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

Dette arbeidet var støttes delvis av en tverrfaglig avtale mellom NIOSH og NIEHS (AES12007001-1-0-6).

1. klargjør NIOSH aerosol sampler
Merk: Den NIOSH aerosol sampler er en totrinns syklon aerosol sampler som samler bioaerosols med to utvalg rør og filtere polytetrafluoroethylene (PTFE).
<ol><li>Før montering, nøye kontrollere sampler. Kontroller sampler å sikre at det ikke er skadet, alle skruene er på plass og lun og tetting tape rundt skjøten dannet av de to halvdelene er intakt. Inspisere sampler O-ring for å sikre at den ikke har hakk, sprekker eller tårer og at den har en vel...

<ol>
	<li>Mendell, M. J., Mirer, A. G., Cheung, K., Tong, M., Douwes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+and+allergic+health+effects+of+dampness,+mold,+and+dampness-related+agents:+a+review+of+the+epidemiologic+evidence.">Respiratory and allergic health effects of dampness, mold, and dampness-related agents: a review of the epidemiologic evidence.</a> Environ Health Perspect. 119 (6), 748-756 (2011).</li><li>Green, B. J., Lemons, A. R., Park, Y., Cox-Ganser, J. M., Park, J. H. <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+fungal+diversity+in+a+water-damaged+office+building.">Assessment of fungal diversity in a water-damaged office building.</a> J Occup Environ Hyg. 14 (4), 285-293 (2017).</li><li>Green, B. J., Lemons, A. R., Park, Y., Cox-Ganser, J. M., Park, J. H. <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+fungal+diversity+in+a+water-damaged+office+building.">Assessment of fungal diversity in a water-damaged office building.</a> J Occup Environ Hyg. , (2016).</li><li>Pitkaranta, M., 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=Analysis+of+fungal+flora+in+indoor+dust+by+ribosomal+DNA+sequence+analysis,+quantitative+PCR,+and+culture.">Analysis of fungal flora in indoor dust by ribosomal DNA sequence analysis, quantitative PCR, and culture.</a> Appl Environ Microbiol. 74 (1), 233-244 (2008).</li><li>Rittenour, W. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+and+validation+of+some+ITS+primer+pairs+useful+for+fungal+metabarcoding+studies.">Comparison and validation of some ITS primer pairs useful for fungal metabarcoding studies.</a> PLoS One. 9 (6), e97629 (2014).</li><li>Toju, H., Tanabe, A. S., Yamamoto, S., Sato, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-coverage+ITS+primers+for+the+DNA-based+identification+of+ascomycetes+and+basidiomycetes+in+environmental+samples.">High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples.</a> PLoS One. 7 (7), e40863 (2012).</li><li>Lemons, A. R., 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=Microbial+rRNA+sequencing+analysis+of+evaporative+cooler+indoor+environments+located+in+the+Great+Basin+Desert+region+of+the+United+States.">Microbial rRNA sequencing analysis of evaporative cooler indoor environments located in the Great Basin Desert region of the United States.</a> Environ Sci Process Impacts. , (2017).</li><li>Cao, G., Noti, J. D., Blachere, F. M., Lindsley, W. G., Beezhold, D. H. <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+improved+methodology+to+detect+infectious+airborne+influenza+virus+using+the+NIOSH+bioaerosol+sampler.">Development of an improved methodology to detect infectious airborne influenza virus using the NIOSH bioaerosol sampler.</a> J Environ Monit. 13 (12), 3321-3328 (2011).</li><li>Soo, J. C., Lee, T., Kashon, M., Kusti, M., Harper, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quartz+in+coal+dust+deposited+on+internal+surface+of+respirable+size+selective+samplers.">Quartz in coal dust deposited on internal surface of respirable size selective samplers.</a> J Occup Environ Hyg. 11 (12), D215-D219 (2014).</li><li>Wang, C. H., 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=Field+evaluation+of+personal+sampling+methods+for+multiple+bioaerosols.">Field evaluation of personal sampling methods for multiple bioaerosols.</a> PLoS One. 10 (3), e0120308 (2015).</li><li>Lindsley, W. G., Schmechel, D., 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=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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+DNA+extraction+methodologies+used+for+assessing+fungal+diversity+via+ITS+sequencing.">Comparison of DNA extraction methodologies used for assessing fungal diversity via ITS sequencing.</a> J Environ Monit. 14 (3), 766-774 (2012).</li><li>ACGIH. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Documentation of the threshold limit values and biological exposure indices. Appendix C: Particle size-selective sampling criteria for airborne particulate matter. , (2001).</li><li>Rodes, C. E., Thornburg, J. W., Ruzer, L. S., Harley, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aerosols handbook: measurement, dosimetry, and health effects. , (2005).</li><li>Broadwater, K., 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=Investigating+a+persistent+odor+at+an+aircraft+seat+manufacturer.">Investigating a persistent odor at an aircraft seat manufacturer.</a> J Occup Environ Hyg. 13 (10), D159-D165 (2016).</li><li>Couch, J., 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=.">.</a> Health Hazard Evaluation Program: Evaluation of Potential Hazards during Harvesting and Processing Cannabis at an Outdoor Organic Farm. Report No. 2015-0111-3271. , (2017).</li><li>Feinstein, L. M., Sul, W. J., Blackwood, C. 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>

Bestemme fungal mangfoldet i en yrkesmessig miljøbruk sekvensering tilnærminger har forbedret fungal fare identifikasjon og eksponering vurdering. Bruker denne tilnærmingen har tillatt for påvisning av mange sopp vokser det er ofte ikke oppdaget kultur eller mikroskopi-baserte metoder for vurdering. En metode for prøvetaking bioaerosols fra arbeids- og innendørs miljøer og utvinning av genomisk DNA fra Luftprøver forsterkning og sekvensering presenteres her. Bestemmelse av sopp mangfold ved hjelp av disse metoden...

Fungal eksponeringer innendørs og yrkesmessig kan føre til åndedretts morbidities, inkludert økt følsomhet og astma1. Identifikasjon av sopp farer er viktig for å vurdere risiko og forebygge arbeidstaker eksponering. Disse sopp farer kan være et resultat av innendørs forurensning, uteluft inntrenging eller miljømessige forstyrrelser som fører til transport av sopp materialer i områder der arbeidstakere er tilstede2. Metoder for å vurdere fungal eksponering har inkludert levedyktig kultur prøvetaking samt mikroskopiske identifikasjon av sopp sporer. Disse metodene har flere begrensninger og ofte overse mange fungal arter som kan være medvirkende til total fungal byrden3. Kultur-baserte tilnærminger kan bare skille dem levedyktig fungal organismer som kan bli dyrket på næringsstoffer media. Identifisere fungal sporer arter nivå via mikroskopi kan bli forvirret av sporer dele lignende morphologies. Begge metodene er svært avhengige mycologists å analysere og identifisere fungal arter, med mange gjenværende uidentifisert.
For å forbedre eksisterende metoder som brukes i yrkesmessig Faremomenter og eksponering vurderinger, har mange forskere slått til molekylær-baserte teknologier. Sekvensering tilnærminger for å vurdere mikrobiell mangfold i innendørs og yrkesmessig har avdekket et bredere spekter av sopp oppdaget sammenlignet med metoder som mikroskopi og levedyktig kultur3,4 ,5. Metoden som presenteres her beskriver luften prøvetaking yrkesmessig miljøer og utvinning av genomisk DNA til å identifisere potensielle fungal farer. Faremomenter oppnås ved sekvensering av kjernefysisk ribosomal interne transkribert spacer, eller dens, områder som er svært variabel blant sopp og har blitt brukt til å skille fungal arter6,7, 8,9. Mange arter finnes i yrkesmessig innstillinger, som noen arter som tilhører rekken Basidiomycota, ikke er identifiserbare i levedyktig kultur og er vanskelig å skille mikroskopisk. Disse sopp er observert i høy relative overflod i innendørs og yrkesmessig miljøer vurdert av sekvensering fungal ITS regioner3,4,10. DENS sekvensering har gitt større kunnskap i mangfoldet av sopp oppstått innenfor innendørs og yrkesmessig miljøer.
Protokollen beskrevet her detaljer metodene brukes til å samle, trekke ut og forsterke fungal ITS regioner fra bioaerosols for sekvensen analyse. Denne tilnærmingen benytter National Institute for Occupational Safety og helse (NIOSH) totrinns syklon aerosol sampler samle partikler i luften. Denne sampler ble utviklet for å samle bioaerosols og egen respirabelt (≤4 µm aerodynamisk diameter) og ikke-respirabelt (> 4 µm aerodynamisk diameter) partikler, som gir mulighet for identifisering av sopp organismer i innendørs miljøer som er mest sannsynligvis pustes inn av en arbeidstaker11. Andre luft samplere, inkludert syklon samplere, er tilgjengelige på markedet som kan samle partikler innen respirabelt (< 4 µm) bruker filtrerer12,13. Derimot skiller NIOSH to-trinns syklon aerosol sampler fungal arter basert på deres aerodynamisk diameter til disponibel, polypropylen rør som kan behandles umiddelbart for nedstrøms programmer14.
Prosessene med utpakking genomisk DNA og forsterke fungal ITS regionene er beskrevet i denne protokollen. De utvinning metodikkene som presenteres er utviklet spesielt for utvinning av genomisk DNA fra sopp og bakterier, som mange kommersielle kits målrette pattedyrceller, bakterier eller gjær15spesielt. Primere brukt i denne studien er valgt basert på deres samlede dekning av både fungal sin 1 og dens 2 regioner4,5. Sekvensering av disse regionene tillater sammenligning av mange banked ITS sekvenser, inkludert den sekvensen regionen sin 1, dens 2 regionen eller både sin 1 og dens 2 regioner. Fungal mangfoldet av Luftprøver samlet i en innendørs bruker disse metodene vises, avslører et betydelig antall sekvenser plassert i stamtreet Ascomycota og Basidiomycota samt andre sekvenser tilhører mindre dominerende fungal rekker, som Zygomycota. Det brede mangfoldet av sopp sekvenser identifisert ved hjelp av denne tilnærmingen vil ikke hentes med tradisjonelle fare identifikasjon metoder som dyrking eller mikroskopi. Sekvensering av sopp ITS områder gir en forbedret metode for å identifisere fungal farer og gi en bedre forståelse av innendørs og yrkesmessig fungal eksponeringer.

<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

Tradisjonelle metoder for å identifisere fungal eksponeringer i yrkesmessig miljøer som kultur og mikroskopi-baserte tilnærminger, har flere begrensninger som har resultert i utelukkelse av mange arter. Fremskritt innen de siste to tiårene har ført helse forskere å slå til molekylær-baserte tilnærminger for å identifisere fungal farer. Disse metodene har resultert i deteksjon av mange arter innen innendørs og yrkesmessig miljøer som ikke ble funnet ved hjelp av tradisjonelle metoder. Denne protokollen detaljer tilnærming for å bestemme fungal mangfold i luften prøver gjennom genomisk DNA utvinning, forsterkning, sekvensering og taksonomisk identifikasjon av sopp interne transkribert spacer (ITS) regioner. Sekvensering resultatene i deteksjon av mange fungal arter som oppdaget eller er vanskelig å identifisere arter nivå med kultur eller mikroskopi. Mens disse metodene ikke gir kvantitative tiltak av sopp byrden, de tilbyr en ny tilnærming til Faremomenter og kan brukes til å fastsette generelle arter rikdom og mangfold i en yrkesmessig miljø.

Distribusjon innen et miljø kan vurderes med relative overflod ved å bestemme antall kloner av hver OTU i luften prøvene. Figur 6 er en krone diagram representerer taksonomisk plassert arter i et innemiljø etter 60 minutter i luften prøvetaking. Det kan være observert at miljøet inneholder en rekke arter innen to store fungal rekker, Ascomycota og Basidiomycota, samt arter som tilhører rekken Zygomycota (Rhizopus microsporus). Tradisjonelle m...

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

Avgjøre fungal mangfoldet i et miljø er en metode som brukes i helse studier å identifisere helsefare. Denne protokollen beskriver DNA utvinning fra yrkesmessig Luftprøver for forsterkning og sekvensering av sopp dens regioner. Denne tilnærmingen oppdager mange fungal arter som kan bli oversett av tradisjonelle vurderingsmetoder.

Innsamling og utvinning av yrkesmessig Air prøver for analyse av sopp DNA

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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