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The plant species Gloriosa superba and Colchicum autumnale produce extremely poisonous colchicine as a major toxic metabolite. Almost all previous studies on colchicine poisoning have focused on drug analysis and clinical and pathological aspects. In this study, we developed a rapid, highly sensitive method to identify G. superba and C. autumnale. This method, which can distinguish between G. superba and C. autumnale using even minute amounts of plant material, is based on duplex real-time PCR in combination with melting curve analysis. To discriminate between the two genera of colchicine-containing plants, we designed new primer pairs targeting the region of the ycf15 gene, which is present in C. autumnale but not G. superba. By producing PCR amplicons with easily distinguishable melting temperatures, we were able to rapidly and accurately distinguish G. superba from C. autumnale. The new primer pairs generated no PCR amplicons from commercially available human DNA or various plant DNAs except for G. superba and C. autumnale. Sensitivity testing indicated that this assay can accurately detect less than 0.031 ng of DNA. Using our method in conjunction with colchicine drug analysis, we successfully identified G. superba in the stomach contents of a suicide victim who ingested massive quantities of a colchicine-containing plant. According to these results, duplex real-time PCR analysis is very appropriate for testing forensic samples, such as stomach contents harboring a variety of vegetables, and enables discrimination between G. superba and C. autumnale in forensic and emergency medical fields.

Environmental factors such as outside temperature at the time of death are very important for forensic diagnoses and police investigations. In particular, death in a cold environment is associated with factors of forensic interest, including hypothermia, drowning in cold water, or postmortem body movement by a suspect. Hypothermia raises a special problem because of the difficulty of evaluation during autopsy. We describe here a unique method of estimating antemortem environmental temperature, involving the immunohistochemical analysis of HSP70 expression patterns in glomerular podocytes. Using this method, we found that HSP70 was present in glomerular podocytes at autopsy and that HSP70 was highly expressed, mainly in the nucleus of podocytes, in deaths associated with exposure to cold. Interestingly, this expression pattern was specific to death in a cold environment, including hypothermia and drowning in cold water. Analysis of the pattern of HSP70 expression in glomeruli may therefore be very useful in forensic diagnosis, for determining whether the antemortem environmental temperature was low. Moreover, immunohistochemical and real-time PCR assays of the molecular mechanism of HSP70 and HSF1 expression in glomeruli following exposure to cold indicated that HSP70 was rapidly translocated to the nucleus of podocytes following exposure to cold, but without new protein synthesis.