School of Veterinary Medicine, University of California, Davis, California, USA.
J Clin Microbiol. 2011 Feb;49(2):618-23. doi: 10.1128/JCM.00807-10. Epub 2010 Dec 15.
Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens.
目前缺乏快速、灵敏的大象分枝杆菌检测诊断方法。聚合酶链反应（PCR）分析的 DNA 提取方法对许多物种的结核病筛查都很有用，但尚未在象鼻冲洗样本中得到验证。我们评估了三种 DNA 提取方法检测象鼻冲洗样本中结核分枝杆菌复合体的分析灵敏度和特异性。在三种不同处理条件下（热处理、苯酚处理和污染分枝杆菌），评估了 ZR 土壤微生物 DNA 试剂盒（ZR）和传统的盐和乙醇沉淀（TSEP）方法。第三种方法是使用柱过滤法，用于评估受土壤污染的样本。未感染大象的象鼻冲洗样本中混入了不同浓度的牛分枝杆菌细胞，并在 DNA 提取前进行了所述处理条件。提取的 DNA 使用 IS6110 靶向 PCR 分析进行扩增。ZR 和 TSEP 方法在所有三种条件下，每 1.5ml 象鼻冲洗中可检测到低至 1 至 5 个牛分枝杆菌细胞和 10 个牛分枝杆菌细胞。根据土壤量的不同，柱过滤法每 1.5ml 象鼻冲洗中可检测到低至 5 至 50 个牛分枝杆菌细胞。通过使用相同的 PCR 技术从非结核分枝杆菌物种中提取 DNA 并进行扩增，评估了分析特异性。仅扩增出牛分枝杆菌 DNA，表明该 PCR 技术具有 100%的分析特异性。我们的结果表明，这些 DNA 提取技术有望成为检测象鼻冲洗样本中结核分枝杆菌复合体的有用方法。
