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.
