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 culture3,4,5. 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 species6,7,8,9. 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 regions3,4,10. 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 filters12,13. 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 regions4,5. 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
Representative Results
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported in part by an interagency agreement between NIOSH and NIEHS (AES12007001-1-0-6).
Materials
| 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 |
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