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

Hello, I'm Tim Duso of Agriculture and AgriFood Canada. In this video, we'll demonstrate a protocol for determining the presence and abundance of specific microorganisms in a complex clinical or environmental sample using a bio plex instrument. This protocol uses fluorescent polystyrene beads that are chemically coupled to a species specific oligonucleotide probe.

An amplicon is generated from the sample using universal PCR primers and the amplicon is hybridized to the probe coupled beads. Two signals are generated with the bio plex instrument. One identifies the bead and one quantifies the hybridization signal.

The overall goal of the procedure is to determine a profile of the microbiota of interest in a rapid and semi-quantitative manner. We've applied this technique to determine bacterial profiles in vaginal swabs and use the data generated to diagnose bacterial vaginosis a clinical condition that is characterized by a change in the microbiota in which lactobacillus organisms become less prevalent and other organisms such as Gardnerella, vais, and apoian vae become detectable in vaginal swabs. It's important to keep in mind however, that this technique can be applied to any other microbiota for which a rapid multiplex assay is desirable.

Let's begin by describing how the procedure works. A microbial profile is generated by amplifying a protein and coating gene chaperone in 60 from all organisms present in A DNA extract of the swab. One of the universal amplification primers is modified by the addition of biotin, as well as the inclusion of phospho ate modified bases.

At the five prime end, the amplicon is made single stranded using T seven exon nuclease, which degrades only the antisense strand. Since the sense strand is protected by a phospho ate modified PCR primer, oligonucleotide probes complementary to the remaining strand are covalently coupled to fluorescent polystyrene beads. With each unique bead color containing a probe detecting a different organism, up to a hundred different beads can be used for a single sample.

The single stranded PCR product from the vaginal swab is hybridized to a probe coupled bead mixture containing probes for all of the organisms of interest. A fluorescent reporter FICO ethin is added that binds to the biotinylated probes through a strep din conjugate. Finally, the hybridization mixture is analyzed on a luminex or bio plex instrument, which examines each bead individually for identity and hybridization signal.

The results of this analysis indicate the presence of specific microorganisms and the intensity of the hybridization signal correlates to the abundance of that organism. In the sample, the presence of BV related organisms can be used to diagnose bv. The main advantage of this technique over existing methods like the grand stain, is that information is generated on the presence of specific microorganisms and their abundance.

Demonstrating the procedure will be Jennifer Town, a research assistant In our laboratory, Re suspend the microspheres by vortexing for approximately 20 seconds. Transfer 400 microliters of microspheres to an einor tube centrifuge at 14, 000 times G for one minute, remove and discard the snat Resus. Suspend the microspheres in 50 microliters of 0.1 molar MES pH 4.5.

Add one animo of capture oligo to the microspheres and mix by vortexing. Add 2.5 microliters of fresh EDC solution to the microspheres. Incubate the reaction at room temperature for 30 minutes in the dark.

Discard the EDC solution that was prepared earlier and prepare a fresh sample of 10 milligrams per milliliter EDC in water. As above, add another 2.5 microliters of fresh EDC solution to the microspheres and vortex for five seconds. Incubate again at room temperature for 30 minutes in the dark.

Wash the beads by adding one milliliter of 0.02%Tween 20 vortex to resuspend the beads centrifuge at 14, 000 times G.For one minute, remove and discard the supernatant. Wash the beads again by adding one milliliter of 0.1%SDS Resus. Suspend the beads in 100 microliters of te buffer.

Enumerate the beads in a hemo cytometer To determine the stock concentration. Prepare a microsphere master mix by diluting each bead to a final concentration of 100 beads per microliter in te buffer pool the coupled beads according to the desired plex of the assay Store the microsphere master mix at four degrees in the dark. This mixture can be stored for months if kept under these conditions, generate PCR product for each sample.

Include the phospholate and biotin modified five prime primer set immediately after the PCR is complete. Add two microliters of T seven exonuclease to each PCR tube incubate at room temperature for 40 minutes. At the end of this incubation, add 12.5 microliters of 0.5 molar E-D-T-A-P-H 8.0 in mix.

This gives a total of about 64.5 microliters of single stranded PCR product. Resus Bend Microsphere master mix with a pipette. Dispense an appropriate amount into an einor tube.

Cap the tube and sonicate in a water bath sonica for two minutes. Dispense 17 microliters of single stranded PCR product into the appropriate wells of a low profile 96. Well plate add 33 microliters of Resuspended Sonicated bead mixture to each.

Well cover the plate with a silicone cover and tap. Gently put the plate into thermocycler with a program of 95 degrees for five minutes, 60 degrees for 10 minutes, 60 degree hold or pause 60 degrees for five minutes. End, start the program.

Make fresh strep Aden FICO Ethin solution. You'll need 25 microliters per well. Make strep and FICO ethin by diluting the stalk at one milligram per milliliter.

One in 50 to 20 micrograms per milliliter with one times tmac buffer. When Thermocycler gets to the 60 degrees hold step, open the lid, remove the silicone cover and add strep din FICO ethin solution directly to each. Well replace the silicone cover close thermocycler lid and resume the program.

When the program is complete, take the plate out and quickly transfer it to the bio plex machine to read. The plate should be read within 10 minutes. Read the plate at 60 degrees.

Ensure that the bio plex has been prewarm to this temperature. This shows the results of corresponding gram stains and luminex profiles of longitudinal samples from a single individual at time zero. The gram stain is consistent with a clinical diagnosis of bv, and this is corroborated by a Fiveplex Luminex assay that shows positive hybridization signals for BV associated organisms, including Gardner Relic, vais, and Apoian Vae.

Lactobacillus Iners is also detected at low levels as time progresses. This individual transitions from a BV microbiota to a normal microbiota as shown by gram stains. Luminex analysis of these same samples shows that Gardnerella Vaginalis abundance peaks and wanes while lactobacillus iners increases to become the dominant organism by the final time point.

Using this method for profiling the microbiota information is generated on the presence of specific organisms as well as their abundance. Since the method is sequence based, probe design can be informed by deep sequencing analysis and microorganisms that are identified by next generation Sequencing methods can be tracked in samples in a rapid, relatively low cost manner. Watching this video should have given you a good understanding of how to generate a microbial profile from a sample using Luminex or Bio Plex instrument.

We've shown you how to couple oligonucleotide probes to the fluorescent beads, generate single stranded alicon from a clinical or environmental sample, perform hybridization, and determine the results on Luminex or Bio Plex instrument. Good luck with your experiments.