Tuberculosis (TB) caused by Mycobacterium tuberculosis is the seventh leading cause of death worldwide, second only to human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) among infectious diseases. In 2004, an estimated 8.9 million people developed TB and 1.7 million died. This situation is compounded by the HIV pandemic with almost 13 million people currently co-infected with HIV and TB, and the emergence of multidrug resistant TB (MDR-TB). In the Unites States, there are about 15,000 new cases of TB reported each year, and approximately 10% of them have resistance to drugs. Rapid diagnosis of TB infections and identification of drug resistance is key to disease control and treatment. In order to accomplish the Healthy People 2010 goal of reducing the time required for the laboratory confirmation of the diagnosis of tuberculosis to 48 hours, rapid tests to detect Mycobacterium tuberculosis or its products are needed. In addition, rapid tests that can reduce the turnaround-time for detection of drug- resistance are needed as well. It is clear that no test is yet available that meets target specifications, and new methods that can overcome limitations and respond to the challenges posed will be well received. Our proposed work will employ state-of-the-art technology to provide differential diagnostics to identify Mycobacterium tuberculosis infection and determine if it has mutations that cause resistances to the most commonly used antibiotics for TB. The goal is to achieve sample-to-result testing in less than 90 minutes. After sample preparation using automated nucleic acid (NA) extraction instrumentation, the NA template will be drawn into an in-line ultra-fast PCR reaction where a highly multiplexed amplification assay (covering 25 mutation/targets) will take place. This powerful multiplex amplification technology from our collaborator overcomes the obstacles of traditional multiplex PCR assay development. It allows for specific and sensitive amplification of multiple targets in a single reaction. The PCR products, mixed with hybridization buffer, will then be transported downstream in a closed format to perform an extremely simple microfluidic based multiplexed detection. The PCR product will be passed over a linear glass capillary substrate with spatially addressable capture zones that are modified with different oligonucleotide capture probes. The smaller diameter linear glass substrate will be housed in a slightly larger diameter polymeric tubing to provide an "array-in-a tube" conditions with microfluidic characteristics for rapid and sensitive fluorescence detection utilizing an extremely simple and inexpensive setup. In Phase I, a breadboard system will be developed and the technology feasibilities will be demonstrated with the nucleic acid templates provided by collaborators. Phase I results will enable the subsequent design and development of an automated clinical diagnostic assay that will fulfill a crucial need in TB care, as well as the detection of other diseases. [unreadable] [unreadable] Tuberculosis (TB), which claims nearly 2 million lives each year, is the seventh leading cause of death worldwide, second only to HIV/AIDS among infectious diseases. Making the condition worse, the emergence of multidrug resistant TB (MDR-TB) makes the treatment more difficult and costly. In the Unites States, there are about 15,000 new cases of TB reported each year, and approximately 10% of them have resistance to drugs. A quick and accurate diagnostics tool for TB and MDR-TB is key to disease control and treatment and could save up to 625,000 of those lives each year (Nature S1, 46-57; 2006). [unreadable] [unreadable]