Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Summary

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Abstract

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Introduction

Fungal exposures in indoor and occupational environments can result in respiratory morbidities, including allergic sensitization and asthma¹. Identification of fungal hazards is important for assessing risk and preventing worker exposure. These fungal hazards may be a result of indoor contamination, outdoor air intrusion, or environmental disturbances that result in the transport of fungal materials into areas where workers are present². Methods to assess fungal exposure have included viable culture sampling as well as microscopic identification of fungal spores. These approaches have several limitations and often overlook many fungal species that could be contributing to the overall fungal burden³. Culture-based approaches can only differentiate those viable fungal organisms that can be cultivated on nutrient media. Identifying fungal spores to species level via microscopy can be confounded by spores sharing similar morphologies. Both methods are highly dependent on mycologists to analyze and identify the fungal species, with many remaining unidentified.

To improve upon existing methodologies used in occupational hazard identification and exposure assessments, many researchers have turned to molecular-based technologies. Sequencing-based approaches for assessing microbial diversity within indoor and occupational environments have revealed a broader spectrum of fungal species encountered compared to methods such as microscopy and viable culture^3,4,5. The method presented here describes the air sampling of occupational environments and extraction of genomic DNA for the identification of potential fungal hazards. Hazard identification is accomplished by sequencing the nuclear ribosomal internal transcribed spacer, or ITS, regions that are highly variable among fungi and have been commonly used to differentiate fungal species^6,7,8,9. Many species found in occupational settings, such as some species belonging to the phylum Basidiomycota, are not identifiable in viable culture and are difficult to differentiate microscopically. These fungi have been observed in high relative abundance within indoor and occupational environments assessed by sequencing fungal ITS regions^3,4,10. ITS sequencing has provided greater knowledge into the diversity of fungi encountered within indoor and occupational environments.

The protocol described here details the methods used to collect, extract, and amplify fungal ITS regions from bioaerosols for sequence analysis. This approach utilizes the National Institute for Occupational Safety and Health (NIOSH) two-stage cyclone aerosol sampler to collect particulates in the air. This sampler was developed to collect bioaerosols and separate respirable (≤4 µm aerodynamic diameter) and non-respirable (>4 µm aerodynamic diameter) particles, which allows for identification of fungal organisms within indoor environments that are most likely to be inhaled by a worker¹¹. Other air samplers, including cyclone samplers, are available on the market that have the ability to collect particles within the respirable range (<4 µm) using filters^12,13. In contrast, the NIOSH two-stage cyclone aerosol sampler separates fungal species based on their aerodynamic diameter into disposable, polypropylene tubes that can be immediately processed for downstream applications¹⁴.

The processes of extracting genomic DNA and amplifying the fungal ITS regions are detailed in this protocol. The extraction methodologies presented have been developed specifically for the extraction of genomic DNA from fungi and bacteria, as many commercial kits target mammalian cells, bacteria, or specifically yeasts¹⁵. The primers used in this study are selected based on their overall coverage of both the fungal ITS 1 and ITS 2 regions^4,5. Sequencing of these regions allows for the comparison of many banked ITS sequences, including those that sequence the ITS 1 region, the ITS 2 region, or both the ITS 1 and ITS 2 regions. The fungal diversity of air samples collected in an indoor setting using these methods are shown, revealing a substantial number of sequences placed in the phyla Ascomycota and Basidiomycota as well as other sequences belonging to less dominant fungal phyla, such as Zygomycota. The broad diversity of fungal sequences identified using this approach would not be captured using traditional hazard identification methodologies like cultivation or microscopy. Sequencing of fungal ITS regions provides an enhanced method to identify fungal hazards and allow for a better understanding of indoor and occupational fungal exposures.

Protocol

1. Preparing the NIOSH aerosol sampler

NOTE: The NIOSH aerosol sampler is a two-stage cyclone aerosol sampler that collects bioaerosols using two sampling tubes and a polytetrafluoroethylene (PTFE) filter.

1. Before assembly, carefully inspect the sampler. Check the sampler to ensure it is not damaged, all screws are in place and snug, and the sealing tape around the seam formed by the two halves is intact. Inspect the sampler O-ring to ensure it does not have any nicks, cracks or tears and that it has a very light coating of silicone grease.
   NOTE: The sampler includes a 37 mm 3 µm PTFE filter in a polystyrene or polypropylene three-piece filter cassette.
   1. Assemble the filter cassette by placing a cellulose or plastic filter support pad (included with the filters) on the gridded surface of the base piece (see Figure 1). Use filter forceps to put the filter on top of the filter support pad with the collection side facing up.
   2. Seal the filter cassettes using a manual or pneumatic press. Hand closure will allow air to leak around the filter and reduce the collection efficiency. Insert the ring-shaped piece (the extension cowl), and use the press to push it down tightly and evenly. Press down the extension cowl onto the filter tightly enough to ensure air does not leak around the filter.
      NOTE: The top piece of the cassette is not used while sampling but should be saved to cover the filter once sampling is complete.
   3. Fit the assembled cassette onto the top of the sampler and push it down as far as possible. Wrap a piece of 19 mm (3/4 inch) tape around the outside of the filter cassette and sampler to hold the filter cassette in place and to act as a backup seal to prevent leaks.
   4. Screw each tube tightly and fully into the sampler until it bottoms out and wrap sealing tape around each tube to act as a secondary seal against leaks.
      NOTE: The first tube, a 15 mL polypropylene tube, collects non-respirable particles with an aerodynamic diameter of >4 µm. The second tube, a 1.5 mL polypropylene microcentrifuge tube, collects respirable particles between 1 and 4 µm. The remaining smaller particles, <1 µm, are collected on the PTFE filter (see Figure 2).
2. Calibrate the airflow through the NIOSH sampler with a calibration jar before each use as differences in the filters and samplers will cause the airflow to vary.
   1. To use a calibration jar, insert the jar's Luer fitting into the top of the filter cassette, place the sampler in the jar, and seal the jar.
   2. Connect the calibration jar to a calibration flow meter and the sampling pump. Turn the flow meter on and allow it to warm up for a few minutes. Note that the flow meter must always have a filter attached to its inlet to avoid contaminating the flow meter and the sampler.
   3. Turn the sampling pump on and allow it to run for a few minutes to warm up and stabilize.
   4. Adjust the pump to set the correct flow rate at 3.5 L/min.
      NOTE: The flow rate for the NIOSH aerosol sampler is usually set to 3.5 L/min. At this flow rate, the sampler conforms to the ACGIH/ISO criteria for respirable particle sampling¹⁶. Other flow rates can be used if different cut-off sizes are desired or to reduce the noise level, but be aware that if a different flow rate is used, then the sampler will no longer conform to the respirable sampling criteria.
   5. When calibration is finished, turn off the pump and flow meter.

2. Static and personal aerosol sampling

1. Set up the NIOSH aerosol sampler for either personal or static area sampling.
   NOTE: Static sampling refers to using the aerosol sampler while it is attached to a tripod or other holding device (see Figure 3), as versus personal sampling when the sampler and pump are mounted on the person being studied (see Figure 4).
   1. For static area sampling, keep the samplers as close as possible to the specific area being studied. Keep the samplers away from air inlets, room entrances and places that might be in the path of the airflow.
      NOTE: Aerosol concentrations can vary considerably even over short distances, especially if the aerosol source is in the room¹⁷.
   2. For personal sampling, set up the sampler within the person's breathing zone. Attach the sampler to the lapel, shoulder or chest, and place the sampling pump at the waist or in a backpack.
      NOTE: The breathing zone is commonly assumed to be a 30 cm hemisphere surrounding a person's nose from which the majority of air is drawn during inhalation¹⁷.
2. Make sure that the sampler tubing is well clear of the sampler inlets and that nothing is obstructing the inlets or interfering with the airflow into the sampler. The sampler tubing must not be pinched or kinked. Any blockage will cause the pump to stop.
3. Turn on the pumps. After a minute or two, check if the pumps are still running.
   NOTE: Conduct air sampling for time periods ranging from as little as 10 min to a full 8 h work shift, depending on the environment^10,18,19. The results presented in Figure 6 are representative of a 60 min sampling period in an indoor environment.
4. After air sampling is completed, collect the sample tubes and filter. Remove the sealing tape, unscrew the tubes, and cap them. Place the third piece of the filter cassette over the filter. Store samples at 4 °C until ready for processing.

3. Extraction of genomic DNA from air samples

1. Extract genomic DNA (gDNA) from each stage of the NIOSH BC251 sampler. Process the air sampling collection tubes and filter separately to allow for determination of respirable and non-respirable microbial aerosols in the sample.
   NOTE: Alternatively, the three stages can be combined and extracted if sample inoculum is too small.
   1. In a class II biological safety cabinet or laminar flow clean bench, aseptically remove the filter. Wipe the sampling cassette down using 70% ethanol and pry the sampling cassette open using a cassette-opening tool. Use a filter lifter to push the filter and support pad upwards. Grasp the filter using filter forceps and place it in a sterile petri dish. Cut the filter into 6 equal pieces and place them in a 2 mL reinforced tube containing 300 mg glass beads (0.2 - 0.5 mm).
   2. Place the tube containing the filter in liquid nitrogen for 30 s and immediately place it in a bead mill homogenizer set at 4.5 m/s for 30 s.
   3. Repeat step 3.1.2 once or twice until the filter is shredded. Small pieces of intact filter may remain. Add 0.5 mL of lysis buffer (4 M urea, 200 mM Tris, 20 mM NaCl, 200 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4).
   4. Add 0.3 mL of lysis buffer to the 15 mL and 1.5 mL air sampler tubes. Vortex the tube for 10-  15 s while upright and then another 10 - 15 s inverted. Transfer the contents of each tube to 2 mL reinforced tubes containing 300 mg glass beads.
      NOTE: Most of the material collected in each sampler tube will accumulate near the top of the tube.
   5. Process all tubes in the bead mill homogenizer at 4.5 m/s for 30 s and centrifuge them at 20,000 x g for 1 min at 22 °C. Transfer supernatants to sterile 1.5 mL microcentrifuge tubes and centrifuge the tubes at 20,000 x g again for 1 min at 22 °C. Repeat this step.
2. Add 30 µL of lysis reagent (see Table of Materials) to each tube and incubate the tubes at 37 °C for 15 min.
3. Add 0.2 mL of binding buffer (10 M urea, 6 M guanidine-HCl, 10 mM Tris-HCl, 20% Triton X-100, pH 4.4) and proteinase K (100 µg/mL) to each tube and incubate the tubes at 70 °C for 10 min. Add 100 µL of isopropanol to each tube.
4. Transfer the extract solutions into glass fiber filter tubes (700 µL capacity) placed in 2 mL collection tubes and centrifuge the collection tubes at 20,000 x g for 30 s at 22 °C. Discard the collection tubes and place the filter tubes into new 2 mL collection tubes.
5. Add 0.5 mL of inhibitor removal buffer (5 M guanidine-HCl, 20 mM Tris-HCl, pH 6.6, 38% ethanol) to each tube and centrifuge the collection tubes at 20,000 x g for 30 s at 22 °C. Discard the collection tubes and place the filter tubes into new 2 mL collection tubes.
6. Add 0.5 mL of wash buffer (20 mM NaCl, 2 mM Tris-HCl, pH 7.5, 80% ethanol) to each tube and centrifuge the collection tubes at 20,000 x g for 30 s at 22 °C. Discard the collection tubes and place the filter tubes into new 2 mL collection tubes. Repeat this process.
7. Centrifuge the collection tubes at 20,000 x g for an additional 1 min at 22 °C to remove any residual wash buffer. Discard the collection tubes and place the filter tubes into new collection tubes.
8. Add 100 µL warm (≥70 °C) elution buffer (10 mM Tris-HCl, pH 8.5) to each tube and incubate them at room temperature for 1 - 2 min. Centrifuge the collection tubes at 20,000 x g for 30 s at 22 °C.Transfer the empty filter tubes to a sterile 1.5 mL microcentrifuge tube and re-apply eluates from step 3.8. Incubate at room temperature for 1 - 2 min. Centrifuge at 20,000 x g for 30 s at 22 °C.
   NOTE: Eluates can be used immediately for step 4 or stored at -20 °C until ready to use. Genomic DNA can be stored for up to a year at -20 °C. It is recommended that DNA be stored at -80 °C if long-term storage is required.

4. Amplification of fungal ribosomal DNA

1. Use universal fungal primers to amplify ITS regions 1 and 2 from the extracted gDNA.
   NOTE: For this protocol, the primer pair Fun18Sf (5'-TTGCTCTTCAACGAGGAAT-3')/ITS4R (5'-TCCTCCGCTTATTGATATGC-3') are used to provide the greatest coverage of the ITS regions^4,5. Other primer sets, such as those that amplify ITS1 or ITS2 regions alone, can be used.
   1. Set up polymerase chain reactions (PCR) for each sample in triplicate 50 µL reactions in sterile 0.5 mL PCR tubes or 96-well PCR plates as follows: 5 µL of extracted template DNA (from step 3), 33.3 µL of PCR grade water, 5 µL of 10x PCR buffer, 1.5 µL of 50 mM MgCl₂, 1 µL of 10 mM 2'-deoxynucleoside 5'-triphosphates, 0.5 µL of 20 µM Fun18Sf forward primer, 0.5 µL of 20 µM ITS4R reverse primer, and 0.2 µL of Taq DNA polymerase.
   2. Perform reactions in a thermal cycler: denaturation at 95 °C for 3 min; 6 cycles of denaturation (96 °C, 30 s) annealing (50 °C, 45 s) and primer extension (72 °C, 3 min); 20 cycles of denaturation (96 °C, 30 s), annealing (50 °C, 45 s), and primer extension (72 °C,1 min); and primer extension at 72 °C for 10 min. Keep the reaction mixtures at 4 °C until next step.
2. Combine the triplicate PCR reactions purify using a silica membrane-based purification kit.
   NOTE: This procedure allows for the removal of PCR components (primers, dNTPs, enzymes, salts, etc.) and other contaminants from the amplified DNA preparations.
   1. Combine the three reactions for each sample (150 µL total) in sterile 1.5 mL microcentrifuge tubes. Add binding buffer (5 M guanidine-HCl, 30% isopropanol) at a 5x volume (750 µL) and mix the solution by pipetting up and down ten times.
   2. Add 450 µL of the mixture to spin columns placed in collection tubes and centrifuge the tubes at 17,900 x g for 30 s. Discard the filtrates. Add the remaining 450 µL to the columns and centrifuge at 17,900 x g for 30 s at 22 °C.
      NOTE: The spin columns contain a silica membrane that allows for adsorption of the DNA to the columns.
   3. Discard the filtrates and add 750 µL washing buffer (10 mM Tris-HCl, pH 7.5, 80% ethanol) to the spin columns. Centrifuge the columns at 17,900 x g for 30 s at 22 °C.
      NOTE: This process removes the contaminants from the PCR reaction mentioned above.
   4. Discard the filtrate and centrifuge the empty columns at 17,900 x g for 1 min at 22 °C.
      NOTE: This step is to remove any residual washing buffer that may interfere with downstream applications.
   5. Transfer the spin columns to sterile 1.5 mL microcentrifuge tubes. Add 45 µL of elution buffer (10 mM Tris-Cl, pH 8.5) and incubate the tubes at room temperature for 5 min. Centrifuge the spin columns at 17,900 x g for 1 min at 22 °C.
   6. Use the eluted DNA immediately for steps 4 and 5 or store at -20 °C until ready for use.
      NOTE: The amplicons can be stored for up to a year at -20 °C. It is recommended that DNA be stored at -80 °C if long-term storage is required.

5. Verification of fungal ITS amplification using agarose gel electrophoresis

1. Cast a 1% agarose gel containing 1 µg/mL ethidium bromide. Dissolve agarose in boiling 1x Tris-acetate-EDTA (TAE) buffer. Once the solution has cooled to approximately 50 °C, add the ethidium bromide and pour into gel cast.
2. After the agarose gel has solidified, immerse the gel in 1x TAE and prepare the amplicons to be loaded on the gel. To 8 µL of amplified DNA, add 2 µL of 5x loading buffer.
   NOTE: These volumes can be adjusted depending on the concentration of the desired loading buffer.
3. Load the samples, all 10 µL, onto the gel along with a DNA ladder for size reference. Run the gel at 75 V (6 V/cm) for approximately 90 min and visualize bands, typically seen between 750 and 1,000 base pairs, using ultraviolet light (see Figure 5).

6. Sequencing and analysis of fungal ITS regions

1. Sequence the extracted gDNA or amplified fungal ITS regions and analyze the sequences using current and appropriate methods.

Results

The species distribution within an environment can be assessed using relative abundance by determining the number of clones of each OTU identified in the air samples. Figure 6 is a Krona chart representing the taxonomically placed species within an indoor environment following 60 min of air sampling. It can be observed that the environment contains a variety of species within two major fungal phyla, Ascomycota and Basidiomycota, as well as species belonging t...

Discussion

Determining the fungal diversity within an occupational environment using sequencing-based approaches has improved fungal hazard identification and exposure assessment. Using this approach has allowed for the detection of many additional fungal species that are often not detected using culture or microscopy-based methods of assessment. A method for sampling bioaerosols from occupational and indoor environments and the extraction of genomic DNA from air samples for ITS amplification and sequencing is presented here. Deter...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NIOSH BC251 bioaerosol cyclone sampler	NIOSH	BC251	The NIOSH sampler is not yet commercially available. Please contact William Lindsley, PhD (wlindsley@cdc.gov) for information on obtaining the NIOSH sampler
Fisherbrand Sterile Microcentrifuge Tubes with Screw Caps	Fisher Scientific	02-681-373	1.5 mL polypropylene microcentrifuge tubes for air sampling; screw top threading must match the threading of the NIOSH sampler
Falcon 15 mL Conical Centrifuge Tubes	Corning	352096	15 mL polypropylene tubes for air sampling
Clean Room Vinyl Tape, Easy-Remove, 1/4" Width	McMaster-Carr	76505A1	sealing tape
Filter Cassette, Clear Styrene, 37 mm	SKC Inc.	225-3LF	3-piece sampling cassette (no filter). Contains: cassette base, extension cowl, cassette cap and inlet/outlet plugs
PTFE hydrophobic fluoropore membrane filters, 3.0 µm, 37 mm	EMD Millipore	FSLW03700	Contains: 37 mm, 3.0 µm PTFE filters and support pads
Fisherbrand filter forceps	Fisher Scientific	09-753-50	filter forceps
Model 502 Precision PanaPress	PanaVise	502	pneumatic cassette press is constructed from this precision arbor press
Scotch Super 33+ vinyl electrical tape	McMaster-Carr	76455A21	19 mm tape
Multi-purpose Calibration Jar, Large	SKC Inc.	225-112	calibration jar
Universal PCXR4 Sample Pump	SKC Inc.	224-PCXR4	sampling pump
Mass Flowmeter 4140	TSI Inc.	4140	flow meter
Roche High Pure PCR Template Kit	Roche Diagnostics	11796828001	Kit used for genomic DNA extraction. Contains: Lysis buffer, Binding buffer, Proteinase K, Inhibitor removal buffer, Wash buffer, Elution buffer, Glass fiber filter tubes and 2 ml collection tubes
Fisherbrand 2 mL Reinforced Polypropylene Screw Cap Tubes with Caps	Fisher Scientific	15340162	2 mL reinforced tubes for bead homogenization
Glass beads, acid washed, 212-300 µm	Sigma-Aldrich	G1277	glass beads
Fisher Scientific Bead Mill 24 Homogenizer	Fisher Scientific	15-340-163	bead homogenizer
CelLytic B Cell Lysis Reagent, 10X	Sigma-Aldrich	C8740	lysis reagent
Platinum Taq polymerase	Invitrogen	10966-018	Contains: Platinum Taq polymerase, 10X PCR buffer (no MgCl2), 50 mM MgCl2, KB Extender
dNTP Mix	Invitrogen	18427-088	10 mM dNTP mix
QIAquick PCR Purification Kit	Qiagen	28106	Kit used to purify fungal amplicons. Contains: Buffer PB (binding buffer), Buffer PE (washing buffer), Buffer EB (elution buffer), pH Indicator dye (optional), and GelPilot loading dye
Owl EasyCast Mini Gel Electrophoresis System	Thermo Fisher	B1 or B2
TrackIt 1 KB Plus DNA Ladder	Thermo Fisher	10488-085	DNA ladder