Dental calculus offers a rich source of information about the life and death of past populations. The objective of this study was to assess the efficacy of a novel approach for concurrently analyzing the microbiome and INNUL human DNA markers from dental calculus samples, with the aim of maximizing the information yield from skeletal human remains whereas minimizing their damage. To this end, we integrated high-throughput 16S rDNA sequencing with the InnoTyper®21 system. This method enabled the identification of at least 14 Alu-based INNUL markers and the amelogenin marker for sex determination from all eight ancient samples. Furthermore, numerous bacterial species were identified and associated with specific human microbiomes and potential health conditions. Findings of this study provide compelling evidence that dental calculus can serve as a viable source for both human microbiome analysis and genetic identity profile determination, while simultaneously minimizing damage to skeletal remains.

Wildlife forensic analysis frequently deals with highly degraded DNA samples, including those from tanned hides, processed products of traditional Chinese medicine, and specimens stored in preservation fluids. These samples present significant challenges for traditional DNA barcoding due to the difficulty of amplifying sufficiently long DNA fragments. To overcome this limitation, we designed five primer pairs to amplify short mitochondrial DNA (mtDNA) fragments (~100 base pairs). When aligned collectively, these fragments span approximately 500 bp of the cytochrome C oxidase subunit I (COI) gene, which is one of the most common species-barcoding targets. We subjected the primers to a comprehensive validation process, including in silico analysis and experimental verification using various non-degraded and degraded samples of animal tissues. Here, we demonstrate the efficacy and reliability of our DNA minibarcoding method. By addressing the limitations of traditional DNA barcoding, this method improves the accuracy and success of species identification, thereby supporting wildlife conservation, management, and forensic investigations.

Background/Objectives The leopard (Panthera pardus), an apex predator listed in CITES Appendix I and classified as Vulnerable by the IUCN, is undergoing severe population declines driven by habitat loss, human-wildlife conflict, and illegal trade. Rapid and reliable species and individual identification is critical for conservation and forensic applications, particularly when analyzing highly processed or degraded seized wildlife products, where morphological identification is often impossible. We aimed to develop and validate a robust multiplex quantitative real-time PCR (qPCR) assay combined with a short tandem repeat (STR) system for the species-specific detection and individual identification of P. pardus. Methods The qPCR assay (Ppar Qplex) was designed to target a mitochondrial Cytochrome b (Cyt b) fragment for species confirmation, a nuclear marker (PLP) for general Feliformia detection and quantification, and an artificial internal positive control (IPC) to monitor PCR inhibition. The assay's performance was validated for robustness, specificity, sensitivity, repeatability, and reproducibility, utilizing DNA extracted from 30 P. pardus individuals (hair and feces) and tested against 18 related Feliformia species and two outgroups. Individual identification was achieved using a set of 18 STR loci and a sex determination system adapted from previously published Panthera panels. Results Validation demonstrated high specificity for the Ppar Qplex: mitochondrial amplification occurred exclusively in P. pardus samples. The nuclear marker consistently amplified across all 18 tested Feliformia species but not the outgroups. The assay showed high analytical sensitivity, successfully detecting DNA at concentrations as low as 1 pg/µL, with consistent results confirmed across different sample types, replicates, and independent users. Furthermore, the STR multiplex successfully generated 30 unique individual profiles using the 18 polymorphic loci and the sex determination system. Conclusions The combined qPCR assay and STR system provide a fast, sensitive, and highly specific molecular framework for rapid leopard detection, quantification, and individual identification from a wide range of sample types. These tools strengthen forensic capacity to combat wildlife crime and provide critical data to support evidence-based conservation management of P. pardus. P. pardus, an apex predator listed in CITES Appendix I and classified as Vulnerable by the IUCN, is undergoing severe population declines driven by habitat loss, human-wildlife conflict, and illegal trade. Rapid and reliable identification of seized specimens is therefore critical for conservation and forensic applications, mainly when products are highly processed. We developed and validated a multiplex quantitative real-time PCR (qPCR) assay targeting the mitochondrial gene Cytochrome b (Cyt b) for species-specific detection. The assay was tested on verified leopard individuals and validated across 18 Feliformia and two outgroup species (Homo sapiens, Canis lupus familiaris). Analytical performance was assessed through robustness, specificity, sensitivity, repeatability, and reproducibility. Mitochondrial amplification occurred exclusively in leopard samples, while nuclear markers amplified consistently across Feliformia but not in outgroup species. The assay's limit of DNA detection is 1 pg/µL and produces consistent results across replicates, tested types of samples (hair, feces), and independent users, with internal controls confirming the absence of inhibition. In addition, we present the results of successful individual identification using the set of 18 STR loci and the sex determination system. The developed qPCR and STR systems provide a fast, sensitive, and specific solution for leopard detection and quantification, reinforcing forensic efforts against wildlife crime and supporting conservation of P. pardus.

Biological samples of non-human origin, commonly encountered in wildlife crime investigations, present distinct challenges regarding forensic DNA analysis efforts. Although the types of samples encountered in human identity testing can vary to some degree, analyzing DNA from one species is facilitated by unified processes, common genetic marker systems, and national DNA databases. In contrast, non-human animal species identification is confounded by a diverse range of target species and a variety of sampling materials, such as feathers, processed animal parts in traditional medicine, and taxidermy specimens, which often contain degraded DNA in low quantities, are contaminated with chemical inhibitors, and may be comingled with other species. These complexities require specialized analytical approaches. Compounding these issues is a lack of validated non-human species forensic sampling and typing kits, and the risk of human DNA contamination during evidence collection. Markers residing on the mitochondrial genome (mtDNA) are routinely sought because of the large datasets available for comparison and their greater sensitivity of detection. However, the barcoding results can be complicated at times for achieving species-level resolution, the presence of nuclear inserts of mitochondrial DNA (NUMTs), and the limitation of mtDNA analysis alone to detect hybrids. Species-specific genetic markers for identification have been developed for a few high-profile species; however, many CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora)-listed organisms lack specific, validated forensic analytical tools, creating a significant gap in investigative enforcement capabilities. This deficiency stems in part from the low commercial nature of wildlife forensics efforts, a government research-driven field, the difficulty of obtaining sufficient reference samples from wild populations, limited training and education infrastructure, and inadequate funding support.

The Eurasian lynx (Lynx lynx) is listed in CITES Appendix II and is protected under the Bern Convention and the EU Habitats Directive, yet it remains a frequent target of wildlife crime, highlighting the urgent need for reliable identification methods. This study focuses on determination and DNA quantification of the Lynx spp. using quantitative real-time PCR (qPCR). The Llynx Qplex quantification multiplex system effectively distinguishes Lynx spp. from other Feliformia species by targeting mitochondrial and nuclear markers. Additionally, we present the results of the developmental validation of the Llyn STRplex system for individual identification and databasing using six STR loci. This study followed ISFG recommendations for non-human DNA testing and developmental validation guidelines. Both systems demonstrate high sensitivity (5 pg genomic DNA for Llynx Qplex and 30 pg of mtDNA for Llyn STRplex) and high specificity to Lynx spp., confirmed by testing against 16 related Feliformia species. Robustness was evaluated, showing sensitivity to temperature variation, and both repeatability and reproducibility were successfully tested across replicates and conditions. Given that forensic casework often involves degraded and limited biological material, molecular tools must be both sensitive and specific to ensure accurate results. Developing precise and efficient tools is essential for supporting investigations of wildlife crime involving the Eurasian lynx, as well as efforts aimed at conserving the species.

This article examines the use of chip electrophoresis in wildlife crime investigations through three mock case studies. Specifically, we analysed DNA extracted from the tanned hide of Panthera pardus, a species protected under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), at various stages of the tanning process (Case study #1). Tanned hides present a unique challenge due to the detrimental effects of tanning on DNA integrity, resulting in highly degraded DNA extracts. Therefore, assessing DNA integrity before performing standard DNA analyses is critical to conserving laboratory resources. One of the conventional methods for evaluating DNA integrity involves determining the degradation index using quantitative polymerase chain reaction (qPCR). This study explored whether chip electrophoresis, a faster and simpler alternative to qPCR, could provide comparable assessments of DNA integrity. In addition, its applicability for use in established assays, such as the Triplex assay, which is used for rapidly classifying unknown biological material (Case study #2) and species identification based on mitochondrial DNA lenght polymorphism (Case study #3). The findings indicate that while chip electrophoresis is effective in established assays, it is unsuitable for assessing the quality of DNA extracted from tanned hides.

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.

In scenarios when the morphological identification of samples is not feasible, the utilization of molecular analyses becomes an imperative. However, that can often be challenging in conditions where standard molecular laboratories cannot be established. In this study, we present a simplified and field-friendly protocol of the previously published Triplex PCR Assay for the identification of mammalian and plant sample origin using a fully portable molecular laboratory from Bento Lab (Bento Bioworks Ltd., London, UK), effectively eliminating the need for a traditional laboratory setup. The protocol in combination with correctly selected kits enables DNA extraction, result evaluation based on electrophoresis, and direct use of the PCR products for downstream analyses such as Sanger sequencing and third-generation sequencing; therefore, enabling the use of molecular analysis directly in situ or for educational purposes in a classroom.

This study aimed to provide an overview of the methodological approach used for the species determination of big cats. The molecular system described herein employs mitochondrial DNA control region (CR-mtDNA)-length polymorphism in combination with highly sensitive and precise capillary electrophoresis. We demonstrated that the described CR-mtDNA barcoding system can be utilized for species determination where the presence of biological material from big cats is expected or used as a confirmatory test alongside Sanger or massive parallel sequencing (MPS). We have also addressed the fact that species barcoding, when based on the analysis of mtDNA targets, can be biased by nuclear inserts of the mitochondrial genome (NUMTs). The CR-mtDNA barcoding system is suitable even for problematic and challenging samples, such as hair. CR-mtDNA-length polymorphisms can also distinguish hybrids from pure breeds.

The study aimed to analyze the entomological material collected during 13 autopsies performed on the unidentified cadavers revealed at different stages of decay in the Upper Silesia Region(Poland)over 2016-2022.During the preparation of human tissues for genetic identification,we revealed larvae,puparia,and adult insects in previously undescribed locations:costal cartilage,femur nutrient canals(foramen nutrients),and tooth cavities.The taxonomical assessment was done using morphological examination or DNA barcoding,where necessary.Based on our observations,we conclude that the apical constriction,foramen,and cavities may serve as migration paths inside teeth,and the femur nutrient canals to the bone marrow.The study also revealed that the beetle Necrobia ruficollis(Fabricius,1775)and the moth family Pyralidae Latreille,1802(Phycitinae)moths can form pupal chambers inside the costal cartilage,indicating that these insects can complete their life cycle inside this cache.We believe that the newly reported locations of carrion insects in human remains may be relevant to forensic entomology,as they provide new opportunities to collect insect evidence.

We investigated the development of a simple and sensitive assay for the identification of minute amounts of animal and human biological material. The suggested system uses isothermal amplification of DNA in a rapid assay format, which returns the results in as little as 40 minutes from sampling. The assay used for the proof of concept employs the novel handheld SaLux19 device (Max-Planck Innovation prototype, Germany). This well-established infield detection system could also be utilized for forensic scenarios. The results presented here demonstrated that the assay is sufficiently specific and sensitive and can detect the presence of Sus scrofa nucleic acids.

Our research has developed a highly sensitive and simple assay to detect small amounts of animal and human biological material in less than 40 min. The handheld SaLux19 device developed at the Max Planck Institute of Experimental Medicine in Göttingen, Germany, was used to validate our concept. The proposed system uses isothermal amplification of DNA in a rapid assay format. Our results show that the assay can detect Sus scrofa nucleic acids with very high sensitivity and specificity. This detection system has potential for forensic scenarios.

Illegal wildlife trade is currently on the rise, and it is becoming one of the most lucrative crime sectors. The rarer the species, the higher the demand. Wildlife trade falls under international regulations, such as the CITES convention. Proving that this convention has been violated is a complex process and can be very difficult to do. DNA analysis methods remain (in many cases) the only way to determine whether a certain specimen originated from a protected animal species, a specific individual, or a species in which it is legal to trade. Tanned animal hides are a specific type of specimen. With this type of biological material, obtaining amplifiable DNA is often difficult. This pilot study aimed to map the effect of the chemicals used in the tanning process on the degradation of the DNA yielded from such samples. The DNA was quantified using two different approaches: qPCR and Qubit fluorometry. The degree of DNA fragmentation was assessed by determining the degradation index. The results indicate that reagents containing chromium have the greatest influence on DNA degradation. However, by using the presented protocol, enough amplifiable DNA can be obtained from hides treated with aluminum-based reagents.

The aim of this study is to provide an overview of ongoing research on and the development of identification tools for big cats (Panthera tigris, Panthera leo, Panthera pardus, …). The set of tools includes a species-specific RT PCR quantitation system (nuclear and mitochondrial), STR multiplexes, a rapid system for big cat species determination, and a database solution.

The aim of this technical note is to provide an overview of methodical approaches used to develop molecular systems for species determination/DNA quantification called Ptig Qplex and individual identification called Ptig STRplex of Panthera tigris samples. Both systems will help to combat the illegal trade of endangered species and create a worldwide shared database of DNA profiles.

Research on ancient and forensic DNA is related in many ways, and the two fields must deal with similar obstacles. Therefore, communication between these two communities has the potential to improve results in both research fields. Here, we present the insights gained in the ancient DNA community with regard to analyzing DNA from aged skeletal material and the potential use of the developed protocols in forensic work. We discuss the various steps, from choosing samples for DNA extraction to deciding between classical PCR amplification and massively parallel sequencing approaches. Based on the progress made in ancient DNA analyses combined with the requirements of forensic work, we suggest that there is substantial potential for incorporating ancient DNA approaches into forensic protocols, a process that has already begun to a considerable extent. However, taking full advantage of the experiences gained from ancient DNA work will require comparative studies by the forensic DNA community to tailor the methods developed for ancient samples to the specific needs of forensic studies and case work. If successful, in our view, the benefits for both communities would be considerable.

Skeletal remains are among the most difficult types of samples encountered in forensic DNA casework and historical investigations due to prolonged exposure to environmental insults. DNA extracted from bone often is degraded, in low quantities, and contains co-purified inhibitors from the surrounding soil and/or burial vault material. When sexually dimorphic skeletal elements are not recovered, determining the sex of a decedent can be challenging. With unidentified human skeletal remains, genetic data often are evaluated in concert with anthropological analyses, as well as other types of metadata, to improve confidence in making associations or for positive identifications. This study evaluated a multi-faceted molecular genetic approach to increasing the amount of data that can be recovered from degraded skeletal remains. Results demonstrate that using a newer-generation multiplex (GlobalFiler™) with an expanded set of highly discriminatory DNA markers - combined with co-amplification of three different sex-determining loci, one additional PCR cycle, and testing multiple cuttings from the same bone or multiple regions within a skeleton - can improve reliability and accuracy in skeletal remains identifications by providing data concordance.

The aim of this paper is to present the STRAND (STR ANimal Database) cloud expert system for non-human DNA analysis. The cloud expert system (CES) combines the cross-referenced registries of STR markers for different species and a DNA database for comparison of DNA profiles, with a repository of scientific papers and a dashboard for unpublished data, protocols, negative results and announcements related to animal DNA typing.

The aim of our study was to monitor the quality and quantity of DNA in bone samples that were boiled for 48 h. Bos taurus bone disks were sampled every hour for 48 h. The subsequent DNA analysis used multiple mitochondrial DNA (mtDNA) targets (100-700 bp) to evaluate the quality and quantity of the DNA extracted. The DNA extracted from bone disks remained typeable after boiling for 48 h. We have proven that DNA typing results can be obtained even after long-term boiling.

Bones are a valuable source of DNA in forensic, anthropological, and archaeological investigations. There are a number of scenarios in which the only samples available for testing are highly degraded and/or skeletonized. Often it is necessary to perform more than one type of marker analysis on such samples in order to compile sufficient data for identification. Lineage markers, such as Y-STRs and mitochondrial DNA (mtDNA), represent important systems to complement autosomal DNA markers and anthropological metadata in making associations between unidentified remains and living relatives or for characterization of the remains for historical and archaeological studies. In this comparative study, Y-STR typing with both Yfiler™ and Yfiler™ Plus (Thermo Fisher Scientific, Waltham, MA, USA) was performed on a variety of human skeletal remains, including samples from the American Civil War (1861–1865), the late nineteenth century gold rush era in Deadwood, SD, USA (1874–1877), the Seven Years’ War (1756–1763), a seventeenth-century archaeological site in Raspenava, Bohemia (Czech Republic), and World War II (1939–1945). The skeletal remains used for this study were recovered from a wide range of environmental conditions and were extracted using several common methods. Regardless of the DNA extraction method used and the age/condition of the remains, 22 out of 24 bone samples yielded a greater number of alleles using the Yfiler™ Plus kit compared to the Yfiler™ kit using the same quantity of input DNA. There was no discernable correlation with the degradation index values for these samples. Overall, the efficacy of the Yfiler™ Plus assay was demonstrated on degraded DNA from skeletal remains. Yfiler™ Plus increases the discriminatory power over the previous generation multiplex due to the larger set of Y-STR markers available for analysis and buffer modifications with the newer version kit. Increased haplotype resolution is provided to infer or refute putative genetic relationships.