A highly multiplexed convection flow RT-qPCR assay for rapid and

Project Summary Respiratory tract infections RTI are the th leading cause of the morbidity and mortality in the United States and seventh worldwide There are between and 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 andlt min identification and discrimination of 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 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 or more different DNA targets In this Phase I SBIR application we will design and validate a Donut PCR assay for the DNA of common respiratory disease pathogens