|dc.description.abstracteng||The control of meat and its products is essential for sales and consumer protection. Due to personal preferences, but also religious and health reasons, the correct labelling is crucial. For meat species inspection, samples have to be sent to a well-equipped laboratory, where a qualified technician extract the DNA from the meat and conduct real-time PCR. The extraction alone takes at least five hours. The real-time polymerase chain reaction (PCR) is used as the gold standard method for the detection of animal species in meat products. The mechanism of real-time PCR is based on three different temperature steps for DNA denaturation (95 °C), primer annealing (60 °C) and elongation (72 °C) of the DNA string. In addition, the use of a fluorescence-labelled probe enables real-time detection of positive signals. In real-time PCR, highly sophisticated, big and expensive devices are required. Furthermore, the run time is around 90 minutes. For the above mentioned reasons, the aim of this doctoral thesis was to determine a rapid detection method for the identification of animal species in meat and meat products in order to simplify on site screening during production or in the sales outlets to enable immediate execution.
An isothermal DNA amplification, recombinase polymerase amplification (RPA), was chosen as the detection method. The RPA amplifies its target gene at a constant temperature between 39 and 42 ° C using enzymes and recombinant protein. Similar to the real-time PCR, the successful amplification is visualized by a fluorescence-labelled probe in a maximum of 15 minutes.
For the development of the RPA assays for the identification of animal species in meat products, primers and probes were targeting the mitochondrial genes of pork, horse, chicken, turkey, cattle and sheep. The sensitivity of each assay was evaluated by performing eight independent runs using serial concentration of molecular DNA standard of each species (102 to 100 DNA molecules/reaction) and the datasets were subjected to probit-regression analysis. The selected primers were able to amplify their target species with a sensitivity between one and 30 DNA molecules/reaction in a maximum of 11 minutes. No cross-reactions were observed, in other words, each primer combination detects only its target animal species. For field validation of the developed assays, meat and salami mixed samples spiked with various concentration (10, 5, 1, 0.5 and 0.1%) of foreign meat were produced. Each RPA
assay was successfully able to detect meat concentration down to of 0.1% in tested samples. The 0.1 % is the lower recommended value by the German Food and Feed Code §64 (LFGB). Moreover, two different fluorescent dyes (FAM and ROX) were applied to detect meat contamination in duplex, whereby one sample can be tested for up to two species in one reaction. No loss of sensitivity in the duplex assays was noticed. This step was important to reduce the assay running costs while maintaining the same productivity.
Another important issue in meat industry is the freedom from food borne pathogens. Infectious agents can be ingested through the consumption of contaminated meat and lead to food poisoning in humans. In Africa, however, eating "bush meat" can lead to more contagious and deadly infection like monkey pox (MPXV). In order to diagnose such an infection as early as possible and to start the control measures, the use of a rapid test is beneficial. To make this possible, another RPA assay that detects the monkeypox tumor necrosis factor (TNF) binding protein gene was developed. Both monkey pox clades can be determined with a sensitivity of 16 DNA molecules/μl in 10 minutes. With the selected primer pairs, there is no cross-reaction with the closely related tested viruses or monkey genome. The clinical performance of the MPXV-RPA-assay was tested, revealing a specificity of 100% (50/50), while the sensitivity was 95% (43/45). This assay will pave the way for the identification of food borne infectious agents at low resource settings.
Upon presenting my data to the scientific community and end user in international meetings, many individuals have raised the importance of screening more than six animal species. Both RPA and real-time PCR is restricted to the number of the developed assays as well as the fluorescence channels in the detection devices. On other hand, next generation sequencing represents a method with no target limit. For the identification of an unknown adulteration animal source an Oxford nanopore sequencing protocol was tested. The method was combined with offline BLAST search to allow sequencing and data analysis in less than one hour. The developed procedure was successfully detected the contamination of the mock pork sample with 0.1% beef, sheep, goat, horse, donkey, chicken, turkey, duck and rabbit meat. The specificity of the technology was challenged with sequences of exotic animal species as dog, camel, lion, impala, bison and japanese quail. The nanopore sequencing
combined with the Offline BLAST search has proven adequate sensitivity and specificity for species identification and represent the future of molecular diagnostics.
In summary, in the PhD thesis, not only a rapid on-site detection system for the identification of six animal species in meat products based on the recombinase polymerase amplification were developed, but also a rapid sequencing protocol for the use of the Oxford Nanopore technologies. Both detection methods can be combined with an easy to perform DNA extraction method and all methods can be carried out in a mobile suitcase lab, whereby screening of meat and its products can be performed at point of need. The current work will pave the way for the implementation of such technologies for the benefit of the community and consumers.||de