The overall goal of the following experiment is to genotype multi-drug resistant microbacterium tuberculosis, using an amplification microarray. This is achieved by performing a multiplex asymmetric PCR over the top of a microarray within a single microfluidic reaction chamber. There the sample is simultaneously amplified, labeled size selected, made single stranded, and hybridized to the microarray.
Next, after the microarray is washed, a reader shows if microbacterium tuberculosis is present, and if it contains a DNA mutation known to confer resistance to rifampin iso, niazi ethambutol, or streptomycin. The made advantage of this method over existing microarray line probe or nucleic acid hybridization methods is that an amplification microarray significantly simplifies the user interaction with the test. Still, while providing the analytical sensitivity of PCR and the multiplexing capacity of a microarray For this procedure, have a clean workspace wiped down with decontaminate solution and have nucleic acids pure enough for multiplex PCR.
After thawing and mixing all the stock solutions, assemble the PCR master mix. When preparing this mix, make at least one extra volume of mixture for the TB test shown here. Also, add extra T polymerase to the master mix.
In addition to the amount of tack provided with the stock reagents mix one microliter aliquots of sample DNA to 49 microliters of master mix. Carefully add 48 microliters of each reaction mixture to the center of the appropriate microarray. Do not touch the microarray itself.
Then carefully cover and seal the microarray Improperly. Applying the cover slip can introduce bubbles that may lead to artifacts, evaporation, poor amplification, or non-uniform hybridization. Any of these conditions can lead to an erroneous result.
Next, load the micro rate into the thermal cycler. With the substrates against the heat block, the heated lid option must be off. For laboratories that follow universal PCR precautions, it is more efficient to include several amplification microarrays per slide and process multiple tests simultaneously.
Now, run the appropriate thermal cycling program. This program has 50 amplification cycles, which may seem high but needed because of the predominantly single stranded amplicon population created by the reaction. When the reaction is complete, remove the microarrays and shut down the thermal cycler.
Carefully open the microarray chamber with flat and forceps. Remove the slip, cover and gasket. The microarray can be damaged by this step, so be careful Improperly.
Removing the gasket assembly can lead to physical damage of the microarray. Be careful not to bend or flex the cover slip and take great care to avoid scratching the surface of the microarray with the forceps, Now transfer the microarray substrates to a histology slide holder. Bathe the substrates in the wash buffer for 10 minutes at room temperature with gentle agitation, follow the wash solution bath with two or three more washes, all in deionized water.
Dry the microarrays before proceeding to imaging. A gentle stream of compressed air can be used for this purpose in this demonstration, parameters are specific to the MDR TB test and an Econ DX 2100 field portable imager. A blue light on the imager indicates that it is ready.
Turn on the computer. Open the system software provided by a conni and wait for the software to announce that it has established a connection with the camera. Next, insert the dried microarray into the imager.
With the microarray towards the lens and away from the user. The software will now provide a preview image from the dropdown menu. Select the MDR TB analysis script.
Enter an exposure time between 100 and 500 milliseconds. Overexposed pixels will be read. Adjust the exposure to minimize them.
When the saturation is set to minimize over exposure, start the analysis. The software places an MDRT TB grid on the image scores, samples and background intensities, and then reports drug resistance and genotyping results. Once the analysis is complete and saved, remove the chip from the imager and start a new analysis session to analyze the next array.
Continue the analysis for each of the processed microarrays. Saving the data each time. Qualitative image analysis can provide insight into sources of experimental noise or variability that is challenging to identify in data tables generated by automated image analysis software.
A high quality array lacking artifacts is seen here. Several artifacts can lead to the introduction of bubbles which create halos. Despite array artifacts, the software will still retrieve values from being scanned.
A diagnostic reporting algorithm would, despite getting values recognized this array as invalid. A dilution series of wild type H 37 RA genomic DNA was used on a microarray PCR template at 6.25 picograms the equivalent of 1, 250 cells. All probes were detected.
An SNR value above three is a positive result, and registered values were much higher. Next, world Health Organization reference isolates were genotyped. Single nucleotide polymorphisms present on the array were detected as the corresponding mutation in the MDR TB genome.
Several examples of detecting near neighbor mutations were also evident. A discussion is provided in the text protocol. Once mastered, this technique can be done in about three and a half to six and a half hours, depending on the concentration of DNA, the specific assay, the efficiency and uniformity of the thermal cycler, and the desired analytical or clinical sensitivity and specificity.
Also, the number of amplification cycles and post amplification hybridization times can be readily shortened or lengthened to get the desired performance characteristics.
