Abstract
Infectious diseases are a leading cause of global morbidity and mortality, accounting for 29% of worldwide
deaths. Next-generation sequencing (NGS) is a useful tool in pathogen detection, strain identification, and drug
susceptibility testing (among other applications). A primary issue for NGS for rapid pathogen genomic analysis
is that raw patient samples typically have a low bacterial load, requiring culturing that can take weeks to months
before a sufficient microbial load is generated. However, culturing is economically and logistically unsustainable
and presents with other biological issues that may confound results. Additionally, enrichment of the pathogen-
specific genes is highly dependent on sample extraction efficiency. Using nucleic acid testing (NAT) and NGS
methods, efficient DNA extraction is essential for the successful and accurate identification of microorganisms
or populations of microbes. Poor DNA extraction when analyzing clinical and environmental samples consisting
of resilient microbes leads to inconclusive or inaccurate diagnostic results. There is a need for high-efficiency
extraction of nucleic acids from hard-to-lyse microorganisms in direct patient samples to facilitate reliable clinical
diagnostic workflows. Triangle Biotechnology (Triangle Bio) is developing a novel and proprietary
technology for efficient, high-throughput, reproducible, and unbiased microbial lysis, based on a
cavitation-enhancing nanodroplet reagent for use with low-cost sonication devices. The proposed
nanodroplets preferentially target to microbes with resilient cell walls and deliver focused mechanical shear
forces. In Phase I, Triangle Bio demonstrated a 6-100x and 2-5x improvement in DNA extraction from
Mycobacterium smegmatis (a model for Mycobacterium tuberculosis [Mtb]) and Enterococcus faecalis (a Gram-
positive bacteria), respectively, compared to commonly used commercial kits. In Phase II, the company will
establish a platform of nanodroplet formulations applicable to a wide range of infectious pathogens with
significant clinical impact. Triangle Bio will accomplish this research through the following three aims: 1) Identify
targeting ligand candidates compatible with 12 representative microbial species and three clinical sample
matrices (Y1), 2) Validate binding and cavitation performance of candidate formulations and optimize workflow
conditions for clinical sample matrices spiked with four target microbial species (Y2-Y3), and 3) Evaluate
workflows by demonstrating improved performance of targeted NGS for diagnosis of drug-resistant Mtb (Y3).
Successful implementation of this technology could have significant impacts on a wide range of applications
requiring reliable microbial lysis techniques, including but not limited to NGS for infectious disease detection and
diagnosis, NGS based food safety testing for infectious pathogens, and clinical and environmental microbiome
studies where resilient microbes can be underrepresented in metagenomic analysis.
