Date of this Version
The traditional diagnostic tests for tuberculosis consist of an acid fast stain and a culture test from a sputum sample. With the emergence of drug resistant strains of tuberculosis, nucleic acid amplification has become the diagnostic test of choice. The nucleic acid amplification test consists of four steps: sputum sample collection, lysis of bacilli to release DNA, DNA amplification by PCR and detection of PCR products. The DNA extraction step has been largely overlooked and this study describes a systematic approach to measure the kinetics of cell lysis in a Tris–EDTA buffer. Mycobacterium smegmatis is a saphorytic, fast-growing mycobacterium that is often used as a surrogate of Mycobacterium tuberculosis in laboratory studies. M. smegmatis cells have been transformed with green fluorescent protein (GFP) genes. Transformed cells are lysed in a temperature-controlled cuvette that is equipped with optical input/output. The fluorescence signal increases when the GFP is released from lysed cells, and the extent of lysis of the loaded cells can be followed in real time. The experimental results are complemented by two theoretical models. The first model is based on a Monte Carlo simulation of the lysis process and the accompanying probability density function, as described by the Fokker–Planck equation. The second model follows a chemical reaction engineering approach: the cell wall is modeled as layers, where each layer is made up of “blocks.” Blocks can only be removed if they are exposed to the lysis solution and the model describes the rate of block exposure and removal. Both models are consistent with the experimental results. The main findings are: (1) the activation energy for M. smegmatis lysis in Tris–EDTA buffer is 22.1 kcal/mol, (2) cells lyse on the average after 14–17% loss in cell wall thickness locally, (3) with the help of the models, the initial distribution in cell wall thickness of the population can be resolved and (4) near complete lysis of the cells is accomplished in 200 s at 80 °C (90 s at 90 °C). The results can be used to design an optimal lysis protocol that compromises between shorter processing times at higher temperature and reduced thermal damage to DNA at lower temperature.
Supplementary data tables A-F are attached below as "Related files."
Supplementary data table A
8212008 50 x TE 70 C 1.xls (164 kB)
Supplementary data table B
8212008 50 x TE 75 C 1.xls (270 kB)
Supplementary data table C
8212008 50 x TE 80 C 1.xls (194 kB)
Supplementary data table D
8212008 50 x TE 85 C 1.xls (173 kB)
Supplementary data table E
8212008 50 x TE 90 C 1.xls (129 kB)
Supplementary data table F