Name: detection of viruses from bioaerosols using anion exchange resin
Uploaded: 2018-08-22T14:45:00
Description: Watch this Scientific Journal Video about Detection of Viruses from Bioaerosols Using Anion Exchange Resin at JoVE.com

This work was supported by funding from the CDC/NIOSH High Plains Intermountain Center for Agricultural Health and Safety (5U54OH008085) and the Colorado Bioscience Discovery Evaluation Grant Program (14BGF-16).

1. Setup of the Bioaerosol Chamber (See Figure 2)
<ol>
	<li>Pre-load the liquid impingers with 20 mL of 0.01 M phosphate buffered saline, pH 7.5 (PBS).
	<ol>
		<li>Add 0.5 g of anion exchange resin and suspend within the PBS of one of the liquid impingers, with another liquid impinger serving as a control.</li>
	</ol>
	</li>
	<li>Position liquid impingers in parallel inside the bioaerosol chamber using clamp stands with aerosol inlets facing the nebulizer. 
	NOTE: See...

<ol>
	<li>Pirtle, E. C., Beran, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+survival+in+the+environment.">Virus survival in the environment.</a> Revue scientifique et technique (International Office of Epizootics). 10 (3), 733-748 (1991).</li><li>Nguyen, T. 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=A+community-wide+outbreak+of+legionnaires+disease+linked+to+industrial+cooling+towers--how+far+can+contaminated+aerosols+spread?.">A community-wide outbreak of legionnaires disease linked to industrial cooling towers--how far can contaminated aerosols spread?.</a> The Journal of Infectious Diseases. 193 (1), 102-111 (2006).</li><li>Marks, P. 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=Evidence+for+airborne+transmission+of+Norwalk-like+virus+(NLV)+in+a+hotel+restaurant.">Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant.</a> Epidemiology and Infection. 124 (3), 481-487 (2000).</li><li>Marks, P. 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+school+outbreak+of+Norwalk-like+virus:+Evidence+for+airborne+transmission.">A school outbreak of Norwalk-like virus: Evidence for airborne transmission.</a> Epidemiology and Infection. 131 (1), 727-736 (2003).</li><li>Corzo, C. A., Culhane, M., Dee, S., Morrison, R. B., Torremorell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airborne+detection+and+quantification+of+swine+influenza+a+virus+in+air+samples+collected+inside,+outside+and+downwind+from+swine+barns.">Airborne detection and quantification of swine influenza a virus in air samples collected inside, outside and downwind from swine barns.</a> PLoS One. 8 (8), e71444 (2013).</li><li>Anderson, B. D., 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=Bioaerosol+sampling+in+modern+agriculture:+A+novel+approach+for+emerging+pathogen+surveillance.">Bioaerosol sampling in modern agriculture: A novel approach for emerging pathogen surveillance.</a> The Journal of Infectious Diseases. 214 (4), 537-545 (2016).</li><li>Hietala, S. K., Hullinger, P. J., Crossley, B. M., Kinde, H., Ardans, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+air+sampling+to+detect+exotic+Newcastle+disease+virus+in+two+California+commercial+poultry+flocks.">Environmental air sampling to detect exotic Newcastle disease virus in two California commercial poultry flocks.</a> Journal of Veterinary Diagnostic Investigation. 17 (2), 198-200 (2005).</li><li>Jonges, 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=Wind-mediated+spread+of+low-pathogenic+avian+influenza+virus+into+the+environment+during+outbreaks+at+commercial+poultry+farms.">Wind-mediated spread of low-pathogenic avian influenza virus into the environment during outbreaks at commercial poultry farms.</a> PLoS One. 10 (5), e0125401 (2015).</li><li>Otake, S., Dee, S. A., Jacobson, L., Torremorell, M., Pijoan, C. <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+aerosol+transmission+of+porcine+reproductive+and+respiratory+syndrome+virus+under+controlled+field+conditions.">Evaluation of aerosol transmission of porcine reproductive and respiratory syndrome virus under controlled field conditions.</a> The Veterinary Record. 150 (26), 804-808 (2002).</li><li>Wu, Y., 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=Aerosolized+avian+influenza+A+(H5N6)+virus+isolated+from+a+live+poultry+market,+China.">Aerosolized avian influenza A (H5N6) virus isolated from a live poultry market, China.</a> The Journal of Infection. 74 (1), 89-91 (2017).</li><li>Choi, M. 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=Live+animal+markets+in+Minnesota:+A+potential+source+for+emergence+of+novel+influenza+A+viruses+and+interspecies+transmission.">Live animal markets in Minnesota: A potential source for emergence of novel influenza A viruses and interspecies transmission.</a> Clinical Infectious Diseases. 61 (9), 1355-1362 (2015).</li><li>Haig, C. W., Mackay, W. G., Walker, J. T., Williams, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioaerosol+sampling:+Sampling+mechanisms,+bioefficiency+and+field+studies.">Bioaerosol sampling: Sampling mechanisms, bioefficiency and field studies.</a> The Journal of Hospical Infection. 93 (3), 242-255 (2016).</li><li>Anderson, B. D., Lednicky, J. A., Torremorell, M., Gray, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+bioaerosol+aampling+for+airborne+virus+surveillance+in+swine+production+facilities:+A+mini+review.">The use of bioaerosol aampling for airborne virus surveillance in swine production facilities: A mini review.</a> Frontiers in Veterinary Science. 4, 121 (2017).</li><li>Verreault, D., Moineau, S., Duchaine, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+sampling+of+airborne+viruses.">Methods for sampling of airborne viruses.</a> Microbiology and Molecular Biology Reviews. 72 (3), 413-444 (2008).</li><li>Hogan, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling+methodologies+and+dosage+assessment+techniques+for+submicrometre+and+ultrafine+virus+aerosol+particles.">Sampling methodologies and dosage assessment techniques for submicrometre and ultrafine virus aerosol particles.</a> Journal of Applied Microbiology. 99 (6), 1422-1434 (2005).</li><li>Yu, K. -. P., Chen, Y. -. P., Gong, J. -. Y., Chen, Y. -. C., Cheng, C. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+collection+efficiency+of+the+liquid+impinger+for+ultrafine+particles+and+viral+aerosols+by+applying+granular+bed+filtration.">Improving the collection efficiency of the liquid impinger for ultrafine particles and viral aerosols by applying granular bed filtration.</a> Journal of Aerosol Science. 101, 133-143 (2016).</li><li>Perez-Mendez, A., 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=Evaluation+of+a+simple+and+cost+effective+filter+paper-based+shipping+and+storage+medium+for+environmental+sampling+of+F-RNA+coliphages.">Evaluation of a simple and cost effective filter paper-based shipping and storage medium for environmental sampling of F-RNA coliphages.</a> J Virol Methods. 194 (1-2), 60-66 (2013).</li><li>Chandler, J. C., 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-based+evaluation+of+a+male-specific+(F+)+RNA+coliphage+concentration+method.">Field-based evaluation of a male-specific (F+) RNA coliphage concentration method.</a> Journal of Virological Methods. 239, 9-16 (2017).</li><li>Perez-Mendez, A., Chandler, J. C., Bisha, B., Goodridge, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+of+enteric+viruses+from+tap+water+using+an+anion+exchange+resin-based+method.">Concentration of enteric viruses from tap water using an anion exchange resin-based method.</a> Journal of Virological Methods. 206, 95-98 (2014).</li><li>Perez-Mendez, A., Chandler, J. C., Bisha, B., Goodridge, L. D. <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+an+anion+exchange+resin-based+method+for+concentration+of+F-RNA+coliphages+(enteric+virus+indicators)+from+water+samples.">Evaluation of an anion exchange resin-based method for concentration of F-RNA coliphages (enteric virus indicators) from water samples.</a> Journal of Virological Methods. 204, 109-115 (2014).</li><li>Kammerer, J., Carle, R., Kammerer, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+and+ion+exchange:+Basic+principles+and+their+application+in+food+processing.">Adsorption and ion exchange: Basic principles and their application in food processing.</a> Journal of Agricultural and Food Chemistry. 59 (1), 22-42 (2011).</li><li>Chandler, J. C., 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+method+for+the+improved+detection+of+aerosolized+influenza+viruses+and+the+male-specific+(F+)+RNA+coliphage+MS2.">A method for the improved detection of aerosolized influenza viruses and the male-specific (F+) RNA coliphage MS2.</a> Journal of Virological Methods. 246, 38-41 (2017).</li><li>Friedman, S. D., Cooper, E. M., Calci, K. R., Genthner, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+assessment+of+a+real+time+reverse+transcription-PCR+method+to+genotype+single-stranded+RNA+male-specific+coliphages+(Family+Leviviridae).">Design and assessment of a real time reverse transcription-PCR method to genotype single-stranded RNA male-specific coliphages (Family Leviviridae).</a> Journal of Virological Methods. 173 (2), 196-202 (2011).</li><li>Selvaraju, S. B., Selvarangan, R. <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+three+influenza+A+and+B+real-time+reverse+transcription-PCR+assays+and+a+new+2009+H1N1+assay+for+detection+of+influenza+viruses.">Evaluation of three influenza A and B real-time reverse transcription-PCR assays and a new 2009 H1N1 assay for detection of influenza viruses.</a> Journal of Clinical Microbiology. 48 (11), 3870-3875 (2010).</li><li>Cademartiri, 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=Immobilization+of+bacteriophages+on+modified+silica+particles.">Immobilization of bacteriophages on modified silica particles.</a> Biomaterials. 31 (7), 1904-1910 (2010).</li><li>Michen, B., Graule, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoelectric+points+of+viruses.">Isoelectric points of viruses.</a> Journal of Appled Microbiology. 109 (2), 388-397 (2010).</li><li>Turgeon, N., Toulouse, M. J., Martel, B., Moineau, S., Duchaine, C. <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+five+bacteriophages+as+models+for+viral+aerosol+studies.">Comparison of five bacteriophages as models for viral aerosol studies.</a> Applied and Environmental Microbiology. 80 (14), 4242-4250 (2014).</li><li>Vergara, G. G., 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=Evaluation+of+FRNA+coliphages+as+indicators+of+human+enteric+viruses+in+a+tropical+urban+freshwater+catchment.">Evaluation of FRNA coliphages as indicators of human enteric viruses in a tropical urban freshwater catchment.</a> Water Research. 79, 39-47 (2015).</li><li>Tung-Thompson, G., Libera, D. A., Koch, K. L., de Los Reyes, F. L., Jaykus, L. 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This protocol outlines a method for sensitive viral capture from bioaerosols using modified liquid impingers. The method is optimized for detection and quantification of the viral load in bioaerosols. The specific modification demonstrated here involves the addition of anion exchange resin to liquid contained within a common liquid impinger. This method was developed for its simplicity in downstream sample processing, whereas other sample processing techniques such as centrifugation, filtration, and precipitation-based m...

The purpose of this method is to provide a highly sensitive bioaerosol sampling platform to facilitate molecular detection of negatively-charged viruses from bioaerosols. Microorganisms, including viral particles, can survive in bioaerosols for extended periods of time1. Bioaerosols can travel over relatively long distances and maintain viability and infectivity, as evidenced by an outbreak of Legionnaires&#39; disease that originated from industrial cooling towers located at a distance of 6 km from the affected individuals and resulted in 18 fatalities2. Indirect transmission of viruses to humans mediated by bioaerosols can occur in multiple settings and has been demonstrated for norovirus outbreaks in schools and restaurants3,4. Similarly, bioaerosol transmission of viruses can occur in agricultural settings such as in swine and poultry farms, with this transmission route being considered as a major factor in the movement of viruses between production facilities5,6,7,8,9.
Effective sampling of virus-laden bioaerosols allows for improvement in rapid diagnostics and preparedness for outbreak prevention, as shown in demonstrations in which H5 influenza A viruses were detected from bioaerosols in live animal markets in China and the United States10,11. Current bioaerosol sampling technologies involve a number of different particle capture principles, and can be broadly categorized into impingers, cyclones, impactors, and filters12. It is beyond the scope of this protocol to exhaustively cover all advantages and disadvantages of these platforms for sampling of viruses from bioaerosols; however, it can be stated that the majority of these sampling devices have not been optimized for the collection of viruses and bacteriophages13. Furthermore, infectivity of viral particles is often negatively affected, with liquid impingers considered to maintain viral infectivity more effectively than sampling devices such as solid impactors or filters14. However, one disadvantage of liquid impingement is the target dilution effect, which occurs because viruses are collected in relatively large volumes (typically &#8805;20 mL) of liquid in the collection vessel. Another important disadvantage involves the suboptimal efficiency of liquid impingers to concentrate particles &#60;0.5 &#181;M in size15. However, capture efficiency of these devices can be improved by immobilization on solid matrices, as immobilization can enhance preservation of viral nucleic acids and viral infectivity16,17.
We have previously demonstrated that anion exchange resin is an effective tool for the capture and concentration of viruses from liquid matrices, including F-RNA bacteriophages, hepatitis A virus, human adenovirus, and rotavirus18,19,20. As defined by the manufacturer, the anion exchange resin utilized in this work is a macroreticular polystyrene strong base anion exchange resin in which functionalized quaternary amine groups mediate attraction and capture of anions in a liquid medium21. Consequently, the anion exchange resin is expected to capture viruses with net-negative surface charges, including many enteric viruses, influenza viruses and other viruses relevant to public and animal health.
The current protocol involves the addition of anion exchange resin to a liquid impinger. In this system, the resin serves as a secondary concentration step for viral particles captured in the impinger liquid. Nucleic acids can then be directly eluted in small volumes, providing a concentrated sample for molecular analyses. Thus, the main advantage of this method is the improvement in viral detection sensitivity, primarily through reduction in sample volume. Additionally, due to the inherent non-specific capture of negatively-charged viruses, the method is likely applicable for detection of a large number of viruses of interest. Here, the method is demonstrated for vaccine strains of type A and type B influenza viruses and the FRNA coliphage MS2 (MS2). These viruses are subsequently detected using standard qRT-PCR assays as previously described22. The end-point user should not expect to encounter difficulties in performing this method, because modifications to currently existing equipment do not constitute major disruptions to the conventional flow of bioaerosol sampling and analysis.

<table>
	<tbody>
		<tr>
			<td>Escherichia coli bacteriophage MS2 (ATCC 15597-B1)</td>
			<td>American Type Culture Collection</td>
			<td>ATCC 15597-B1</td>
			<td></td>
		</tr>
		<tr>
			<td>FluMist Quadrivalent</td>
			<td>AstraZeneca</td>
			<td>Contact manufacturer</td>
			<td>Viral constitutents of this vaccine are subject to change on an annual basis</td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>1855195</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers and probes</td>
			<td>Integrated DNA Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#181;M sterile filter</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1 L pyrex bottles or equivalent</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL pipet tips</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL pipettor</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL serological pipet</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-aid or equivalent</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>52904</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect Probe RT-PCR Kit</td>
			<td>Qiagen</td>
			<td>204443</td>
			<td></td>
		</tr>
		<tr>
			<td>Amberlite IRA-900 chloride form</td>
			<td>Sigma-Aldrich</td>
			<td>216585-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Water (molecular biology grade)</td>
			<td>Sigma-Aldrich</td>
			<td>W4502-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf DNA LoBind Microcentrifuge Tubes</td>
			<td>ThermoFisher</td>
			<td>13-698-791</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL Conical Centrifuge Tubes&#160;</td>
			<td>ThermoFisher</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Polypropylene Centrifuge Tubes</td>
			<td>ThermoFisher</td>
			<td>05-538-62</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Platinum One-Step qRT-PCR Kit w/ROX</td>
			<td>ThermoFisher</td>
			<td>11745100</td>
			<td></td>
		</tr>
		<tr>
			<td>SKC Biosampler 20 mL, 3-piece glass set</td>
			<td>SKC Inc.</td>
			<td>225-9593</td>
			<td></td>
		</tr>
		<tr>
			<td>Vac-u-Go sample pumps</td>
			<td>SKC Inc.</td>
			<td>228-9695</td>
			<td></td>
		</tr>
		<tr>
			<td>Collison nebulizer (6-jet)</td>
			<td>BGI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPA capsule</td>
			<td>PALL</td>
			<td>12144</td>
			<td></td>
		</tr>
		<tr>
			<td>Q-TRAK indoor air quality monitor 8554</td>
			<td>TSI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Alnor velometer thermal anemometer AVM440-A</td>
			<td>TSI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>SidePak AM510 personal aerosol monitor</td>
			<td>TSI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioaerosol chamber</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of viruses from bioaerosols using anion exchange resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

Figure 1 demonstrates the principle behind charge-based capture of viruses from bioaerosols via inclusion of resin in liquid-based impingers. Figure 2 shows the setup of the custom-built bioaerosol chamber. Figure 3 describes the steps involved in setting up the aerosolization experiment and measures to ensure quality control. Figure 4 shows amplification curves for qRT-...

Watch this Scientific Journal Video about Detection of Viruses from Bioaerosols Using Anion Exchange Resin at JoVE.com

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

This method can help answer key questions about the type, level, and distribution of viruses in bioaerosols that are important for public and veterinary health. The main advantages of this method are its improvement on the sensitivity of molecular detection of viruses from bioaerosols, its field amenability, and its ease of use. Create suspensions of the experimental concentrations of viral particles of interest in 100 milliliter volumes of PBS within the glass vessels of a six-jet collision nebulizer.To set up the bioaerosol chamber, preload two liquid impingers with 20 milliliters of 0.01 molar PBS followed by the addition of 0.5 grams of anion exchange resin to a single impinger. Assemble the impinger. Install the suspensions within the chamber.Use clamp stands to position liquid impingers in parallel inside the bioaerosol chamber with the aerosol sampler inlets facing the nebulizer. Place a direct read instrument near the liquid impingers to monitor the environmental variables and place an additional direct read aerosol monitor near the liquid impingers to measure the mass concentration of the bioaerosol. Then, run the axial fans in the corners of the bioaerosol chamber to facilitate mixing of the aerosol.When the chamber is ready, start the instruments for measuring the variables within the bioaerosol chamber. Use a primary flow standard to calibrate the system to maintain the consistency between the sampling volumes. Seal the chamber for a 10 minute purge with HEPA filtered air.Deliver filtered, dried air to the nebulizer at 6.9 kilopascals of pressure to generate a target concentration of viral bioaerosols. When the target mass concentration of the aerosol has been reached, stop the nebulization. Then, use a HEPA filtered line located near the nebulizer to supply diluted air, taking care to maintain a constant pressure within the chamber and actively sample the bioaerosol chamber atmosphere for 40 minutes to achieve a sampling volume of 500 liters per sample.Calibration of the instrument is critical for determining the concentration of air contaminants. The sampling train should be calibrated before and after each sampling event. Pre and post calibrations should be within 5%of one another.Turn off the pumps at the end of the sampling. To isolate nucleic acids directly from the anion exchange resin, transfer all of the anion exchange resin containing liquid from the liquid impingers into individual, sterile 50 milliliter conical tubes. After allowing the resin to settle, slowly decant the liquid sample from the anion exchange resin, using a one milliliter pipette tip to remove any remaining liquid from the resin.Next, add 560 microliters of viral lysis buffer containing carrier RNA from a viral RNA isolation kit directly to the anion exchange resin, for a 10 minute incubation at room temperature with periodic mixing. At the end of the incubation, use a one milliliter pipette tip to transfer the viral lysate to a sterile 1.5 milliliter conical tube and proceed with the RNA isolation according to the manufacturer's instructions. Then, perform quantitative real-time PCR for detection of MS2 bacteriaphage and influenza A and B viruses using five microliters of nucleic acid eluent per sample.As shown in these representative qRT-PCR amplification curves, the detection of Type A influenza viruses can be achieved up to 3.26 cycles sooner when anion exchange resin is used, compared to direct testing of the impingement liquid. In a qRT-PCR that is 100%efficient, a gain of one threshold cycle corresponds to a two-fold increase in target concentration, thus the representative results indicate a 9.58 fold improvement in detection sensitivity. Once mastered, this technique can be completed in less than three hours if performed properly.While attempting this procedure, it's important to remember that when air sampling is conducted in the field, parameters such as relative humidity and temperature may affect collection efficiencies and that collection buffers may have to be optimized for different viruses. Following this procedure, other culture based methods can be utilized to answer additional questions about viral infectivity. After its development, this technique has paved the way for researchers and other professionals in fields of agricultural and public health, to explore alternative methods for viral capture and concentration from bioaerosols for risk assessment in viral disease surveillance.After watching this video, you should have a good understanding of how to capture and concentrate viruses from bioaerosols using modified liquid impingers, extract nucleic acids directly from the anion exchange resin and detect viruses using qRT-PCR. Don't forget that working with viral pathogens can be extremely hazardous and precautions such as using personal protective equipment and bio-safety level two or higher should always be taken when performing this procedure.

detection-of-viruses-from-bioaerosols-using-anion-exchange-resin

Research

JoVE Journal

Environment

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the ...

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A multidimensional gas chromatography method for the analysis of dissolved hydrogen sulfide in liquid crude oil samples is presented. A Deans switch is used to heart-cut light sulfur gases for separation on a secondary column and detection on a sulfur chemiluminescence detector.

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Building Double-decker Traps for Early Detection of Emerald Ash Borer

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions of forest and landscape ash (Fraxinus spp.) trees. Several artificial trap designs to attract and capture EAB beetles have been developed to detect, delineate, and monitor infestations. Double-decker (DD) traps consist of two corrugated plastic prisms, one green and one purple, attached to a 3 m tall polyvinyl chloride (PVC) pipe supported by a t-post. The green prism at the top of the PVC pipe is baited with cis-3-hexenol, a compound produced by ash foliage. Surfaces of both prisms are coated with sticky insect glue to capture adult EAB beetles. Double-decker traps should be placed near ash trees but in open areas, exposed to sun. Double-decker trap construction and placement are presented here, along with a summary of field experiments demonstrating the efficacy of DD traps in capturing EAB beetles. In a recent study in sites with relatively low EAB densities, double-decker traps captured significantly more EAB than green or purple prism traps or green funnel traps, all of which are designed to be suspended from a branch in the canopy of ash trees. A greater percentage of double decker traps were positive, i.e., captured at least one EAB, than the prism traps or funnel traps that were hung in ash tree canopies.

Effective traps to attract and capture the emerald ash borer (EAB) are a key element of detecting and managing this invasive pest. Double-decker traps, placed in full sun near ash trees, incorporate visual and olfactory cues and were more likely to capture EAB than other trap designs in field trials.

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions ...

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and organiclastic sulfate reduction (OSR) in marine sediments. However, unravelling the complex pyritization sequence is a challenge because of the coexistence of different sequentially formed pyrite phases. This manuscript describes a sample preparation procedure that enables the use of secondary ion mass spectroscopy (SIMS) to obtain in situ &#948;34S values of various pyrite generations. This allows researchers to constrain how SO4-AOM affects pyritization in methane-bearing sediments. SIMS analysis revealed an extreme range in &#948;34S values, spanning from -41.6 to +114.8&#8240;, which is much wider than the range of &#948;34S values obtained by the traditional bulk sulfur isotope analysis of the same samples. Pyrite in the shallow sediment mainly consists of 34S-depleted framboids, suggesting early diagenetic formation by OSR. Deeper in the sediment, more pyrite occurs as overgrowths and euhedral crystals, which display much higher SIMS &#948;34S values than the framboids. Such 34S-enriched pyrite is related to enhanced SO4-AOM at the sulfate-methane transition zone, postdating OSR. High-resolution in situ SIMS sulfur isotope analyses allow for the reconstruction of the pyritization processes, which cannot be resolved by bulk sulfur isotope analysis.

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Analyses of the sulfur isotopic composition (&#948;34S) of pyrite from methane-bearing sediments have typically focused on bulk samples. Here, we applied secondary ion mass spectroscopy to analyze the &#948;34S values of various pyrite generations to understand the diagenetic history of pyritization.

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and ...

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf mottling, vein yellowing, defoliation, root decay, and ultimately, the death of the citrus plant. When infected, the citrus plants have stunted growth and produce flowers out of season. These flowers rarely yield fruit, and those that do yield small, bitter, irregularly shaped citrus fruit that are not desirable. This disease is spread by the Asian citrus psyllid, Diaphorina citri, and by the grafting of infected citrus tissue. The pathogen has a long and variable incubation period within the citrus plant&#8212;sometimes years, before symptoms appear. Attempts to culture this pathogen in vitro have been unsuccessful, possibly due to the low and uneven concentration of the pathogen within infected citrus tissue, or because it is difficult to replicate the environmental conditions conducive to growth of the pathogen. It is very difficult to identify the disease before it has spread, due to its long incubation period and researchers&#39; inability to culture the pathogen. As a result, the disease only becomes apparent after suddenly destroying a citrus farmer&#39;s entire yield. Presented here is a method for the accurate and specific detection of the citrus greening pathogen, Candidatus liberibacter spp. using a genomic DNA extraction kit and PCR. This method is simple, efficient, cost effective, and adaptable for quantitative analysis. This method can be adapted for use on any citrus tissue; however, it is potentially limited by the amount of pathogen present in the tissue. Nevertheless, this method will allow citrus farmers to identify infected citrus plants earlier, and curb the spread of this destructive disease before it can further spread.

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus Greening is a particularly destructive disease affecting citrus crops globally. Presented here is a simple method using PCR and genomic DNA extraction of citrus leaf tissue for the accurate and precise identification of the citrus greening pathogen, Candidatus liberibacter spp.

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf ...

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...