Developing a qPCR – Melt Curve Analysis protocol to identify Indian Antelopes and related species

Akshay, Simha K. J. (2018) Developing a qPCR – Melt Curve Analysis protocol to identify Indian Antelopes and related species. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Akshay Simha K. J. (13MS056)) 13MS056.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

Detecting the presence or absence of species in a habitat is important in designing conservation and management programs, particularly for rare and endangered species. When such organisms are not easily sighted, presence needs to be inferred from secondary evidence such as footprints or fecal samples. At the landscape scale such presence/absence data can be used to model species distributions and abundance. In recent years, molecular genetic methods for species identification from non-invasively obtained DNA material have been developed and used. Such methods have the advantage of samples being collected rapidly from large areas and analyzed with high precision in the laboratory. Accurate species identification methods are useful in other applications such as wildlife forensics, to identify & detect contamination in animal products and to prevent illegal trafficking. Here we attempt to design a rapid screening method for identification of several wild ungulate species that are of conservation importance. We use qPCR and melt-curve analysis, the characteristics of which are specific to the DNA sequence. We amplify the regions in presence of a fluorescent dye that binds to dsDNA to produce fluorescence. The fluorescence decreases when the dsDNA is melted, releasing the dye. These melting profiles and melting temperatures are used as signatures to identify species. This removes need for post-PCR handling of samples, making it costeffective. While the results so far have not yielded any significant leads, we intend to subject the amplified products to sequencing to verify the specificity of primers used. Results from this endeavor will help us refine our technique further. Indian antelopes and related bovids were once widespread in the Indian subcontinent but many are now rare and restricted to small protected areas. Assessing their distributions and abundance is important for wildlife management. Here we study Blackbuck, Chinkara, Four-horned antelope, and Nilgai, and develop a species identification protocol using regions on mitochondrial 16S rRNA and Cytochrome b genes. The protocol is validated using tissue and fecal samples collected from known individuals. We will use this protocol to obtain presence/absence data for a subset of species from a protected area in central India to develop habitat occupancy models for population estimation.

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