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. 2025 Apr 17;15(4):664.
doi: 10.3390/life15040664.

Developmental Validation of DNA Quantitation System, Extended STR Typing Multiplex, and Database Solutions for Panthera leo Genotyping

Lenka Vankova  1   2 , Johana Alaverdyan  1   2 , Daniel Vanek  1   2   3   4
Affiliations

Developmental Validation of DNA Quantitation System, Extended STR Typing Multiplex, and Database Solutions for Panthera leo Genotyping

Lenka Vankova et al. Life (Basel). .

Abstract

This study describes the development of a species determination/DNA quantification system called Pleo Qplex and an individual identification STR multiplex called Pleo STRplex using Panthera leo samples. Pleo Qplex enables us to measure the quantity of extracted nuclear and mitochondrial DNA and detect the presence of co-purified inhibitors. Pleo STRplex, consisting of seven loci, enables the determination of the DNA profile from a sample of Panthera leo based on the analysis of short tandem repeats (STRs). The Pleo STRplex provides additional loci on top of previously published STR loci in Ptig STRplex and contains a specific STR marker that confirms Panthera leo. An allelic ladder of all STR markers was prepared to enable reliable allele calling. The STR loci can also be used to type the DNA of other members of the genus Panthera. The work on the resulting STR profiles is performed using GenoProof Suite, which offers databasing, matching, and relationship analysis.

Keywords: CITES organism; DNA quantitation; STR multiplex; databasing; forensic genetics; matching analysis; species determination; wildlife.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 3
Figure 3
The qPCR plots generated using Pleo Qplex compare nuclear DNA quantitation (blue curve) and mitochondrial DNA (red curve) for various members of the genus Panthera (P. tigris, P. pardus, P. uncia, P. onca) and a tiger/lion hybrid. IPC DNA (green curve) is an internal positive control for qPCR.
Figure 1
Figure 1
The qPCR plot generated using Pleo Qplex on a Panthera leo sample shows nuclear DNA quantitation (blue curve) and mitochondrial DNA (red curve). IPC DNA (green curve) is an internal positive control for qPCR.
Figure 2
Figure 2
The qPCR plot generated using Pleo Qplex on a Homo sapiens sample shows nuclear DNA quantitation (blue curve) and mitochondrial DNA (red curve). IPC DNA (green curve) is an internal positive control for qPCR.
Figure 4
Figure 4
The results of Pleo Qplex sensitivity study.
Figure 5
Figure 5
Resulting electropherogram (EPG) with a DNA profile consisting of 7 STRs from the Panthera leo positive control sample. The peak colors on the EPG (blue, black, red) reflect the fluorophore used for the 5′ primer fluorescent label (see Table 3).
Figure 6
Figure 6
Resulting electropherogram (EPG) of the Pleo STRplex allelic ladder. This allelic was prepared to reflect the common alleles of other individuals belonging to the genus Panthera. The peak colors on the EPG (blue, black, red) reflect the fluorophore used for the 5’ primer fluorescent label (see Table 3).
Figure 7
Figure 7
Resulting electropherograms of the Pleo STRplex with decreasing concentrations of input DNA extracted from P. leo. The peak colors on the EPG (blue, black, red) reflect the fluorophore used for the 5’ primer fluorescent label (see Table 3).
Figure 8
Figure 8
Reduction in peak height (5 pg) and allelic dropout (1 pg) in Pleo STRplex and other big cats. 5 pg plot was magnified 2×; 1 pg plot was magnified 10×. The peak colors on the EPG (blue, black, red) reflect the fluorophore used for the 5’ primer fluorescent label (see Table 3).
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