Multilabel hybridization probes for sequence-specific detection of sepsis-related drug resistance genes in plasmids

• Published in: Talanta open Vol. 3; p. 100034
• Main Authors: Hanson, Robert L., Lazalde, Elaine, Knob, Radim, Harris, David H., Akuoko, Yesman, Nielsen, Jacob B., Woolley, Adam T.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2021; Elsevier
• Subjects: DNA hybridization; Drug resistance; Multilabel fluorescence; Plasmid detection; Sequence-specific hybridization; Plasmid detection; DNA hybridization; Drug resistance; Sequence-specific hybridization; Multilabel fluorescence; multilabel fluorescence; drug resistance; plasmid detection; sequence-specific hybridization
• ISSN: 2666-8319; 2666-8319
• DOI: 10.1016/j.talo.2021.100034

•Multilabel probes increase fluorescence signal from labeled bacterial plasmids by 3-fold.•Bacterial plasmid from a dilute sample is concentrated on a porous acrylate monolith in a sequence-specific manner.•Fluorescence signal is linear with loaded plasmid concentration between 1 pM and 1 nM.•This system can serve to increase signal for single-molecule counting and DNA hybridization technologies. Emerging antimicrobial drug resistance is increasing the complexity involved in treating critical conditions such as bacterial induced sepsis. Methods for diagnosing specific drug resistance tend to be rapid or sensitive, but not both. Detection methods like sequence-specific single-molecule analysis could address this concern if they could be adapted to work on smaller targets similar to those produced in traditional clinical situations. In this work we demonstrate that a 120 bp double stranded polynucleotide with an overhanging single stranded 25 bp probe sequence can be created by immobilizing DNA with a biotin/streptavidin magnetic bead system, labeling with SYBR Gold, and rinsing the excess away while the probe retains multiple fluorophores. These probes with multiple fluorophores can then be used to label a bacterial plasmid target in a sequence-specific manner. These probes enabled the detection of 1 pM plasmid samples containing a portion of an antibiotic resistance gene sequence. This system shows the possibility of improving capture and fluorescence labeling of small nucleic acid fragments, generating lower limits of detection for clinically relevant samples while maintaining rapid processing times. [Display omitted]