Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

For laboratories that follow universal PCR precautions, it is operationally more efficient to include several amplification microarrays and gaskets per substrate and wash all amplification microarrays simultaneously in a bulk container, as described here. Consumable formats are available for performing post-amplification microarray washing steps in an entirely sealed, closed amplicon test, as reported elsewhere^30,33.

1. Setup

1. Extract and purify nucleic acids from the sample under appropriate biosafety conditions with a method of choice. The requirement is that nucleic acids be of sufficient purity for asymmetric, multiplex PCR amplification.
2. Prepare workspace by wiping surfaces and equipment with decontaminating solutions.
3. Place amplification reagents on ice or cold block.
4. Label a sterile 1.5 ml microfuge tube for the amplification master mix, and label a sterile 0.2 ml microfuge tube for each sample.
5. After reagents are thawed, briefly vortex and collect contents to the bottom of the tube with a pulse-spin in a mini-centrifuge. Do not vortex Taq polymerase. Gently flick the tube to mix and follow with a pulse-spin in mini-centrifuge.
6. Prepare an amplification master mix using the per-sample reaction volumes shown in Table 1. To account for possible pipetting inaccuracies, prepare at least one more reaction volume than the total number of samples being processed. Note that the master mix contains additional Taq polymerase, over and above what is provided with the muliplex PCR buffer. Only add the amplification/inhibition control to the master mix after all other reagents are combined, and the stock reagent tubes returned to storage.

Table 1. MDR-TB amplification microarray master mix composition.

7. Vortex the master mix and collect contents to the bottom of the tube with a pulse-spin in a mini-centrifuge.
8. Combine 49 µl of master mix and 1 µl of M. tuberculosis DNA to each of the sample tubes from step 1.4, above. For an external, no template control (NTC), use 1 µl of molecular biology grade water in place of the M. tuberculosis DNA sample. Vortex and pulse-spin the tube(s) when finished.

2. Load Amplification Microarrays

1. Add 48 µl of each master mix/sample onto the center of their respective microarrays, being careful to not touch the microarray itself.
2. Place a cover slip on top of the microarray gasket and seal, being careful not to trap any air bubbles in the microarray chamber. Air bubbles can negatively affect amplification efficiency and may create artifacts or noise in the subsequent microarray image.

3. Thermal Cycling

1. Once all microarrays are loaded with sample, place substrates on the flat block thermal cycler. Make sure that the heated lid option is turned “Off”. Equipment obtained from Akonni Biosystems will already be prequalified for use. Otherwise, it is very important to verify that the flat block thermal cycler(s) provide(s) uniform, consistent heating across all microarray substrates.
2. Open the thermal cycler software, select the appropriate program, enter “50 µl” for the reaction volume, and initiate the run. The thermal cycling protocol described here consists of:

   Thermal Cycling Steps
   1	88 °C	5 min
   2	88 °C	30 sec
   3	55 °C	1 min
   4	65 °C	30 sec
   5	Repeat steps (2-4) for 50 cycles
   6	65 °C	3 min
   7	55 °C	3 hr

3. The total number of thermal cycles and 55 °C post-amplification hold time are independent variables that the user may modify, as needed or appropriate for the specific experiment. Because the MDR-TB master mix creates asymmetric (predominantly single-stranded) amplicons, it is usually helpful to include more thermal cycles than might otherwise be utilized for a conventional, exponential PCR amplification protocol.
4. When the thermal cycling and hybridization program is complete, end the program, remove the amplification microarrays, close the software, and turn off the thermal cycler(s).

4. Wash and Dry

1. For washing up to 24 substrates simultaneously, prepare at least 250 ml 1x SSPE – 0.1% Triton X-100 wash buffer, and two containers with at least 250 ml deionized or Milli-Q grade water each. The wash buffer can be prepared in advance.
2. Carefully remove the cover slip and gasket from each amplification microarray chamber using flat-end forceps. This step is the point at which there is the greatest risk of physically damaging gel element arrays. Use appropriate PCR contamination controls to prevent the spread of amplified nucleic acids within the work environment.
3. Place microarray substrates in a histology slide holder, and place all slides into the wash bin containing the wash buffer. Cover the container with a lid.
4. Wash microarrays for 10 min at room temperature with gentle agitation.
5. Dip the microarrays three times each in two successive washes of deionized or Milli-Q grade water, and air dry.

5. Imaging

The asymmetric MDR-TB primer mix generates Cy3-labeled amplicons. Gel element microarrays can be imaged with any standard microarray imager capable of imaging Cy3. The following imaging procedure is specific for the MDR-TB master mix, Dx2100 field portable imager, and automated analysis software provided by Akonni.

1. Ensure that the ethernet cable from the imager is connected to the computer.
2. Turn on the imager. The power switch is located on the back panel of the instrument. Blue LEDs on the front of the imager will illuminate when imager is powered on.
3. Turn on the computer.
4. On the computer desktop, double click the software icon to launch the automated image analysis software.
5. Wait for the software to establish a connection with the imager camera. Once the connection is established, the Analyze button becomes available, and the message “Connection Successful” will appear in the lower left corner of the screen.
6. Insert the dried amplification microarray with the microarray facing away from the user, and toward the objective lens. If the microarray is improperly oriented with respect to the lens and camera, the automated analysis software will fail to place a grid on the fluorescence image.
7. Close the access door. A preview image will be available on the computer screen.
8. Select the MDR-TB analysis script from the drop-down menu and enter the desired Exposure Time (in milliseconds). A typical starting point for gel element amplification microarrays is 100-500 msec.
9. Saturated pixels will be displayed on the Preview Image in red. Adjust the exposure time to minimize the number of saturated pixels within individual spots.
10. Click Analyze to acquire and analyze the fluorescence image. The software will automatically place an MDR-TB assay-specific grid onto the image, extract integrated intensity and background values, and report drug resistance and genotyping results. The automated analysis will typically take a few seconds. For challenging images that contain scratches, dust, fluorescent artifacts, or physical damage to the microarray features, the software may need up to 1 min to complete the analysis.
11. Once the analysis is complete, click on Save, select the destination folder, type in a filename, and click OK. Underlying microarray data are saved as an .xls file and can be exported for off-line analyses, if desired.
12. Once the analysis is complete, remove the amplification microarray from the imager, and click on New to initiate the next analysis.
13. When finished collecting data, close the software, shut down the computer, and turn off the imager.