Name: multiplex detection of bacteria in complex clinical and environmental samples using oligonucleotide-coupled fluorescent microspheres
Uploaded: 2011-10-23T14:00:00
Description: Watch this Scientific Journal Video about Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres at JoVE.com

We thank Alberto Severini and Vanessa Goleski for help with assay development and critical comments on this manuscript. This work was funded by the Public Health Agency of Canada and the Industrial Research Assistance Program (National Research Council of Canada). Additional support was obtained from the University of Saskatchewan Publication Fund.

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 b...

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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...

<table border="1">
 <tbody>
 <tr>
 <td>Oligonucleotide name</td>
 <td>Company</td>
 <td>Sequence1</td>
 </tr>
 <tr>
 <td>H279BP</td>
 <td rowspan="3">Invitrogen, IDT, or other</td>
 <td>Biotin-OEFOGAIIIIGCIGGIGAYGGIACIACIAC</td>
 </tr>
 <tr>
 <td>H280</td>
 <td>YKIYKITCICCRAAICCIGGIGCYTT </td>
 </tr>
 <tr>
 <td>H1612BP</td>
 <td>Biotin-OEFOGAIIIIGCIGGYGACGGYACSACSAC</td>
 </tr>
 <tr>
 <td>H1613</td>
 <td>&nbsp;</td>
 <td>CGRCGRTCRCCGAAGCCSGGIGCCTT</td>
 </tr>
 <tr>
 <td colspan="3">1O, phosphorothioate-C; E, phosphorothioate-G; F, phosphorothioate-A; I, inosine; Y, C or T; R, A or G; K, T or G; S, C or G.</td>
 </tr>
 </tbody>
</table>
Table 1. Sequences of the modified oligonucleotides for universal cpn60 PCR.
<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>1-Ethyl-3-(3-dimethylamiopropyl) carbodiimide HCl (EDC)</td>
 <td>Pierce</td>
 <td>22980</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluorescent polystyrene beads: MicroPlex Microspheres (Luminex), or Bio-Plex COOH Bead (Bio-Rad)</td>
 <td>Luminex or Bio-Rad</td>
 <td>Bio-Rad: 171-506xxx where xxx corresponds to the bead identifier</td>
 <td>Magnetic beads are becoming available for oligonucleotide coupling and may offer certain advantages. Have not been tried by these authors.</td>
 </tr>
 <tr>
 <td>Capture oligonucleotides (5' amino C12 modified)</td>
 <td>Invitrogen, IDT, or other</td>
 <td>various</td>
 <td>Desalted purity level is acceptable. Sequences of the capture probes used to characterize vaginal swabs are provided in the manuscript on which this protocol is based13.</td>
 </tr>
 <tr>
 <td>T7 exonuclease</td>
 <td>New England Biolabs</td>
 <td>M0263S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Streptavidin-R-phycoerythrin (SA-PE)</td>
 <td>Invitrogen</td>
 <td>S-866</td>
 <td>Be careful to obtain high purity SA-PE; this catalog number is recommended</td>
 </tr>
 <tr>
 <td>Thermowell 96 well PCR plates</td>
 <td>Fisher</td>
 <td>CS006509</td>
 <td>fit into both 96-well thermocycler and BioPlex machine</td>
 </tr>
 <tr>
 <td>Thermowell sealing mat</td>
 <td>Fisher</td>
 <td>CS006555</td>
 <td>Can be re-used; wash with soapy water, rinse well, and dry</td>
 </tr>
 <tr>
 <td>5M TMAC</td>
 <td>Sigma</td>
 <td>T3411</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bio-Plex or Luminex instrument</td>
 <td>Bio-Rad or Luminex</td>
 <td>Bio-Rad : 171-000201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PrimerPlex software for probe design</td>
 <td>Premier Biosoft</td>
 <td>www.premierbiosoft.com</td>
 <td>Suggested for Luminex probe design, although other software platforms may be used</td>
 </tr>
 </tbody>
</table>
Table 2. Specific reagents and equipment.
<table border="1">
 <tbody>
 <tr>
 <td>component</td>
 <td>&mu;l/assay</td>
 <td>&mu;l/100 assays</td>
 <td>final concentration</td>
 </tr>
 <tr>
 <td>10x PCR buffer (Invitrogen)</td>
 <td>5</td>
 <td>500</td>
 <td>1x</td>
 </tr>
 <tr>
 <td>50 mM MgCl2 (Invitrogen)</td>
 <td>2.5</td>
 <td>250</td>
 <td>2.5 mM</td>
 </tr>
 <tr>
 <td>10 mM dNTP</td>
 <td>1</td>
 <td>100</td>
 <td>0.2 mM each</td>
 </tr>
 <tr>
 <td>H279BP, 25 &mu;M</td>
 <td>0.25</td>
 <td>25</td>
 <td>375 nM</td>
 </tr>
 <tr>
 <td>H1612BP, 25 &mu;M</td>
 <td>0.75</td>
 <td>75</td>
 <td>125 nM</td>
 </tr>
 <tr>
 <td>H280, 25 &mu;M</td>
 <td>0.25</td>
 <td>25</td>
 <td>375 nM</td>
 </tr>
 <tr>
 <td>H1613, 25 &mu;M</td>
 <td>0.75</td>
 <td>75</td>
 <td>125 nM</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>34</td>
 <td>3400</td>
 <td>---</td>
 </tr>
 <tr>
 <td>Totals</td>
 <td>44.5</td>
 <td>4450</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Table 3. Suggested mixtures for PCR with modified cpn60 UT primers (Table 1). The assay is set up for 5 &mu;l of template DNA and 0.5 &mu;l (2.5U) of Taq DNA polymerase (Invitrogen). Normally large volumes are prepared (e.g. sufficient for 100 assays) and stored at -20&deg;C.

multiplex detection of bacteria in complex clinical and environmental samples using oligonucleotide-coupled fluorescent microspheres

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 others1-3. 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 criteria6,7. 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 normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. 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.

Watch this Scientific Journal Video about Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres at JoVE.com

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

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.

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

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