InDevR acknowledges NIH/NIAID U01AI070276 and R43AI077112 for funding this work.

1.	Sample amplification using RT-PCR
<ol>
<li>Extract viral RNA from clinical material or a viral isolate using the Qiagen MinElute Virus Spin Kit in conjunction with the QIAcube automated nucleic acid extraction platform. Extractions are performed on 200 &mu;l specimen with a final elution volume of 60 &mu;l. Store extracts at -70&deg;C or lower for later use.</li>
<li>In a template free-area, prepare the RT-PCR master mix on ice according to the manufacturer's protocol. To incorporate biotin during RT-PCR, use a biotinylated dNTP mixture in place of the manufacturer supplied dNTP mix. The cost of using a biotinylated dNTP mixture is less than $1 USD/assay. Alternative methods of biotin incorporation such as the use of a biotinylated primer can also be used. Add primer mixes at final concentrations of 1.0 &mu;M for Flu A, 1.0 &mu;M for Flu B, and 0.14 &mu;M for the internal control. The Flu A primer set amplifies the matrix gene segment (1032 nt product) and the Flu B primer set amplifies a non-structural gene segment (811 nt product). Each FluChip primer set contains one primer with a 5' phosphoryl group to facilitate the generation of single stranded DNA via enzymatic digestion with lambda exonuclease following PCR. The resulting single stranded product requires much shorter hybridization times than the double stranded equivalent and significantly shortens the overall assay time. A pre-prepared mixture of these primers as well as the internal control template RNA are available from InDevR to interested researchers on a limited basis.</li>
<li>Briefly vortex and distribute 18 &mu;l of the master mix into thin-walled PCR tubes.</li>
<li>Transfer tubes on ice to a suitable work area for template addition, and add 2 &mu;l template to each reaction tube.</li> 
<li>Transfer PCR tubes to a thermal cycler, and run the following thermal profile: reverse transcription at 50&deg;C for 30 min, enzyme inactivation/activation at 95&deg;C for 15 min, 40 PCR cycles of 95&deg;C for 30 s, 55&deg;C for 30s, and 72&deg;C for 1 min, and a final extension at 72&deg;C for 10 min.</li>
</ol>
2.	Hybridization of RT-PCR products to low density microarrays
<ol>
<li>In order to generate single stranded DNA for FluChip hybridization, prepare the enzymatic digestion mixture by combining 1.0 &mu;l lambda exonuclease enzyme, 2.2 &mu;l of the accompanying reaction buffer, and 0.8 &mu;l of nuclease-free water. These amounts are for a single sample, but can be simply scaled for the total number of samples to be digested. Remove samples from the thermal cycler and add 4 &mu;l of the prepared mixture to each reaction to digest the phosphorylated strand of the PCR product. Return samples to the thermal cycler and program the thermal cycler to 37 degrees Celsius for 15 minutes followed by 95 degrees Celsius for 10 minutes to complete enzymatic digestion and heat fragmentation steps.</li>
<li>The custom influenza microarrays used are printed on aldehyde functionalized glass slides by Applied Microarrays Inc. (Tempe, AZ). 5'-amino terminated capture sequences are combined with an optimized spotting buffer and printed at a final concentration of 20 &mu;M (except for the positive control sequence which additionally has a 3'-biotin modification and is spotted at a final concentration of 500 nM). A non-contact spotting method is used, with an optimal spot diameter of 300 &mu;m and center to center pitch of 700 &mu;m.</li> 
<li>Remove microarrays from the storage box and apply single-use hybridization wells around the microarrays by removing the protective sheet and pressing firmly around well perimeter.</li> 
<li>Place slides in a wash bin containing 110 ml of purified water for a 5 min pre-hybridization wash using an orbital shaker at 60-90 rpm. Dry the array by gently touching a tissue wiper to the edge of the well and allowing water to be wicked away.</li>
<li>Combine 22 &mu;l of 2x Hybridization Buffer with each of the fragmented ssDNA products, and pipet 40 &mu;l of the hybridization solutions into the microarray wells.</li>
<li>Allow slides to hybridize in closed humidity chamber for 60 minutes.</li> 
<li>Remove the slides from the humidity chamber, briefly rinse arrays with Wash Buffer D in a rinse bottle before placing into the slide rack. Typically the rinsing volume of Wash Buffer D is 2 ml per array. Place slide rack containing slides in wash bin containing 110 ml Wash Buffer A. Place bin on orbital shaker at 60-90 rpm for 1 minute.</li>
<li>Remove slide rack from Wash Buffer A, briefly rinse with Wash Buffer D, transfer slide rack to a bin containing Wash Buffer B, and shake at 60-90 rpm for 5 minutes.</li>
<li>Gently dry the array on each slide, and place dried slides in humidity chamber in preparation for ampliPHOX Colorimetric Detection steps.</li>
</ol>
3.	ampliPHOX: labeling hybridized product and calibration chips with ampliTAG
<ol>
<li>Combine 10 &mu;l of ampliTAG, 20 &mu;l of 2x ampliTAG buffer, and 10 &mu;l of purified water for each array to be processed. Reagent volumes can be simply scaled for the total number of samples. Make sure to prepare enough labeling mixture to account for all sample arrays and calibration arrays needed. The number of calibration chips needed depends on whether you are performing a full calibration (for a new instrument or new lot of reagents), or just an instrument check. For a full calibration, 3 calibration chips should be labeled, and for a reagent check, just a single calibration chip should be labeled. Please note that before the calibration arrays are labeled, they should go through the pre-hybridization washing step that was previously described for the influenza arrays.</li>
<li>Transfer 40 &mu;l of label mixture to each array and allow labeling reaction to proceed in a closed humidity chamber for 5 minutes.</li> 
<li>Immediately rinse arrays with Wash Buffer D in a rinse bottle before placing slides in a slide rack. Transfer rack into bin containing 110 ml of Wash Buffer C, and shake at 60-90 rpm for 5 minutes.</li> 
<li>Using a second wash bin filled with purified water, perform three consecutive brief water dips to remove salt residue. Dry the arrays by gently touching a tissue wiper to the edge of the wells.</li>
<li>The microarrays are now properly labeled with ampliTAG, and the remainder of the ampliPHOX detection procedure can be performed. Photoactivation and imaging of labeled arrays should be completed within 24 hours, and additional arrays can be stored in a dark slide box until use.</li>
</ol>
4.	ampliPHOX: calibration, signal amplification, and imaging
<ol>
<li>Turn ampliPHOX reader on and ensure ampliVIEW software is ready for photoactivation.</li>
<li>Determine the optimal photoactivation time using calibration chips. In addition to the brief introduction that follows, this procedure is also outlined in detail in the ampliPHOX Operation Manual. The calibration chips contain a series of dilutions of a biotinylated control sequence that are utilized to optimize the sensitivity of the assay, with the goal of maximizing the number of rows of the calibration chip that produce a positive signal as determined by the software.</li>
<li>Remove ampliPHY from 4&deg;C, allow to warm to room temperature, and vortex briefly to mix. Pipet 3 &mu;l of ampliPHY enhancer into the ampliPHY vial, and vortex thoroughly for 10 seconds.</li>
<li>Evenly transfer 40 &mu;l of ampliPHY solution into the microarray well containing the ampliTAG-labeled array, ensuring that no bubbles are present. Close the ampliPHY vial between each application. Insert the microarray slide into the photoactivation bay of the ampliPHOX Reader.</li> 
<li>For the first calibration array, use the default photoactivation time in the 'Time' box on the lefthand panel of the software, and click the green 'Start' button to start the photoactivation. Once completed, remove the array and rinse with purified water to remove excess ampliPHY. Clear polymer formation on some of the spots should now be visible.</li> 
<li>Allow the polymer spots to dry for 2 minutes, then distribute two drops of ampliRED onto the array and allow staining to proceed for 2 minutes.</li> 
<li>Next, quickly rinse microarray with purified water and dry with a tissue wipe.</li>
<li>Insert microarray into the imaging bay of the ampliPHOX Reader, and click the 'Capture New Image' button in the Imaging tab. Once imaged, adjust the crop and save the image.</li> 
<li>In the Analysis tab, select the Calibration Chip mask and the 'Auto Placement' button to start the analysis. The software will automatically produce a 'Summary Record' showing the quantified results. Based on these results, the photoactivation time is adjusted by 10 seconds for the second calibration array, and the procedure is repeated.</li> 
<li>Once the optimal photoactivation time has been determined, the sample arrays can be processed using the same signal amplification and imaging protocol used for the calibration arrays.</li>
<li>Once the image is captured for the sample arrays, a mask for your particular array layout such as the influenza array layout described here can be created.The ampliVIEW software is capable of performing automated image analysis and generating influenza subtyping results for each sample.</li> 
</ol>
5. Representative Results:
<img src="/files/ftp_upload/2682/2682fig1.jpg" alt="Figure 1"/> 
Figure 1. Schematic illustration of the ampliPHOX colorimetric detection method. (A) Biotinylated target DNA is hybridized to each spot in the array, and (B) labeled with ampliTAG. (C) ampliPHY solution is then added, and (D) exposed to light to form visible polymer spots. (E) The polymer spots formed are subsequently stained with a non-toxic dye to improve contrast.
<img src="/files/ftp_upload/2682/2682fig2.jpg" alt="Figure 2"/> 
Figure 2. (A) Influenza low density microarray layout. Sequences 1-7 and 10-13 target influenza A, and sequences 8, 9 target influenza B. (B) ampliPHOX and (C) fluorescence images of a 2009 novel H1N1 ('swine flu') specimen showing the same detection pattern by both methods.
<img src="/files/ftp_upload/2682/2682fig3.jpg" alt="Figure 3"/> 
Figure 3. From left to right, representative ampliPHOX images for influenza A H3N2, human-origin H1N1, 2009 novel H1N1 (swine-origin), and a negative specimen. All 3 subtypes show visually distinct patterns on the array. Notice in the negative that only the MS2 internal control is seen, indicating the RT-PCR amplification was not inhibited.

<ol>
	<li>Kumar, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Widely+Used+Diagnostics+"DNA-Microarray"-A+Review.">The Widely Used Diagnostics "DNA-Microarray"-A Review.</a> Amer J Inf Dis. 5, 207-218 (2009).</li><li>Miller, M. B., Tang, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Concepts+of+Microarrays+and+Potential+Applications+in+Clinical+Microbiology.">Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology.</a> Clin Microbiol Rev. 22, 611-633 (2009).</li><li>Mikhailovich, V., Gryadunov, D., Kolchinsky, A., Makarov, A. A., Zasedatelev, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+microarrays+in+the+clinic:+infectious+diseases.">DNA microarrays in the clinic: infectious diseases.</a> BioEssays. 30, 673-682 (2008).</li><li>Call, 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=Challenges+and+opportunities+for+pathogen+detection+using+DNA+microarrays.">Challenges and opportunities for pathogen detection using DNA microarrays.</a> Crit Rev Microbiol. 31, 91-99 (2005).</li><li>Raoult, D., Fournier, P. E., Drancourt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+the+future+hold+for+clinical+microbiology.">What does the future hold for clinical microbiology.</a> Nat Rev Microbiol. 2, 151-159 (2004).</li><li>Dawson, E. D., Rowlen, K. L., Wang, Q., Tao, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MChip:+A+Single+Gene+Diagnostic+for+Influenza+A.">MChip: A Single Gene Diagnostic for Influenza A.</a> Influenza: Molecular Virology. , (2010).</li><li>Gall, A., Hoffman, B., Harder, T., Grund, C., Ehricht, R., Beer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+hemagglutinin+subtyping+and+pathotyping+of+avian+influenza+viruses+by+a+DNA+microarray.">Rapid hemagglutinin subtyping and pathotyping of avian influenza viruses by a DNA microarray.</a> J Virol Meth. 160, 200-205 (2009).</li><li>Townsend, M. B., Dawson, E. D., Mehlmann, M., Smagala, J. A., Dankbar, D. M., Moore, C. L., Smith, C. B., Cox, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FluChip:+Experimental+evaluation+of+a+diagnostic+influenza+microarray.">FluChip: Experimental evaluation of a diagnostic influenza microarray.</a> J Clin Microbiol. 44, 2863-2871 (2006).</li><li>Wang, Z., Daum, L. T., Vora, G. J., Metzgar, D., Walter, E. A., Canas, L. C., Malanosky, A. P., Lin, B., Stenger, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+influenza+viruses+with+resequencing+arrays.">Identifying influenza viruses with resequencing arrays.</a> Emerg Inf Dis. 12, 638-646 (2006).</li><li>Kessler, N., Ferraris, O., Palmer, K., Marsh, W., Steel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+DNA+Flow-Thru+Chip,+a+three-dimensional+biochip,+for+typing+and+subtyping+of+influenza+viruses.">Use of the DNA Flow-Thru Chip, a three-dimensional biochip, for typing and subtyping of influenza viruses.</a> J Clin Microbiol. 42, 2173-2185 (2004).</li><li>Kuck, L. R., Taylor, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photopolymerization+as+an+innovative+detection+technique+for+low-density+microarrays.">Photopolymerization as an innovative detection technique for low-density microarrays.</a> Biotechniques. 45, 179-186 (2008).</li><li>Avens, H. J., Bowman, C. N. <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+fluorescent+polymerization-based+signal+amplification+for+sensitive+and+non-enzymatic+biodetection+in+antibody+arrays.">Development of fluorescent polymerization-based signal amplification for sensitive and non-enzymatic biodetection in antibody arrays.</a> Acta Biomat. 6, 83-89 (2010).</li><li>Sikes, H. D., Jenison, R., Bowman, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+detection+using+polymerization-based+amplification.">Antigen detection using polymerization-based amplification.</a> Lab on a Chip. 9, 653-656 (2008).</li></ol>

All of the authors are employees of InDevR, Inc., a for-profit entity. InDevR is commercializing the ampliPHOX technology described herein.

The ampliPHOX colorimetric detection technology presented here is a rapid, inexpensive alternative to single color fluorescence detection for lower density microarray applications. Shown schematically in Figure 1, the detection principle is based on the use of a photoinitiator label (1B). In the presence of a monomer-containing solution (1C), light exposure causes the photoinitiator (ampliTAG) to trigger a polymerization reaction only in labeled regions (1D). Although demonstrated here on a DNA array fo...

<table border="1">
	<tbody>
 	<tr>
 		<th>Reagent/equipment</th>
 <th>Manufacturer</th>
 <th>Catalog #</th>
 <th>Comments</th> 
 </tr>
 <tr>
 	<td>Qiagen MinElute Virus Spin Kit</td>
 <td>Qiagen</td>
 <td>57704</td>
 <td>single 60 &mu;l elution</td>
 </tr>
 <tr>
 	<td>QIAcube</td>
 <td>Qiagen</td>
 <td>9001292</td>
 <td>optional</td>
 </tr>
 <tr>
 	<td>ABI 9800 Fast Thermal Cycler</td>
 <td>Applied Biosystems</td>
 <td>4441166</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Qiagen OneStep RT-PCR kit</td>
 <td>Qiagen</td>
 <td>210210</td>
 <td>kit dNTPs not used</td>
 </tr>
 <tr>
 	<td>2x Spotting Buffer</td>
 <td>InDevR Inc.</td>
 <td>MI-5007</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Biotinylated dNTP Mix</td>
 <td>InDevR Inc.</td>
 <td>MI-5009</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Lambda exonuclease</td>
 <td>Epicentre Biotechnologies</td>
 <td>LE032K</td>
 <td>2500 U, 10U/&mu;l</td>
 </tr>
 <tr>
 	<td>FluChip primer mix</td>
 <td>InDevR</td>
 <td>N/A</td>
 <td>not yet available for sale</td>
 </tr>
 <tr>
 	<td>Orbital Shaker</td>
 <td>Madell Technology</td>
 <td>ZD-9556-A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Wash Bins</td>
 <td>InDevR Inc.</td>
 <td>MI-4002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Wash Racks</td>
 <td>InDevR Inc.</td>
 <td>MI-4003</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>2x Hybridization Buffer</td>
 <td>InDevR Inc.</td>
 <td>MI-5004</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Calibration Chips</td>
 <td>InDevR Inc.</td>
 <td>AP-5006</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Wash Buffers A-D</td>
 <td>InDevR Inc.</td>
 <td>MI-5005</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>ampliRED</td>
 <td>InDevR Inc.</td>
 <td>AP-5004</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>ampliTAG</td>
 <td>InDevR Inc.</td>
 <td>AP-5001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>2x ampliTAG Buffer</td>
 <td>InDevR Inc.</td>
 <td>AP-5002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>ampliPHY, ampliPHY enhancer</td>
 <td>InDevR Inc.</td>
 <td>AP-5003	</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

ampliphox colorimetric detection on a dna microarray for influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

Watch this Scientific Journal Video about ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza at JoVE.com

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

ampliphox-colorimetric-detection-on-a-dna-microarray-for-influenza

Research

JoVE Journal

Immunology and Infection

9.9K Views.  InDevR Inc.. ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package. 

Video: ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

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.

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 ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...