Multiplex RPA for Antibiotic Resistance Detection

RPA and DNA-Template: In this study, RPA was performed using the TwistAMP^® Liquid Basic Kit (TwistDxTM Limited; TALQBAS01) according to the manufacturer's instructions. Any modifications are noted at the appropriate point. Amplification took place for 60 min during the incorporation rate studies and for 40 min during the microarray experiments. The volume of the reaction mixture was 25.5 µl. For the multiplex microarray experiments, genomic DNA prepared and provided by the Robert Koch Institute was used with resistance genes from different pathogens (bla_CTX-M15 [E. coli; 735/14–1], bla_NDM [E. coli; 2/10], bla_VIM [P. aeruginosa; 359/11]; bla_KPC [E. coli; 17/11]) and concentrations up to 1 ng/µl (Table 1). The corresponding concentrations are given in the associated results section. Purified PCR template with a concentration of 1 ng/µl to 1.5 ng/µl was used for the general study of incorporation rates and singleplex RPA.Table 1

List of organisms and primer. Illustration of the organisms used in this study including the target resistance gene and the RPA primer.

Organism	Isolate no	Resistance gene	Primer	Sequence	Fragment [bp]
E. coli	2/10	NDM-1	NDM-R *	CAAGCTGGTTCGACAACGCATTGGCAT	220
			NDM-F *	CAACGGTTTGATCGTCAGGGATGGCGG
P. aeruginosa	359/11	VIM-2	VIM-F *	TGGTCTCATTGTCCGTGATGGTGATGAGTTGCT	191
			VIM-R *	TACGTTGCCACCCCAGCCGCCCGAAGGACATC
E. coli	17/11	KPC-2	KPC-F *	CATTCGCTAAACTCGAACAGGACTTTG	809
			KPC-R *	CCAATAGATGATTTTCAGAGCCTTACTG
E. coli	735/14–1	CTX-M15	CTX-M15-F *	TCACGCTGTTGTTAGGAAGTGTGCCGCTGTATGC	141
			CTX-M15-R *	CGATAAAGTATTTGCGAATTATCTGCTGTGT

The isolate number refers to the nomenclature of the Robert Koch Institute. All primer were synthesized and provided by metabion international AG. (*reference: Warmt et al.^{33 (link)}).

Cy5 labelling: For labelling the amplicons with Cy5 fluorescent label, either 5-(3-aminoallyl)-2'-deoxyuridine-5'-triphosphate (Cy5-dUTP) from Jena Bioscience (NU-803-XX-CY5-S) or primer labelled at the 5'-end from metabion international AG were chosen. Again, the respective concentrations are listed at the corresponding position in the results section.