Tuberculosis (TB) caused by Mycobacterium tuberculosis is a global health issue that is responsible for more than 1.6 million deaths annually; majority of which occur in developing nations. In addition to routine TB, often inappropriate use of antibacterial drugs results in multi-drug resistant (MDR) and extensively drug-resistant (XDR) TB strains, which are 500-600 times more expensive to treat. Totally drug-resistant TB is also known. Developing nations often lack the resources to diagnose MDR/XDR TB in time resulting in delayed treatment and further infections. Diagnosis of MDR/XDR TB is challenging because it requires detection of many single nucleotide polymorphisms (SNPs) imparting the resistance. Current strategies for the diagnosis of MDR/XDR TB require expensive instruments costing ~$90,000 for equipment (e.g., GeneXpert®), $20-$50 per test, and well-trained technicians. None of these resources are adequate in developing nations. Our group has developed an inexpensive genetic analysis platform, named Gene-ZTM at a manufacturing cost of $500, which has the potential to carry out real time isothermal amplification assays. It employs fluorescence-based loop-mediated isothermal amplification (LAMP) in microfluidic chips for detection and quantification of nucleic acids markers using an iPod TouchTM as control and visualization software. When fully developed and validated, this platform could become a low-cost and simpler alternative to GeneXpert®. Gene-ZTM also has the ability to be operated using batteries and over time with solar charger, making it the instrument of choice for limited resource settings. Using this platform, the goal of this doctoral work was to develop a simple, sensitive, and inexpensive field deployable microfluidic chip for detecting TB and demonstrate its potential for MDR TB using a model SNP imparting resistance to isoniazid, the resistance that is currently measured using GeneXpert® system. The following four objectives were set. (i) Reduce the detection time and enhance the sensitivity for routine TB. (ii) Stabilize the molecular biology reagents to be stable under field conditions for long periods of time, (iii) Develop a simplified low-cost microfluidic chip design with sample in answer out capabilities, and (iv)Develop an isothermal approach for detection of a model SNP to be used for detection of MDR TB using LAMP. Based on optimization studies carried out using 11 fluorescent DNA-intercalating dyes and Bst polymerase, a protocol was developed to detect 10 copies of M. tuberculosis genomic DNA in less than 15 min. Using trehalose, a protocol was established to stabilize molecular biology reagents for their storage at room temperature for up to one year with no or minimal loss of activity. A polyester-based chip that did not contain any valves and allowed multiplex detection without cross contamination between reaction chambers was also developed. An initial proof-of-concept was demonstrated using an SNP associated with isoniazid resistance. Work with patient sputum was outside the scope of this research because of the requirements associated with a BSL3 facility. Overall, this work provides critical data necessary to detect TB, MDR/XDR-TB using a simple platform suitable for limited resource settings.
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