Drowning Diagnosis by Detecting Diatoms rbcL Genes with PCR-Capillary Electrophoresis

摘要

We established PCR capillary electrophoresis (PCR-CE) method to amplify genes encoding large subunit of ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL) from diatoms for drowning diagnosis. We tested a set of oligonucleotide primers for the specific amplification of rbcL gene segments by PCR. With the primer ND-rbcL, 6 diatoms species (Navicula sp., Nitzschia sp., Cyclotella sp., Melosira varians, Synedra, and Skeletonema) showed positive results with PCR products of 197 bp, and cyanobacteria, green algae, bacteria and human DNA showed negative results. The sensitivity of the diatoms DNA in the above 6 species were 0.502 ng, 0.117 ng, 0.029 ng, 0.042 ng, 0.275 ng and 0.215 ng, respectively (20 mu L PCR system). In this study, we used PCR-CE method to detect 45 corpses (3 corpses from death on land, 2 waterborne corpses from non-drowning cases, and 40 corpses from drowning cases). The detection rate of diatoms in the lungs, livers and kidneys of the corpses was 97.5%, 70.0% and 55.0%, respectively. By counting the positive results in livers or kidneys, we got the total positive rate of 82.5%. When the diatoms (more than 10/10 g tissues) were found in lungs, livers and kidneys, there was no statistical difference (P 0.05) between the 100% total positive rate by microwave digestion-vacuum filtration-automated scanning electron microscopy (MD-VF-Auto SEM) method and 94.2% by PCR-CE. The present study indicated that the PCR-CE method based on the ND-rbcL primer showed a good application prospect in diatom detection, and makes drowning diagnosis easier to popularization.