Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes

Abstract

In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods such as RPA, becomes a promising tool in the detection of pathogens and target specific genes. Actually, RPA even provides more advantages. This isothermal method got popular in point of care diagnostics because of its speed and sensitivity but requires pre-labelled primer or probes for a following detection of the amplicons. To overcome this disadvantages, we performed an labelling of RPA-amplicons with Cy5-dUTP without the need of pre-labelled primers. The amplification results of various multiple antibiotic resistance genes indicating great potential as a flexible and promising tool with high specific and sensitive detection capabilities of the target genes. After the determination of an appropriate rate of 1% Cy5-dUTP and 99% unlabelled dTTP we were able to detect the blaCTX-M15 gene in less than 1.6E−03 ng genomic DNA corresponding to approximately 200 cfu of Escherichia coli cells in only 40 min amplification time.

Investigation and validation of labelling loop mediated isothermal amplification (LAMP) products with different nucleotide modifications for various downstream analysis

“…Microarray detection is a common and valuable tool for the analysis of PCR and isothermal amplification products 25 , 35 , 36 . Recently, we were able to present nucleotide-based labelling during recombinase polymerase amplification (RPA) followed by microarray as a very advantageous method 26 . In this study, we have successfully transferred this technique in a most intriguing way to detect and distinguish LAMP products.…”

“…With a suitable selection of probes, it is possible to distinguish the amplicons not only from false positives, but even from single nucleotide polymorphisms (SNPs). Following this hybridization, the signals of the labelled and hybridized amplicons are read out 25 , 26 .…”

“…In a previous study we demonstrated that this labelling is useful for isothermal methods such as recombinase polymerase amplification (RPA) for a sensitive detection of pathogens and differentiation of single nucleotide polymorphism (SNP) gene variants 26 . Therefore, we were highly motivated to investigate the suitability of this labelling technique for other isothermal methods.…”

Labelling of RPA amplicons: an experimental comparison between primer and nucleotide labelling used for multiplex detection of antibiotic resistance genes

“…We have already shown in previous works that the use of labelled nucleotides can certainly have advantages over primer labelling when isothermal ampli cation methods are used 18,22 . Also, the use of Cy5-labelled dNTPs to highlight amplicons during RPA has already been successfully demonstrated as a proof of concept 29 .…”

Antibiotic resistance mechanism and diagnosis of common foodborne pathogens based on genotypic and phenotypic biomarkers

“…Recently, an alternative labeling technique without pre‐labeled primer sets was proposed by Warmt et al. (2021) (Figure 5). In this labeling system, 5‐(3‐aminoallyl)−2′‐deoxyuridine‐5′‐triphosphate (Cy5‐dUTP) was used to label RPA amplicons for the detection of bla CTX‐M15 gene (beta‐lactam resistance).…”