Name: ampliphox colorimetric detection on a dna microarray for influenza
Uploaded: 2011-06-09T12:00:00
Description: Watch this Scientific Journal Video about ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza at JoVE.com

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 bi...

<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>

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 ...

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