on hydrogel beads that provide for microfluidic free compartmentalization of the sample as they are used as nanoreactors for reverse transcription, PCR amplification and combined real time and digital detection of gene transcripts. We have applied these nanoreactor beads for establishing an assay for the detection and quantification of BCR-ABL1 fusion transcripts. The assay has been characterized for its precision and linear dynamic range. A comparison of the new method against conventional real time RT-PCR analysis (reference method) with clinical samples from patients with chronic myeloid leukemia (CML) revealed excellent concordance with Pearsons correlation coefficient of 0.983 and slope of 1.08.
在许多诊断程序中，如监测治疗反应或检测可测量的残留疾病，在宽动态范围内精确量化生物样本中的分子靶标是一个关键要求。最先进的数字 PCR 检测方法提供了四个数量级的动态范围。然而，数字检测方法很复杂，需要复杂的微流控工具。在这里，我们提出了一种检测方法，该方法能够在单个测量中，在超过六个数量级的动态范围内，对 RNA 靶标进行超精确的定量。该方法基于水凝胶珠，这些水凝胶珠在用作逆转录、PCR 扩增和组合实时和数字检测基因转录本的纳米反应器时，提供了对样品的无微流控分隔。我们已经将这些纳米反应器珠应用于建立一种用于检测和定量 BCR-ABL1 融合转录本的检测方法。该方法已针对其精密度和线性动态范围进行了表征。与来自慢性髓性白血病 (CML) 患者的临床样本的常规实时 RT-PCR 分析（参考方法）进行比较，显示出极好的一致性，皮尔逊相关系数为 0.983，斜率为 1.08。
