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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.

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.

Tématem příspěvku je nová analýza dostupných poznatků o hrobech K1 a K2 v lodi předrománské rotundy sv. Víta, které byly objeveny již v roce 1911 a připisovány několika přemyslovským knížatům. Provedena byla zejména revize antropologických poznatků, analýzy stabilních izotopů uhlíku (δ13C) a dusíku (δ15N), analýza DNA a radiouhlíkové datování. Získaná data nedávají dohromady konzistentní obraz. Archeologie vylučuje připsání ostatků knížeti Bořivoji I. († asi 888/889), antropologie pak knížeti Boleslavu I. († 972). Oba obory by dovolovaly připsat s určitou pravděpodobností kosterní ostatky knížeti Boleslavu II. († 999), tomu ale neodpovídá radiokarbonové datování. Navržena je možnost připsat ostatky písemně doloženému jménem neznámému synovi knížete Boleslava I., který zemřel před rokem 972. Analýza DNA z K2 připsala ostatky ženě, pravděpodobně tedy manželce osoby K1.

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.

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.