An experimental comparison between primer and nucleotide labelling to produce RPA-amplicons used for multiplex detection of antibiotic resistance genes

Direct labelling of amplification products using isothermal amplification is currently done most frequently by incorporating previously labelled primer. Although this method is well proven and widely used, it is not a universal solution due to some weaknesses. Alternatively, labelled nucleotides could be used, whose application and functionality have been already partially demonstrated. It remains to be determined how this method performs in comparison to traditional labelling, in particular combined with isothermal amplification methods. In this work, we show a detailed analysis of the labelling efficiency under different conditions and compare the results with the traditional primer-labelling method in the context of RPA amplification. Impressively, our results showed that using Cy5-labelled dUTPs can achieve much more efficient labelling for fragments above 200 bp, while using them for smaller fragments does not bring any relevant disadvantages, but also no major benefit. Furthermore, this work successfully demonstrate for the first time a quadruplex microarray for the detection of resistance genes using RPA and direct labelling with Cy5-dUTP as a potential application scenario. The sensitivities achieved here extend to SNP discovery for the detection of the proper bla(KPC) variant.