A Benchtop Automated Sputum-to-Genotype System Using a Lab-on-a-Film Assembly for Detection of Multidrug-Resistant Mycobacterium tuberculosis

Abstract

Automated genotyping of drug-resistant Mycobacterium tuberculosis (MTB) directly from sputum is challenging for three primary reasons. First, the sample matrix, sputum, is highly viscous and heterogeneous, posing a challenge for sample processing. Second, acid-fast MTB bacilli are difficult to lyse. And third, there are hundreds of MTB mutations that confer drug resistance. An additional constraint is that MTB is most prevalent where test affordability is paramount. We address the challenge of sample homogenization and cell lysis using magnetic rotation of an external magnet, at high (5000) rpm, to induce the rotation of a disposable stir disc that causes chaotic mixing of glass beads ("MagVor"). Nucleic acid is purified using a pipet tip with an embedded matrix that isolates nucleic acid ("TruTip"). We address the challenge of cost and genotyping multiple mutations using 203 porous three-dimensional gel elements printed on a film substrate and enclosed in a microfluidic laminate assembly ("Lab-on-a-Film"). This Lab-on-a-Film assembly (LFA) serves as a platform for amplification, hybridization, washing, and fluorescent imaging, while maintaining a closed format to prevent amplicon contamination of the workspace. We integrated and automated MagVor homogenization, TruTip purification, and LFA amplification in a multisample, sputum-to-genotype system. Using this system, we report detection down to 43 cfu/mL of MTB bacilli from raw sputum.

Figure 1

A. Photograph of the interior of the analyzer depicting the MagVor for lysis and homogenization, the TruTip for purification, the deep-well plate so that all liquids are part of the consumable, pipetting station, the Lab-on-a-Film Assembly (LFA), the thermal cycler, and the imager. B. Illustration of the interfaces of the analyzer sub-systems with the consumables; the order of the analyzer operations is numbered. (Note, Step 6 - Air Drying occurs at Column 7 of the deep-well plate). The carriage moves the consumable to each interface on the analyzer.

Figure 2.

Layout of the consumable for 6 samples in which the following steps occur. (1) Lysis and homogenization occur in lysis tubes, which include a magnetized stir disc and glass beads. (2) The TruTip aspirates the sample mixed with a binding buffer so that it flows through the pores of the matrix in the tip resulting in DNA bound to the matrix. (3) The porous matrix is washed to remove the impurities. (4) The matrix is dried with air. (5) The bound DNA is eluted into an elution buffer. (6) The purified DNA is amplified with an asymmetric PCR reaction. (7) The product (with fluorescent labels) hybridize to the gel elements. (8) The gel elements are washed to remove unbound product. And, (9) an image of the array is captured and analyzed.

Figure 3.

Illustration of the LFA on the system with the thermal cycler in the (1) “up” (disengaged) position, (2) in the “down” (engaged) position, and (3) with the imager capable of individually imaging and analyzing each array of gel elements.

Figure 4.

Sputum-to-genotype amplification signals across 8 runs with correct detection (genotyping) down to 43 cfu/mL. Also shown are the signals for the external control (1pg H37Ra DNA).