Mag bind total pure ngs kit

PCR products labelled with nucleotides were purified with the Mag-Bind® Total Pure NGS Kit (Omega Bio-Tek; M1378-01) according to the manufacturer's instructions prior to LFA analysis to determine the test strip detection limit. Afterwards, elution with 20 µL ddH₂O was performed. The remaining LFA experiments (i.e. primer labelling) were performed with unpurified amplicons immediately after PCR. For this, 90 µL of the corresponding HybriDetect assay buffer was added to one well of a 96-well plate to perform the HybriDetect Universal Lateral Flow Assay (Milenia Biotec GmbH; MGHD 1). 10 µL of the sample to be analysed was placed on the sample pad of the test strip. Afterwards, with the sample pad facing downwards, the test strip was placed in the prepared well containing the HybriDetect Assay Buffer. The test was removed from the well after 15 min incubation and evaluated. The result of the test strip was considered positive if it showed two lines (control line and test line). If only the control line was visible, the result was negative. If the control line was missing, the test result was invalid. The documentation of the result was done with a smartphone camera (Apple Iphone SE; second generation).

For the detection of the RPA efficiency via gel electrophoresis, the products were purified with the Mag-Bind® Total Pure NGS Kit (Omega Bio-Tek; M1378-01) according to the manufactures instructions with a finishing eluation step in 15 µl ddH₂O and 3 µl DNA Gel Loading Dye (6X) (Thermo Fisher Scientific; R0611) was added. An agarose gel with a 2% concentration in 1 × TAE-buffer (50 × TAE-buffer; PanReac AppliChem; A1691) was used for the separation of the fragments containing 2 µl of peqGreen (VWR; 732–3196) in a volume of 50 ml gel to visualize the band. A total volume of 18 µl of the purified and prepared samples were used for the electrophoresis which ran at 120 V for 1 h before the detection via BioDoc Analyzer (Biometra) including a Canon EOS 1100D was made.
The detection of the Cy5-dUTP labelled amplicons via microarray was performed without any purification of the RPA products.