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Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres
Published on: October 23, 2011
An All-in-One Highly Multiplexed Diagnostic Assay for Rapid, Sensitive, and Comprehensive Detection of Intraocular
Paulo J M Bispo1, Nicole Belanger1, Ashley Li2
1From the Department of Ophthalmology (P.J.M.B., N.B., A.L., R.L., G.S., W.C., J.C., M.S.G., L.S.), Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, USA; Infectious Disease Institute (P.J.M.B., N.B., J.C., M.S.G., L.S.), Harvard Medical School, Boston, Massachusetts, USA.
Purpose:
Intraocular infections are sight-threatening conditions that can lead to vision loss. Rapid identification of the etiologies plays a key role in early initiation of effective therapy to save vision. However, current diagnostic modalities are time consuming and lack sensitivity and inclusiveness. We present here a newly developed comprehensive ocular panel designed to improve diagnostic yields and provide a tool for rapid and sensitive pathogen detection.
Design:
Experimental laboratory investigation.
Methods:
A panel containing 46 pathogens and 2 resistance/virulence markers that are commonly detected in intraocular infections was developed. Genomic targets were scrutinized for stretches predicted to be specific for a particular species while being conserved across different strains. A set of primers for sample enrichment, and two 50mer NanoString compatible probes were then designed for each target. Probe-target hybrids were detected and quantified using the NanoString nCounter SPRINT Profiler. Diagnostic feasibility was assessed in a pilot clinical study testing samples from infectious retinitis (n = 15) and endophthalmitis (n = 12) patients, for which the etiologies were confirmed by polymerase chain reaction (PCR) or culture.
Results:
Analytical studies demonstrated highly sensitive detection of a broad spectrum of pathogens, including bacteria, viruses, and parasites, with limits of detection being as low as 2.5 femtograms per reaction. We also found excellent target specificity, with minimal cross-reactivity detected. The custom-designed NanoString ocular panel correctly identified the causative agent from all clinical specimens positive for a variety of pathogens.
Conclusion:
This highly multiplexed panel for pathogen detection offers a sensitive, comprehensive, and uniform assay run directly on ocular fluids that could significantly improve diagnostics of sight-threatening intraocular infections.
