Name: cost-effective method for microbial source tracking using specific human and animal viruses
Uploaded: 2011-12-03T14:00:00
Description: Watch this Scientific Journal Video about Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses at JoVE.com

This work was partially supported by the Spanish Government "Ministerio de Educaci&oacute;n y Ciencia" (project AGL2008-05275-C01/ALI), by the European Union Research Framework 7 funded projects VIROBATHE (Contract No. 513648), VIROCLIME (Contract No. 243923) and by the Catalan Agency of Water, Ag&egrave;ncia Catalana de l'Aigua (ACA), Departament de Control i Millora dels Ecosistemes Aqu&agrave;tics. During the developed study Marta Rusi&ntilde;ol was a fellow of the Catalan Government "AGAUR" (FI-DGR).

1. Concentration of the viral particles present in water samples
<ol>
	<li>Collection and conditioning of water samples
		<ol>
		<li>Collect a minimum of 2 replicates of 10 L per sample in plastic containers with flat bottom and one extra sample as a process control. This last sample will be spiked with a known amount of viral particles and used as a control. 
		Note: It is recommended to have special separate material (bottles, tube, etc.) for spiked samples.</li>
		<li>Check the calibration of the conductimeter ...

<ol>
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Microbiol. 72 (12), 7894-7896 (2006).</li><li>Calgua, B., Mengewein, A., Grunert, A., Bofill-Mas, S., Clemente-Casares, P., Hundesa, A., Wyn-Jones, A. P., López-Pila, J. M., Girones, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+a+one-step+low+cost+procedure+to+concentrate+viruses+from+seawater+samples.">Development and application of a one-step low cost procedure to concentrate viruses from seawater samples.</a> J. Virol. Methods. 153 (2), 79-83 (2008).</li><li>Hernroth, B. E., Conden-Hansson, A. C., Rehnstam-Holm, A. S., Girones, R., Allard, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+factors+influencing+human+viral+pathogens+and+their+potential+indicator+organisms+in+the+blue+mussel,+Mytilus+edulis:+the+first+Scandinavian+report.">Environmental factors influencing human viral pathogens and their potential indicator organisms in the blue mussel, Mytilus edulis: the first Scandinavian report.</a> Appl. Environ. Microbiol. 68, 4523-4533 (2002).</li><li>Hundesa, A., Bofill-Mas, S., de Motes, M. a. l. u. q. u. e. r., Rodriguez-Manzano, C., Bach, J., Casas, A., M, ., Girones, R. <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+a+quantitative+PCR+assay+for+the+quantitation+of+bovine+polyomavirus+as+a+microbial+source-tracking+tool.">Development of a quantitative PCR assay for the quantitation of bovine polyomavirus as a microbial source-tracking tool.</a> J. Virol. Methods. 163 (2), 385-389 (2010).</li><li>Hundesa, A., de Motes, M. a. l. u. q. u. e. r., Albinana-Gimenez, C., Rodriguez-Manzano, N., Bofill-Mas, J., Suñen, S., E, ., Girones, R. <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+a+qPCR+assay+for+the+quantification+of+porcine+adenoviruses+as+an+MST+tool+for+swine+fecal+contamination+in+the+environment.">Development of a qPCR assay for the quantification of porcine adenoviruses as an MST tool for swine fecal contamination in the environment.</a> J. Virol. Methods. 158 (1-2), 130-135 (2009).</li><li>Hundesa, A., de Motes, M. a. l. u. q. u. e. r., Bofill-Mas, C., Albinana-Gimenez, S., N, ., Girones, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+human+and+animal+adenoviruses+and+polyomaviruses+for+determination+of+sources+of+fecal+contamination+in+the+environment.">Identification of human and animal adenoviruses and polyomaviruses for determination of sources of fecal contamination in the environment.</a> Appl. Environ. Microbiol. 72 (12), 7886-7893 (2006).</li><li>Layton, B. A., Walters, S. P., Lam, L. H., Boehm, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterococcus+species+distribution+among+human+and+animal+hosts+using+multiplex+PCR.">Enterococcus species distribution among human and animal hosts using multiplex PCR.</a> J. Appl. Microbiol. 109, 539-547 (2010).</li><li>de Motes, M. a. l. u. q. u. e. r., Clemente-Casares, C., Hundesa, P., Mart&#237;n, M., Girones, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+bovine+and+porcine+adenoviruses+for+tracing+the+source+of+fecal+contamination.">Detection of bovine and porcine adenoviruses for tracing the source of fecal contamination.</a> Appl. Environ. Microbiol. 70 (3), 1448-1454 (2004).</li><li>Pal, A., Sirota, L., Maudru, T., Peden, K., Lewis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+assays+for+the+detection+of+virus-specific+DNA+in+simples+with+mixed+populations+of+polyomaviruses.">Real-time PCR assays for the detection of virus-specific DNA in simples with mixed populations of polyomaviruses.</a> J. Virol. Methods. 135 (1), 32-42 (2006).</li><li>Stapleton, C. M., Kay, D., Wyer, D. a. v. i. e. s., Watkins, C., Kay, J., McDonald, C., Porter, A. T., J, ., Gawler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+operational+utility+of+a+Bacteroidales+quantitative+PCR-based+MST+approach+in+determining+the+source+of+faecal+indicator+organisms+at+a+UK+bathing+water.">Evaluating the operational utility of a Bacteroidales quantitative PCR-based MST approach in determining the source of faecal indicator organisms at a UK bathing water.</a> Water Res. 43, 4888-4899 (2010).</li><li>Wang, J., Horner, G. W., Keef, O., S, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+molecular+characterization+of+bovine+polyomavirus+in+bovine+sera+in+New+Zealand.">Detection and molecular characterization of bovine polyomavirus in bovine sera in New Zealand.</a> N. Z. Vet. J. 53, 26-30 (2005).</li></ol>

The procedure described would fulfill the conditions for a fitting method for routine environmental and public health laboratories: reproducible, reliable, straightforward and cost-effective. The protocol is simple; however it must be followed carefully. Low conductivity in the samples without adding the requested concentration of artificial seawater salts would dramatically reduce the recovery of viruses as would be the case if the stirring time for flocculation is significantly reduced (less than 5 hours for example).<...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>High speed centrifuge (8,000xg)</td>
 <td>Berckman Coulter</td>
 <td>Avanti J-20XP</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pH-meter, thermometer and conductimeter</td>
 <td>Afora</td>
 <td>LPPC3003</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Plastic tubes 100-200 cm length</td>
 <td>Deltalab</td>
 <td>350059</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sterile graduated disposable pipettes</td>
 <td>Labclinics</td>
 <td>PN10E1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sterile plastic tubes of 1.5 and 10-15 mL (Eppendorf, Falcons, etc.)</td>
 <td>Afora</td>
 <td>KA298/00</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Centrifuge pots (500 mL)</td>
 <td>Fisher Scientific</td>
 <td>SE5753512</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Magnetic stirrers and magnets (one per sample)</td>
 <td>Fisher Scientific</td>
 <td>10510</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass or plastic containers having flat bottoms to allow the use of magnetic stirrers </td>
 <td>Deltalab</td>
 <td>191642</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>A peristaltic pump for removing the supernatant (or a water-jet vacuum pump)</td>
 <td>Watson-Marlow</td>
 <td>323E/D</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Timer to switch-off the stirring after 8-10 hours</td>
 <td>Deltalab</td>
 <td>900400</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hydrochloric acid (1N and 0.1N)</td>
 <td>Panreac</td>
 <td>141020.1611</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium hydroxide (1N)</td>
 <td>Panreac</td>
 <td>131687.1211</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Artificial seawater sea salts </td>
 <td>Sigma</td>
 <td>S9883</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Skimmed milk (SM)</td>
 <td>Difco</td>
 <td>232100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate buffer pH 7,5</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>1:2 v/v of sterile Na2HPO4 0,2M and NaH2PO4 0,2M at pH 7.5</td>
 </tr>
 <tr>
 <td>Thiosulphate </td>
 <td>Panreac</td>
 <td>121879.1209</td>
 <td>Make a 10% solution in water </td>
 </tr>
 <tr>
 <td>QIAamp Viral RNA Mini Kit </td>
 <td>Qiagen</td>
 <td>52904</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>96-well optical reaction plate (500 units)</td>
 <td>Applied Biosystems</td>
 <td>43426659</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Optical adhesive covers (100 units)</td>
 <td>Applied Biosystems</td>
 <td>4311971</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TaqMan Environmental PCR Master Mix 2x</td>
 <td>Applied Biosystems</td>
 <td>4396838</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>

cost-effective method for microbial source tracking using specific human and animal viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

Watch this Scientific Journal Video about Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses at JoVE.com

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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