SBIR-STTR Award

Miniaturized DNA Sequencer for Identification of Microbial Pathogens Abstract. The goal of this project is develop a compact and inexpensive DNA sequencer based on the implementation of sequencing-by-synthesis chemistry on a droplet-based digital microfluidic cartridge. The proposed device would be capable of sequencing 10's to 100's of base pairs using an inexpensive disposable cartridge and a compact and simple piece of equipment. The cartridge will also integrate sample preparation capability, including DNA amplification, to enable widespread use by non-specialists and provide rapid and reliable results at low cost. The initial application for this technology will be microbial pathogen identification. The rationale for this is that even a few tens of base pairs can provide discrimination of microbial pathogens while there is a genuine clinical and medical need for an instrument that can perform this type of analysis rapidly, automatically, and at low cost. As a proof of concept, a prototype system will be developed in Phase I and an evaluation will be performed to assess the ability of the prototype to identify a set of clinically relevant yeasts and moulds.

Public Health Relevance:
A compact and inexpensive instrument to sequence small amounts of DNA for the purpose of identifying microbial pathogens will be developed and tested. The proposed instrument would improve the ability of clinicians to diagnose and treat infectious diseases.

Public Health Relevance Statement:
A compact and inexpensive instrument to sequence small amounts of DNA for the purpose of identifying microbial pathogens will be developed and tested. The proposed instrument would improve the ability of clinicians to diagnose and treat infectious diseases.

NIH Spending Category:
Bioengineering; Biotechnology; Genetics; Human Genome; Infectious Diseases

Project Terms:
Academic Medical Centers; Base Pairing; Biochemistry; Chemistry; Chemistry, Biological; Clinical; Clinical Evaluation; Clinical Testing; Cognitive Discrimination; Collaborations; Communicable Diseases; Core Facility; DNA; DNA Sequence; DNA amplification; Deoxyribonucleic Acid; Development; Devices; Diagnosis; Diagnostic; Discrimination; Discrimination (Psychology); Equipment; Evaluation; Fungi, Filamentous; Goals; Immunoassay; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases and Manifestations; Infectious Disorder; Investigators; Laboratories; Left; Medical; Methods; Methylation; Microfluidic; Microfluidics; Molds; Patients; Performance; Pharmacogenetics; Phase; Preparation; Protein Methylation; Protocol; Protocols documentation; Research; Research Personnel; Researchers; Sampling; Science of Chemistry; Specialist; Synthesis Chemistry; Synthetic Chemistry; System; System, LOINC Axis 4; Technology; Testing; University Medical Centers; Yeasts; abstracting; base; cancer type; clinical applicability; clinical application; clinical relevance; clinical test; clinically relevant; cost; design; designing; digital; improved; instrument; meetings; microbial; miniaturize; pathogen; prototype; public health relevance; research clinical testing; tool development

The ultimate goal of this project is to develop an extremely compact and inexpensive DNA sequence for applications in laboratory and clinical settings. The device will implement pyrosequencing on a disposable droplet based ""digital microfluidic"" cartridge and will be capable of sequencing 10's to 100's of nucleotides at a very low cost per run and at a low initial equipment investment. This platform will also integrate sample preparation, including DNA amplification, with sequencing to provide the integration and ease-of-use demanded of clinicians and other non-specialists who require DNA sequence data. The initial application for this capability will be microbial pathogen identification. In Phase I, a prototype was developed and the microfluidic protocols and biochemical reaction conditions for pyrosequencing were optimized. As a feasibility demonstration 20-40 base pairs within the ITS2 genetic region were sequenced for 13 different molds and yeasts which provided unambiguous identification of each isolate. In Phase II, this success will be built upon by integrating sample preparation steps including DNA concentration, DNA amplification, and template preparation into the system. The integrated system will be evaluated and compared to other commercially available solutions in terms of speed, cost and accuracy of fungal identification. Further optimization of the pyrosequencing biochemistry will be performed to enable longer read lengths on shorter time scales while retaining high accuracy. Finally, a data analysis tool will be developed to automate base calling and database queries. The final product will represent a streamlined approach to fungal species identification, integrating a number of components of the contemporary pathogen identification workflow.

Public Health Relevance:
A compact and inexpensive instrument which can automatically identify fungal isolates by sequencing a small region of their DNA will be developed and tested. This product would greatly improve the ability of clinicians to treat infectious disease.

Thesaurus Terms:
Academic Medical Centers;Base Pairing;Binding;Binding (Molecular Function);Biochemical Reaction;Biochemistry;Biological Chemistry;Chemistry;Clinical;Collaborations;Communicable Diseases;Computer Software;Dna;Dna Sequence;Dna Amplification;Data;Data Analyses;Data Analysis;Data Banks;Data Bases;Databanks;Databases;Deoxyribonucleic Acid;Development;Devices;Diagnostic;Electronic Databank;Electronic Database;Enzymatic Reaction;Equipment;Filamentous Fungi;Genetic;Goals;Immunoassay;Infectious Disease Pathway;Infectious Diseases;Infectious Diseases And Manifestations;Infectious Disorder;Investigators;Investments;Loinc Axis 4 System;Laboratories;Left;Length;Life;Link;Methods;Methylation;Microfluidic;Microfluidics;Molds;Molecular Interaction;Nature;Nucleotides;Patients;Performance;Pharmacogenetics;Phase;Preparation;Protein Methylation;Protocol;Protocols Documentation;Reading;Research;Research Personnel;Researchers;Running;Sampling;Sequence Determination;Software;Solutions;Specialist;Speed;Speed (Motion);System;Technology;Testing;Time;University Medical Centers;Yeasts;Base;Cancer Type;Clinical Applicability;Clinical Application;Clinical Data Repository;Computer Program/Software;Cost;Data Repository;Design;Designing;Developmental;Digital;Flexibility;Flexible;Improved;Instrument;Microbial;Miniaturize;Next Generation;Pathogen;Prototype;Success;Tool;Tool Development