Fig 1.
Schematic description of a microfluidic disk segment (“GeneSlice”) with pre-stored reagents (A), and a compatible rotor holder (“GeneSlice 100 Rotor”), which can hold up to four GeneSlices in one Rotor-Gene Q run (B).
The GeneSlice comprises pre-amplification chambers for sample and no-template control (NTC) liquids, capillary siphon valves for transfer of the pre-amplification product and a centrifugo-thermopneumatic two-stage aliquoting structure [31] for 14 (sample) and 1 (NTC) main-amplification(s), respectively. The aliquoting structures guide excess liquid into waste chambers. All required reagents are pre-stored in lyophilized or air-dried format. The air vent for pressure equalization is covered with a membrane. Siphon valves are rendered hydrophilic by coatings.
Fig 2.
Illustration of microfluidic process flow of a GeneSlice for nested PCR and melt curve analysis on a Rotor-Gene Q.
The microfluidic structures of interest at different points in time (A-E) are shown on top, indicating liquid movement from the dark blue to the light blue position. Corresponding temperatures (red) and rotational speed (black) at each depicted point in time can be read from the given diagram at the bottom. The liquid stays in the position depicted in B during pre-amplification, which consists of 10 thermal PCR cycles at constant 400 RPM. During main-amplification and melt curve analysis, which follow the last (E) depicted fluidic operation, no further fluidic operations take place and liquids stay in the main-amplification cavities (E, light blue).
Fig 3.
Melt curve analysis of sheep and goat amplimers.
1 ng of sheep and goat DNA were amplified on two GeneSlices. The melt curve analysis shows the successful amplification of the 12S rRNA and cytb genes for both species. The different melting behavior is caused by inter-species sequence differences within the Caprinae.
Table 1.
Sensitivity level of species analyzed within the assay for detection of European animal groups on GeneSlices.
Bold: species analyzed in triplicates.
Fig 4.
Melt curve analysis of a mixture of human and pig DNA in different ratios.
Artificial mixtures of human and pig DNA were created from 0% to 100%, each, and amplified. Analysis of the respective animal-group-specific main-amplification and universal 12S rRNA cavities shows resolution of the DNA components down to 1% (12S rRNA) for pig and human; 5% both for pig, and 10% both for human as minor components.