Cepheid Inc. and the New Jersey Medical School have developed an assay system that integrates sample processing and real-time PCR diagnosis into a single, hands free process. During the successful completion of a phase I STTR application, we demonstrated that this system simultaneously detects the presence of Mycobacterium tuberculosis (TB) and diagnose rifampin resistance directly from clinical sputum samples. The assay uses an inexpensive plastic cartridge to collect sputum samples, concentrate the bacteria present in the sputum, perform high efficiency cell lysis and deliver inhibitor-free DNA into a PCR tube without user input. Detection of TB and rifampin resistance is also performed within the cartridge using a real-time PCR assay and a series of molecular beacon probes. The goal of this phase II STTR application is to further refine the sensitivity and robustness of the assay and to develop the commercial reagents, materials, software and protocols so that our system can be tested in larger-scale trials leading to its commercialization. This STTR will result in a desktop system that is sufficiently simple and robust to be performed anywhere with minimal training; and sufficiently sensitive and specific to be performed on patients with smear negative tuberculosis. Specifically we aim to: 1) Modify the assay to function in a new 6-color instrument, and further refine the assay using innovative strategies to increase its sensitivity, specificity and robustness. 2) Convert assay components and protocols into the appropriate format for clinical trials and subsequent commercialization by lyophilizing all reagents for factory packaging, and developing simple methods to load samples and buffers into the cartridge. 3) Perform pre-clinical testing to verify assay sensitivity, specificity and limits of detection and assess the effects of potential interfering substances. The system will then be retested using a large number of clinical sputum specimens. Finally, we will develop complete documentation on the assay including production, and development reports. ? ? ?
