Mammal Research

, Volume 62, Issue 4, pp 405–411 | Cite as

A novel real-time TaqMan™ PCR assay for simultaneous detection of Neotropical fox species using noninvasive samples based on cytochrome c oxidase subunit II

  • M. CosseEmail author
  • F. Grattarola
  • N. Mannise
Methods Paper


Strategies to evaluate and monitor elusive mammal species require the development of genetic techniques and their application to unambiguous biological material for ecological and genetic studies. In order to assess cytochrome c oxidase subunit II gene inter- and intraspecific variations, we compared sequences from different Neotropical canids and domestic dogs. We developed a primer pair to amplify a 154-bp fragment of this gene and a species-specific multiplex TaqMan™ assay for accurate identification of two native fox species occurring in sympatry in South America, the crab-eating fox (Cerdocyon thous) and the pampas fox (Lycalopex gymnocercus). The assays can also distinguish domestic dogs (Canis lupus familiaris) from both wild foxes. The use of different fluorescent reporter dyes for species identification in a multiplex probe PCR-RT assay reduces labor and costs. The methodology presented in this study demonstrates an efficient approach to enable high-performance analysis and represents a reliable cost-effective tool for molecular ecology research to monitor the wild canid populations by noninvasive genetic sampling. This standardized assay will allow large-scale high-throughput analyses in a routine and reliable way.


mtDNA COII gene Molecular ecology Canid Biodiversity monitoring Uruguay 



The research was supported by Programa de Desarrollo de las Ciencias Básicas (PEDECIBA) and grant from Agencia Nacional de Investigación e Innovación (ANII) (FCE_2_20011_1_5700). The authors wish to thank the Monitoreo participativo de fauna en Paso Centurión EFI-UdelaR team, and Cabo Polonio forest ranger team (SNAP) and to acknowledge sample contribution from C. Máspoli, C. Aristimuño, C. Prigioni, M. Baptista, A. Saralegi, O. Lussich, N. Hernandez and C. Pérez, and José M. Venzal.


  1. Ali ME, Razzak MA, Hamid SBA (2014) Multiplex PCR in species authentication: probability and prospects—a review. Food Anal Methods 7(10):1933–1949CrossRefGoogle Scholar
  2. Bardeleben C, Moore RL, Wayne RK (2005a) Isolation and molecular evolution of the selenocysteine tRNA (Cf TRSP) and RNase P RNA (Cf RPPH1) genes in the dog family, Canidae. Mol Biol Evol 22(2):347–359CrossRefPubMedGoogle Scholar
  3. Bardeleben C, Moore RL, Wayne RK (2005b) A molecular phylogeny of the Canidae based on six nuclear loci. Mol Phylogenet Evol 37(3):815–831CrossRefPubMedGoogle Scholar
  4. Beja-Pereira A, Oliveira R, Alves PC, Schwartz MK, Luikart G (2009) Advancing ecological understandings through technological transformations in noninvasive genetics. Mol Ecol Resour 9(5):1279–1301CrossRefPubMedGoogle Scholar
  5. Berta A (1982) Cerdocyon thous. Mamm Species 186:1–4CrossRefGoogle Scholar
  6. Broquet T, Ménard N, Petit E (2007) Noninvasive population genetics: a review of sample source, diet, fragment length and microsatellite motif effects on amplification success and genotyping error rates. Conserv Genet 8(1):249–260CrossRefGoogle Scholar
  7. Chaves PB, Graeff VG, Lion MB, Oliveira LR, Eizirik E (2012) DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples. Mol Ecol Resour 12(1):18–35CrossRefPubMedGoogle Scholar
  8. Courtenay O, Maffei L (2010) Cerdocyon thous. In: Sillero-Zubiri C, Hoffmann M, Macdonald DW (eds) Canids: foxes, wolves, jackals and dogs. Status survey and conservation action plan: IUCN/SSC Canid Specialist Group, IUCN, Gland. IUCN, Cambridge, pp 32–38Google Scholar
  9. Ebert C, Knauer F, Spielberger B, Thiele B, Hohmann U (2012) Estimating wild boar Sus scrofa population size using faecal DNA and capture-recapture modelling. Wildl Biol 18(2):142–152CrossRefGoogle Scholar
  10. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4):783–791CrossRefPubMedGoogle Scholar
  11. Galimberti A, Sandionigi A, Bruno A, Bellati A, Casiraghi M (2015) DNA barcoding in mammals: what’s new and where next? Hystrix 26(1):13–24Google Scholar
  12. González S, Cosse M, del Rosario FM, Emmons L, Vynne C, Duarte JMB, Beccacesi MD, Maldonado JE (2015) Population structure of mtDNA variation due to Pleistocene fluctuations in the South American Maned Wolf (Chrysocyon brachyurus, Illiger, 1815): management units for conservation. J Hered 106(S1):459–468CrossRefPubMedGoogle Scholar
  13. Hausknecht R, Bayerl H, Gula R, Kuehn R (2010) Application of quantitative real-time polymerase chain reaction for noninvasive genetic monitoring. J Wildl Manag 74(8):1904–1910CrossRefGoogle Scholar
  14. Holland PM, Abramson RD, Watson R, Gelfand DH (1991) Detection of specific polymerase chain reaction product by utilizing the 5′----3'exonuclease activity of Thermus aquaticus DNA polymerase. P Natl Acad Sci USA 88(16):7276–7280CrossRefGoogle Scholar
  15. Hughes J, Macdonald DW (2013) A review of the interactions between free-roaming domestic dogs and wildlife. Biol Conserv 157:341–351CrossRefGoogle Scholar
  16. Johansson MK (2006) Choosing reporter-quencher pairs for efficient quenching through formation of intramolecular dimers. In: Fluorescent Energy Transfer Nucleic Acid Probes. Springer, pp 17–29Google Scholar
  17. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16(2):111–120CrossRefPubMedGoogle Scholar
  18. Kohn MH, Wayne RK (1997) Facts from feces revisited. Trends Ecol Evol 12(6):223–227CrossRefPubMedGoogle Scholar
  19. Lucherini M, Luengos Vidal EM (2008) Lycalopex gymnocercus (Carnivora: Canidae). Mamm Species 1–9Google Scholar
  20. Lucherini M, Pessino M, Farias AA (2004) Pampas fox (Pseudalopex gymnocercus). In: Sillero-Zubiri C, Hoffmann M, Macdonald DW (eds) Canids: foxes, wolves, jackals and dogs: status survey and conservation action plan, Survey and conservation action plan, vol Status. IUCN/SSC Canid Specialist Group, Gland, pp 63–68Google Scholar
  21. Mondol S, Ullas Karanth K, Samba Kumar N, Gopalaswamy AM, Andheria A, Ramakrishnan U (2009) Evaluation of non-invasive genetic sampling methods for estimating tiger population size. Biol Conserv 142(10):2350–2360CrossRefGoogle Scholar
  22. Morrone JJ (2014) Biogeographical regionalization of the Neotropical region. Zootaxa 3782(1):1–110CrossRefPubMedGoogle Scholar
  23. Nowak NM (1999) Walker’s mammals of the world, vol 1. JHU Press, BaltimoreGoogle Scholar
  24. O'Meara DB, Sheehy E, Turner PD, O'Mahony D, Harrington AP, Denman H, Lawton C, MacPherson J, O'Reilly C (2014) Non-invasive multi-species monitoring: real-time PCR detection of small mammal and squirrel prey DNA in pine marten (Martes martes) scats. Acta Theriol 59(1):111–117CrossRefGoogle Scholar
  25. O'Neill D, Turner PD, O'Meara DB, Chadwick EA, Coffey L, O'Reilly C (2013) Development of novel real-time TaqMan® PCR assays for the species and sex identification of otter (Lutra lutra) and their application to noninvasive genetic monitoring. Mol Ecol Resour 13(5):877–883CrossRefPubMedGoogle Scholar
  26. Pamilo P, Nei M (1988) Relationships between gene trees and species trees. Mol Biol Evol 5(5):568–583PubMedGoogle Scholar
  27. Petit E, Valiere N (2006) Estimating population size with noninvasive capture-mark-recapture data. Conserv Biol 20(4):1062–1073CrossRefPubMedGoogle Scholar
  28. Procheş Ş, Ramdhani S (2012) The world’s zoogeographical regions confirmed by cross-taxon analyses. Bioscience 62(3):260–270CrossRefGoogle Scholar
  29. Rodriguez-Castro KG, Ciocheti G, Ribeiro JW, Ribeiro MC, Galetti PM (2017) Using DNA barcode to relate landscape attributes to small vertebrate roadkill. Biodivers Conserv:1–18Google Scholar
  30. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406PubMedGoogle Scholar
  31. Schoske R, Vallone PM, Ruitberg CM, Butler JM (2003) Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci. Anal Bioanal Chem 375(3):333–343CrossRefPubMedGoogle Scholar
  32. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefPubMedPubMedCentralGoogle Scholar
  33. Tchaicka L, Freitas TRO, Bager A, Vidal SL, Lucherini M, Iriarte A, Novaro A, Geffen E, Garcez FS, Johnson WE (2016) Molecular assessment of the phylogeny and biogeography of a recently diversified endemic group of South American canids (Mammalia: Carnivora: Canidae). Genet Mol Biol 39(3):442–451CrossRefPubMedPubMedCentralGoogle Scholar
  34. Vieira E, Port D (2007) Niche overlap and resource partitioning between two sympatric fox species in southern Brazil. J Zool 272(1):57–63CrossRefGoogle Scholar
  35. Walker NJ (2002) A technique whose time has come. Science 296(5567):557CrossRefPubMedGoogle Scholar
  36. Wayne RK, Geffen E, Girman DJ, Koepfli KP, Lau LM, Marshall CR (1997) Molecular systematics of the Canidae. Syst Biol 46(4):622–653CrossRefPubMedGoogle Scholar

Copyright information

© Mammal Research Institute, Polish Academy of Sciences, Białowieża, Poland 2017

Authors and Affiliations

  1. 1.Genética de la Conservación, Departamento de Biodiversidad y Genética – IIBCE/MECMontevideoUruguay

Personalised recommendations