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 and recalibrate if necessary. Prepare a negative control by using tap water previously adjusted to the adequate conductivity (see below 1.6) in one extra plastic 10 L container.</li>
		<li>For spiked samples: Add the standard volume of the control virus (approximately 105 genome copies per 10 L of water) to the sample. Mix by stirring avoiding splashing and aerosols. Positive controls could consist in an uncommon strain of adenovirus such as HAdV-35 or a bacteriophage such as MS2.</li>
		<li>If the sample presents high quantity of suspended material (sand or other materials), let it sediment for 15 minutes. Transfer the water into a new container.</li>
		<li>Adjust the pH of the water sample to 3.5 (&plusmn; 0.1) by the addition of 1 N HCl. This step is important for the concentration of the viruses, so make sure the pH has been properly adjusted. Mix the water thoroughly by vigorous stirring while adding the HCl. (Note: If the pH is lower than 3.5 add 1 M NaOH).</li>
		<li>Adjust the conductivity. If the sample has a conductivity of 1500 &mu;S/cm or higher, this step is not needed. If the conductivity is lower than 1500 &mu;S/cm the formation of flocculated material (flocs) is not guaranteed so adjust the conductivity to 1500 &mu;S/cm by the addition of artificial sea salts (Sigma). Mix vigorously by stirring while adding the sea salts.</li>
		<li>Record the pH of the samples before and after conditioning as well as the volume of HCl used. The conductivity should also be recorded after adjusting the pH. Always disinfect the pH-meter and conductimeter electrodes with a fresh HCl solution, dechlorinate with 10% sodium tiosulphate solution and finally rinse with distilled water.</li>
		</ol>
	</li>
	<li>Preparation of pre-flocculated 1 % skimmed milk (PSM)
		<ol>
			<li>Check the calibration of the pH-meter and the conductimeter and recalibrate if necessary. </li>
			<li>Prepare pre-flocculated skimmed milk solution (1 % PSM, w/v) by dissolving 10 g skimmed milk powder (Difco) in 1 L artificial seawater (dissolve 33.3 g of artificial sea salts in 1 L of dechlorinated tap water and autoclave) and carefully adjusting the pH to 3.5 with 1N HCl. The flocs should be visible. Prepare the solution just before to be used or store at 4&deg;C for 24 h. For dechlorination use 1 ml of 10% tiosulphate solution per 100 ml of water.</li>
		</ol>
	</li>
	<li>Flocculation of viral particles present in water samples
		<ol>
		<li>Add 100 ml of 1 % PSM to the 10-L water sample.</li>
		<li>Stir the samples for 8-10 h to allow the viruses to adsorb to the flocs. Use a timer to switch-off the stirring after 8-10 h.</li>
		<li>Stop the stirring and let the flocs sediment by gravity for 8-10 h.</li>
		</ol>
	</li>
	<li>Collecting and re-dissolving the flocs. Centrifugation
		<ol>
		<li>Remove the supernatant using a peristaltic pump and a plastic pipette connected to a plastic tube. For spiked samples the supernatant should be collected into a bottle and disinfected according to internal procedures. In all cases TAKE CARE not to collect the pellet. </li>
		<li>Collect the sediment with the flocs (approximately 500 ml) into a centrifuge bottle. </li>
		<li>Balance the pots by the addition of PSM pH 3.5. </li>
		<li>Centrifuge the pots in a high-speed centrifuge at 8,000 x g for 30 min at 4&deg;C. As soon as the centrifuge stops, carefully remove the centrifuge pots from the centrifuge. </li>
		<li>Very gently pour off and discard the supernatant. Follow appropriate measures for infectious material.</li>
		<li>Add 7 ml of phosphate buffer to dissolve the pellet in each centrifuge bottle.</li>
		<li>Once the flocs have been dissolved, measure and add phosphate buffer to reach a total volume of 10 ml.</li>
		<li>Homogenize the viral concentrate by vortexing and distribute the 10 ml into clean microtubes which should be frozen at -80&deg;C until needed in further analysis.</li>
		</ol>
	</li>
</ol>
2. Nucleic acid extraction
<ol>
	<li>Perform a nucleic acid extraction with the QIAamp Viral RNA Mini Kit following manufacturer instructions. This kit enables the use of an automated platform (such as Qiacube, Qiagen).</li>
</ol>
3. Quantitative PCR of human adenoviruses (HAdV), JC polyomaviruses (JCPyV), porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV)
<ol>
	<li>Quantification of genome copies in the samples
		<ol>
		<li>Prepare the qPCR mix in a clean separate area by using TaqMan Environmental PCR Master Mix 2x (Applied Biosystems) . The reaction takes place in a 96-well optical reaction plate covered with optical adhesive covers. Concentrations of the Master Mix, primers and probes are described in Table 1.</li>
		<li>Once the mix has been prepared, aliquot 15&mu;l into each well including the controls (see 3.2). The total volume for one reaction after addition of target will be 25&mu;l (15&mu;l mix + 10&mu;l sample or standard).</li>
		<li>Add the nucleic acid extractions from the samples (10&mu;l) in a separate area. Run direct and a ten-fold dilution in purified water of each sample in duplicate. Cover the wells containing the samples with part of an adhesive cover, keep the other part of the cover for the following step.</li>
		<li>Add dilutions of the DNA standard suspensions (10&mu;l) from 100 to 106 GC/10&mu;l by triplicate and using a micropipette exclusively used for the standard DNA. It is advisable to add standards in an area equipped with UV radiation for destroying plasmid DNA and to clean the micropipette after each use. Cover the wells containing the standards with the pre-cut adhesive cover. 
		Note: To prepare standard suspensions to be used in the quantification of genome copies, the target DNA region should be cloned into a plasmid and linearized. In the following address you will find a detailed procedure on how to create standard curves with plasmid DNA templates to be used in qPCR: 
		<a href="http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf">http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf</a></li>
		<li>Perform the qPCR into an adequate system selecting the appropriate parameters (considering the use of adhesive cover and the total volume in each well, etc). Following activation of the AmpliTaq Gold for 10 min at 95&deg;C, 40 cycles of amplification are performed as follows: 15 s at 95&deg;C and 1 min at 60&deg;C for HAdV, JCPyV and BPyV, and 15 s at 95&deg;C, 20s at 55&deg;C and 20s at 60&deg;C for PAdV.</li>
		<li>Once the reactions are completed, store data and results as described in the user&#x2019;s manual of the equipment used. The amount of DNA will be defined as the median of the data obtained after correcting the dilution factor when needed.</li>
		</ol>
	</li>
	<li>Controls
		<ol>
		<li>Use positive and negative controls. The assay must include more than one non-template control (NTC) to prove mix does not produce fluorescence. It is advisable to run the positive process control in order to evaluate potential enzymatic inhibition due to inhibitors present in the studied samples.</li>
		<li>Record results of qPCR assays of two different dilutions of the standard DNA and from the process control. Use results to prepare control charts for quality control (QC) programs related to the sensitivity and efficiency of the assays.</li>
		</ol>
	</li>
	<li>Confirmation of results
		<ol>
		<li>Positive results may be further confirmed using nested-PCR and nucleotide sequencing of the amplicons, producing additional data on the nucleotide sequences of the strains detected1,5,6,7,9,12. </li>
		</ol>
	</li>
</ol> 
Following the procedure described, human and animal viruses have been detected and quantified in bathing waters, seawater and river water2,3. As a representative example, ground water samples from areas presenting high levels of nitrates were evaluated to define the sources of the contamination. Ten-liter water samples were collected from 4 different wells in rural areas of a Northeast region in Spain. Five replicates were collected in each well being one replicate seeded with human adenovirus 2 used as process control. Samples were processed according to the protocol represented in Figure 1. The four replicates analyzed in one of the four sites studied showed positive results for PAdV (mean value 7.74x102 GC/L) which would be related to the presence of pig slurries in the areas surrounding the sampling site and would support fecal porcine contamination as the source of nitrates in groundwater (Table 2).
4. Representative Results:
<img src="/files/ftp_upload/2820/2820fig1.jpg" alt="Figure 1" /> 
Figure 1. Procedure for the detection and quantification of viruses in water.
<img src="/files/ftp_upload/2820/2820table1.jpg" alt="Table 1" /> 
Table 1. Concentration of primers and probes for qPCR assays.
<img src="/files/ftp_upload/2820/2820table3.jpg" alt="Table 3" /> 
Table 2. Detection and quantification of animal and human adenoviruses and polyomaviruses in ground water samples. 
n number of replicates analyzed 
% percentage of positive replicates 
(-) non detected

<ol>
	<li>Bofill-Mas, S., Clemente-Casares, P., Major, E. O., Curfman, B., 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=Analysis+of+the+excreted+JC+virus+strains+and+their+potential+oral+transmission.">Analysis of the excreted JC virus strains and their potential oral transmission.</a> J. Neurovirol. 9 (4), 498-507 (2003).</li><li>Bofill-Mas, S., Albinana-Gimenez, N., Clemente-Casares, P., Hundesa, A., Rodriguez-Manzano, J., Allard, A., Calvo, 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=Quantification+and+stability+of+human+adenoviruses+and+polyomavirus+JCPyV+in+wastewater+matrices.">Quantification and stability of human adenoviruses and polyomavirus JCPyV in wastewater matrices.</a> Appl. Environ. 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>

Two patent applications were filed in 2009 to protect the intellectual property of protocols for quantification of PAdV and BPyV.

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><tbody><tr><td>High speed centrifuge (8,000xg)</td><td>Berckman Coulter</td><td>Avanti J-20XP</td><td></td></tr><tr><td>pH-meter, thermometer and conductimeter</td><td>Afora</td><td>LPPC3003</td><td></td></tr><tr><td>Plastic tubes 100-200 cm length</td><td>Deltalab</td><td>350059</td><td></td></tr><tr><td>Sterile graduated disposable pipettes</td><td>Labclinics</td><td>PN10E1</td><td></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></td></tr><tr><td>Centrifuge pots (500 mL)</td><td>Fisher Scientific</td><td>SE5753512</td><td></td></tr><tr><td>Magnetic stirrers and magnets (one per sample)</td><td>Fisher Scientific</td><td>10510</td><td></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></td></tr><tr><td>A peristaltic pump for removing the supernatant (or a water-jet vacuum pump)</td><td>Watson-Marlow Pumps Group</td><td>323E/D</td><td></td></tr><tr><td>Timer to switch-off the stirring after 8-10 hours</td><td>Deltalab</td><td>900400</td><td></td></tr><tr><td>Hydrochloric acid (1N and 0.1N)</td><td>Panreac</td><td>141020.1611</td><td></td></tr><tr><td>Sodium hydroxide (1N)</td><td>Panreac</td><td>131687.1211</td><td></td></tr><tr><td>Artificial seawater sea salts </td><td>Sigma-Aldrich</td><td>S9883</td><td></td></tr><tr><td>Skimmed milk (SM)</td><td>Difco Laboratories</td><td>232100</td><td></td></tr><tr><td>Phosphate buffer pH 7,5</td><td></td><td></td><td>1:2 v/v of sterile Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; 0,2M and NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; 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></td></tr><tr><td>96-well optical reaction plate (500 units)</td><td>Applied Biosystems</td><td>43426659</td><td></td></tr><tr><td>Optical adhesive covers (100 units)</td><td>Applied Biosystems</td><td>4311971</td><td></td></tr><tr><td>TaqMan Environmental PCR Master Mix 2x</td><td>Applied Biosystems</td><td>4396838</td><td></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.

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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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12.1K 조회수.  University of Barcelona. 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.

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

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

바이러스성 병원균에 대한 임상 샘플을 화면에 판 - 바이러스성 Microarray 분석 (Virochip)를 사용

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

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Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

야생 조류에서 조류 독감 바이러스의 신속한 진단 : 현장에서 휴대용 RRT - PCR 및 정지 - 건조 시약의 사용

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

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.

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.

Bioaerosols 음이온 교환 수 지를 사용 하 여 바이러스의 검출

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

환경과학

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

세균 인공 염색체 : RNA 바이러스 양성 가닥의 연구에 대한 기능 유전체학 도구

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Arenavirus 셀 첨부 파일의 측정을위한 고감도 분석

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

나노 PCR에 의한 동물 표본의 호흡기 병원체의 높은 처리량 검출

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA &#8212; including poly(rA) carrier and ribosomal RNA &#8212; from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based &#39;tagmentation&#39; step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.

This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates.

임상 샘플에서 RNA 바이러스의 편견 깊은 시퀀싱

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from ...

의학

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

VirWaTest, 물 샘플에서 바이러스 검출을 위한 사용 시점 방법

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

<meta charset="utf8"></head><body>Dual-Reporter 및 Multiplex 기술을 사용한 SARS-CoV-2 분석

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

<meta charset="utf8"></head><body>저자 스포트라이트: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques (새로운 면역포획 및 질량 분석 기법을 통한 항바이러스 전략 발전)

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Multiplex Dual-Reporter Strategy를 통한 바이러스 입자의 표면 단백질 에피토프 프로파일링

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

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

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바이러스성 병원균에 대한 임상 샘플을 화면에 판 - 바이러스성 Microarray 분석 (Virochip)를 사용

야생 조류에서 조류 독감 바이러스의 신속한 진단 : 현장에서 휴대용 RRT - PCR 및 정지 - 건조 시약의 사용

Bioaerosols 음이온 교환 수 지를 사용 하 여 바이러스의 검출

세균 인공 염색체 : RNA 바이러스 양성 가닥의 연구에 대한 기능 유전체학 도구

Arenavirus 셀 첨부 파일의 측정을위한 고감도 분석

나노 PCR에 의한 동물 표본의 호흡기 병원체의 높은 처리량 검출

임상 샘플에서 RNA 바이러스의 편견 깊은 시퀀싱

VirWaTest, 물 샘플에서 바이러스 검출을 위한 사용 시점 방법

Multiplex Dual-Reporter Strategy를 통한 바이러스 입자의 표면 단백질 에피토프 프로파일링

Multiplex Dual-Reporter Strategy를 통한 바이러스 입자의 표면 단백질 에피토프 프로파일링