With nearly one-third of the world's population (2 billion people) infected, tuberculosis (TB) is a major problem for global public health. The WHO estimates that each year 12 million people progress to active TB and 2 million of these people will die (WHO50/50, 2002). The current diagnosis for TB is cumbersome and involves multiple patient visits and tests which include culturing of sputum for 4-8 weeks, sputum microscopy, nucleic acid (NA) based tests and chest X-ray. There is an urgent need for a rapid, easy diagnostic test that could be performed at the public health clinic and give a diagnostic answer while the patient waits for the result. The long-term objective of this project is to develop a """"""""smartDNA""""""""-based simple, rapid and inexpensive NA diagnostic test for TB that can be used at the point-of-care (POC). Investigen's """"""""smartDNA technology"""""""" is an elegant and innovative approach to detect TB NA. smartDNA is isothermal and colorimetric. Currently Investigen's research protocol for smartDNA TB detection is: (1) conventional sputum treatment, (2) sample disruption, (3) sample processing by peptide nucleic acid (PNA)-microparticle capture of Mycobacterium tuberculosis complex (MTC) NA, and (4) rapid catalytic 'light-activated' color change of a dye when a second PNA probe binds to complementary NA sequences. The successful development of the smartDNA POC TB detection assay requires six steps: (1) develop detection chemistry and reagents, using off the shelf consumables; (2) improve an in-lab activator/reader instrument; (3) develop simple sputum processing chemistry and reagents, using off the shelf consumables. Steps (4) to (6) translate the first three steps into an integrated LabTop smartDNA detection system with specially designed POC consumables. In this Phase II proposal, we address only the first three steps. The development of an easy to use integrated system with sample preparation and NA detection is our long term goal.
The specific aims of this work are to (1) design MTC-specific PNA probes signature sequences; (2) optimize PNA-microparticle conjunction method; (3) identify capture PNAs that specifically capture signature NA sequences in isolated genomic DNA; (4) identify capture PNAs that can capture signature NA sequences from crude cell lysates; (5) identify PNA probes that efficiently promote the """"""""light-activated"""""""" dye conversion; (6) evaluate the developed system with cells spiked into sputum sediments; (7) evaluate methods for simplification of sputum processing; (8) improve the in-lab smartDNA activator/reader instrument; and (9) test the smartDNA system with patient samples. The rapid and accurate diagnosis of TB is critical to the treatment of the disease and the reduction of the large-scale global public health threat it presents. The smartDNA assay developed during this Phase II project will greatly improve the state of TB diagnosis and treatment, and accordingly, is relevant to the NIH mission of reducing the burden of illness through supporting research for the improved diagnosis, prevention and cure of human diseases. While the United States has been largely successful in detecting, treating and controlling TB, in many countries TB is a serious and growing public health issue; international travel and immigration have increased transmission of the disease. The research to be completed in this project will lead to the development of a point of care TB test that will provide a result at the time of testing; a system that can be used in resource limited settings throughout the world, in clinics and emergency rooms in the United States and potentially even at US borders - greatly aiding the public heath services in the detection, treatment and control of TB. ? ? ?
| Wang, M; Holmes-Davis, R; Rafinski, Z et al. (2009) Accelerated photobleaching of a cyanine dye in the presence of a ternary target DNA, PNA probe, dye catalytic complex: a molecular diagnostic. Anal Chem 81:2043-52 |
