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

Abstract

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.

Figure 1

DNA amplicon labelling during RPA. Illustrated are the two most fundamental options for labelling amplicons directly during RPA. In addition to the DNA-polymerase, recombinase and ssDNA binding proteins commonly used for RPA, as well as the unlabelled dNTPs and primer, dUTPs or primer labelled with fluorophores are also required (blue dashed box). After being amplified, there are either (several) nucleotide labels (left side) or one primer label per ssDNA in the RPA product.

Figure 2

Detection of fluorophore incorporation into RPA amplicons by gel electrophoresis. Shown is the incorporation of Cy5-dUTP with using different concentrations during RPA (2 µM to 80 µM) and by replacing the unlabelled with labelled forward primer (F), reverse primer (R) and both primer (F/R) simultaneously. A=bla_CTX-M15 amplicon and C=bla_KPC amplicon on common gel images using the intercalator SYBR-green are shown. B=bla_CTX-M15 amplicon and D=bla_KPC amplicon: all RPA products in which the dye Cy5 has been incorporated are visible on the same gels.

Figure 3

Comparison of Cy5 incorporation rates as a function of RPA fragment length. The Cy5 incorporation rates of the two fragments of varying lengths for the detection of bla_CTX-M15 (141 bp) and bla_KPC (809 bp) are shown. A: indicates the incorporation rates per 1000 nucleotides used for the formation of DNA amplicons during RPA as a function of the Cy5-dUTP concentration applied. B: shows the absolute incorporation rate of a double-stranded RPA amplicon relative to the respective fragment length and Cy5-dUTP concentration used per 25µl RPA reaction. The dashed lines indicate the amounts of Cy5-dUTP required to theoretically achieve the same incorporation rate as when using one labelled primer.

Figure 4

Comparison of nucleotide and primer labelling with varying fragment size. Shown is a comparison of the fluorescence intensities of two fragments of different sizes (CTX-M15 fragment = 141 bp; KPC fragment = 809 bp) after RPA and detection via microarray. (A)/(C): Microarray false colour representation for the detection of blaCTX-M15 and blaKPC after labelling with 2 µM, 20 µM and 80 µM Cy5-dUTP and labelled reverse primer (F/R*); orange frame marks fragment-specific probes. (B)/(D): Graphic representation of the microarrays shown on the left (probes marked with orange asterisk) supplemented with the negative controls (NTC) and the remaining primer labelling methods (F* forward primer; F*/R* both primer; scan parameter: gain 500, laser power 50, filter standard red).

Figure 5

Duplex and Multiplex detection of four resistance genes by Cy5-dUTP labelling. Depicted are multiplex RPA assays combined with microarray detection for the simultaneous detection of up to four different resistance genes from four different organisms (bla_CTX-M15 [E. coli; 735/14-1], bla_NDM [E. coli; 2/10], bla_VIM [P. aeruginosa; 359/11] and bla_KPC [E. coli; 17/11]). (A): Duplex experiments to validate possible cross-reactions are shown; (+) indicates which primer combinations and associated templates were used during the RPA; (−) indicates the negative controls using the respective primer but without templates. The positions of the respective probes are shown in the singleplex figure (B). For each microarray probe, a fivefold determination (five spots in a row or in a line) was performed. (B): Results of the quadruplex approach. The positions marked in the quadruplex for the probes of the respective amplicons are identical in all figures. Both in the single and multiplex approach, the bla_KPC-2 variant can be detected via the high signal probe (left outer probe in the KPC block; orange arrow guides to KPC-2). Microarray: false colour representation. (C): Schematic representation of KPC variants based on varying SNPs according to Chen L. et al., 2011.