High-throughput homogenous assay for the direct detection of Listeria monocytogenes DNA

Authors: Armstrong, Cheryl; Capobianco, Joseph; Nguyen, Sarah; Guragain, Manita; Liu, Yanhong

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/11/2024
Publication Date: 3/25/2024
Citation: Armstrong, C.M., Capobianco Jr, J.A., Nguyen, S.C., Guragain, M., Liu, Y. 2024. High-throughput homogenous assay for the direct detection of Listeria monocytogenes DNA. Scientific Reports. 14:7026. https://doi.org/10.1038/s41598-024-56911-8.
DOI: https://doi.org/10.1038/s41598-024-56911-8

Interpretive Summary: The Amplified Luminescent Proximity Homogenous Assay-linked Immunosorbent Assay (AlphaLISA) is a high throughput, bead-based diagnostic assay that has been used in both the medical and agricultural fields. Typical AlphaLISAs are based upon protein interactions, hence requiring the development of antibodies. Here, we developed a novel AlphaLISA technology (oligo-AlphaLISA) that allowed direct detection of DNA. We applied this novel approach towards the identification of an important foodborne pathogen, Listeria monocytogenes. This newly developed oligo-AlphaLISA allowed the differentiation of eight L. monocytogenes strains, which are currently regulated in food, from other closely related species of Listeria through the use of only a single nucleotide difference that existed within the 16S rDNA region. Post-optimization, the assay could detect as low as ~250 attomole or 1.5 x 10^8 copies of target DNA. The assay successfully detected L. monocytogenes in two food matrices (non-fat milk and apple juice). Because genotypic differences can now be identified with this assay, oligo-AlphaLISAs have a strong potential for use as tools for the rapid detection of targets with known genome sequences, without the time and expense associated with the development of antibodies.

Technical Abstract: The bead-based immunoassay known as the Amplified Luminescent Proximity Homogenous Assay-linked Immunosorbent Assay (AlphaLISA), has been used for the detection of various protein targets; however, its ability to detect specific nucleic acid sequences is not well established. Here, the capabilities of the AlphaLISA technology were expanded to include direct detection of DNA and was applied to the detection of Listeria monocytogenes. Parameters were defined that allowed the newly developed oligo-AlphaLISA to differentiate L. monocytogenes from other species of Listeria through the use of only a single nucleotide polymorphism that existed within the 16S rDNA region. Investigations into the applicability of this assay with different matrices demonstrated its utility in both milk and juice, although differences in signal intensities were observed. One remarkable feature of the oligo-AlphaLISA is that greater sensitivity could be achieved through the use of multiple acceptors compared to only a single acceptor, even when only a single donor was employed. Additional acceptors are easily incorporated into the assay and a 10-fold change in the detection limit was readily achieved here, with detection limits of 250 attomole of target being recorded. In summary, replacement of antibodies with oligonucleotides allows us to take advantage of genotypic difference(s) and implement the AlphaLISA as a tool for the detection of additional biological markers important for human health.