ABSTRACT
We have developed a sensitive and specific polymerase chain reaction (PCR) method for identifying phytoplankton in cases of death by drowning, and we have designed four primer pairs, EG1, EG2, SK1 and SK2, for chlorophyll-related genes of Euglena gracilis and Skeletonema costatum, which are commonly distributed in all types of water. In order to evaluate the usefulness of this method for diagnosis of drowning, we have used this method for detection of plankton genes in non-drowned rabbits submerged after death and in decomposed drowned rabbits. Plankton DNA was identified in lung samples obtained from the non-drowned rabbits because of postmortem plankton penetration into the respiratory system, and plankton DNA was identified in liver and kidney samples obtained from the decomposed drown rabbits. The results show that our new PCR method is a useful method for diagnosing drowning.
Subject(s)
DNA/isolation & purification , Diatoms/genetics , Drowning/diagnosis , Euglena gracilis/genetics , Phytoplankton/genetics , Polymerase Chain Reaction , Adult , Animals , Female , Humans , Kidney/microbiology , Kidney/pathology , Liver/microbiology , Liver/pathology , Lung/microbiology , Lung/pathology , Male , Middle Aged , Rabbits , Sensitivity and Specificity , Water MicrobiologyABSTRACT
We present a new PCR method for identifying plankton in cases of death by drowning. We designed four primer pairs for chlorophyll-related genes of Euglena gracilis (EG) and Skeletonema costatum (SK), which are commonly distributed in water. The primers were selected from sequences coding chloroplast/chlorophyll apoprotein of EG (EG1 and EG2) and fucoxanthin-chlorophyll a/c harvesting protein of SK (SK1 and SK2). With EG1 or EG2, up to 2 fg of EG-DNA was identified, and 0.2 pg of SK-DNA was detectable with SK1 or SK2. No PCR products were amplified from green vegetables (komatsuna, spinach, parsley) or human DNA with the four primer pairs. Regardless of the origin, seawater or fresh water, most diatoms were detectable with primer pairs of EG1 and EG2. With SK1, only Centrales diatoms were identified, and five diatom strains originating from seawater were detectable with SK2. EG1 and EG2 gave rise to PCR products from most water samples. By using Percoll, plankton was easily isolated from human tissue or blood samples and good results of PCR analysis were obtained in cases of death by drowning.