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. 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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 for influenza identification and subtyping, the technology can be extended to detect any biotinylated product captured on a microarray. As an example, our earlier work on photopolymerization-based detection using a different reagent chemistry was demonstrated on both nucleic acid and antibody-based arrays.12 Others have also shown proof of concept for antibody and protein-based applications of a photopolymerization-based system. In these cases, different reagent chemistries requiring purging with an inert gas were utilized.13,14
A schematic of the influenza low-density microarray and representative images can be seen in Figure 2. Shown in Figure 2A, the array contains a spatial marker/spotting control (in red) and 14 unique capture sequences (in blue), each spotted in triplicate. The capture sequences are amino-terminated synthetic short (&tilde;25 mer) oligonucleotides designed to capture influenza targets that are genetically representative of specific types or subtypes of influenza. The capture sequences are designed to produce distinctly different patterns on the chip for different influenza A subtypes. Figures 2B and 2C show a direct comparison of the detection of a 2009 novel H1N1 specimen by ampliPHOX and traditional fluorescence, respectively. The fluorescence result was generated using a confocal fluorescence microarray scanner (Genetix aQuire). The same overall detection pattern can be easily seen for both methods, however, the ampliPHOX result is visible to the naked eye. 
Shown in Figure 3 are ampliPHOX results from three representative influenza A positive specimens and one negative specimen as processed by the protocol described here. Figures 3A, 3B, and 3C show results for a human-origin H3N2, human-origin H1N1, and swine-origin H1N1 (2009 novel H1N1), respectively. A distinct difference in the overall detection pattern can easily be seen for these 3 images. For instance, it can be seen that sequences 2 and 3 produce signal for all of the influenza A subtypes shown (3A-3C), but that sequences 10, 12, and 13 only produce signal for the swine-origin H1N1 specimen (3C). This approach has been used previously to successfully type and subtype influenza viruses on a microarray.6,8,10 Importantly, the negative specimen shown in Figure 3D shows signal on internal control sequence, indicating no inhibition or failure of the RT-PCR reaction. 
The interpretation of these images is easily automated by the ampliVIEW software. The user first uses the software to define the microarray layout, and then to describe which targets should be present to generate a certain "answer" for this layout. For example, the influenza layout shown in Figure 2A could generate logical results such as "Flu A positive", "Flu B positive", "non-seasonal influenza A", "seasonal influenza A", and "negative", depending on the desired outcomes. Once these logical assignments are made and saved, the software can automatically interpret the images to provide a result with little user input.
ampliPHOX detection technology generates visual, colorimetric results for lower density microarrays with similar sensitivity to fluorescence in minutes. We believe the combination of easy-to-use, inexpensive reagents with a low-cost instrument provides an attractive alternative to traditional microarray detection methods, particularly as more targeted, lower density genomic/diagnostic microarrays become increasingly used in a variety of applications.

<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

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

Nanyang Technological University

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

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 ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Developmental Biology

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

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

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.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Biochemistry

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, ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

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.

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

Bioengineering

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 ...

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 ...

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

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

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.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

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

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

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.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

Biology

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

Source: Khan, S., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59973">Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes.</a> J. Vis. Exp. (2019)
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.

Test mikromacierzy antygenu wirusowego do wykrywania izotypów przeciwciał w surowicy przeciwko antygenowi wirusowemu

Source: Khan, S., et al., Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes. J. Vis. Exp. (2019)
In ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

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

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Test mikromacierzy antygenu wirusowego do wykrywania izotypów przeciwciał w surowicy przeciwko antygenowi wirusowemu