title: Schrodinger's scat: a critical review of the currently available tiger (Panthera Tigris) and leopard (Panthera pardus) specific primers in India, and a novel leopard specific primer.
abstract: BACKGROUND
  Non-invasive sampling has opened avenues for the genetic study of elusive species, which has contributed significantly to their conservation. Where field based identity of non-invasive sample is ambiguous (e.g. carnivore scats), it is essential to establish identity of the species through molecular approaches. A cost effective procedure to ascertain species identity is to use species specific primers (SSP) for PCR amplification and subsequent resolution through agarose gel electrophoresis. However, SSPs if ill designed can often cross amplify non-target sympatric species. Herein we report the problem of cross amplification with currently published SSPs, which have been used in several recent scientific articles on tigers (Panthera tigris) and leopards (Panthera pardus) in India. Since these papers form pioneering research on which future work will be based, an early rectification is required so as to not propagate this error further.
  
  
  RESULTS
  We conclusively show cross amplification of three of the four SSPs, in sympatric non-target species like tiger SSP amplifying leopard and striped hyena (Hyaena hyaena), and leopard SSP amplifying tiger, lion (Panthera leo persica) and clouded leopard (Neofelis nebulosa), with the same product size. We develop and test a non-cross-amplifying leopard specific primer pair within the mitochondrial cytochrome b region. We also standardize a duplex PCR method to screen tiger and leopard samples simultaneously in one PCR reaction to reduce cost and time.
  
  
  CONCLUSIONS
  These findings suggest the importance of an often overlooked preliminary protocol of conclusive identification of species from non-invasive samples. The cross amplification of published primers in conspecifics suggests the need to revisit inferences drawn by earlier work.
authors: Wildlife Institute of India, Chandrabani, Dehradun, 248001, India. pranay.amrut@gmail.com.
authors: Pranay Amruth
authors: PA
authors: Maroju
authors: Wildlife Institute of India, Chandrabani, Dehradun, 248001, India. sonu.bsc90@gmail.com.
authors: Sonu
authors: S
authors: Yadav
authors: Wildlife Institute of India, Chandrabani, Dehradun, 248001, India. kolipakam@gmail.com.
authors: Vishnupriya
authors: V
authors: Kolipakam
authors: Wildlife Institute of India, Chandrabani, Dehradun, 248001, India. singhshweta1090@gmail.com.
authors: Shweta
authors: S
authors: Singh
authors: Wildlife Institute of India, Chandrabani, Dehradun, 248001, India. qnq@wii.gov.in.
authors: Qamar
authors: Q
authors: Qureshi
authors: Wildlife Institute of India, Chandrabani, Dehradun, 248001, India. jhalay@wii.gov.in.
authors: Yadvendradev
authors: Y
authors: Jhala
mesh_terms: D000818:Animals; D045303:Cytochromes b / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism; D007194:India; D046009:Panthera / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013045:Species Specificity; D046010:Tigers / Q000145:classification / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial; D045303:Cytochromes b