BioPoo Assay on eDNA
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BioPoo Assay on eDNA

Overview

This field protocol takes you through the steps of testing fresh or marine water for fecal contamination using QPCR specific for Enterococcus faecalis (universal stool marker) and HF183 Bacteriodes 16S rRNA (human stool specific). These markers were selected based on extensive water contamination research carried out by the CDC, which you can read about here, and are available as a multiplexed QPCR assay (together with an internal positive control) sold by Biomeme Inc. as the BioPoo assay. Finally, this protocol uses the Biomeme portable QPCR platform and consumables, which allows the entire assay to be carried out in the field, without electricity or access to standard lab equipment.

Supply list for packing

General Tips

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Workflow in the field- It worked well to either run through the protocol until the sample was in the homogenization tube or simply collect water samples to bring back to the lab.
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Make sure to change your gloves in between each sample.
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It is advisable to wear shoes that can get wet (or go barefoot :) )

Sample collection

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Use your cell phone to record your GPS coordinates! This will allow you to connect QPCR results with geospatial data later on. You can easily do this without an internet connection by simply snapping a photo from your phone (GPS coordinates are embedded in the pic and can be retrieved later). You may also find it useful to include a sheet of paper in the pic that notes the location, sample number, etc.
  • Set up filter apparatus
  • Fill one 50 ml tube with a fresh or marine water sample.
  • Insert strainer into a second 50 ml tube, and pour water through the strainer and into the collection tube.
    • note- make sure to avoid collecting sand as this clogged some samples taken at the beach. Also, to minimize the number of 50ml tubes used, you can use the same tube per sample site and simply pour through the strainer into the collection tube.
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if you don't have strainers, you can simply let the collected sample sit undisturbed for a few minutes to allow sand and debris to settle before pouring into the filter funnel
  • Pour water sample into filter funnel and use hand-operated vacuum pump to draw this volume through the filter.
  • While sample is being pumped, repeat the collection above at least one more time. Additional samples can be collected if desired. Record volume of water filtered.
    • note- 100ml seems to be a good quantity of water to filter. It was sufficient to detect contamination. It was impossible to filter more than 300ml of ocean water without clogging the filter. Filtering 100ml using the hand pump takes less than 2min.
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Tubes could be rinsed throughly with tap or bottled water between samplings and reused (assuming you are relatively confident that this water doesn't have fecal contamination 🤢)

DNA extraction

You can view a video here

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Set Biomeme sample tray on a flat surface and prepare DNA extraction supplies.
  • Remove the filter cup from the vacuum flask by grabbing the base of the filter cup (blue) with one hand, and the clear sides of the cup with the other hand. Twist to release the blue base (contains the filter). Use a gloved hand to pull the filter out of the base
    • Note: the filter sits on top of a thicker piece of paper. You only want the filter, not the underlying paper.
  • Carefully recover the filter from the funnel, and roll up with capture side facing inward.
    • use tweezers to roll up the filter. Sterilize the tweezers using 70% ethanol between each sample.
  • Slide the rolled filter into empty homogenization vial and orient so that the filter is flat against the inside wall of the vial.
  • Use the plastic transfer pipette to transfer 2ml of homogenization buffer to the homogenization tube.
    • note- if you do not touch the sample with the transfer pipette it can be reused between samples
  • Recap the homogenization tube and shake vigorously for at least 1 minute to properly lyse the sample
  • Attach a 1cc syringe to a filter tip, and use the tip to puncture the foil on the M1 cartridge on the red 'start' location. Best to put two holes in the foil, so that the second hole allows for air to escape as you process the sample.
  • Set this syringe with attached filter aside. You're going to come back to it later.
  • Use a separate 1cc syringe to draw up 1ml of the homogenate.
  • Carefully insert the blunt end of this syringe into one of the holes you just made in the cartridge and slowly dispense the full 1ml volume into the cartridge. Discard this syringe.
  • Now use the syringe with the attached filter tip that you set aside after puncturing the seal. Insert the end into one of the holes in the cartridge and slowly pump up and down 10 times.
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If it becomes hard to pump this volume, it is because your sample tip has clogged, which can happen if the water sample had a lot of particulate that was captured on filter. In the event of a clog, your best bet is to move the filter tip to a new syringe and move on to the next step in the cartridge.
  • Finish moving through each section of the M1 cartridge to complete DNA extraction.
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Never transfer liquid from one part of the M1 cartridge to another!
  • Use your syringe and sample tip to transfer the ~1ml of purified DNA to a self-standing tube. Label tube with sample ID, and record location and other details in your lab notebook and/or on the Notion log book.
  • Repeat this process for up to 8 more samples so that you have a total of nine DNA purifications completed and stored in separate self-standing tubes.
  • At this point you can store samples for later analysis (preferably at -20C, but 4ºC is also OK), or proceed with QPCR immediately

qPCR reaction set-up

You can view a video on adding loading purified sample to Go-Strips here and then placing Go-Strips into your Franklin thermocycler here.

  • We will be using the Biomeme eDNA 'BioPoo' Enterococcus assay. Each tube contains a triplex assay that detects a general fecal marker, Enterococcus faecalis (green channel), the human-specific fecal marker, HF183 Bacteriodes 16S rRNA (amber channel), and an Internal positive control or IPC (red channel).
  • Remove three GO-strips (9 tubes) from packs, set strips in your Biomeme sample tray with the tube connections facing away from you and the tab on the foil seal to the left. Carefully (and with a gloved hand) pull seal off of each GO-strip.
  • Working with one DNA extract at a time, use pipette to transfer 20 ul of DNA to a single tube in a GO-strip.
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Raise GO-strip to eye level to make it easy to see what you're doing. Pipette up and down slowly to resuspend and mix lyophilized reagent throughly. Avoid creating bubbles, particularly at the bottom of the tube. Change tips between tubes to avoid assay cross-contamination.
  • Once DNA has been added to all three tubes in a GO-strip, add rubber caps.
  • Repeat this process with the remaining two GO-strips.
  • You should now have three GO-strips (9 tubes) that contain your 9 DNA extracts and are well mixed and capped.
  • Close the lid on the machine (this pushes the void-filling caps deep into the tubes), pair your phone with the device, and follow the instructions on the phone for setting up your run.
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Login to our lab's Biomeme account on the app using the username beiting@vet.upenn.edu and the password Biomeme1234! (case sensitive). For the 'team' be sure that 'hostmicrobe-three9' is selected.
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When using your phone to set-up the QPCR run, if the QR code on the Go-Strip package isn't scanning properly, try to this fix. After you've logged in as described above, from the app home screen: 1. Click the settings gear icon (top right). 2. Select "Settings" 3. Scroll down and select "Advanced Network Settings" 4. Select "Sync Protocols"

Interpreting your results

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Perhaps the most important thing to keep in mind when interpreting your results is that, since the Biopoo assay is based on detection of DNA, a positive result only means that the target was detected, not that there are live organisms present. For this reason, no conclusions about water potability or safety of recreational water should be based on results from the Biopoo assay. If you’re interested in EPA-approved assays for microbial detection in water, you may want to take a look at the ‘EPA Method 1600’, or Enterolert and Colilert assays from IDEXX.
  • A good strategy is to run all 9 of your samples, take time to review the results on your phone, then decide which, if any, samples need to be rerun.
  • Because the IPC is so bright, it is best to view each assay 'color' separately in the software (simply touch to toggle colors on/off), otherwise the IPC signal (red) can overwhelm and dwarf the Entero (green) or HF183 (amber) signals on the graph.
  • When viewing your data, be sure to also toggle between 'baseline' and 'raw' views. If you see a weak signal and are wondering if it is real, you should see it in both views. On the other hand, if a particular sample only appears to be positive in baseline view, but not raw, then it is probably not real.

Possible assay outcomes

NameEnterococcus (green)HF183 (amber)IPC (red)Interpretation
Outcome 1 (most likely)

Water sample was NOT contaminated with human or animal feces

Outcome 2 (possible)

Water sample was contaminated with human feces

Outcome 3 (possible)

Water sample was contaminated with feces, but likely from animals rather than humans

Outcome 4 (possible)

IPC (and all assays) failed. Sample needs to be rerun with less input (see below for more info)

Outcome 5 (unlikely)

This would be an unusual result and may require a rerun

Outcome 6 (unlikely)

IPC failed. Sample needs to be rerun with less input (see below for more info)

Outcome 7 (unlikely)

IPC failed. Sample needs to be rerun with less input (see below for more info)

Outcome 8 (unlikely)

IPC failed. Sample needs to be rerun with less input (see below for more info)

  • A common cause that can lead to the IPC failing is that natural inhibitors present in the water sample may have made it through extraction and are blocking your PCR reaction from working. This often happens if a lot of biomass was captured on the filter funnel and/or the syringe filter tip begins to clog during DNA extraction.
  • To address a failed IPC reaction, repeat the assay with 4 µl of DNA instead of 20 µl of DNA. This 5-fold reduction in input often dilutes the inhibitors enough that the reaction proceeds normally. If you still get poor results, try using 2 µl or 1 µl of DNA as input.
  • Using a new GO-strip, resuspend lyophilized assay reagent with 16 µl of molecular grade water, then add 4 µl, 2 µl , or 1 µl of DNA sample. Repeat QPCR as above.