The overall goal of this air sampling and DNA extraction methodology is to obtain fungal genomic DNA that can amplified, sequenced, and taxonomically placed to identify potential fungal hazards within occupational settings. This method can help answer key questions in the occupational exposure assessment field, such as what fungal hazards are workers exposed to that may result in negative health effects. The main advantage of this technique is that fungal species that remain undetected using culture or microscopy-based methods can be elucidated using sequencing-based approaches.
To assemble the air-sampling filter cassette, place a cellulose filter support pad on the gridded surface of the base piece, then, with the help of filter forceps, place a filter on top of the filter support pad with the collection side facing upwards. After assembling, seal the filter cassette by inserting the extension cowl and pressing it down tightly and evenly with a manual or pneumatic press. Then, fit the assembled cassette on top of the NIOSH aerosol sampler and push down as much as possible.
Use a piece of 19-millimeter tape to wrap around the outside of the filter cassette and sampler holding the filter cassette in place and acting as a back-up seal to prevent leaks. Screw the collection tubes tightly and fully into the sampler until they bottom out and wrap the sealing tape around the tubes acting as secondary seal against leakage. To conduct personal aerosol sampling, place the sampler within the person's breathing zone.
Attach the sampler to the lapel, shoulder, or chest and then keep the sampling pump at the waist level or in a backpack. Turn the pumps on and check after a minute if the pump is functioning. Upon completing air sampling, collect the air sampler from the subject.
Remove the sealing tape, unscrew the tubes, and then put a cap on them. Place the third piece of filter cassette over the filter. First, wipe the sampling cassette with 70%ethanol, then use a cassette opening tool to open the sampling cassette while working in a Class II biological safety cabinet.
Next, use a filter lifter to lift the support pad and filter upwards. Then, use filter forceps to remove the filter and place it in a sterile Petri dish. Cut the filter into six equal pieces with a steril scalpel and then place them in a two-milliliter reinforced tube containing 300 milligrams of 200 to 500 micrometer glass beads.
Immerse the filter-containing tube in liquid nitrogen for 30 seconds and then quickly place it in a bead mill homogenizer at a speed of 4.5 meters per second for 30 seconds. Add 0.5 milliliters of lysis buffer to tubes containing the remaining small pieces of intact filter, then add 0.3 milliliter of lysis buffer to the 15 and 1.5 milliliter air sampler collection tubes and vortex the tubes in an upright and inverted position for 10 to 15 seconds respectively. Transfer the lysis buffer from each collection tube to two-milliliter reinforced tubes containing 300 milligrams of glass beads.
Process the tubes in the bead mill homogenizer for 30 seconds, then centrifuge the samples at 20, 000 times gravity for one minute at 22 degrees Celsius. After centrifugation, transfer the supernatant in a sterile 1.5-milliliter microcentrifuge tube and again centrifuge the supernatant at 20, 000 times gravity for one minute at 22 degrees Celsius. After adding 30 microliters of lysis reagent, incubate the tubes at 37 degrees Celsius for 15 minutes.
Then, add 0.2 milliliter of binding buffer and 100 micrograms per milliliter of Proteinase K to the tube, then leave the tubes for incubation in a block heater at 70 degrees Celsius for 10 minutes. Finally, add 100 microliters of isopropanol to each tube. Then, place glass fiber filter tubes with a capacity of 700 microliters in two-milliliter collection tubes and transfer the extract solutions into it.
Following transfer, centrifuge the collection tubes at 20, 000 times gravity for 30 seconds at 22 degrees Celsius. After centrifugation, insert the glass filter fiber in fresh collection tubes and discard the collection tube. To each tube add 0.5 milliliter of inhibitor removal buffer and conduct centrifugation at 20, 000 times gravity for 30 seconds at 22 degrees Celsius.
Discard the collection tube after centrifugation and place the filter tubes into new collection tubes. Then, add 0.5 milliliter of wash buffer to each tube and again subject to centrifugation at 20, 000 times gravity for 30 seconds at 22 degrees Celsius. Discard the collection tube and replace with fresh ones after the centrifugation.
Again, add 0.5 milliliter of wash buffer to each tube and centrifuge at 20, 000 times gravity for 30 seconds at 22 degrees Celsius. To remove any residual wash buffer, finally centrifuge the collection tubes at 20, 000 times gravity for one minute at 22 degrees Celsius and discard the collection tubes and replace them with fresh ones. After adding 100 microliters of warm elution buffer, incubate the tubes on the lab bench at room temperature for one to two minutes and centrifuge the collection tubes at 20, 000 times gravity for 30 seconds at 22 degrees Celsius.
Place the filter tubes in a sterile 1.5-milliliter microcentrifuge tube and reapply the eluates for another round centrifugation. Use 0.5 milliliter PCR tubes to set up 50-microliter reactions in triplicate with five microliters of already-extracted DNA for each reaction, then program the thermocycler and start the cycle. Mix all three PCR reactions together in sterile 1.5 milliliter microcentrifuge tubes.
Add 750 microliters of binding buffer and mix thoroughly by pipetting up and down 10 times. Add 450 microliters of the total mixture to spin columns inserted in collection tubes and start centrifuging at 17, 900 times gravity for 30 seconds, then discard the filtrate and add the remaining 450 microliters of the mixture for a second round of centrifugation. Again, discard the filtrate and add 750 microliters of wash buffer to the spin columns and centrifuge at 17, 900 times gravity for 30 seconds at 22 degrees Celsius.
Finally, discard the filtrate and re-spin the empty columns at 17, 900 times gravity for one minute at 22 degrees Celsius. After transferring the spin columns to 1.5-milliliter sterile microcentrifuge tubes, pipette 45 microliters of elution buffer and incubate the tubes on the lab bench at room temperature for five minutes. Then, centrifuge the columns at 17, 900 times gravity for one minute at 22 degrees Celsius.
Immerse a 1%agarose gel in one-fold TAE buffer in an electrophoresis chamber, then mix two microliters of five-fold loading buffer with eight microliters of amplified DNA. Finally, load the entire 10 microliters of the sample on the gel along with the DNA ladder. Run the gel at 75 volts for approximately 90 minutes.
A representative agarose gel demonstrates amplification of internal transcribed spacer regions of fungal genomic DNA derived from occupational air samples. The lane on the extreme left side represents the DNA marker lane. The bands on the right represent the internal transcribed spacer regions amplified from different occupational air samples with sizes ranging from 750 to 1, 000 base pairs.
A representative Krona chart shows the presence of diverse taxonomic fungal species in an indoor environment. The NIOSH bio-aerosol cyclone sampler was used in homes to collect bio-aerosols for one hour to determine the fungal species present. Tabular data represents the diversity and richness indices of fungal species in indoor environments.
Data shows higher Chao-1 Richness indices, Shannon Diversity indices, and Bray-Curtis dissimilarity coefficients in air-conditioned and evaporative cooler environments. After watching this video, you should have a good understanding of how to collect indoor and occupational air samples and extract genomic DNA for the analysis of microbial populations. While attempting this procedure, it is important to remember to utilize the best aseptic methods to prevent microbiological contamination of the samples.
