Molecular Identification of Siberian tiger Target Gene (Cytb gene) Quantitative PCR: A Study in Wildlife Conservation Genetics

Background: The Siberian Tiger (Panthera tigris altaica) is a critically endangered keystone species. Accurate molecular identification of biological traces is essential for effective conservation, anti-poaching enforcement, and ecological monitoring. Objective: The main objective of the current study was to validate the performance of a cytb-targeted quantitative PCR assay for the molecular identification of Siberian Tiger DNA and to assess the utility of droplet digital PCR as a complementary confirmatory tool. Methods: The current study validated a standardized quantitative PCR (qPCR) assay targeting the mitochondrial cytochrome *b* (cytb) gene for specific detection of Siberian Tiger DNA. Assay performance was evaluated using the YouSeq Siberian Tiger qPCR Test Kit on known reference samples, non-target species (Arabian sand cat, cheetah), and serial dilutions. Confirmatory analysis was performed using droplet digital PCR (ddPCR) to ensure specificity and absolute quantification. Results: The qPCR assay demonstrated high sensitivity, with a limit of detection below 100 target copies per reaction, and high specificity, showing no cross-reactivity with non-target species. Siberian Tiger samples produced robust, early amplification (Cq 15.74–18.03). ddPCR provided absolute quantification, confirming target presence and resolving a case of ambiguous qPCR amplification from a White Tiger sample, highlighting ddPCR’s utility in eliminating false positives. Serial dilution analysis confirmed excellent dynamic range and reproducibility. Conclusion: This study validated a sensitive, specific, and reliable cytb-based qPCR/ddPCR assay for the molecular identification of Siberian Tiger DNA. The integrated approach provides a robust tool for wildlife forensics and conservation genetics, enabling accurate detection from trace and degraded samples to support legal enforcement and biodiversity monitoring.