Double specific nested PCR and diagnostic SNP assay for species identification in lynx fecal critical samples


The single lynx species present in the Iberian Peninsula—Lynx pardinus (Temmink, 1827)—is one of the most threatened felines in the world so, any biological indication of its presence is significant. However, it is a very elusive species and physical evidence through direct visualization or camera trapping is difficult to obtain. Genetic noninvasive methods, in particular, molecular scatology, are a reliable alternative to unequivocally identify the presence of a species in a given geographical area. We have developed a highly specific and sensitive molecular method to identify the presence of this critically endangered lynx species from severely deteriorated fecal samples. The method consists in a double specific nested PCR of mitochondrial D-loop sequences and two diagnostic SNPs detected by a multiplex primer extension reaction.

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This work was funded by the Ministerio de Agricultura y Pesca, Alimentación y Medio Ambiente (MAGRAMA), Project 16MNSV002. Thanks are due to the Center El Acebuche in the Doñana National Park (MAGRAMA - Junta de Andalucía) for providing fecal samples from animals bred in captivity, and to Dr. José Antonio Dávila (Instituto de Investigación en Recursos Cinegéticos-Universidad de Castilla La Mancha, IREC-UCLM) and Dr. Eva Martinez Nevado (Zoo-Aquarium, Madrid) for kindly providing additional lynx samples.

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Correspondence to Pilar Arana.

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The authors declare that they have no conflict of interest.

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Alejandra Cruz and Beatriz Matallanas have equally contributed to the article.

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Cruz, A., Matallanas, B., Lobón-Rovira, J. et al. Double specific nested PCR and diagnostic SNP assay for species identification in lynx fecal critical samples. Conservation Genet Resour 11, 173–175 (2019).

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  • Iberian lynx
  • Fecal DNA
  • Threatened species
  • Nested PCR
  • Diagnostic SNPs