The pig is believed to be one of the most important livestock species for humans. It is an excellent animal model for studying the molecular background of several human diseases [1], and above all, it is a meaningful source of global meat production. Pork is widely used in the food industry, but one problem is determining the food’s authenticity, especially in terms of the species. Meat adulteration involves replacing or partially replacing high-value meat with cheaper quality meat [2], which involves economic, quality, safety, and socio-religious issues [3]. Together with pork, products may introduce veterinary drugs banned from the food chain, such as ractopamine, which is forbidden in many countries [4,5]. Moreover, undeclared pork may be contaminated with harmful microorganisms, such as the endoparasites Toxoplasma and Trichinella [6]. The presence of pork in food may also conflict with religious and cultural practices. Thus, the fraud of undeclared pork in food can affect consumer trust in the meat industry. Because of such illegal food practices, many governments have enacted laws prohibiting similar fraud. To ensure existing regulations are followed, laboratories worldwide have designed and developed various methods to identify animal species in food products, including methods to detect porcine components. According to scientific reports, efforts have also been made to provide a cost-efficient diagnostic tool for distinguishing wild boar (Sus scrofa), domestic pig (Sus scrofa domestica), and their hybrids (Figure 1). This is not easy, as the pig and the wild boar are evolutionarily closely related [7], and thus share many genetic markers. However, the identification methods have changed over time, accompanied by the development of innovative techniques that are now based on DNA testing.

Possibilities of species identification of pigs and wild boars (top of the figure) and distinguishing between both subspecies (bottom of the figure) using different molecular techniques.

A considerable number of animal breeds on Earth originated from diverse wild ancestors which, after being domesticated, exhibit a large phenotypic variety [8]. In the study by Groenen et al. [7], a comprehensive analysis of the phylogenetic background of the subspecies structure of wild boars and pigs was provided. The genomes of wild boars and domestic pigs from Europe and Asia revealed separate European and Asian lineages. The study also gave an insight into the history of population size changes. Both lines of wild boars diverged about 1.6–0.8 Myr (million years) ago, resulting in alleles divergence in the millions of loci [7]. Wild boars are the ancestors of modern domestic pigs, and it is believed that multiple domestication events occurred separately for species in Asia and Europe [7,9].

After domestication, many incidents of the admixture of domestic populations with local wild boars occurred because both species are found in the same habitat [9]. Moreover, there are farms (e.g., in Bulgaria and Sardinia) where pigs are kept in semi-wild conditions. It is highly likely that domestic pigs sporadically crossed with wild boars [10]. The frequent hybridisation of these two breeds was found in the studies on the European population [10,11,12,13]. Studies of the MC1R (melanocortin 1 receptor) gene in the Polish population of wild boars also proved the existence of admixed genotypes [14,15]. Crossbreeding domestic pigs with wild boars is documented and has served hunting purposes, better meat production, better taste, and reduced aggressive behaviour [11,12]. However, crossbreds have a high risk of pathogen transmission (e.g., ASF, African Swine Fever), which is the primary reason crossbreeding farms are under strict legislation in the European Union [12]. It is highly likely that some wild boars farmed in Poland were released or escaped from the farms and became a potential source of genome admixture in the wild population. Other studies have also suggested such a scenario [11,12,14]. The domestication of the pig and the mixing of domestic pig and wild boar populations cannot be ignored, as they provide the background to the problem of distinguishing between these subspecies.

The authentication of domestic pig meat is mainly based on mitochondrial DNA (mtDNA). The well-known sequences of the mitochondrial genome allow comparison of the porcine mtDNA with the genome of any species. The advantages of mitochondrial DNA over genomic DNA are due to its properties. The mitochondrial genome is well understood and described in many databases (GenBank, EST, GSS). It is also species-specific and is present in many copies in every organism cell. Moreover, mtDNA is resistant to unfavourable factors, such as high temperature or pressure. This last feature is essential because it allows the identification of DNA in preserved meat products, the production of which involves thermal processing. Properties of mtDNA translate into biological specificity and high sensitivity of pork identification methods, which is very important when investigating highly processed products such as sausages, canned pork, pork fat, or gelatine. However, to date, it is impossible to distinguish pork from venison based on mtDNA variability only.

The heat treatment of food causes fragmentation of the DNA. This may reduce the chance of extracting good quality DNA sufficient for further analysis. In the literature, we can find many reports on the influence of individual types of heat treatment on DNA quality. Traditional cooking, heating in a microwave [16], or processing pork for canned meal influence the amount of DNA obtained, and the isolate rate remains at a reasonable level. In contrast, DNA extracts from gelatine or meat meal had much worse properties. In the first case, the quantity and quality of DNA described by the absorbance ratio had the values of 400–600 ng/µL and 1.7–1.9, respectively. However, only 15–145 ng/µL were obtained for gelatine or meat meal, with a purity often below 1.6 or above 1.9. These are the difficulties any laboratory faces in food product species identification.

The obtained DNA can be analyzed by many methods. Most of them are based on the PCR reaction, which can be used in several variants such as classical PCR, real-time PCR, sequencing, DNA barcoding and NGS (next-generation sequencing). The advantages and limitations of these methods are presented in Table 1.