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

The scope of our research really involves performing molecular analysis that we can use to characterize microbes that are involved in remediation, mining, corrosion prevention, and really helps us answer questions about sustainability. This research addresses a number of research and accessibility gaps through the use of reliable and field-portable molecular biological workflows that are simple to use. Uncap both ends of the filter and connect a three-milliliter syringe filled with air to the filter using the locking luer connection.

Push the air through to expel residual water. Disconnect the syringe and pull in more air before repeating. Then, recap the red outlet cap.

Invert the solution a syringe to mix. Remove the Luer locking syringe cap and connect the Solution A syringe to the encapsulated 0.2 micrometer filter inlet via the Luer locking tip. Depress the plunger to slowly add the Solution A.Then, vigorously hand-shake the closed filter for five minutes.

Now, invert the Solution B syringe several times to mix. Remove the Luer locking cap and connect the syringe to the encapsulated filter inlet via the Luer locking tip. After recapping the inlet filter cap, vigorously hand-shake the closed filter for five minutes.

Remove the inlet cap and invert the filter. Use a three-milliliter syringe attached to the encapsulated filter unit to push one milliliter of air into the filter. Then, pull back on the plunger to remove all of the lysate.

Transfer the lysate into a five-milliliter bead tube. After sealing it with parafilm, vigorously hand-shake the bead tube for five minutes before placing it upright. Place the sample prep cartridge on a flat surface and attach a Luer lockable column to a one-milliliter syringe.

Use the pointed tip of the column to poke two holes into the first section of the sample prep cartridge. Pipette one milliliter of lysate from the bead tube into the newly punched hole into the first section of the cartridge. Insert the syringe column back into the red section of the cartridge.

Then, pull the fluid completely into the syringe and then push it out 10 times. Now, position the pointed tip of the syringe column over the red-orange section and puncture two holes through the foil barrier. Using the plunger, pull the full syringe volume of fluid all the way up into the syringe and pump the fluid completely back out.

Similarly, use the syringe column to puncture and pump the orange, yellow, blue, air-dry, and the green sections, pulling and pushing the plunger the number of times listed on the section. Then, use the one-milliliter syringe and the sample preparation column to transfer all solution from the green section into a well labeled one-milliliter microtube. Place up to three field-extracted DNA samples into the pure sample tray lots of a field-based qPCR instrument, ensuring one sample per designated section.

Place the assay strips for each sample into the corresponding A, B, or C strip slots on the tray, aligning the first well of each strip with the first slot on the tray. Unlock the phone and open the application named after the trademarked qPCR instrument. When the application opens, tap the Start Run button.

On the next screen, tap the Generate IDs button. Enter sample information by tapping under Sample ID and typing the sample name. Tap sample units and select the appropriate unit, then tap under sample amount and enter the volume used during extraction.

After entering information for all samples, tap the Continue button. On the next screen, tap the folder icon and then tap Save to Current Folder. Type a run name and tap Confirm.

Turn on the instrument by pressing and holding the power button until all front lights illuminate. Tap Confirm in the application to proceed. Press connect via integrated short range wireless in the application.

Press the integrated short-range wireless button on the instrument until the blue light flashes. Then tap Confirm and select the instrument name to connect. Remove the caps from the go strip tubes for the first sample.

Add 18 microliters of sterile water to each well and gently pipette to dissolve the pellet. Add two microliters of sample to each well and seal the tubes with rubber assay strip caps, ensuring correct orientation. Tap Confirm in the application once all strips are prepared.

Remove each assay strip. Check for bubbles at the bottom of the wells, then gently rock the strip until bubbles rise. Tap Confirm to proceed.

Open the instrument lid and place the assay strips into the instrument in the correct order and orientation. Close the lid until it locks, and tap Confirm. Tap the Start Run button in the application to begin the run.

When the run finishes, tap Email Results. Once connected to wireless internet, tap Send Again to transmit results. Clean the tray thoroughly between runs to prevent contamination.

Successful completion of DNA isolation using the biological extraction field kit was confirmed by consistent qPCR results when compared to a validated laboratory-based method. Standard culture dilution of Dehalococcoides analyzed using the field-based qPCR unit showed low deviation across 10 repetitions at both 21 degrees Celsius and 30 degrees Celsius. Precision testing of the field instruments showed relative standard deviation below 5%across all runs.

So our findings really advance the field by enabling environmental monitoring to happen in these remote locations, and it really fosters the accessibility, the community engagement, and makes very impactful scientific research. Our results paved the way for understanding how samples change during shipment versus immediate extraction and analysis. Future research will focus on defining the limits of these methods for extreme samples like brine, as well as extreme weather conditions.