Rapid diagnosis of ill patients is critical to treat infected patients and to control the spread of disease. While current tests can be rapid, they test for only a single or small panel of the 1500 known human pathogens. The goal of our proposal is to create a panel that will rapidly and specifically detect any of the known human pathogens and the presence of drug resistance genes. Analyzing the data from this assay will enable a confident diagnosis and guide proper treatment decisions.
Current methods of diagnosing patients only test for a single or small set of pathogens out of the 1500 known human pathogens. This requires a clinician to make a decision on which tests to run. If none of the tests return a positive test, a second set of tests are run. This becomes an iterative process that can take a long time because it takes many hours to perform some of these tests. In addition to time waiting for a positive result, this process is be extremely expensive if many tests are ordered. The current method of unbiased pathogen detection relies on sequencing all of the RNA in a patient sample, bioinformatically removing all of the human sequences, and then BLASTing against the nt database to identify pathogens. The fastest this can be currently done is 24 hours and requires a lot of hands-on library preparation, actual sequencing time, and analysis time. It will be impossible to improve the current method to diagnose patients within an hour. To meet this one-hour timepoint, we propose to develop a rapid test that can identify the presence of any of the 1500 known human pathogens by using a highly multiplexed barcoded probe ligation approach. To develop this test in the R21 phase through the following aims: 1. Validate and benchmark our ligation- dependent isothermal detection strategy using a pilot panel of pathogen targets, 2. Design and experimentally validate a pilot-scale pathogen and drug resistance panel, and 3. Design and experimentally validate a multiplex barcode LAMP readout strategy. Once these aims have been achieved, we will expand the pilot panel and develop a field-deployable system in the R33 phase through the following aims: 1. Scale our platform from pilot scale to full representation of ~1500 pathogens and antibiotic resistance genes, and 2. Develop a low cost, field-compatible turn-key version of this platform, including a custom readout device and kitted reagents. Relevance: This technology is has the potential to revolutionize pathogen detection and diagnosis. The anticipated costs for each test would be under $10 through the use of traditional molecular biology reagents and no requirement for expensive lab equipment. This method can be adapted to simplify and decrease the cost of any nucleic-acid based multiplex assay.