Kane M, Fukunaga T, Maeda H, Nishi K
Department of Legal Medicine, Shiga University of Medical Science, Otsu, Japan.
Int J Legal Med. 1996;108(6):323-6. doi: 10.1007/BF02432130.
Picoplankton belonging to the Synechococcus genus in cyanobacteria (approximately 1 micron in size) are found ubiquitously in Lake Biwa, Japan. However, they could not be morphologically discriminated from other bacteria by microscopy. In this study we attempted to use picoplankton for the diagnosis of drowning by PCR analysis of the 16S ribosomal DNA (rDNA). We designed primers complementary to the variable regions of 16S rDNA of the picoplankton we had sequenced. A comparison was made of the PCR products from the three picoplanktons, five other cyanobacteria, Melosira (diatom), Staurdstrum (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 lung tissue, at least 10 ng of template DNA was needed to obtain a PCR product. The isolation of the picoplankton from human lung tissue increased the sensitivity of PCR more than a hundred-fold. The specific PCR products of the picoplankton were obtained from formalin-fixed drowning tissue. Molecular biological diagnosis of drowning was successful using picoplankton 16S rDNA.
属于蓝藻门聚球藻属的微微型浮游生物（大小约为1微米）在日本琵琶湖随处可见。然而，通过显微镜观察，它们在形态上无法与其他细菌区分开来。在本研究中，我们尝试通过对16S核糖体DNA（rDNA）进行PCR分析，利用微微型浮游生物来诊断溺水。我们设计了与我们已测序的微微型浮游生物16S rDNA可变区互补的引物。对来自三种微微型浮游生物、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产物进行了比较。微微型浮游生物皮克级的模板DNA能被引物特异性扩增。当微微型浮游生物的模板与人类肺组织混合时，至少需要10纳克模板DNA才能获得PCR产物。从人类肺组织中分离出微微型浮游生物使PCR的灵敏度提高了一百多倍。从福尔马林固定的溺水组织中获得了微微型浮游生物的特异性PCR产物。利用微微型浮游生物16S rDNA成功实现了溺水的分子生物学诊断。
