Classification of venomous snakes in Thailand based on the melting temperature specificity of target genes using SYBR Green real-time PCR

The SYBR Green real-time PCR was developed for the rapid and accurate classification of major venomous snakes in Thailand. The method relies on melting temperature (Tm) discrimination of target genes from mitochondrial ribosomes. Short single-stranded primers were designed to specifically amplify synthetic DNA of venomous snakes in a single reaction (singleplex), producing distinct melting peaks. Neurotoxic snakes such as cobras (Naja spp.), king cobra (Ophiophagus hannah) and kraits (Bungarus spp.) exhibited melting peaks ranging from 81.00 to 86.00°C, whereas hematotoxic snakes, including the Siamese Russell's viper (Daboia siamensis), Malayan pit viper (Calloselasma rhodostoma), and green pit vipers (Trimeresurus spp.), generated peaks between 77.00 and 80.00°C. The optimal melting rate for singleplex was 0.5°C/sec, with a sensitivity of approximately 10 copies/µL, which is 10-100 times higher than that of agarose gel-based PCR. Primer specificity was confirmed using DNA from snakebite sites on parafilm sheets, demonstrating reliable species-level identification. Multiplex analysis of several snake groups yielded less consistent results due to high genetic similarity among species; only Naja spp. could be reliably differentiated from Bungarus spp. by applying a slower melting rate of 0.1°C/sec, which improved the resolution of the melting temperature. The coefficient of variation (CV) was less than 5% for both intra- and inter-specific assays, ensuring reproducible outcomes. Overall, SYBR Green real-time PCR provides a sensitive, rapid, and practical tool for distinguishing venomous from non-venomous snakes, supporting accurate identification of snake species from bite wound samples and facilitating appropriate clinical management of snakebite victims.