Lab-on-a-Film disposable for genotyping multidrug-resistant Mycobacterium tuberculosis from sputum extracts

Abstract

We describe a Lab-on-a-Film disposable that detects multidrug-resistant tuberculosis (MDR-TB) from sputum extracts. The Lab-on-a-Film disposable consists of 203 gel elements that include DNA sequences (probes) for 37 mutations, deletions, or insertion elements across 5 genes (including an internal control). These gel elements are printed on a flexible film, which costs approximately 500 times less than microarray glass. The film with printed gel elements is then laminated to additional rollable materials (films) to form a microfluidic flow cell. We combined multiplex amplification and hybridization steps in a single microfluidic chamber, without buffer exchanges or other manipulations up to and throughout hybridization. This flow cell also incorporates post hybridization wash steps while retaining an entirely closed-amplicon system, thus minimizing the potential for sample or amplicon cross-contamination. We report analytical sensitivity of 32 cfu mL-1 across all MDR-TB markers and detection of MDR-TB positive clinical specimens using an automated TruTip workstation for extraction and the Lab-on-a-Film disposable for amplification and detection of the extracts.

Figure 1

Amplification, hybridization, and washing take place on a Lab-on-a-Film disposable, which includes 203 hemispherical gel elements on a thin film substrate. Initially, master mix and sample are introduced into the reaction chamber. Subsequently, asymmetric PCR occurs in the chamber, resulting in a mix of fluorescently-labeled, single-stranded amplicons. Hybridization of the labeled amplicons to the gel elements follows, but this does not require a fluidic transfer step. The unbound molecules are then washed away using a pipettor, and the liquid is imbibed into an absorbent in the waste chamber. The last step is fluorescence imaging and analysis.

Figure 2.

(A) Bright field image showing the morphology of the gel elements on a transparent film after UVB polymerization for 30 minutes, (B) “hydrophilic footprints” (residue) of gel elements after attempting to physically remove the gel elements; the remaining residues indicate that 30 minutes of UVB polymerization resulted in covalent attachment to the film substrate and (C) fluorescence images of gel elements after hybridization; gel elements outside of the red rectangle contain immobilized Cy3-labeled markers, which serve as fidicuals for the automated image analysis algorithm, and gel elements inside the red rectangle contain immobilized probes, which hybridized to complementary Cy3-labeled target.

Figure 3.

Analytical sensitivity study of Lab-on-a-Film disposables, showing correct detection down to 32 cfu/ml. Error bars are standard deviations for n=6 replicates per titer. NTC is the no template control. M13 is an internal control. UN is a universal probe on the array that represents amplification and hybridization efficiency for each of the gene targets. All probes for mutations, deletions, and insertion elements on the array correctly reported wild-type for all titers and all replicates.