Molecular Tools for qPCR Identification and STR-Based Individual Identification of Panthera pardus (Linnaeus, 1758)

Area of Science:

• Molecular Biology
• Conservation Genetics
• Forensic Science

Background:

• Leopards (Panthera pardus) are apex predators facing severe population declines due to habitat loss, human-wildlife conflict, and illegal trade.
• Accurate species and individual identification is crucial for conservation and forensic applications, especially for processed or degraded wildlife products.
• Morphological identification is often impossible for highly processed or degraded samples.

Purpose of the Study:

• To develop and validate a robust multiplex quantitative real-time PCR (qPCR) assay for species-specific detection of Panthera pardus.
• To establish a short tandem repeat (STR) system for individual identification of P. pardus.
• To create a molecular framework for rapid leopard detection, quantification, and individual identification.

Main Methods:

• Developed a multiplex qPCR assay (Ppar Qplex) targeting mitochondrial Cytochrome b (Cyt b) for species confirmation and a nuclear marker (PLP) for Feliformia detection.
• Validated the assay's robustness, specificity, sensitivity, repeatability, and reproducibility using DNA from 30 P. pardus individuals and 18 related Feliformia species.
• Employed a set of 18 STR loci and a sex determination system for individual identification.

Main Results:

• The Ppar Qplex assay demonstrated high specificity, with mitochondrial amplification exclusive to P. pardus samples.
• The assay exhibited high analytical sensitivity, detecting DNA at concentrations as low as 1 pg/µL, with consistent results across various sample types and users.
• The STR multiplex successfully generated 30 unique individual profiles, confirming its efficacy for individual identification.

Conclusions:

• The combined qPCR and STR system offers a fast, sensitive, and highly specific molecular solution for leopard detection and individual identification.
• These tools enhance forensic capacity to combat wildlife crime involving P. pardus.
• The developed methods provide critical data for evidence-based conservation management of leopards.