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A Benchtop Automated Sputum-to-Genotype System Using a Lab-on-a-Film Assembly for Detection of Multidrug-Resistant
Alexander V Kukhtin1, Ryan Norville1, Arial Bueno1
1Akonni Biosystems, Inc., 400 Sagner Avenue, Suite 300, Frederick, Maryland 21701, United States.
Analytical Chemistry
|March 7, 2020
Summary
This study presents a novel automated system for rapid drug-resistant Mycobacterium tuberculosis (MTB) genotyping directly from sputum. The innovative approach enhances sample processing and nucleic acid purification for accurate and affordable diagnostics.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Biotechnology
Background:
- Automated genotyping of drug-resistant Mycobacterium tuberculosis (MTB) from sputum faces challenges due to sample viscosity, difficult cell lysis, and numerous resistance mutations.
- High prevalence of MTB in resource-limited settings necessitates affordable and efficient diagnostic solutions.
Purpose of the Study:
- To develop and validate an integrated, automated system for direct sputum-to-genotype analysis of drug-resistant MTB.
- To overcome limitations in sample processing, cell lysis, and cost-effective multi-mutation genotyping.
Main Methods:
- Utilized magnetic rotation (MagVor) for sample homogenization and chaotic mixing with glass beads for efficient cell lysis.
- Employed a pipet tip with an embedded matrix (TruTip) for nucleic acid purification.
- Developed a microfluidic laminate assembly (Lab-on-a-Film) with 203 gel elements for amplification, hybridization, washing, and fluorescent imaging in a closed system.
Main Results:
- Successfully integrated MagVor homogenization, TruTip purification, and Lab-on-a-Film amplification into a closed, automated multisample system.
- Achieved MTB detection down to 43 colony-forming units/mL directly from raw sputum samples.
- Demonstrated a viable platform for cost-effective, multi-mutation genotyping of drug-resistant MTB.
Conclusions:
- The developed automated system effectively addresses key challenges in direct sputum-based MTB genotyping.
- This technology offers a promising solution for rapid, affordable, and accurate diagnosis of drug-resistant TB, particularly in resource-limited settings.
