Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Summary

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Abstract

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others^1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition⁴, and it can be difficult to manage clinically⁵. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria^6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota⁸. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal^9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV^11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform¹³. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample¹⁴. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

Protocol

This method was used in the research reported in Dumonceaux et al. J. Clin. Microbiol. 47, 4067-4077, doi:10.1128/jcm.00112-09 (2009).

A schematic diagram depicting the overall procedure is presented in Figure 1.

1. Bead coupling

This describes the methods to be used for coupling oligonucleotide probes to polystyrene Luminex beads (see Table 2). Volumes are adapted slightly for evaluation of new capture probes on a trial basis; these volumes are indicated in parentheses.

1. Remove 1-Ethyl-3-(3-dimethylamiopropyl) carbodiimide HCl (EDC) powder from -20°C dessi....

Discussion

The specificity of signal generation is of critical importance; you must be confident that the signal observed truly reflects the detection of amplicon generated from that organism. Software such as PrimerPlex (Premier Biosoft) can help to design probes that will hybridize efficiently, but they may or may not cross-hybridize to non-target species. When universal PCR primers are used as described in this protocol, it is important to keep in mind that the amplicon generated represents all of the organisms present in the sa.......