Name: collection and extraction of occupational air samples for analysis of fungal dna
Uploaded: 2018-05-02T13:00:00
Description: Watch this Scientific Journal Video about Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA at JoVE.com

This work was supported in part by an interagency agreement between NIOSH and NIEHS (AES12007001-1-0-6).

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.
<ol>
	<li>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 tea...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-stage+cyclone+using+microcentrifuge+tubes+for+personal+bioaerosol+sampling.">A two-stage cyclone using microcentrifuge tubes for personal bioaerosol sampling.</a> J Environ Monit. 8 (11), 1136-1142 (2006).</li><li>Rittenour, W. R., Park, J. H., Cox-Ganser, J. M., Beezhold, D. H., Green, B. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+bias+associated+with+incomplete+extraction+of+microbial+DNA+from+soil.">Assessment of bias associated with incomplete extraction of microbial DNA from soil.</a> Appl Environ Microbiol. 75 (16), 5428-5433 (2009).</li><li>Keswani, J., Kashon, M. L., Chen, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+interference+to+conventional+and+real-time+PCR+for+detection+and+quantification+of+fungi+in+dust.">Evaluation of interference to conventional and real-time PCR for detection and quantification of fungi in dust.</a> J Environ Monit. 7 (4), 311-318 (2005).</li><li>Bellemain, E., Carlsen, T., Brochmann, C., Coissac, E., Taberlet, P., Kauserud, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ITS+as+an+environmental+DNA+barcode+for+fungi:+an+in+silico+approach+reveals+potential+PCR+biases.">ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases.</a> BMC Microbiol. 10, 189 (2010).</li><li>Farrelly, V., Rainey, F. A., Stackebrandt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genome+size+and+rrn+gene+copy+number+on+PCR+amplification+of+16S+rRNA+genes+from+a+mixture+of+bacterial+species.">Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species.</a> Appl Environ Microbiol. 61 (7), 2798-2801 (1995).</li><li>Vesper, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+Environmental+Relative+Moldiness+index+for+US+homes.">Development of an Environmental Relative Moldiness index for US homes.</a> J Occup Environ Med. 49 (8), 829-833 (2007).</li></ol>

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...

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 (&#8804;4 &#181;m aerodynamic diameter) and non-respirable (&#62;4 &#181;m 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 (&#60;4 &#181;m) 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.

<table>
	<tbody>
		<tr>
			<td>NIOSH BC251 bioaerosol cyclone sampler</td>
			<td>NIOSH</td>
			<td>BC251</td>
			<td>The NIOSH sampler is not yet commercially available. Please contact William Lindsley, PhD (wlindsley@cdc.gov) for information on obtaining the NIOSH sampler</td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile Microcentrifuge Tubes with Screw Caps</td>
			<td>Fisher Scientific</td>
			<td>02-681-373</td>
			<td>1.5 mL polypropylene microcentrifuge tubes for air sampling; screw top threading must match the threading of the NIOSH sampler</td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>Corning</td>
			<td>352096</td>
			<td>15 mL polypropylene tubes for air sampling</td>
		</tr>
		<tr>
			<td>Clean Room Vinyl Tape, Easy-Remove, 1/4&#34; Width</td>
			<td>McMaster-Carr</td>
			<td>76505A1</td>
			<td>sealing tape</td>
		</tr>
		<tr>
			<td>Filter Cassette, Clear Styrene, 37 mm</td>
			<td>SKC Inc.</td>
			<td>225-3LF</td>
			<td>3-piece sampling cassette (no filter). Contains: cassette base, extension cowl, cassette cap and inlet/outlet plugs</td>
		</tr>
		<tr>
			<td>PTFE hydrophobic fluoropore membrane filters, 3.0 &#181;m, 37 mm</td>
			<td>EMD Millipore</td>
			<td>FSLW03700</td>
			<td>Contains: 37 mm, 3.0 &#181;m PTFE filters and support pads</td>
		</tr>
		<tr>
			<td>Fisherbrand filter forceps</td>
			<td>Fisher Scientific</td>
			<td>09-753-50</td>
			<td>filter forceps</td>
		</tr>
		<tr>
			<td>Model 502 Precision PanaPress</td>
			<td>PanaVise</td>
			<td>502</td>
			<td>pneumatic cassette press is constructed from this precision arbor press</td>
		</tr>
		<tr>
			<td>Scotch Super 33+ vinyl electrical tape</td>
			<td>McMaster-Carr</td>
			<td>76455A21</td>
			<td>19 mm tape</td>
		</tr>
		<tr>
			<td>Multi-purpose Calibration Jar, Large</td>
			<td>SKC Inc.</td>
			<td>225-112</td>
			<td>calibration jar</td>
		</tr>
		<tr>
			<td>Universal PCXR4 Sample Pump</td>
			<td>SKC Inc.</td>
			<td>224-PCXR4</td>
			<td>sampling pump</td>
		</tr>
		<tr>
			<td>Mass Flowmeter 4140</td>
			<td>TSI Inc.</td>
			<td>4140</td>
			<td>flow meter</td>
		</tr>
		<tr>
			<td>Roche High Pure PCR Template Kit</td>
			<td>Roche Diagnostics</td>
			<td>11796828001</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Fisherbrand 2 mL Reinforced Polypropylene Screw Cap Tubes with Caps</td>
			<td>Fisher Scientific</td>
			<td>15340162</td>
			<td>2 mL reinforced tubes for bead homogenization</td>
		</tr>
		<tr>
			<td>Glass beads, acid washed, 212-300 &#181;m</td>
			<td>Sigma-Aldrich</td>
			<td>G1277</td>
			<td>glass beads</td>
		</tr>
		<tr>
			<td>Fisher Scientific Bead Mill 24 Homogenizer</td>
			<td>Fisher Scientific</td>
			<td>15-340-163</td>
			<td>bead homogenizer</td>
		</tr>
		<tr>
			<td>CelLytic B Cell Lysis Reagent, 10X</td>
			<td>Sigma-Aldrich</td>
			<td>C8740</td>
			<td>lysis reagent</td>
		</tr>
		<tr>
			<td>Platinum Taq polymerase</td>
			<td>Invitrogen</td>
			<td>10966-018</td>
			<td>Contains: Platinum Taq polymerase, 10X PCR buffer (no MgCl2), 50 mM MgCl2, KB Extender</td>
		</tr>
		<tr>
			<td>dNTP Mix</td>
			<td>Invitrogen</td>
			<td>18427-088</td>
			<td>10 mM dNTP mix</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Owl EasyCast Mini Gel Electrophoresis System</td>
			<td>Thermo Fisher</td>
			<td>B1 or B2</td>
			<td></td>
		</tr>
		<tr>
			<td>TrackIt 1 KB Plus DNA Ladder</td>
			<td>Thermo Fisher</td>
			<td>10488-085</td>
			<td>DNA ladder</td>
		</tr>
	</tbody>
</table>

collection and extraction of occupational air samples for analysis of fungal dna

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.

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...

Watch this Scientific Journal Video about Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA at JoVE.com

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

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.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

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.

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