Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Summary

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Abstract

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Introduction

Early case detection and rapid treatment are considered the most effective control strategies to reduce Mycobacterium tuberculosis (MTB) transmission¹, and there is now a broad consensus in the TB community that a point of care (POC) or near POC test to simultaneously diagnose TB and drug resistance (DR) is needed. Technologies such as Cepheid’s GeneXpert and other nucleic acid amplification tests reduce the time to diagnosis for many TB patients, and provide a rapid read-out indicating resistance to rifampin or selected mutations conferring resistance to other first or second line drugs². Although real-time and isothermal nucleic acid amplification tests are designed to identify the drug resistance mutations that lead to MDR-TB, the spectrum of mutations they detect is often inadequate to design an individualized drug regimen corresponding to the drug resistance profile of the patient, and technical constraints related to optical cross-talk or the complexity of amplification and reporting chemistries^3-7 may limit the number of loci or mutations that are detected. Thus, detection technologies with higher multiplexing capacity are required to address known gaps in MDR-TB POC diagnostics.

Microarrays and the WHO-endorsed Hain line probe assays can address the “multiple gene, multiple mutations” challenge of diagnosing MDR-TB^8-29. Unfortunately, these hybridization-based, multiplexed detection platforms use multistep, complicated, and open-amplicon protocols that require significant training and proficiency in molecular techniques. The amplification microarray³⁰ was designed to address some of these microarray work-flow and operational concerns. The simplifying fluidic principles are to amplify, hybridize, and detect nucleic acid targets within a single microfluidic chamber. The user introduces the nucleic acid and amplification master mix into a fluidic chamber with a pipette and starts the thermal cycling protocol. For the batch processing method shown here, microarrays are subsequently washed in bulk solution, dried, and imaged. This study demonstrates the functionality of an amplification microarray using an MDR-TB microarray test for rpoB (30 mutations), katG (2 mutations), inhA (4 mutations), rpsL (2 mutations), embB (1 mutation), IS1245, IS6110, and an internal amplification and hybridization control. At least one matched pair of microarray probes (wildtype (WT) and single-nucleotide mutant (MU)) is included for each mutation of interest. Purified nucleic acids from multi-drug resistant M. tuberculosis are from the TDR Tuberculosis Strain Bank³¹. Gel element microarrays are manufactured on glass substrates by copolymerization essentially as described elsewhere³², except that we use 4% monomer and 0.05 mM each probe in the polymerization mixture. Arrays are surrounded with a 50 ml gasket prior to use. After thermal cycling, hybridization, and wash steps, amplification microarrays are imaged on an Akonni portable analyzer. Background-corrected, integrated signal intensities are obtained from the raw .tif images using a fixed circle algorithm. Noise for each gel element is calculated as three times the standard deviation of the local spot backgrounds. Gene targets are typically considered detectable for signal to noise ratio (SNR) values ≥3. In order to determine the presence or absence of a specific mutation in each gene or codon, a discriminant ratio is calculated from the SNR values as (WT-MU)/(WT+MU). Discriminant ratios <0 are indicative of a drug-resistance mutation at the locus, whereas ratios >0 are indicative of the wild-type sequence.

Protocol

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.

Reagent	Per- Sample Volume (µl)	Final [ ]
Mulitplex PCR Buffer with HotStar Taq Plus	25	1x
Bovine serum albumin (BSA)	0.55	0.6 mg/ml
Formamide	3.8	7.6%
Additional Taq polymerase	0.8	units/µl (4 units total)
MDR-TB primer mix	15.75	-
RNase-free H2O	2.1	-
Amplification/Inhibition Control	1	5.0 fg/µl
Total	49

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.

Results

Qualitative image analysis can provide insight into sources of experimental noise or variability that are challenging to identify in data tables generated by automated image analysis software. Thus, it can be useful to visually ascertain that 1) all gel elements are intact and undamaged, 2) the global background is free from fluorescent artifacts that might affect individual signal to noise ratio (SNR) values, 3) there is no evidence for bubble formation or nonuniform amplification/hybridization across the array, and 4) ...

Discussion

The extent of multiplexing with an amplification microarray is ultimately dictated by the efficiency of multiplex asymmetric PCR, not the microarray. In our experience, 10-12 unique primer pairs can be readily multiplexed in an amplification microarray format. Conventional primer and probe design criteria therefore apply to new assays, except that one also needs to consider potential interactions between solution-phase nucleic acids and immobilized microarray probes, the thermal efficiency of the thermal cycler, and prob...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MDR-TB amplification microarrays, with applied gasket and pre-cut cover slips	Akonni Biosystems	Inquire
Multiplex PCR kit, containing 2X PCR buffer with HotStar Taq plus	Qiagen	#206143
Taq polymerase	Qiagen	#201207
RNAse-free water	Qiagen	#206143
Formamide	Thermo Fisher Scientific, Inc.	#BP227-500
20 mg mL-1 non-acetlyated bovine serum albumin (BSA)	Sigma-Aldrich	#3B6917
5X concentrated MDR-TB primer mix	Akonni Biosystems	Inquire
500 fg uL-1 amplification and inhibition control	Akonni Biosystems	Inquire
20X SSPE	Thermo Fisher Scientific, Inc.	#BP1328-4
Triton X-100	Thermo Fisher Scientific, Inc.	#BP151-500
Disinfecting Spray	Current Technologies, Inc.	#BRSPRAY128
70% Isopropyl Alcohol	Decon Labs, Inc.	#8416
Microarray imager, with automated image and data analysis software	Akonni Biosystems	100-20011
Thermal cycler with flat block insert	Akonni Biosystems	100-10021
High-throughput wash station and slide holder	ArrayIt	HTW
Dissecting forceps	Thermo Fisher Scientific, Inc.	#10-300
Mini Vortexer	VWR	#3365040
Mini-centrifuge	VWR	#93000-196
Airbrush Compressor Kit	Central Pneumatic	#95630