We present the centrifugal microfluidic implementation of a four-plex digital droplet polymerase chain reaction (ddPCR). The platform features 12 identical ddPCR units on a LabDisk cartridge, each capable of generating droplets with a diameter of 82.7 ± 9 µm. By investigating different oil–surfactant concentrations, we identified a robust process for droplet generation and stabilization. We observed high droplet stability during thermocycling and endpoint fluorescence imaging, as is required for ddPCRs. Furthermore, we introduce an automated process for four-color fluorescence imaging using a commercial cell analysis microscope, including a customized software pipeline for ddPCR image evaluation. The applicability of ddPCRs is demonstrated by the quantification of three cancer-associated KRAS point mutations (G12D, G12V and G12A) in a diagnostically relevant wild type DNA background. The four-plex assay showed high sensitivity (3.5–35 mutant DNA copies in 15,000 wild type DNA copies) and linear performance (R² = 0.99) across all targets in the LabDisk.
Multiplexing of analyses
is essential to reduce sample and reagent
consumption in applications with large target panels. In applications
such as cancer diagnostics, the required degree of multiplexing often
exceeds the number of available fluorescence channels in polymerase
chain reaction (PCR) devices. The combination of photobleaching-sensitive
and photobleaching-resistant fluorophores of the same color can boost
the degree of multiplexing by a factor of 2 per channel. The only
additional hardware required to create virtual fluorescence color
channels is a low-cost light-emitting diode (LED) setup for selective
photobleaching. Here, we present an assay concept for fluorescence
color multiplexing in up to 10 channels (five standard channels plus
five virtual channels) using the mediator probe PCR with universal
reporter (UR) fluorogenic oligonucleotides. We evaluate the photobleaching
characteristic of 21 URs, which cover the whole spectral range from
blue to crimson. This comprehensive UR data set is employed to demonstrate
the use of three virtual channels in addition to the three standard
channels of a commercial dPCR device (blue, green, and red) targeting
cancer-associated point mutations (KRAS G12D and
G12V). Moreover, a LOD (limit of detection) analysis of this assay
confirms the high sensitivity of the multiplexing method (KRAS G12D: 16 DNA copies/reaction in the standard red channel
and KRAS G12V: nine DNA copies/reaction in the virtual
red channel). Based on the presented data set, optimal fluorogenic
reporter combinations can be easily selected for the application-specific
creation of virtual channels, enabling a high degree of multiplexing
at low optical and technical effort.
There is an increasing demand for optimization-free multiplex assays to rapidly establish comprehensive target panels for cancer monitoring by liquid biopsy. We present the mediator probe (MP) PCR for the quantification of the seven most frequent point mutations and corresponding wild types (KRAS and BRAF) in colorectal carcinoma. Standardized parameters for the digital assay were derived using design of experiments. Without further optimization, the limit of detection (LoD) was determined through spiking experiments with synthetic mutant DNA in human genomic DNA. The limit of blank (LoB) was measured in cfDNA plasma eluates from healthy volunteers. The 2-plex and 4-plex MP ddPCR assays showed a LoB of 0 copies/mL except for 4-plex KRAS G13D (9.82 copies/mL) and 4-plex BRAF V600E (16.29 copies/mL) and allele frequencies of 0.004% ≤ LoD ≤ 0.38% with R2 ≥ 0.98. The quantification of point mutations in patient plasma eluates (18 patients) during follow-up using the 4-plex MP ddPCR showed a comparable performance to the reference assays. The presented multiplex assays need no laborious optimization, as they use the same concentrations and cycling conditions for all targets. This facilitates assay certification, allows a fast and flexible design process, and is thus easily adaptable for individual patient monitoring.
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