Name: ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza (Video) | JoVE Journal
Uploaded: 2011-06-09T12:00:00.000+00:00
Duration: 9 min 32 s
Description: Learn ampliPHOX colorimetric detection for influenza viral subtypes using photopolymerization on DNA microarrays. This rapid method offers cost-effective signal amplification with visible results comparable to fluorescence sensitivity.

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5 minutes

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 μl specimen with a final elution volume of 60 μl. Store extracts at -70°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 μM for Flu A, 1.0 μM for Flu B, and 0.14 μM 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 μl 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 μl template to each reaction tube.</li> 
<li>Transfer PCR tubes to a thermal cycler, and run the following thermal profile: reverse transcription at 50°C for 30 min, enzyme inactivation/activation at 95°C for 15 min, 40 PCR cycles of 95°C for 30 s, 55°C for 30s, and 72°C for 1 min, and a final extension at 72°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 μl lambda exonuclease enzyme, 2.2 μl of the accompanying reaction buffer, and 0.8 μl 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 μl 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 μM (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 μm and center to center pitch of 700 μm.</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 μl of 2x Hybridization Buffer with each of the fragmented ssDNA products, and pipet 40 μl 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 μl of ampliTAG, 20 μl of 2x ampliTAG buffer, and 10 μl 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 μl 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°C, allow to warm to room temperature, and vortex briefly to mix. Pipet 3 μl of ampliPHY enhancer into the ampliPHY vial, and vortex thoroughly for 10 seconds.</li>
<li>Evenly transfer 40 μl 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="DNA immobilization diagram, PCR spotter setup, microarray formation, sequencing process." data-altupdatedat="1788235205000" data-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="FluChip layout diagram; DNA detection method; ampliPHOX and fluorescence images; data analysis." data-altupdatedat="1788235205000" data-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="Pathogen detection using dot blot technique; diagram shows H3N2, H1N1 assay results and control." data-altupdatedat="1788235205000" data-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/pubmed?term=((The+Widely+Used+Diagnostics+%22DNA-Microarray%22-A+Review%5BTitle%5D)+AND+%22Amer+J+Inf+Dis%22%5BJournal%5D)">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/pubmed?term=((Basic+Concepts+of+Microarrays+and+Potential+Applications+in+Clinical+Microbiology%5BTitle%5D)+AND+%22Clin+Microbiol+Rev%22%5BJournal%5D)">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/pubmed?term=((DNA+microarrays+in+the+clinic%3A+infectious+diseases%5BTitle%5D)+AND+%22BioEssays%22%5BJournal%5D)">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/pubmed?term=((Challenges+and+opportunities+for+pathogen+detection+using+DNA+microarrays%5BTitle%5D)+AND+%22Crit+Rev+Microbiol%22%5BJournal%5D)">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/pubmed?term=((What+does+the+future+hold+for+clinical+microbiology%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">What does the future hold for clinical microbiology.</a> Nat Rev Microbiol. 2, 151-159 (2004).</li>
<li>Dawson, E. D., Rowlen, K. L. MChip: A Single Gene Diagnostic for Influenza A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDawson%2C+ED+%22Influenza%3A+Molecular+Virology%22">Influenza: Molecular Virology</a>. Wang, Q., Tao, Y. J. , Caister Academic Press. Norfolk, UK. (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/pubmed?term=((Rapid+hemagglutinin+subtyping+and+pathotyping+of+avian+influenza+viruses+by+a+DNA+microarray%5BTitle%5D)+AND+%22J+Virol+Meth%22%5BJournal%5D)">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/pubmed?term=((FluChip%3A+Experimental+evaluation+of+a+diagnostic+influenza+microarray%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Identifying+influenza+viruses+with+resequencing+arrays%5BTitle%5D)+AND+%22Emerg+Inf+Dis%22%5BJournal%5D)">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/pubmed?term=((Use+of+the+DNA+Flow-Thru+Chip%2C+a+three-dimensional+biochip%2C+for+typing+and+subtyping+of+influenza+viruses%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Photopolymerization+as+an+innovative+detection+technique+for+low-density+microarrays%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">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/pubmed?term=((Development+of+fluorescent+polymerization-based+signal+amplification+for+sensitive+and+non-enzymatic+biodetection+in+antibody+arrays%5BTitle%5D)+AND+%22Acta+Biomat%22%5BJournal%5D)">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/pubmed?term=((Antigen+detection+using+polymerization-based+amplification%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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><tbody><tr><td>Qiagen MinElute Virus Spin Kit</td><td>Qiagen</td><td>57704</td><td>single 60 &amp;#x03BC;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></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></td></tr><tr><td>Biotinylated dNTP Mix</td><td>InDevR Inc.</td><td>MI-5009</td><td></td></tr><tr><td>Lambda exonuclease</td><td>Epicentre Biotechnologies</td><td>LE032K</td><td>2500 U, 10U/&amp;#x03BC;l</td></tr><tr><td>FluChip primer mix</td><td>InDevR Inc.</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></td></tr><tr><td>Wash Bins</td><td>InDevR Inc.</td><td>MI-4002</td><td></td></tr><tr><td>Wash Racks</td><td>InDevR Inc.</td><td>MI-4003</td><td></td></tr><tr><td>2x Hybridization Buffer</td><td>InDevR Inc.</td><td>MI-5004</td><td></td></tr><tr><td>Calibration Chips</td><td>InDevR Inc.</td><td>AP-5006</td><td></td></tr><tr><td>Wash Buffers A-D</td><td>InDevR Inc.</td><td>MI-5005</td><td></td></tr><tr><td>ampliRED</td><td>InDevR Inc.</td><td>AP-5004</td><td></td></tr><tr><td>ampliTAG</td><td>InDevR Inc.</td><td>AP-5001</td><td></td></tr><tr><td>2x ampliTAG Buffer</td><td>InDevR Inc.</td><td>AP-5002</td><td></td></tr><tr><td>ampliPHY, ampliPHY enhancer</td><td>InDevR Inc.</td><td>AP-5003</td><td></td></tr></tbody></table>

ampliphox colorimetric detection on a dna microarray for influenza

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.

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 ...

The overall goal of the following experiment is to utilize a new and rapid signal amplification method to detect and characterize influenza viral subtypes on the flu chip, low density microarray platform. This is achieved by incorporating biotinylated DTPs and using primers with a five prime phosphol end to amplify the matrix gene segment and a non-structural gene segment from influenza RNA extracts. PCR is followed by enzymatic digestion and fragmentation to generate short single stranded biotinylated and amplified viral product, which is then hybridized to the array after hybridization.Fox detection uses photo polymerization to grow large polymer spots where hybridization occurs, and differential hybridization patterns are used to detect influenza viral subtypes. The main advantage of this technique over existing methods like standard fluorescence detection, is that it's rather inexpensive and provides a quick result visible to the naked eye with similar analytical sensitivity to fluorescence. Though this method can provide insight into the characterization of influenza viruses using the flu chip because it's a universal detection method.Ampl Fox can also be applied to a wide variety of other systems, such as other DNA and protein-based low density microarrays. In theory, any biotinylated product should be able to be detected using amox. Demonstrating the procedure will be Amber Taylor, an associate scientist, and Kevin Moulton, a research assistant here at Endeavor who have both been central in the development of the Fox instrument and detection chemistry over the last several years.Extract viral RNA from 200 microliters of viral isolate using a viral RNA extraction kit and the manufacturer's protocol. Elute 60 microliters of final volume and store extracts at minus 70 degrees Celsius or lower for later use. Then prepare the R-T-P-C-R master mix on ICE according to the manufacturer's protocol.Using a BIOTINYLATED DNTP mixture add the flu chip primer mix, which contains the concentrations shown to the master mix. One primer from each set should contain a five prime phosphol group to facilitate enzymatic digestion. The flu a primer set produces a 1032 base pair product, and the flu B primer set produces an 811 base pair product.Briefly vortex and distribute 18 microliters of master mix into thin walled PCR tubes. Then add two microliters of RNA template to each reaction tube. Transfer PCR tubes to a thermal cycler and use a cycling profile with an appropriate a kneeling temperature.The cycling profile used in this assay is shown here. Prepare the enzymatic digestion mix by combining 2.2 microliters of the enzyme reaction buffer, 0.8 microliters of nuclease free water and 1.0 microliters of lambda exonuclease enzyme per PCR reaction. Remove the samples from the thermal cycler and add four microliters to each PCR tube.Return the samples to the thermal cycler and program the thermal cycler as shown to 37 degrees Celsius for 15 minutes, followed by 95 degrees Celsius for 10 minutes. Next, apply single use hybridization wells around the microarrays to be hybridized. Then wash the slides in 110 milliliters of purified water on an orbital shaker for five minutes.Dry the slides by gently touching a tissue wipe to the edge of the well. Combine 22 microliters of two x hybridization buffer with each of the fragmented single strand DNA products mixed briefly and pipette 40 microliters into the microarray wells. Allow the slides to hybridize for one hour in a humidity chamber after hybridization, remove the slides from the humidity chamber and briefly rinse each slide with two milliliters of wash buffer D.Place the slides into a slide rack.Next, add 110 milliliters of wash buffers A and B to separate containers using an orbital shaker. Wash the slide rack in buffer A at 60 to 90 RPM, the speed used for all subsequent washes for one minute. Then remove the slide rack from wash buffer A and briefly rinse with wash buffer D.Transfer the slide rack to wash buffer B and wash on the orbital shaker for five minutes.After washing, gently dry the arrays on each slide with a tissue wipe and place dried slides in the humidity chamber. In preparation for fox color metric detection steps, combine 10 microliters of amplitude, 20, microliters of two x amplitude buffer, and 10 microliters of purified water for each array to be processed, transfer 40 microliters of label mixture to each array and allow the labeling reaction to proceed in a closed humidity chamber For five minutes During hybridization, prepare 110 milliliters of wash buffer C in a container. After labeling, immediately rinse each array with wash buffer D.Place each slide in the slide rack and wash and wash buffer C on the orbital shaker for five minutes after buffer sea wash, briefly immersed the slide rack three times in a container of purified water.To remove salt residues dry the arrays as previously shown. Store the arrays in a dark slide box until photo activation and imaging, which should be completed within 24 hours. Next, turn on the Amli Fox reader and ensure the Amli view software is ready for photo activation prior to array sample detection.First, determine the optimal photo activation time for all samples by following a short calibration procedure. Once per daily use, then warm the Amplify solution to room temperature and vortex briefly to mix pipette three microliters of amplify enhancer into the amplify vial and vortex thoroughly. For 10 seconds evenly apply 40 microliters of Amplify solution to only one slide at a time ensuring that no bubbles are present.Insert the microarray slide into the photo activation bay of the Amplify Reader. Start photo activation by selecting, start in the PL view software using the photo activation time determined in the calibration process. Once completed, rinse the microarray with two milliliters of purified water to remove excess amplify clear polymer formation has now occurred on spots containing target DNA but is better visualized.After staining, allow the polymer spots to dry for two minutes. Then distribute two drops of amply red onto the array and allow staining to proceed for two minutes After staining quickly rinse the slide with purified water and dry as previously shown, insert the slide into the imaging bay of the Amli Fox reader to collect an image using amli software to process results. The left image shows detection of a 2009 novel H one N one specimen by Amli Fox.While the image on the right uses traditional fluorescence generated by a confocal fluorescence microarray scanner, the same overall detection pattern can be easily seen for both methods. However, the Amli fox result is visible to the naked eye. These images show flu chip results from a human origin, H three N two human origin, H one N one, swine origin, H one N one, and a negative fox array lacking detectable influenza.A sequences two and three outlined in blue produce signal for all of the influenza. A subtypes shown, but the sequences outlined in red in the lower right only produce signal for the swine origin, H one N one specimen. The negative specimen shows signal on an internal control sequence outlined in green, indicating no inhibition or failure of the R-T-P-C-R reaction.Once mastered the entire process from vial extraction through amox detection and final result interpretation can be performed in approximately six hours. The Ampl Fox detection portion of the procedure, including instrument calibration, can be completed in roughly 30 minutes Following this procedure. Other methods like partial or full genome sequencing can be performed in order to answer additional questions.This influenza assay is intended as an initial screen, particularly for samples which result in a unique hybridization pattern. Because detection is a general assay, we hope researchers will find new and interesting low density microarray applications for which detection could add value.

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

Research

JoVE Journal

Immunology and Infection

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.

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral 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 μl volume of test material and has the potential for concurrent screening of multiple pathogens.

High-throughput Detection Method for Influenza Virus

In this video, we demonstrate the process for detecting antibody isotypes in multiple human serum samples using a microarray slide printed with antigenic subtypes of influenza virus strains.

Viral Antigen Microarray Assay to Detect Antibody Isotypes in Serum Against Viral Antigen

The video demonstrates an assay for quantifying influenza viruses A and B. This assay employs nicking endonuclease amplification with influenza-specific primers and target detection using molecular beacon probes.

A Molecular-Based Assay for Quantifying Influenza A and B Viruses

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Microfluidic Chip Fabrication and Method to Detect Influenza

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

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.

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

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.

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Learn ampliPHOX colorimetric detection for influenza viral subtypes using photopolymerization on DNA microarrays. This rapid method offers cost-effective signal amplification with visible results comparable to fluorescence sensitivity.

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

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