Valente Wj, Pienaar E, Fast A, Fluitt A, Whitney Se, Fenton Rj, Barletta Rg, Chacon O, Viljoen Hj
Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, NE 68588-0643.
Chem Eng Sci. 2009 May 1;64(9):1944-1952. doi: 10.1016/j.ces.2008.12.015.
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 by Tris-EDTA buffer is 22.1kcal/mole, (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, (4) near complete lysis of the cells is accomplished in 200 seconds at 80°C (90 seconds 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.
传统的结核病诊断测试包括抗酸染色和对痰液样本进行培养测试。随着耐多药结核菌株的出现，核酸扩增已成为首选的诊断测试方法。核酸扩增测试包括四个步骤：痰液样本采集、裂解杆菌以释放DNA、通过聚合酶链反应（PCR）进行DNA扩增以及检测PCR产物。DNA提取步骤在很大程度上被忽视了，本研究描述了一种系统方法来测量在Tris-EDTA缓冲液中细胞裂解的动力学。耻垢分枝杆菌是一种腐生、快速生长的分枝杆菌，在实验室研究中常被用作结核分枝杆菌的替代物。耻垢分枝杆菌细胞已用绿色荧光蛋白（GFP）基因进行了转化。将转化后的细胞在配备有光学输入/输出的温度控制比色皿中裂解。当GFP从裂解的细胞中释放出来时，荧光信号会增加，并且可以实时跟踪加载细胞的裂解程度。实验结果得到了两个理论模型的补充。第一个模型基于对裂解过程的蒙特卡罗模拟以及由福克-普朗克方程描述的伴随概率密度函数。第二个模型遵循化学反应工程方法：细胞壁被建模为多层，其中每层由“块”组成。只有当“块”暴露于裂解溶液时才能被去除，该模型描述了“块”暴露和去除的速率。两个模型都与实验结果一致。主要发现如下：（1）Tris-EDTA缓冲液裂解耻垢分枝杆菌的活化能为22.1千卡/摩尔，（2）细胞在局部细胞壁厚度损失14 - 17%后平均发生裂解，（3）借助这些模型，可以解析群体细胞壁厚度的初始分布，（4）在80°C下200秒（90°C下90秒）可实现细胞几乎完全裂解。这些结果可用于设计一种最佳裂解方案，该方案在较高温度下较短的处理时间与较低温度下对DNA的热损伤减少之间进行权衡。
