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 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 ˜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.
1. Sample amplification using RT-PCR
2. Hybridization of RT-PCR products to low density microarrays
3. ampliPHOX: labeling hybridized product and calibration chips with ampliTAG
4. ampliPHOX: calibration, signal amplification, and imaging
5. Representative Results:
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.
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.
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.
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 (˜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.
The authors have nothing to disclose.
InDevR acknowledges NIH/NIAID U01AI070276 and R43AI077112 for funding this work.
Reagent/equipment | Manufacturer | Catalog # | Yorumlar |
---|---|---|---|
Qiagen MinElute Virus Spin Kit | Qiagen | 57704 | single 60 μl elution |
QIAcube | Qiagen | 9001292 | optional |
ABI 9800 Fast Thermal Cycler | Applied Biosystems | 4441166 | |
Qiagen OneStep RT-PCR kit | Qiagen | 210210 | kit dNTPs not used |
2x Spotting Buffer | InDevR Inc. | MI-5007 | |
Biotinylated dNTP Mix | InDevR Inc. | MI-5009 | |
Lambda exonuclease | Epicentre Biotechnologies | LE032K | 2500 U, 10U/μl |
FluChip primer mix | InDevR | N/A | not yet available for sale |
Orbital Shaker | Madell Technology | ZD-9556-A | |
Wash Bins | InDevR Inc. | MI-4002 | |
Wash Racks | InDevR Inc. | MI-4003 | |
2x Hybridization Buffer | InDevR Inc. | MI-5004 | |
Calibration Chips | InDevR Inc. | AP-5006 | |
Wash Buffers A-D | InDevR Inc. | MI-5005 | |
ampliRED | InDevR Inc. | AP-5004 | |
ampliTAG | InDevR Inc. | AP-5001 | |
2x ampliTAG Buffer | InDevR Inc. | AP-5002 | |
ampliPHY, ampliPHY enhancer | InDevR Inc. | AP-5003 |