Enhancing Snakebite Surveillance Through Molecular Tools: Development of Species-Specific Polymerase Chain Reaction Primers for Snakes in Japan

Polymerase chain reaction (PCR)-based species identification methods could enhance snakebite surveillance, especially in regions where multiple venomous snake species with similar clinical presentations coexist. We developed species-specific PCR primers targeting mitochondrial cytochrome b gene sequences for five snake species commonly found in Japan: Elaphe climacophora, Elaphe quadrivirgata, Gloydius blomhoffii, Rhabdophis tigrinus, and Protobothrops flavoviridis. The PCR method successfully detected snake DNA from tissue samples with high specificity, showing minimal crossreactivity between species. Only P. flavoviridis primers showed crossreaction with E. climacophora DNA at the target size, whereas G. blomhoffii primers produced weak amplification of E. climacophora DNA at a different size than expected. Some nonspecific bands were detected with human DNA, but these were substantially weaker and different in size compared with specific amplification products. The method also successfully detected snake DNA from noninvasive samples, including oral swabs, samples from venom collection containers (where snakes had deposited saliva during venom extraction procedures), and bite sites on dead mice. The minimum detectable DNA concentrations ranged from 0.02 to 4.5 pg/µL across different species. Although our current method requires separate PCR reactions for each species and needs further validation with clinical specimens, it provides a foundation for molecular-based snake species identification. This approach could particularly benefit regions in which accurate species identification is crucial for appropriate antivenom selection, effective clinical management, and well-informed interventional research. Future studies should focus on validating this method with clinical specimens and developing multiplex PCR systems for more practical surveillance applications.