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 arbejde blev støttet i en del af en tværinstitutionelle aftale mellem NIOSH og NIEHS (AES12007001-1-0-6).

1. forberedelse NIOSH aerosol sampler
Bemærk: NIOSH aerosol sampler er en to-trins cyklon aerosol sampler, der indsamler bioaerosols ved hjælp af to stikprøver rør og en polytetrafluorethylen (PTFE) filter.
<ol><li>Før montering, omhyggeligt inspicere sampler. Kontrollere sampler til at sikre, at det ikke er beskadiget, alle skruer er på plads og LUN og forsegling båndet rundt sømmen dannet af de to halvdele er intakt. Inspicere sampler O-ring til at sikre, at det ikke har nogen rift...

<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. 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=Internal+transcribed+spacer+rRNA+gene+sequencing+analysis+of+fungal+diversity+in+Kansas+City+indoor+environments.">Internal transcribed spacer rRNA gene sequencing analysis of fungal diversity in Kansas City indoor environments.</a> Environ Sci Process Impacts. 16 (1), 33-43 (2014).</li><li>Martin, K. J., Rygiewicz, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal-specific+PCR+primers+developed+for+analysis+of+the+ITS+region+of+environmental+DNA+extracts.">Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts.</a> BMC Microbiol. 5, 28 (2005).</li><li>Monard, C., Gantner, S., Stenlid, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+ITS1+and+ITS2+to+study+environmental+fungal+diversity+using+pyrosequencing.">Utilizing ITS1 and ITS2 to study environmental fungal diversity using pyrosequencing.</a> FEMS Microbiol Ecol. 84 (1), 165-175 (2013).</li><li>Op De Beeck, M., Lievens, B., Busschaert, P., Declerck, S., Vangronsveld, J., Colpaert, J. 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>

Bestemmelse af svampe mangfoldigheden inden for en erhvervsmæssig miljø ved hjælp af sekventering-baserede tilgange har forbedret svampe farevurderingen identifikation og eksponering. Ved hjælp af denne fremgangsmåde har tilladt til påvisning af mange yderligere svampe arter, der er ofte ikke registreret bruger kultur eller mikroskopi-baserede metoder til vurdering. En metode for prøveudtagning bioaerosols fra arbejdspladsen og indendørs miljøer og udvinding af genomisk DNA fra Luftprøver for dens forstærkning...

Svampe engagementer i indendørs og faglige miljøer kan resultere i respiratoriske morbiditet, herunder allergisk sensibilisering og astma1. Identifikation af svampe farer er vigtig for vurderingen af risikoen og forebygge arbejdstagernes eksponering. Disse svampe farer kan være et resultat af indendørs forurening, udendørs luft indbrud eller miljømæssige forstyrrelser, der resulterer i transporten af svampe materialer i områder, hvor arbejdstagerne er til stede2. Metoder til at vurdere svampe eksponering har inkluderet bæredygtig kultur prøveudtagning samt mikroskopisk identifikation af svampesporer. Disse tilgange har flere begrænsninger og ofte overser mange svampe arter, der kan bidrage til den samlede svampe byrde3. Kultur-baserede tilgange kan kun skelne de levedygtige fungal organismer, der kan dyrkes på næringsstof medier. At identificere svampesporer til artsniveau via mikroskopi kan blive forvirret af sporer deling lignende morfologier. Begge metoder er stærkt afhængige af mycologists til at analysere og identificere de svampe arter, med mange resterende uidentificerede.
For at forbedre eksisterende metoder, der anvendes i arbejdsskade identifikation og eksponering vurderinger, mange forskere har henvendt sig til molekylære-baserede teknologier. Sekventering-baserede metoder til vurdering af mikrobiel diversitet inden for indendørs og faglige miljøer har afsløret et bredere spektrum af svampe arter fundet i forhold til metoder som mikroskopi og levedygtige kultur3,4 ,5. Metoden præsenteres her beskriver luft prøvetagning af faglige miljøer og udvinding af genomisk DNA til identifikation af potentielle svampe farer. Fareidentifikation opnås ved sekventering nuklear ribosomale interne transskriberede spacer eller ITS, regioner, der er meget varierende blandt svampe og har været almindeligt brugt at differentiere svampe arter6,7, 8,9. Mange arter findes i erhvervsmæssig indstillinger, såsom nogle arter tilhører phylum Basidiomycota, ikke kan identificeres i levedygtige kultur og er vanskeligt at skelne mikroskopisk. Disse svampe er blevet observeret i høj relativ overflod inden for indendørs og faglige miljøer vurderet af sekventering svampe ITS regioner3,4,10. DENS sekventering har givet større viden i mangfoldigheden af svampe fundet inden for indendørs og faglige miljøer.
Protokollen beskrevet her detaljer metoderne, der anvendes til at indsamle, uddrage og forstærke svampe ITS områder fra bioaerosols til Sekvensanalyse. Denne metode udnytter det nationale Institut for Occupational Safety and Health (NIOSH) to-trins cyklon aerosol sampler at indsamle partikler i luften. Denne sampler blev udviklet til at indsamle bioaerosols og separat respirabelt (≤4 µm aerodynamisk diameter) og ikke-respirabelt (> 4 µm aerodynamisk diameter) partikler, der giver mulighed for identifikation af fungal organismer som indendørs miljøer, der er mest tilbøjelige til at blive indåndes af en arbejdstager11. Andre luft samplere, herunder cyklon samplere, er tilgængelige på markedet, som har mulighed for at indsamle partikler inden for respirabelt rækkevidde (< 4 µm) ved hjælp af filtre12,13. Derimod adskiller NIOSH to-trins cyklon aerosol sampler svampe arter baseret på deres aerodynamisk diameter til engangstændere, polypropylen rør, der umiddelbart kan behandles for downstream programmer14.
Processer til udvinding genomisk DNA og forstærke de svampe ITS regioner er beskrevet i denne protokol. Udvinding metoder præsenteret er blevet udviklet specielt til udvinding af genomisk DNA fra svampe og bakterier, som mange kommercielle kits målrette pattedyrceller, bakterier eller specifikt gær15. Primere anvendes i denne undersøgelse er udvalgt på grundlag af deres samlede dækning af både svampe dens 1 og dens 2 regioner4,5. Sekventering af disse regioner tillader til sammenligning af mange krænges ITS-sekvenser, herunder dem, denne sekvens regionen dens 1, dens 2 region eller både dens 1 og dens 2 regioner. De svampe mangfoldighed af Luftprøver indsamlet i en indendørs indstilling ved hjælp af disse metoder er vist, afslører et betydeligt antal sekvenser placeret i phyla Sæksvampe og Basidiomycota samt andre sekvenser tilhører mindre dominerende svampe phyla, som Zygomycota. Den brede mangfoldighed af svampe sekvenser identificeret ved hjælp af denne fremgangsmåde ville ikke blive fanget ved hjælp af traditionelle fare identifikation metoder som dyrkning og mikroskopi. Sekventering af svampe ITS områder giver en forbedret metode til at identificere svampe farer og giver mulighed for en bedre forståelse af indendørs og erhvervsmæssig svampe engagementer.

<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

Traditionelle metoder til at identificere svampe engagementer i faglige miljøer, såsom kultur og mikroskopi-baserede tilgange, har flere begrænsninger, der har ført til udelukkelsen af mange arter. Fremskridt inden for området i de sidste to årtier har ført arbejdsmiljø forskere at henvende sig til molekylære-baserede tilgange til at identificere svampe farer. Disse metoder har ført til påvisning af mange arter inden for indendørs og faglige miljøer, der ikke har været påvist ved hjælp af traditionelle metoder. Denne protokol detaljer en fremgangsmåde til bestemmelse af svampe mangfoldighed inden for luft prøver gennem genomisk DNA-ekstraktion, forstærkning, sekventering og taksonomisk identifikation af svampe interne transskriberet spacer (ITS) regioner. Sekventering resultaterne i afsløring af mange svampe arter, der er enten ikke fundet eller vanskeligt at identificere arter niveau ved hjælp af kultur eller mikroskopi. Mens disse metoder ikke giver kvantitative foranstaltninger af svampe byrde, de tilbyder en ny tilgang til farlighedsidentifikation og kan bruges til at bestemme samlede arter rigdom og mangfoldighed i en erhvervsmæssig miljø.

Arter distribution i et miljø kan vurderes ved hjælp af relative forekomst af fastsættelsen af antallet af kloner af hver OTU identificeret i Luftprøver. Figur 6 er en krone diagram, der repræsenterer arternes taksonomisk placeret inden for en indendørs miljø efter 60 min i luften prøvetagning. Det kan konstateres, at miljøet indeholder en bred vifte af arter i to store svampe phyla, Sæksvampe og Basidiomycota, samt arter tilhører phylum Zygomycota...

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

Bestemmelse af svampe mangfoldigheden inden for et miljø er en metode, der udnyttes i arbejdsmedicinske undersøgelser at identificere sundhedsmæssige farer. Denne protokol beskriver DNA-ekstraktion fra erhvervsmæssig Luftprøver til forstærkning og sekventering af svampe ITS regioner. Denne metode registrerer mange svampe arter, der kan blive overset af traditionelle beregningsmetoder.

Kollektion og udvinding af erhvervsmæssig Luftprøver til analyse af svampe 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.

Research

Education

JoVE Journal

JoVE Core

Environment

Biology

Nursing

Ecology

Unit 1

Introduction to Health

Biodiversity and Conservation

SCIENTISTS IN ACTION

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615. Måling af Enterovirus og norovirus Forekomst i vand efter kultur og RT-qPCR. I. Indsamling af virus prøver

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

En simpel metode til Automated Solid Phase Extraction af vandprøver for Immunologisk analyse af små miljøgifte

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Air-sampled Filter Analysis for Endotoxins and DNA Content

Air-samplet Filter Analyse for Endotoxiner og DNA indhold

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Samtidig DNA-RNA-ekstraktion fra kystsedimenter og kvantificering af 16S rRNA-generne og udskrifter af Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Ekstraktion og analyse af Mikrobiel Phospholipid fedtsyrer i Jordbunden

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Ændring og anvendelse af en løvblæser-vac for Field Sampling af leddyr

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Anvendelse af en filterpatron til filtrering af vandprøver og Udvinding af Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Automatiseret, høj opløsning Mobile Collection System for Nitrogen isotopanalysen af ​​NO<sub&gt; x</sub

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

En lipidekstraktions- og analysemetode til karakterisering af jordmikrober i forsøg med mange prøver

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Ekstraktion af organiske chlorpesticider fra plastpellets og plasttypeanalyse

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...