Cepeda C, Santos Y
Department of Microbiology and Parasitology, Faculty of Biology, University of Santiago de Compostela, Spain.
Int Microbiol. 2000 Dec;3(4):235-8.
We describe a rapid, low-toxicity and simple method for the detection of the bacterial fish pathogen Flavobacterium psychrophilum. The method, based on the polymerase chain reaction (PCR), combined the electrophoresis of PCR products in a vertical agarose gel and a modified methylene blue stain. DNA was amplified directly either from bacterial suspensions or from tissues experimentally infected with F. psychrophilum, using different non-toxic commercial DNA extraction kits. The protocol allowed to detect 15 to 150 cells of the pathogen in bacterial suspension, without prior DNA extraction, and 7500 to 75,000 cells in seeded spleen tissue and ovarian fluid using Dynabeads DNA DIRECT extraction system. This method, which has the advantage of not using hazardous products, is proposed as a fast tool for routine identification of F. psychrophilum.
我们描述了一种用于检测细菌性鱼类病原体嗜冷黄杆菌的快速、低毒且简单的方法。该方法基于聚合酶链反应（PCR），将PCR产物在垂直琼脂糖凝胶中进行电泳，并采用改良的亚甲基蓝染色法。使用不同的无毒商业DNA提取试剂盒，直接从细菌悬液或经嗜冷黄杆菌实验感染的组织中扩增DNA。该方案无需预先提取DNA，即可检测细菌悬液中15至150个病原体细胞，使用磁珠DNA直接提取系统可检测接种脾脏组织和卵巢液中7500至75000个细胞。该方法具有不使用危险产品的优点，被提议作为嗜冷黄杆菌常规鉴定的快速工具。
