Simple detection of bacterioplankton using a loop-mediated isothermal amplification (LAMP) assay: First practical approach to 72 cases of suspected drowning

Abstract

We developed a novel molecular tool for assisting the diagnosis of death by drowning and evaluated its validity in forensic practical cases. Two novel sets of loop-mediated isothermal amplification (LAMP) primers were designed to detect either representative freshwater (Aeromonas) or marine (Vibrio, Photobacterium, Listonella) bacterioplankton (aquatic bacteria) in one tube using the LAMP technique. The assay involves only mixing template DNA with seven reagents and incubating at 64°C for 80min and does not require special or expensive equipment because detection is based on visual observation under natural light. The assay’s excellent specificity was also demonstrated using 17 standard (control) strains and 124 other bacterial strains cultured from drowning and non-drowning victims in our previous studies. We then assayed 299 specimens (135 lung, 164 blood) from 72 victims, including 45 who had drowned in rivers, ditches, seas, and around estuaries. LAMP assay results could provide effective information to assist the diagnosis of death by drowning in practical cases. The LAMP assay would be useful for suspected drowning cases, as it is a less-laborious and less-expensive minimal test when death by drowning is sufficiently confirmed or negated from only autopsy findings and environmental data or when diatom testing is not performed due to logistic, personnel, or budgetary limitations. Moreover, the assay could serve as a simple additional test when the density of diatoms in the lungs is very low due to low density in the water.