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 basis; these volumes are indicated in parentheses.
<ol>
 <li>Remove 1-Ethyl-3-(3-dimethylamiopropyl) carbodiimide HCl (EDC) powder from -20&deg;C dessicator and warm it to room temperature.</li>
 <li>Resuspend the microspheres by sonciation in a waterbath sonicator for 20 seconds, then vortexing approximately 20 seconds.</li>
 <li>Transfer 400 &mu;l (100 &mu;l) of microspheres to an Eppendorf tube (this corresponds to 5x106 or 1.25x106 microspheres). Luminex suggests the use of low protein binding microcentrifuge tubes (e.g. Eppendorf Protein LoBind tubes, catalog number 0030 108.094) to prevent the uncoupled beads from sticking to the tubes and interfering with bead recovery. We have not found this to be a problem using standard polypropylene microcentrifuge tubes, which we typically use (e.g. VWR catalog number 87003-298).</li>
 <li>Pellet at 14000 x g for 1 minute. Remove and discard the supernatant.</li>
 <li>Resuspend the microspheres in 50 &mu;l (12.5 &mu;l) of room-temperature 0.1 M 2-(N-Morpholino)ethanesulfonic acid (MES) pH 4.5.</li>
 <li>Prepare a fresh solution of EDC at 10 mg/ml in water. 
 <ol>
 <li>Prepare less than 1 ml of this solution by weighing 5-10 mg of EDC on an analytical scale, then adding water to 10 mg/ml.</li>
 </ol>
 </li>
 <li>Add 1 nmol of 5'-amino C12-modified capture oligonucleotide (Table 2) to microspheres and mix by vortexing. 1 nmol is 5 &mu;l of 200 &mu;M oligonucleotide.</li>
 <li>Add 2.5 &mu;l of fresh EDC solution to the microspheres and mix by vortexing for 5 seconds.</li>
 <li>Incubate at room temperature for 30 minutes in the dark.</li>
 <li>Discard the EDC solution (step 1.6) and prepare a fresh sample of 10 mg/ml EDC in water as above (step 1.6).</li>
 <li>Add another 2.5 &mu;l of fresh EDC solution to the microspheres and vortex for 5 seconds.</li>
 <li>Incubate at room temperature for 30 minutes in the dark.</li>
 <li>Wash the beads by adding 1 ml of 0.02% Tween 20. Vortex (optionally sonicate for 20 seconds as well) to resuspend the beads.</li>
 <li>Centrifuge 14000 x g 1 minute. Remove and discard the supernatant.</li>
 <li>Wash the beads again by adding 1 ml of 0.1% sodium dodecyl sulfate (SDS). Vortex (optionally sonicate for 20 seconds as well) to resuspend the beads.</li>
 <li>Centrifuge 14000 x g 1 minute. Remove and discard the supernatant.</li>
 <li>Resuspend the beads in 100 &mu;l (25 &mu;l) of Tris-EDTA (TE) buffer [10 mM Tris-Cl pH 8.0, 1 mM EDTA, pH 8.0]. </li>
 <li>Enumerate the beads in a haemocytometer or Coulter counter to determine the stock concentration.</li>
 <li>Prepare a Microsphere Master Mix by diluting each bead to a final concentration of 100 beads/&mu;l in TE buffer. Pool coupled beads corresponding to the desired plex of the assay (e.g. for a 10-plex assay, mix 10 different coupled beads at a final concentration of 100/&mu;l of each bead).</li>
 <li>Store the Microsphere Master Mix at 4&deg;C in the dark. The mixture can be stored for months if kept under these conditions.</li>
</ol>
2. Chaperonin 60 universal target (cpn60 UT) amplicon production and generation of single strands.
<ol>
 <li>Generate polymerase chain reaction (PCR) product for each sample. Include the phosphorothioate- and biotin-modified 5' primer set (Table 1). See Table 3 for volumes to mix and concentrations.</li>
 <li>Immediately after the PCR is complete, add 2 &mu;l (20 units) of T7 exonuclease to each PCR tube (the PCR buffer will suffice for the T7 reaction). Incubate the reaction at room temperature (~22-25&deg;C) for 40 minutes. </li>
 <li>At the end of this incubation, add 12.5 &mu;l of 0.5 M ethylene diamine tetraacetic acid (EDTA) pH 8.0 and mix. This gives a total of ~64.5&mu;l of single-stranded PCR product.</li>
</ol>
3. Hybridization of single-stranded PCR product to oligonucleotide-coupled polystyrene beads.
<ol>
 <li>Prewarm the instrument to 60&deg;C to maintain hybridization temperature during analysis. Turn on the platform heater using the instrument's software and be sure to use the brass heating block that fits the PCR-style plate that contains the hybridized bead mixture.</li>
 <li>Resuspend Microsphere Master Mix (step 1.20) with a pipette, dispense an appropriate amount into an Eppendorf tube, cap the tube, and sonicate in a waterbath sonicator for 2 minutes. Alternatively, to ensure absolute consistency in bead resuspension, the microsphere master mix can be resuspended by vortexing and pipetting, then sonication as above, then dispensing the desired amount into a polypropylene Eppendorf tube.</li>
 <li>Dispense 17 &mu;l of single-stranded PCR product (step 2.2) into the appropriate wells of a low-profile 96-well Thermowell PCR plate (Table 2). Add 33 &mu;l of resuspended, sonciated bead mixture to each well. Cover with silicone cover (Table 2) and tap gently.</li>
 <li>Put the plate into the thermocycler with a program of: 95&deg;C for 5 min, 60&deg;C for 10 min, 60&deg;C hold, 60&deg;C for 5 min, end. Start the program.</li>
 <li>Make fresh streptavidin-phycoerythrin (SA-PE) solution; you will need 25 &mu;l per well (make several wells extra). Make SA-PE solution by diluting stock SA-PE (1 mg/ml) 1:50 to 20 &mu;g/ml with 1x tetramethylammonium chloride (TMAC) buffer (3 M TMAC; 0.1% Sarkosyl; 50 mM Tris-HCl, pH 8.0; 4 mM EDTA, pH 8.0).</li>
 <li>When the thermocycler gets to the 60&deg;C hold step, open the lid, remove the silicone cover and add SA-PE solution directly to each well (DO NOT take the plate out of the thermocycler). Replace the silicone cover, close the thermocycler lid and resume the program.</li>
 <li>When the program is complete, take the plate out and quickly transfer it to the BioPlex machine to read. Plate must be read within 10 minutes. Read at 60&deg;C; ensure that the BioPlex has been prewarmed to this temperature. Be certain that the probe height has been adjusted to accommodate the plate utilized, as described in the user&#x2019;s manual for the instrument used.</li>
 <li>For accurate bead and signal detection, the gate settings on the BioPlex must be set according to the type of microspheres being used. BioRad polystyrene beads require a gate setting of 4,335-10,000 while magnetic microspheres require a setting of 5,000-25,000. There is also the option of running the plate using the High PMT setting which increases the reporter gain value. This can increase the intensity of lower signals however it is important include an appropriate negative control as background signal will also be increased.</li>
</ol>
4. Representative results: 
One of our goals in developing and implementing this assay was to make it as simple and streamlined as possible. We therefore optimized the critical post-amplification steps, including T7 exonuclease treatment time and hybridization time in order to develop an assay that can be completed in a reasonable time frame. As shown in Figure 2A, T7 treatment of the amplicon is essential for signal generation, as most probes had little or no signal with short or no T7 treatment. The signal increased linearly until approximately 40 minutes, at which time the signal increase slowed. No degradation of the signal was observed even at 2 hours of T7 treatment time, indicating that the phosphorothioate modification of the primers is highly effective in preventing target strand degradation as described15. We chose 40 minutes for T7 treatment time to minimize the overall protocol time, but it is clear from Figure 2A that T7 treatment can go on for much longer. We also determined the effect of hybridization time on signal generation (Figure 2B) and found that 10 minutes was sufficient for maximum signal, as no further increase in signal was observed even after 4 hours of hybridization. Therefore, a 10 minute hybridization step was chosen, again to minimize the overall assay time. With these results in mind, and with rapid DNA extraction techniques such as InstaGene (Bio-Rad), the overall assay including DNA extraction, PCR, and Luminex analysis can be completed in 4-5 hours.
On a cautionary note, the level of aggregation of the polystyrene beads during a Luminex or BioPlex run can have a major impact on the efficiency of the assay. The BioPlex software will display the level of bead aggregation, which occurs when more than one bead is detected in the laser path and results in the exclusion of the hybridization signal from the aggregate. Apparent bead aggregation can also be caused by any particulate matter that is not the proper size for a single bead, and can even be caused by air bubbles. In any of these events, the signal from the bead aggregate, air bubble, or particle is discarded. In most cases, we find that bead aggregation is minimal (Figure 3A) and the targeted level of 100 counting events for each bead is easily achievable. Occasionally, however, the beads show a moderate (Figure 3B) or severe (Figure 3C) level of aggregation. In these cases, since most of the data is discarded, the instrument may have trouble reaching 100 events per bead type and the results may be questionable. The sonication step (step 3.2) is meant to minimize bead aggregation. In addition, storing the beads as a dilute microsphere master mix (step 1.20) may help. We have noticed that excluding TMAC from the hybridization buffer &#x2013; adding it to the SA-PE diluent instead (step 3.5) &#x2013; helps to minimize bead aggregation. Moreover, while we have not tested this, the newer magnetic beads available from Luminex or BioRad are thought to display less of a tendency to aggregate.
The results of the application of a 5-plex Luminex array targeting G. vaginalis, A. vaginae, L. iners, L. crispatus, and L. gasseri along with corresponding Gram stained vaginal swab smears are shown in Figure 4. These samples were taken from a single individual at multiple time points. At time 0, the individual was diagnosed with BV based on Gram stain (Figure 4A) and the Luminex results from this same sample (Figure 4B) show that of the organisms represented in the array, G. vaginalis was most prevalent, while A. vaginae and L. iners were also positive. Nine days later, the individual was still BV positive and the signal for G. vaginalis had increased substantially while A. vaginae and L. iners were still positive. Importantly, after this time the individual began to transition to a normal microbiota as Gram positive bacilli became detectable in the smears (Figure 4A) while the signal for G. vaginalis waned and the signal for L. iners increased (Figure 4B). By our original definition of BV with this method (samples positive for G. vaginalis and/or A. vaginae were considered BV positive)13 , this individual was BV positive at all time points, although the Gram stain failed to detect G. vaginalis in the latter two time points. With the Luminex method described here, trends can be easily compared to sequencing-based methods, and the results of the Luminex assay typically corroborate Gram stains13 while providing additional information on organism identity and abundance.
<img src="/files/ftp_upload/3344/3344fig1.jpg" alt="Figure 1" /> 
Figure 1. Schematic diagram of the Luminex protocol for determining the microbiota profile of a complex clinical or environmental sample. The protocol starts with template DNA that has been extracted from the sample of interest. (1) Generate PCR product from template DNA using strand-specific biotinylated, phosphorothioate-modified cpn60 UT PCR primers coupled with unmodified cpn60 UT PCR primers (Table 1); (2) This generates a PCR product pool representing the microbiota with the biotin-phosphorothioate modification on one strand; (3) Digest the double-stranded PCR product with T7 exonuclease, which cannot degrade the phosphorothioate-modified strand and therefore generates single-stranded DNA that is modified at the 5' end with biotin; (4) Couple polystyrene beads to species-specific cpn60 UT probes &#x2013; each bead has a unique spectral address (indicated by bead color) that is discernable by the Luminex or Bio-Plex instrument; (5) Hybridize the single-stranded PCR product generated from the sample of interest to the suite of species-specific oligonucleotide-coupled beads; (6) Add streptavidin-phycoerythrin conjugate that binds to the biotinylated single-stranded PCR product and acts as an indicator of hybridization; (7) Determine the spectral address (bead identity) and hybridization signal intensity using a Luminex or Bio-Plex instrument. At least 100 beads are counted for each bead identity and the median fluorescence intensity (MFI) of the phycoerythrin signal is reported as the output. Up to 100 different bead types can be evaluated simultaneously but an assay showing the discrimination of three bead types is illustrated. (8) When the MFI of PCR replicates generated from the same sample is significantly greater than the negative control for a given bead (one-tailed student's t-test, p&lt;0.05), the sample is considered to be positive for that organism.
<img src="/files/ftp_upload/3344/3344fig2.jpg" alt="Figure 2" /> 
Figure 2. Optimization of Luminex assay parameters. Five different probes targeted to Peptostreptococcus anaerobius were used with amplicons generated from a mixed template comprising plasmids containing the cloned cpn60 UT of 20 bacteria known to be associated with the vagina, including P. anaerobius. (A). Effect of T7 exonuclease treatment time. The protocol described above was followed but the time of treatment with T7 exonuclease was varied prior to hybridization and the median fluorescence intensity (MFI) was determined for all probes. (B). Effect of hybridization time. The protocol described above was followed with a variety of hybridization times. The same sets of probes were used with an amplicon generated from the same template as in A and the median fluorescence intensity (MFI) was determined for all probes.
<img src="/files/ftp_upload/3344/3344fig3.jpg" alt="Figure 3" /> 
Figure 3. Determination of bead aggregation using BioPlex software. Three examples are given of BioPlex runs in which the level of bead aggregation is (A) acceptable (5%); (B) borderline (50%); and (C) unacceptable (80%).
<img src="/files/ftp_upload/3344/3344fig4.jpg" alt="Figure 4" /> 
Figure 4. Application of a 5-plex Luminex array to BV diagnosis in sequential samples taken from the same individual. (A). Gram-stained slides showing the traditional diagnostic used to assess each sample for BV. (B). Application of a 5-plex Luminex array prepared and executed as described in this protocol to the same four samples shown in (A). Bacterial targets whose MFI signal was significantly positive by our definition (one-tailed student's t-test, p&lt;0.05) are indicated by an asterisk (*).

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No conflicts of interest declared.

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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Research

JoVE Journal

Immunology and Infection

16.1K visualizações.  Agriculture and Agri-Food Canada. Nós descrevemos um método multiplex para a detecção de microorganismos em uma amostra usando oligonucleotídeos-coupled partículas fluorescentes. Amplicon de todos os organismos dentro de uma amostra é hibridizada com um painel de sonda acoplada contas. Um instrumento Luminex ou Bio-Plex é usado para consultar cada grânulo para o tipo de grânulo e sinal de hibridização.

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

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to the best of these authors' knowledge, no method has been developed to allow for the in situ visualization of the 
bacteria in histological sections.
While bacterial localization has been determined in vivo in previous studies using plate counts 3, radiometric labeled 4, 
or bioluminescent bacteria 5, most of these studies have only been performed at the gross organ level, with one exception 6. This limitation 
is of particular concern because E. ictaluri has a complex infection cycle 1,7, and it has a variety of virulence factors 8,9. 
The complex interaction of E. ictaluri with its host is similar in many respects to Salmonella typhi 10, which is in the same taxonomic family.
Here we describe a technique allowing for the detection of bacteria using indirect immuno-histochemistry using the monoclonal Ed9 antibody described by Ainsworth et al.11.
Briefly, a blocking serum is applied to paraffin embedded histological sections to prevent non-specific biding. Then, the 
sections are incubated with the primary antibody: E. ictaluri specific monoclonal antibody Ed9. Excess antibodies are rinsed away and the FitC 
labeled secondary antibodies are added. After rinsing, the sections are mounted with a fluorescent specific mounting medium. 
This allowed for the detection of E. ictaluri in situ in histological sections of channel catfish tissues.

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

Here we describe a procedure allowing the labeling of Edwardsiella ictaluri in situ in histological sections from channel catfish Ictalurus punctatus using indirect immunohistochemistry with monoclonal antibodies Ed9 as a primary, and fluorescent FitC labeled antibodies as a secondary. This allowed for the detection of the bacterium using fluorescent microscopy.

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.