Polymerase chain reaction-based snake origin tracing in commercial venom crystals by targeting the mitochondrial D-loop.

Snake venom is a valuable raw material for numerous therapeutic formulations because of its life-saving pharmacological potential. However, due to their high price, fake "snake venoms" have captured a significant portion of the global market, and there is currently no reliable reported DNA-based method available for quickly distinguishing between fakes and originals. Therefore, in this study, a set of newly designed snake-specific universal primers targeting mitochondrial D-loop fragments were employed to detect snake origins in commercial venom crystals by only simplex polymerase chain reaction analysis. Under the optimal thermal cycling conditions, only the 145-149 bp snake-specific mitochondrial D-loop fragments from pure and mixed backgrounds were amplified by the newly designed primers. Specificity was achieved by confirming no DNA amplification occurred in the DNA admixture of ten different chordates, and universality by individual DNA amplification of nine different snakes. The primers that efficiently amplified the minimum mitochondrial DNA contained in a total of 10 ng in a 10.0 μl reaction were also successfully able to detect the snake origin in commercial cobra venom crystals. These findings suggest that the newly designed primers can be used to differentiate the original and fake commercial snake venom crystals in order to achieve the highest standards of snake venom-based medications through amplifying the snake-specific mitochondrial D-loop fragments.