Direct and rapid PCR amplification using digested tissues for the diagnosis of drowning

Abstract

We developed a direct and rapid method for the diagnosis of death by drowning by PCR amplification of phytoplankton DNA using human tissues. The primers were designed based on the DNA sequence of the 16S ribosomal RNA gene (16S rDNA) of Cyanobacterium. Samples of lung, liver and kidney tissues were collected from 53 autopsied individuals diagnosed as death by drowning. Without DNA extraction, the tissue fragments were incubated directly in a digest buffer developed in this study, for 20 min. Using 1 microL of the tissue digest solution in PCR, the 16S rDNA was successfully amplified. The specific 16S rDNA fragment was identified from the standard picoplankton Euglena gracilis, the tissues of bodies died from drowning and water samples from the drowning scenes. On the other hand, no PCR products were found in the tissues of individuals who died from causes other than drowning. Various quantities of tissue weighing 1, 5, 10, 20 and 30 mg were tested, and the PCR amplification detected the specific 16S rDNA fragment from all the quantities of tissue tested. This method was found to be more reliable, sensitive, specific and rapid when compared to the conventional diagnosis of death by drowning using the diatom test by acid digestion method.