Conventional methods to identify and test the antibiotic susceptibility of Mycobacterium tuberculosis are both slow and insensitive. Current nucleic acid amplification-based techniques present a partial solution by offering a more rapid result, but these methods require a relatively high degree of technical sophistication. Furthermore, assay sensitivity is often limited because there are no reliable methods either to concentrate the bacilli within a sputum sample into a small volume for polymerase chain reaction (PCR) amplification, or to extract M. tuberculosis DNA from sputum samples that is free of PCR-reaction inhibitors. We have developed an extremely sensitive and specific single-tube PCR assay that identifies M. tuberculosis and detects resistance to rifampin directly from clinical sputum samples. In this phase I STIR, we will develop a low-cost cartridge-based PCR system that will combine dramatically increased sensitivity with completely """"""""hands-free"""""""" operation and analysis.
The specific aims are 1) to develop a low-cost cartridge system that will concentrate all of the M. tuberculosis bacilli within an entire sputum sample into a small volume that can be used in a single PCR reaction, then wash the concentrated cells free of PCR inhibitors and completely lyse the cells; 2) to develop a whole-cell mycobacterial reporter that will serve as a quality control for all assay steps from sputum processing through PCR and analysis; and 3) to test the system using clinical sputum specimens. This project will result in a rapid system that is sensitive and specific enough to be performed on patients with smear-negative tuberculosis, and that is simple and inexpensive enough be performed almost anywhere with minimal training.
