(62av) A Kinetic Study of in Vitro Lysis of Mycobacterium Smegmatis

The emergence of drug-resistant strains of tuberculosis has made nucleic acid amplification the diagnostic test of choice, replacing the traditional acid-fast stain and culture test. The amplification test consists of 1) sputum sample collection, 2) lysis of bacilli to extract DNA, 3) amplification of DNA by PCR, and 4) detection of PCR products. We describe the kinetics of lysis in the DNA extraction step, using Mycobacterium smegmatis in a Tris-EDTA buffer. M. smegmatis, often used as a surrogate of M. tuberculosis, was transformed with green fluorescent protein (GFP) genes and lysed at different temperatures in a cuvette. The extent of lysis was tracked by measuring the increase in the fluorescence signal as the cells lysed. Two theoretical models were formulated to provide a quantitative description of lysis. First, a Monte Carlo simulation modeled the cell wall as layers of blocks, in which blocks exposed to the lysing solution are removed at random. The model produced a time-varying probability distribution function that was analyzed using the Fokker-Planck equation. Second, in a reaction engineering approach, a differential equation describing the change in the number of exposed blocks on each layer was solved. Both models yielded results consistent with the experiments. The most important findings were that 1) the activation energy of lysis is 22 kcal/mol, 2) cells lyse after 14-17% loss in cell wall thickness, 3) the initial distribution of cell wall thicknesses was determined, and 4) near-complete lysis is achieved in 200 s at 80°C or 90 s at 90°C. The results will aid the design of a lysis protocol for nucleic amplification tests.