Respiratory tract infections (RTI) are the 4th-leading cause of the morbidity and mortality in the United States and seventh worldwide. There are between 10 and 20 well-known bacterial and viral pathogens (not including numerous subtypes and genotypes) which are considered as the main causes of RTI. Current in-vitro diagnostic (IVD) tests for RTI pathogens are based on PCR (RT-PCR) amplification of photogenic nucleic acids (NA). Detection of the amplified target genes can occur in real-time regime (real-time PCR) that provides fast turnaround time but can usually identify not more than five pathogens or pathogen genotypes per assay. On the other hand, employing post-amplification techniques such as hybridization with solid-phase spatially separated oligonucleotide probes (microarrays) can significantly enlarge the number of pathogens to be detected, but requires open-tube manipulation with PCR amplification products, such as the transfer of the PCR products to microarray chamber and microarray washings. These open-tube procedures greatly complicate assays protocol, extend analysis time, and increase risks of sample cross-contamination. Here, the PI proposes to develop and demonstrate a proof-of-concept assay for fast (<30 min) identification and discrimination of 10 common RTI pathogens using an innovative multiplexed RT-PCR platform, named the Donut PCR. The platform includes a disposable microfluidic reaction chamber where amplification occurs due to convection-based thermocycling. The chamber equipped with an embedded label-free microarray allowing real-time detection of amplification products without opening the reaction chamber. To run the assay, the team has developed a complementary PCR instrument which is characterized by the easiness of operation, small footprint and low energy consumption (functional prototype complete). The team anticipates that the demonstrated assay will constitute a solid basis for further development of cost-effective, rapid IVD systems capable of fast identification of a large variety of genetic targets.
Public Health Relevance Statement: Project Narrative Rapid detection and classification of infectious disease pathogens can inform timely intervention that improves patient outcomes and shortens patient recovery times. We have developed the Donut PCR, an affordable, highly- multiplexed, and portable platform for simultaneous real-time analysis of 30 or more different DNA targets. In this Phase I SBIR application, we will design and validate a Donut PCR assay for the DNA of 10 common respiratory disease pathogens.
Project Terms: Adenoviruses; Aliquot; amplification detection; analog; antimicrobial drug; Antiviral Agents; Attention; Award; Bacteria; base; Biological Assay; Cations; Characteristics; Chlamydophila pneumoniae; Classification; Communicable Diseases; Complementary DNA; Consumption; Convection; cost effective; Data; design; Detection; Development; Diagnostic; Diagnostic Procedure; Diagnostic tests; Digit structure; Disadvantaged; Discrimination; DNA; DNA Viruses; Engineering; Ensure; Enzymes; Gene Targeting; Genetic; Genomic DNA; Genotype; Gold; Growth; Home environment; Human; human DNA; Immunologics; improved; In Vitro; innovation; insight; instrument; Intervention; Label; light weight; Lung diseases; Methods; Microfluidics; molecular diagnostics; Morbidity - disease rate; mortality; Mycoplasma pneumoniae; Names; Nose; novel; Nucleic Acid Amplification Tests; Nucleic Acids; Oligonucleotide Probes; One-Step dentin bonding system; operation; pathogen; Patient-Focused Outcomes; Patients; Pharmacy facility; Phase; phase 2 study; Physicians; point of care; portability; Predisposition; Primer Extension; Procedures; Protocols documentation; prototype; rapid detection; Reaction; Recovery; Respiratory syncytial virus; Respiratory Tract Diseases; Respiratory Tract Infections; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Rhinovirus; Risk; RNA; RNA Viruses; Running; Sampling; Small Business Innovation Research Grant; Solid; Specimen; Sputum; Swab; System; Techniques; Testing; Time; Tube; United States; uptake; Viral; viral DNA; viral RNA; Work
