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 assay?s 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

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.

Agency
National Institute of Health (NIH)
Institute
National Center for Immunication and Respiratory Diseases (NCIRD)
Type
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
Project #
1R43IP001131-01
Application #
9680597
Study Section
Special Emphasis Panel (ZRG1)
Project Start
2019-09-01
Project End
2020-02-29
Budget Start
2019-09-01
Budget End
2020-02-29
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Nuprobe USA, Inc.
Department
Type
DUNS #
081085522
City
Cambridge
State
MA
Country
United States
Zip Code
02140