Trost Anja, Graf Barbara, Eucker Jan, Sezer Orhan, Possinger Kurt, Göbel Ulf B, Adam Thomas
Institute for Microbiology and Hygiene, Medical Faculty of Humboldt University, Charité, Dorotheenstr. 96, 10117 Berlin, Germany.
J Microbiol Methods. 2004 Feb;56(2):201-11. doi: 10.1016/j.mimet.2003.10.007.
For molecular diagnosis of fungal disease using DNA amplification procedures in the routine laboratory, choice of appropriate target structures and rapid and inexpensive identification of amplification products are important prerequisites. Most diagnostic procedures described thus far are characterized by limited applicability, considerable cost for laboratory equipment or low power of discrimination between species. This study aimed at identification of a PCR target appropriate for diagnosis of clinically relevant yeasts and an affordable procedure for characterization of the PCR products to the species level. Here, we describe a PCR-based system using amplification of intergenic spacers ITS1 and ITS2 and restriction length polymorphism of PCR products after sequence-specific enzymatic cleavage. We show the evaluation of the system for clinically relevant Candida species. The simple and inexpensive procedure should be instrumental for rapid identification of medically important yeasts.
对于在常规实验室中使用DNA扩增程序进行真菌疾病的分子诊断,选择合适的靶标结构以及快速且低成本地鉴定扩增产物是重要的先决条件。迄今为止所描述的大多数诊断程序的特点是适用性有限、实验室设备成本高昂或物种间鉴别能力低。本研究旨在鉴定适合诊断临床相关酵母的PCR靶标,并找到一种经济实惠的方法将PCR产物鉴定到物种水平。在此,我们描述了一种基于PCR的系统,该系统利用基因间隔区ITS1和ITS2的扩增以及序列特异性酶切后PCR产物的限制性片段长度多态性。我们展示了该系统对临床相关念珠菌属物种的评估。这种简单且经济实惠的程序应有助于快速鉴定医学上重要的酵母。