Microbial DNA Analysis in the Field Using a Biological Extraction Field Kit and a Field qPCR Unit

When considering molecular biological analyses, like qPCR, there are several limitations for sample management. A clear example is the demand for simple, reliable tools to quantify contaminant-degrading bacteria at remote sites impacted by train derailments or petroleum releases, supporting timely evaluation of environmental risk and impact^1,2,3,4,5. The primary limitation is that no matter how cold you keep the sample, microbial communities can (and will) shift over time^6,7. To ensure data accurately reflects the original environment from which the samples were collected, it is recommended that the DNA be extracted from those samples within 24 h⁸. For an engineer collecting microbial samples on an offshore oil platform, or a scientist deep in the interior of a rain forest, this timeline is not feasible. Getting a water or soil sample from one of these remote locations to a shipping company within the same day and then overnighting to a laboratory (all while maintaining refrigeration), where the DNA can then be extracted prior to analysis, is simply impossible.

Once the DNA is extracted, the clock stops, and the sample's entire genetic information is preserved indefinitely with proper storage techniques⁹. This makes extracted DNA incredibly unique compared to the vast majority of chemical, metallurgical, and biological samples - most of which have a limited shelf life. Archived DNA can be retrieved years or decades later and analyzed for new targets on new platforms. For this reason, it is crucial that the extracted DNA truly represents the original microbial community, and not the altered one from delayed transit.

Additionally, the requirement of a laboratory to extract and analyze the samples greatly limits both flexibility and consistency in sampling programs. The cost of logistics alone can be prohibitory for certain projects¹⁰. The ability to analyze samples, accurately quantifying genetic targets of interest within hours or even minutes of sampling, opens many doors for environmental projects around the globe.

NOTE: The following protocol was performed using a Bio-Extract Kit (biological extraction field kit) and the FieldQuant qPCR (Field-based qPCR).

1. Biomass sampling from groundwater and a DNA isolation protocol using an encapsulated filter and a biological extraction field kit

1. Wear gloves and safety glasses for all steps in step 1.
2. Prepare the peristaltic pump and tubing and purge the well using standard volume purging, following the Environmental Protection Agency (EPA) Groundwater Sampling guidance¹¹.
3. Remove the encapsulated filter, adapter, clamp, and caps from the provided centrifuge tube (Figure 1).
4. Pass the tubing (1/4-5/16 in inner diameter [I.D.]) through the provided clamp and tighten the clamp to secure the hose to the locking luer adapter (Figure 2).
5. Attach the filter's inlet to the Luer-Lock adapter installed in the previous step.
6. Place the filter within a receiving container so that the amount of water filtered can be accurately measured.
7. Filter up to 1 L of water using low-flow rates (<0.5 L/min). The amount of water filtered will vary depending on the turbidity of the water.
   1. If the filter clogs before 1 L is filtered, stop and continue to the next step.
      NOTE: DNA analysis is sensitive and can be performed with as little as 10 mL, depending on the biomass of the system.
8. Manually record the volume that passed through the filter in milliliters (mL). Discard the filtered water.
9. Cap the filter on both ends. Ensure that the thinner end is closed with the red cap and the thicker end is closed with the white Luer-Lock plug.
10. Place the filter back into the plastic tube provided.
11. Affix the label to the centrifuge tube and notate the amount of water that passed through the filter, the location, sampling date, and the analyses requested. Store at 1-4 °C until extracted.
    NOTE: Filters must be extracted within 24 h of sampling.
12. Using the included alcohol prep wipes, clean a flat surface that can be used to stage the extraction (e.g., a wooden board, truck tailgate, etc.)
13. To extract the DNA, lay out all components of the biological extraction field Kit onto the flat, clean surface (Figure 3).
    1. Uncap both ends of the filter and set the caps aside for use later. Find and open one of the 3 mL syringes included in the kit. Pull air into the 3 mL syringe and connect it to the filter via the locking luer connection.
    2. Push the air out of the syringe to push any remaining water out of the filter. Disconnect the syringe and pull in more air before repeating.
    3. Repeat several times, ensuring that as much water as possible is pushed out of the filter. Recap the red outlet cap.
14. Invert the closed Solution A syringe several times to ensure it is well mixed. Remove the luer-locking syringe cap.
15. Connect the Solution A syringe to the encapsulated 0.2 µm filter inlet via the luer-locking tip and slowly add all of Solution A by depressing the plunger. Pushing too fast can cause back pressure to form, so be sure to lower the plunger slowly. If filter clogging is preventing the addition of Solution A, remove the red outlet cap of the filter and try again. Recap the filter.
16. Vigorously hand-shake the closed filter for 5 min.
    NOTE: To prevent fatigue, multiple samples can be prepped up to this step, and all can be hand shaken at the same time. If available, a rechargeable vortex mixer can be used to perform this step rather than hand-shaking.
17. Invert the closed Solution B syringe several times to ensure it is well mixed. Remove the luer-locking cap from the syringe.
18. Remove the encapsulated filter inlet cap. Connect the Solution B syringe to the encapsulated filter inlet via the luer-locking tip and slowly add all of Solution B by depressing the plunger. Pushing too fast can cause back pressure to form, so be sure to lower the plunger slowly. Recap the filter inlet cap.
19. Vigorously hand-shake the closed filter for 5 min.
    NOTE: To prevent fatigue, multiple samples can be prepped up to this step and all be hand shaken at the same time. If available, a rechargeable vortex mixer can be used to perform this step rather than hand-shaking.
20. Remove the inlet cap.
21. With the filter inverted, push 1 mL of air into the filter using a 3 mL syringe attached to the encapsulated filter unit and then pull back on the plunger to remove as much of the lysate (liquid) as possible.
    NOTE: If the lysate is particularly viscous, it may be necessary to push and pull the plunger several times to remove the solution from the filter.
22. Add the lysate to a 5 mL bead tube. Cap the tube and seal it with parafilm on the top.
23. Vigorously hand-shake the tube for 5 min. Place the tube upright while continuing with the steps.
    NOTE: To prevent fatigue, multiple samples can be prepped up to this step, and all can be hand shaken at the same time. If available, a rechargeable vortex mixer can be used to perform this step rather than hand-shaking.
24. While avoiding contamination of the foil top, place the sample prep cartridge onto a flat surface.
25. Remove the 1 mL syringe from its packaging and screw the luer-lockable column onto the end of the syringe. Use the pointed tip of the column to poke two holes into the first section of the sample prep cartridge.
    CAUTION: Some sections of the cartridge contain chaotropic salts and/or ethanol. Wear gloves and safety glasses.
26. Using a plastic volumetric pipette, transfer 1 mL of lysate (liquid only, avoid transferring beads as much as possible) from the bead tube into the newly punched hole of the first sample prep cartridge section. For best results, slowly add the lysate at a slight angle when pipetting into the hole in the foil.
27. Insert the syringe-column back into the red section of the cartridge. Pull the fluid completely into the syringe and then push it out. Perform this pump action 10 times.
28. Push all the fluid out of the syringe and back into the red section of the cartridge before moving to the next step. No liquid should be transferred from one section to another - this applies to each remaining step of the sample DNA isolation protocol.
29. Position the pointed tip of the syringe-column over the red-orange section and puncture two holes through the foil barrier. Use the plunger to pull the full syringe volume the fluid all the way up the syringe and pump all the way back out. Repeat this step once for a total of 2 pumps, pushing all liquid out. Do not transfer any liquid to the next step.
30. Position the pointed tip of the syringe-column over the orange section and puncture two holes through the foil barrier. Use the plunger to pull the full syringe volume of fluid into the syringe and pump all the way back out. Do not transfer any liquid to the next step.
31. Position the pointed tip of the syringe-column over the yellow section and puncture twice through the foil barrier. Use the plunger to pull the full syringe volume of fluid into the syringe and pump all the way back out. Do not transfer any liquid to the next step.
32. Position the pointed tip of the syringe-column over the blue air-dry section and puncture through the foil barrier only once. Use the plunger to pull the full syringe volume of air into the syringe and quickly pump back out. Quickly repeat this pumping action a minimum of 20 times until the column appears dry.
33. Position the pointed tip of the syringe-column over the final green section and puncture through the foil barrier twice. Use the plunger to pull the full syringe volume of fluid into the syringe and pump all the way back out. Repeat this step for a total of 5 pumps.
34. Using the 1 mL syringe and sample prep column, transfer all the solution from the green section into a well-labelled 1 mL microtube - this is the field-extracted DNA sample. Store at 1-10 °C (e.g., in a cooler using ice/ice packs, or in a refrigerator, etc.) for up to a week, or until use.
    NOTE: For long-term storage, DNA samples can be stored at -20 °C indefinitely.

2. Field-based qPCR analysis protocol using a field qPCR unit

NOTE: The field-based qPCR instrument and the phone that controls the instrument rely on rechargeable batteries. Ensure that both are fully charged before traveling to the field.

1. Place up to three field-extracted DNA samples into the pure sample slots of the tray. Ensure that there is one sample per section: one sample in slots 1, 2, or 3, one sample in slots 4, 5, or 6, and one sample in slots 7, 8, or 9.
2. Place the assay strips for each sample into the corresponding A, B, or C strip slots on the tray. Line up the first well on the strip with the first slot on the tray.
3. Unlock the phone and open the application named after the trademarked qPCR instrument. The phone and the field qPCR unit connect via integrated short-range wireless. When the app opens, tap the Start Run button.
   NOTE: The application is pre-loaded on the included phone and cannot be downloaded elsewhere.
4. On the next screen, tap the Generate IDs button.
5. This next screen is where the sample information for each sample is entered; tap under Sample ID and type in the sample name.
   1. Tap sample units and select the appropriate unit of volume for the sample type that was used in the extraction (grams [g], milliliters [mL], or swab).
   2. Then tap under sample amount and type in the volume of sample that was used in the field extraction above (e.g., milliliters of water filtered). Repeat this process for the remaining two samples.
   3. When entering the sample information for all the samples is complete, tap the continue button.
      NOTE: If using less than 3 samples, leave the sample ID as the default, select any unit, and type a 1 in the sample amount line for any unused samples to proceed.
6. On the next screen, tap on the folder. Move to the next screen and tap save to current folder.
7. On the next screen, type in a run name and tap confirm.
8. Turn on the instrument by pressing and holding down the power button, located on the top left of the instrument, until all the lights on the front of the instrument are lit (~5 s). Then, tap confirm in the app to continue.
9. On the next screen, tap connect via Integrated short-range wireless. Press the integrated short-range wireless button (a familiar trademarked symbol) on top of the instrument until the blue light begins to flash. Tap confirm and wait until the app and instrument sync together. Once they are synced, tap on the instrument name in the app to select it, and then tap the connect button to proceed to loading the assay strips.
   NOTE: If the instrument and app do not sync the first time, make sure the integrated short-range wireless light on the instrument is still flashing and tap scan again.
10. Before removing the caps of the assay strips, check to make sure the lyophilized pellets are not at the top of any of the assay strip wells. If they are, gently flick the bottom of the well to make the pellet drop lower into the well.
11. Remove the caps from the go strip tubes for the first sample. Add 18 µL of sterile water to the first well of the assay strip and gently pipette up and down to dissolve the pellet. Repeat for the remaining 2 wells.
12. Add 2 µL of the sample to each well and seal the tubes by inserting a set of rubber assay strip caps into the tubes with the notches between the caps facing the back of the tray. Repeat steps 2.10-2.12 for the remaining samples if necessary. Once all assay strips have been filled and sealed with strip caps, tap confirm in the app.
13. Remove each assay strip one at a time and check for bubbles at the bottom of the wells. If there are bubbles at the bottom of any of the wells, gently rock the strip back and forth until the bubbles move up towards the top of the well. Once all wells have been checked, tap confirm in the app and proceed to loading the strips in the instrument.
14. Open the lid of the instrument and place the assay strips into the field qPCR instrument in the same order and orientation that they are on the tray, so that the hinges of the well and notches between the caps are facing the back of the instrument. After all strips have been added, gently push the lid down until it locks into place and tap confirm.
15. Tap the start run button in the app.
    NOTE: The run will begin and will take ~45 min to complete using preset run conditions. These conditions are not editable.
16. When the run finishes, tap email results. Once the phone is on Wifi, results will be sent for analysis and reporting; until then, the results are stored on the instrument.
    1. Once connected to the Wifi, the user will need to hit the send button again to send the results.
17. Clean the tray between runs to avoid contamination between samples.
    NOTE: The output is an Excel file containing cycle threshold (Ct) values for each well. These results are sent to the laboratory, which checks the amplification quality and converts the Ct values to abundances using validated standard curves specific to each qPCR target and based on the volume and matrix of the original sample. These results are sent to the user as an electronic deliverable as well as a PDF.

DNA extraction from representative environmental samples was performed using both the biological extraction field kit and a highly validated laboratory-based method previously described4. As shown in Figure 4, successful completion of DNA isolation using the field kit was confirmed when comparing qPCR results to a highly validated laboratory-based extraction method. This consistency confirms that the method preserved the DNA integrity of environmental samples in the field.

The components of the field qPCR kit are shown in Figure 5. Analysis of a standard culture dilution of Dehalococcoides (DHC) using the field qPCR unit was performed at both indoor (21 °C) and outdoor (30 °C) temperatures to illustrate the robustness of the instrument under real-world field conditions. The results, seen in Figure 6, highlight the low deviation for 10 different repetitions of each. For full context, DHC is an organism capable of degrading chlorinated solvents and is commonly quantified via qPCR within the environmental remediation industry4.

To validate the precision of the instruments, DHC was analyzed at three different concentrations using ten field qPCR instruments performed in duplicates (Figure 7). Even at low abundances, the relative standard deviation remained low (below 5%) across all runs. Note that, compared to standard deviation, relative standard deviation is important for logarithmic data as it normalizes the results through the following equation:

Where RSD is Relative Standard Deviation, σ is the standard deviation of the dataset, and µ is the mean of the dataset.

Together, both methods within this paper provide reproducible results across replicate field extractions and qPCR assays. The agreement between field and laboratory extractions and qPCR analysis demonstrates that reliable, high-quality data can be obtained without reliance on laboratory infrastructure, enabling microbial analyses to be carried out in remote or resource-limited environments.

Figure 1: Encapsulated filter kit components. The encapsulated filter is used to filter biomass out of the water sample. By connecting to a peristaltic pump via tubing, water is pushed through the filter. After notating the total volume of water, the water is discarded and the biomass collected on the filter is ready for DNA extraction. Components: a. sterile tube that holds the filter and components before and after use, b. inlet cap, c. hose clamp, d. luer-locking tubing connection adapter, e. encapsulated filter, f. rubber outlet cap. Image used with permission from Microbial Insights, Inc.12. Please click here to view a larger version of this figure.

Figure 2: Filter and tubing connection. The tubing is pushed over the luer-locking tubing connection adapter and is held in place using the hose clamp. The other end of the tubing is connected to a peristaltic pump (this connection and the pump operation are outside the scope of this protocol). Image used with permission from Microbial Insights, Inc.12. Please click here to view a larger version of this figure.

Figure 3: DNA isolation field kit components. All components are sterile prior to use. The components include: (1) encapsulated filter kit (see Figure 1 for more details), (2) 3 mL syringe, (3) 1 mL syringe pre-loaded with Solution A, capped, (4) 1 mL syringe pre-loaded with Solution B, capped, (5) 3 mL syringe, (6) silicone bead tube, (7) 1 mL syringe fitted with isolation column, (8) 1 mL disposable transfer pipette, (9) DNA isolation sample prep cartridge, (10) 1.5 mL microcentrifuge tube. Not pictured: parafilm squares for sealing during shaking, and alcohol prep wipes used to clean the surface used for extraction. Image used with permission from Microbial Insights, Inc.12. Please click here to view a larger version of this figure.

Figure 4: Field extraction comparison using real-world samples. Comparative qPCR results of DNA isolated from two representative samples using a laboratory method (red bars) versus the biological extraction field kit (blue bars). These were not analyzed in replicate, as would be expected in a standard qPCR analysis. Gene targets (x-axis) show abundance (y-axis) within an order of magnitude for both methods. Information on each specific gene target is not included as it is outside of the scope of this paper. Please click here to view a larger version of this figure.

Figure 5: Field-based qPCR instrument and components. The components include: A. The Field-based qPCR instrument, B. sample prep tray, C. assay strip containing lyophilized reagents, D. phone pre-loaded with application used to run the instrument. Image used with permission from Microbial Insights, Inc.12. Please click here to view a larger version of this figure.

Figure 6: Demonstration of field qPCR robustness. Dehalococcoides (DHC) standard analyzed in ten replicates under outdoor (30 °C, red bars) and indoor (21 °C, blue bars) temperature conditions, highlighting the reliability of the instrument for the field. Replicate run number is listed along the x-axis and the calculated DHC abundance is listed along the y-axis. The Inside (20 °C) mean was calculated to be 4.92 × 10² cell/mL, SD = 1.14 × 10² cells/mL (n = 10), and the Outside (30 °C) mean was calculated to be 5.89 × 10² cells/mL, SD = 1.80 × 10² cell/mL (n = 10). Measurements obtained at 20 °C and 30 °C did not differ significantly (Welch's t-test, p = 0.17). Please click here to view a larger version of this figure.

Figure 7: Demonstration of field qPCR precision. Precision performance between 10 different instruments, run in duplicate at three different standard concentrations. The true value and replicate number (n = 2 for 10 instruments at each standard concentration) is listed along the x-axis, with colored bars specific to the field unit. The y-axis shows the calculated abundance for each run. Percent relative standard deviation for each standard concentration is shown in the dotted lines above the bars. Please click here to view a larger version of this figure.

A critical feature of this protocol is that it enables microbial DNA extraction and qPCR analysis under field conditions without the need for laboratory infrastructure or electricity¹². The method has been optimized to balance portability with reliability, providing reproducible results that are consistent with laboratory-based workflows even when performed outdoors under a range of ambient temperatures¹². The suggested range of use is 4 °C to 95 °C (39 °F to 104 °F), and side-by-side comparison at standard outdoor temperatures (30 °C, 86 °F) for a field crew show reliable consistency (Figure 6). This consistency is essential to ensure that field results are interpretable alongside laboratory datasets.

Several steps are crucial for protocol success. During the extraction process, complete removal of liquid from filters and thorough mixing of reagents are important to ensure efficient lysis and DNA recovery. Likewise, careful transfer of lysate into the sample prep cartridge while minimizing bead carryover is key to preventing clogging or column failure. Troubleshooting strategies include slowing plunger depression to reduce back-pressure and repeating syringe pumping if viscous lysates impede fluid movement. For qPCR, avoiding bubbles in assay wells and ensuring complete pellet resuspension are critical to accurate amplification.

Despite its advantages, the method has limitations. DNA yields may be lower than those achieved with benchtop centrifugation-based kits, and highly turbid or organic-rich samples can challenge column performance. Although the isolation kits are simple and easy for anyone to use - including users with no previous laboratory experience - there is additional risk of error when comparing an inexperienced person in the field. Additionally, while the field qPCR unit provides rapid results, it is restricted to a limited number of simultaneous reactions (currently limited to 9 gene targets per run) compared to standard thermocyclers. These limitations are offset, however, by the speed of analysis and the ability to capture microbial community profiles in the field. There is no inherent limitation on what qPCR targets can be analyzed besides the catalog of the laboratory that offers the unit.

The significance of this method lies in its capacity to mobilize molecular analyses, enabling real-time monitoring of microbial populations in environments such as oilfields, agricultural sites, or remote ecosystems. By preserving DNA integrity at the point of sampling and generating results on-site, this approach reduces logistical barriers, enhances data quality, and allows rapid decision-making. Future applications may include pathogen surveillance in public health, monitoring of bioremediation progress, or microbial risk assessment in water systems. With minor modifications, the platform can be continually adapted for additional molecular targets and emerging qPCR assays.