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.