Fully Automated FISH
Fluorescence in-situ hybridization or FISH is an established technique to detect and localize specific sequences of nucleic acids. The challenge often faced by labs using this technique is the manual intervention and multiple devices necessary to perform the sample preparation, permeabilization and hybridization, microscopy, and data analysis. We change all of that.
The FISH technology developed for the Accelerate Pheno™ system creates a hands-free experience for the lab by automating the entire process. The technology enables sample preparation, rapid hybridization of several mono-labeled DNA oligonucleotides that target ribosomal RNA. It includes signal capture using digital microscopy and data analysis within a single system. And it does this all automatically with results available in approximately 2 hours.
Identification methods today frequently rely on purified samples of one organism, adding 24 hours or more of subculturing before testing. Techniques using mass-spectrometry or biochemical reactions often fail when presented with a sample of mixed bacteria or fungi. Many highly sensitive PCR techniques offer multi-species detection, but for a limited menu of species.
Designed to address these complex issues, Accelerate scientists and engineers developed an approach involving an array of simultaneous FISH tests. For each test, signal from a target probe is compared with signal from universal bacterial and eukaryotic probes. Colocalization of of the target probe signal with universal probe signal confirms the presence and identity of the target while differentiating from non-specific staining.
Comparison of dark-field, universal, and target signals informs organism detection and identification.
Signal generated from the automated FISH technology enables quantitation of individual cells and provide, where relevant, an organism concentration or CFU/mL.
A monomicrobial sample is suspected when only one target organism is detected across each FISH assay and universal probe signals colocalize with target probe signals. The monomicrobial result is confirmed by comparing the relative quantities of target and non-target organisms in a sample using a universal nucleic acid stain.
When one organism is detected and identified and its relative quantity matches that of the universal bacterial or eukaryotic probe and nucleic acid stain, the analysis flags the result as monomicrobial.
By leveraging data from each target probe signal, its colocalization with universal signal, and comparing their relative quantities with nucleic acid staining, the fully automated FISH technology by Accelerate allows for non-target or off-panel organism detection and polymicrobial identification.
Red: universal probe signal / Green: target organism signal