Ivan Francisco LoncarevicID1*, Susanne Toepfer1, Stephan Hubold1, Susanne Klingner1, Lea Kanitz1, Thomas Ellinger1, Katrin Steinmetzer1, Thomas Ernst2, Andreas Hochhaus2, Eugen Ermantraut1


1 BLINK AG, Jena, Germany, 2 Universita¨tsklinikum Jena, Klinik fu¨r Innere Medizin II, Abteilung Haematologie und Internistische Onkologie, Jena, Germany


Chemistry, Molecular Biology


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.



Images were taken using a 5x objective (field of view 416mm x 2.774mm), pE-4000 (CoolLED Ltd.) light source and a CMOS camera (UI-3260CP-M-GL, IDS).

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The 256 wavelength combinations from 16 LEDs (365 nm -770 nm) of the pE-400 offers a high level of flexibility. In this case of assay development, the researchers could therefore use a variety of fluorophores including FAM, Cy5 and Rox to monitor their PCR method taking place on nanoreactor beads.

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PloS one

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