Kane M, Yamamoto Y, Ushiyama I, Nishimura A, Nishi K
Department of Legal Medicine, Shiga University of Medical Science, Otsu, Japan.
Electrophoresis. 2000 Jan;21(2):351-4. doi: 10.1002/(SICI)1522-2683(20000101)21:2<351::AID-ELPS351>3.0.CO;2-T.
Three strains of picoplankton designated as brown, green, and pink belonging to the Synechococcus genus in cyanobacteria (approximately 1 microm in size) are found ubiquitously in Lake Biwa, Japan. However, they could not be morphologically discriminated from other bacteria such as Proteobacteria and Bacillus by microscopy. In this study, we attempted to use the polymerase chain reaction (PCR) analysis of the 16S ribosomal DNA (rDNA) from picoplankton for the diagnosis of death by drowning. A segment of 16S rDNA was sequenced in order to investigate their phylogenetic relationships and to design the specific primers. The PCR products from three picoplanktons were compared with those from five other cyanobacteria, Melosira (diatom), Staurastrum (green alga), bacteria from Lake Baikal, and humans. The picogram order of template DNA from picoplankton was specifically amplified by the primers. When the template of picoplankton was mixed with human tissue, at least 10 ng of template DNA was needed to obtain a PCR product. The efficiency of PCR was increased more than hundredfold by isolating the picoplankton from human lung tissue. The specific PCR products of the picoplankton were obtained from a formalin-fixed drowning body (lung and liver) that was found in a downstream river and Lake Biwa. The PCR analysis of the picoplanktion 16S rDNA is considered useful for the diagnosis of death by drowning.
在日本琵琶湖普遍发现了三种属于蓝藻门聚球藻属（大小约为1微米）的微微型浮游生物菌株，分别命名为棕色、绿色和粉色。然而，通过显微镜观察，它们在形态上无法与其他细菌（如变形菌和芽孢杆菌）区分开来。在本研究中，我们尝试利用对微微型浮游生物的16S核糖体DNA（rDNA）进行聚合酶链反应（PCR）分析来诊断溺水死亡。对一段16S rDNA进行测序，以研究它们的系统发育关系并设计特异性引物。将三种微微型浮游生物的PCR产物与其他五种蓝藻、直链藻（硅藻）、十字藻（绿藻）、贝加尔湖细菌以及人类的PCR产物进行比较。特异性引物能特异性扩增微微型浮游生物皮克级的模板DNA。当微微型浮游生物的模板与人体组织混合时，至少需要10纳克模板DNA才能获得PCR产物。通过从人肺组织中分离微微型浮游生物，PCR效率提高了一百多倍。在一条下游河流和琵琶湖中发现的一具经福尔马林固定的溺水尸体（肺和肝脏）中获得了微微型浮游生物的特异性PCR产物。微微型浮游生物16S rDNA的PCR分析被认为对溺水死亡的诊断有用。
