Simple Nested Allele-Specific approach with penultimate mismatch for precise species and sex identification of tiger and leopard


Accurate species and sex identification of non-invasive and forensic samples of the tiger and leopard is still confusing when using the allele-specific methods. We designed allele-specific methods with penultimate nucleotide mismatch in a nested manner for the exact identification and double-checking of forensic samples. The mismatch design is a novel concept in species and sex identification, making the allele-specific targeting precise. We developed three sets of markers, a 365 bp outer and a 98 bp inner marker for nested tiger species identification assay, 136 bp leopard specific marker, and carnivore sex identification markers. We validated the method with tissue/blood forensic samples of various felids and herbivorous available in our lab and on known fecal samples from Vandalur Zoo. We also collected 37 scat samples at diverse stages of deterioration from the Mudumalai Tiger Reserve, Tamil Nadu, India. The 365 bp targeted markers resulted in 70.2% (n = 22; 22/37) amplification success, while the 98 bp FAM-labelled marker amplified 89% (n = 33; 33/37) scat samples independently. The 136 bp leopard markers answered four scat samples (11%) unrequited by the tiger specific markers. We evaluated species and the sex identification with these markers in another 190 non-invasive samples provided by the Mudumalai Tiger Reserve authorities. Among which 56.3% (n = 107) of samples were recognized as tiger (64 male and 43 female) and 38.9% (n = 74) as leopard (41 male and 33 female). The method supersedes any other previous methods in this regard by its high accuracy and simplicity.

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Data availability

The NCBI GenBank accessions used in the publication is acknowledged appropriately and provided in the supplementary material S1a. The samples were collected for the study following the regulations of the authority.

Code availability

All the software used in the present study are open source software and downloaded accordingly.


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The authors acknowledge the Tamil Nadu Forest Department and Mudumalai Tiger Conservation Foundation for giving permission and samples vide permission number WL5 (A) 034236/2015 Dated 15-12-2015, Permit No. 71/2015 and Ref. No. WL (A)/ 3386/2019 dated 05-02-2019 Permit No 08/2019. The support of Dr. Shekhar Kumar Niraj IFS, Mr. Srinivas R Reddy IFS, Dr. Raghuram Singh IFS, Mr. Ameer Haja and Dr. S Nandini are deeply acknowledged.


No funding was received for conducting this study.

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The first author (GN), and second author (PMB) implemented the work in the lab and prepared basic manuscript. The third author (TTS), forth author (BR) and eighth author (GM) done the field works. The fifth author (RA) done the bioinformatics work and lab support. The sixth author (KKK), seventh author (GDK) and the ninth author (MJ) developed and evaluated the concepts and corrected the manuscript. The last author (RS) obtained permission, finalised and approved the manuscripts and concepts.

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Correspondence to Raveendranathanpillai Sanil.

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The authors do not have any financial or non financial conflict of interest related to this publication.

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We obtained permission from the Tamil Nadu forest Department via order numbers WL5(A) 034236/2015 dated 15–12-2015 and Ref.No.WL (A)/ 3386/2019 dated 05-02-2019 Permit No 08/2019.

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Nittu, G., Bhavana, P.M., Shameer, T.T. et al. Simple Nested Allele-Specific approach with penultimate mismatch for precise species and sex identification of tiger and leopard. Mol Biol Rep 48, 1667–1676 (2021).

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  • AS technique
  • Tiger species identification
  • Leopard species identification
  • Nested PCR
  • Molecular gender identification