Troedsson Christofer, Lee Richard F, Stokes Vivica, Walters Tina L, Simonelli Paolo, Frischer Marc E
Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, GA 31411, USA.
Appl Environ Microbiol. 2008 Jul;74(14):4336-45. doi: 10.1128/AEM.02131-07. Epub 2008 May 23.
Increasingly, diseases of marine organisms are recognized as significant biotic factors affecting ecosystem health. However, the responsible disease agents are often unknown and the discovery and description of novel parasites most often rely on morphological descriptions made by highly trained specialists. Here, we describe a new approach for parasite discovery, utilizing denaturing high-performance liquid chromatography (DHPLC) reverse-phase ion-pairing technology. Systematic investigations of major DHPLC variables, including temperature, gradient conditions, and target amplicon characteristics were conducted to develop a mechanistic understanding of DNA fragment separation by DHPLC. As a model system, 18S rRNA genes from the blue crab (Callinectes sapidus) and a parasitic dinoflagellate Hematodinium sp. were used. Binding of 18S rRNA gene PCR amplicons to the DNA separation column in the presence of triethylammonium acetate (TEAA) was inversely correlated with temperature and could be predicted based on the estimated DNA helicity of the PCR amplicon. Amplicons of up to 498 bp were resolved as single chromatographic peaks if they had high (>95%) DNA helicity. Amplicons that differed by as few as 2 bp could be resolved. Separation of 18S rRNA gene PCR amplicons was optimized by simultaneous manipulation of both temperature and solvent gradients. The optimal conditions included targeting regions of high DNA helicity (>95%), temperatures in the range of 57 to 63 degrees C, and a linear acetonitrile gradient from 13.75 to 17.5% acetonitrile in 0.1 M TEAA (55 to 70% buffer B) over a 9-min period. Under these conditions, amplicons from a variety of parasites and their hosts can be separated and detected by DHPLC.
海洋生物疾病日益被视为影响生态系统健康的重要生物因素。然而，致病病原体往往不明，新型寄生虫的发现和描述大多依赖训练有素的专家所作的形态学描述。在此，我们描述一种利用变性高效液相色谱（DHPLC）反相离子对技术发现寄生虫的新方法。对包括温度、梯度条件和目标扩增子特征在内的主要DHPLC变量进行了系统研究，以形成对DHPLC分离DNA片段的机理认识。作为模型系统，使用了来自蓝蟹（Callinectes sapidus）和寄生性甲藻Hematodinium sp.的18S rRNA基因。在乙酸三乙铵（TEAA）存在下，18S rRNA基因PCR扩增子与DNA分离柱的结合与温度呈负相关，并且可以根据PCR扩增子估计的DNA螺旋度进行预测。如果具有高（>95%）DNA螺旋度，长达498 bp的扩增子可解析为单一色谱峰。相差仅2 bp的扩增子也能被解析。通过同时调节温度和溶剂梯度优化了18S rRNA基因PCR扩增子的分离。最佳条件包括靶向高DNA螺旋度（>95%）区域、温度在57至63摄氏度范围内以及在9分钟内0.1 M TEAA（55至70%缓冲液B）中乙腈从13.75%线性梯度至17.5%的乙腈梯度。在这些条件下，来自多种寄生虫及其宿主的扩增子可通过DHPLC分离和检测。
