Nanoreactor beads for POC TB resistance testing - ABSTRACT Rapid and sensitive point of care tests that can detect all forms of drug resistance in tuberculosis (TB) are urgently needed to address the rise of drug resistance. We propose to develop a novel mutation detection system that uses recently developed nanoreactor bead chemistry and the Blink Diagnostic’s testing platform to identify both the presence of Mycobacterium tuberculosis (Mtb) and all of the clinically important mutations associated with isoniazid (INH), rifampin (RIF), ethambutol (EMB), pyrazinamide (PZA), fluoroquinolone (FQ), linezolid (L), bedaquiline (B), and pretomanid/delamanid (Pa/De) resistance. The new system will include integrated sample processing and rapid thermal cycling technologies to produce results within 30 minutes at the point of care. Mutations will be detected using digital and real-time PCR in novel addressable nanoreactor beads followed by melting temperature analysis enabling robust detection of hundreds of different resistance mutations with the sensitivity of current molecular diagnostics and the quantitation and hetero-resistance detection capacity of digital PCR. This proposal will build upon an established partnership between Blink scientists and engineers who previously developed highly successfully instruments and assays at Alere (currently Abbott Rapid Diagnostics), and the academic team responsible for the suite of Xpert TB assays previously developed in collaboration with Cepheid to perform the following specific aims: 1. Develop mis-match tolerant or “sloppy” molecular beacons (SMBs) that identify mutations associated with INH, RIF, EMB and FQ resistance that are optimized to nanobead format and fully functional on the Blink platform. 2. Finalize a SMB assay that queries the entire Mtb pncA gene to identify mutations causal of PZA resistance specifically adapted for nanobead format in the BLINK system. 3. Finalize development of cartridge based mechanical nucleic acid extraction process from sputum and other matrixes for Mtb. 4. Expand the nanobead assay to test for all clinically relevant mutations causing with resistance to Pa, B and L using the principals and chemistries developed in aims 1 – 2. 5. Perform initial laboratory and clinical validation studies of the final aim 1-4 assays using stored clinical samples.