Improving diagnostic sensitivity for difficult-to-lyse microbial samples with nanodroplet technology

利用纳米液滴技术提高难以裂解的微生物样品的诊断灵敏度

• 批准号: 10628013
• 负责人: Sandeep Kasoji
• 金额: $ 99.84万
• 依托单位: TRIANGLE BIOTECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-03 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10628013
• 关键词: Accounting; Acinetobacter baumannii; Acoustics; Antibodies; Bacteria; Binding; Biological; Biological Assay; Biotechnology; Blood; California; Candida albicans; Cell Nucleus; Cell Wall; Cell surface; Cells; Cessation of life; Chemicals; Clinical; Communicable Diseases; Communities; Cytolysis; DNA; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic Sensitivity; Drug resistance; Drug resistant Mycobacteria Tuberculosis; Enterococcus faecalis; Fluorometry; Food Safety; Formulation; Foundations; Genes; Genus Mycobacterium; Gram-Positive Bacteria; Health; Human; Lead; Lectin; Ligand Binding; Ligands; Measures; Mechanics; Metagenomics; Methodology; Methods; Microbe; Modeling; Molecular Analysis; Molecular Diagnosis; Morbidity - disease rate; Mycobacterium smegmatis; Mycobacterium tuberculosis; Nucleic Acid Amplification Tests; Nucleic Acids; Pathogen detection; Patients; Peptides; Performance; Pharmaceutical Preparations; Phase; Polymerase Chain Reaction; Population; Predisposition; Protocols documentation; Public Health; Reagent; Reproducibility; Research; Resistance; Sampling; Shock; Sonication; Sputum; Techniques; Technology; Testing; Thick; Tuberculosis diagnosis; Universities; Urine; cellular resilience; clinical diagnostics; clinically relevant; cost; design; diagnostic platform; direct application; drug testing; improved; in-vitro diagnostics; innovation; mechanical force; microbial; microbiome; microbiome research; microorganism; mortality; nanoDroplet; next generation sequencing; novel; novel diagnostics; pathogen; pathogen genomics; preservation; resilience; safety testing; screening; statistics; technology validation; tool

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.

摘要