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Contributos para a Implementação de ensaios moleculares específicos visando a garantia de autenticidade em rações (DNA de Ruminantes)
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A encefalopatia espongiforme bovina (EEB) é uma doença priónica neurológica, fatal e progressiva, que afeta o sistema nervoso central dos bovinos, provocando lesões degenerativas no cérebro. Foi identificada pela primeira vez no Reino Unido em 1986 e nos finais da década de 80 e início dos anos 90, surgiu em humanos sob a forma de uma nova variante da doença de Creutzfeldt-Jakob, associada ao consumo de produtos contaminados derivados de bovinos. A encefalopatia espongiforme bovina tem origem na alimentação animal, sendo propagada principalmente através da incorporação de farinhas de carne e ossos ou de proteínas animais transformadas contaminadas nas rações. Estas substâncias, frequentemente obtidas a partir da reciclagem de restos de animais mortos expostos ao agente infecioso — o prião — foram responsáveis pela disseminação da doença entre os bovinos. Com o objetivo de garantir a segurança alimentar e proteger a saúde pública, foram implementadas normas rigorosas, incluindo a remoção de matérias de risco específico e a proibição do uso de determinadas proteínas animais transformadas na alimentação de animais de criação — medida conhecida como Feed Ban. Paralelamente, foram estabelecidos métodos de controlo para assegurar o cumprimento dessas restrições. Com o avanço das tecnologias, foram desenvolvidos novos métodos analíticos mais sensíveis e específicos. Entre eles, destaca-se a reação em cadeia da polimerase em tempo real, que foi posteriormente reconhecido como método oficial para este tipo de controlo. Este trabalho teve como principal objetivo a implementação de ensaios moleculares para a deteção de ácido desoxirribonucleico (DNA) de ruminantes em rações destinadas à alimentação animal, recorrendo inicialmente ao método de reação em cadeia da polimerase em tempo real, seguido da sua otimização para a versão digital da técnica. Para tal, foram analisadas amostras de alimentos compostos para animais, preparadas com diferentes concentrações de DNA, simulando diversos níveis potenciais de contaminação. Paralelamente, foi realizada a análise por PCR digital, permitindo a comparação entre os dois métodos em termos de desempenho e limites de deteção.
Bovine spongiform encephalopathy (BSE) is a fatal and progressive neurological prion disease that affects the central nervous system of cattle, causing degenerative lesions in the brain. It was first identified in the United Kingdom in 1986 and in the late 80s and early 90s, it emerged in humans in the form of a new variant of Creutzfeldt-Jakob disease, associated with the consumption of contaminated products derived from cattle. Bovine spongiform encephalopathy originates in animal feed and is spread mainly through the incorporation of meat and bone meal or contaminated processed animal proteins into feed. These substances, often obtained from the recycling of dead animal remains exposed to the infectious agent — the prion — were responsible for the spread of the disease among cattle. In order to ensure food safety and protect public health, strict standards have been implemented, including the removal of specific risk materials and the prohibition of the use of certain processed animal proteins in the feed of farmed animals – a measure known as the Feed Ban. At the same time, control methods have been established to ensure compliance with these restrictions. With the advancement of technologies, new, more sensitive and specific analytical methods have been developed. Among them, the real-time polymerase chain reaction (PCR) stands out, which was later recognized as the official method for this type of control. The main objective of this work was the implementation of molecular assays for the detection of deoxyribonucleic acid (DNA) from ruminants in feed, initially using the real-time polymerase chain reaction method, followed by its optimization for the digital version of the technique. To this end, samples of compound feed were analyzed, prepared with different concentrations of DNA, simulating different potential levels of contamination. At the same time, digital PCR analysis was carried out, allowing the comparison between the two methods in terms of performance and detection limits.
Bovine spongiform encephalopathy (BSE) is a fatal and progressive neurological prion disease that affects the central nervous system of cattle, causing degenerative lesions in the brain. It was first identified in the United Kingdom in 1986 and in the late 80s and early 90s, it emerged in humans in the form of a new variant of Creutzfeldt-Jakob disease, associated with the consumption of contaminated products derived from cattle. Bovine spongiform encephalopathy originates in animal feed and is spread mainly through the incorporation of meat and bone meal or contaminated processed animal proteins into feed. These substances, often obtained from the recycling of dead animal remains exposed to the infectious agent — the prion — were responsible for the spread of the disease among cattle. In order to ensure food safety and protect public health, strict standards have been implemented, including the removal of specific risk materials and the prohibition of the use of certain processed animal proteins in the feed of farmed animals – a measure known as the Feed Ban. At the same time, control methods have been established to ensure compliance with these restrictions. With the advancement of technologies, new, more sensitive and specific analytical methods have been developed. Among them, the real-time polymerase chain reaction (PCR) stands out, which was later recognized as the official method for this type of control. The main objective of this work was the implementation of molecular assays for the detection of deoxyribonucleic acid (DNA) from ruminants in feed, initially using the real-time polymerase chain reaction method, followed by its optimization for the digital version of the technique. To this end, samples of compound feed were analyzed, prepared with different concentrations of DNA, simulating different potential levels of contamination. At the same time, digital PCR analysis was carried out, allowing the comparison between the two methods in terms of performance and detection limits.
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Ruminantes Encefalopatia espongiforme bovina Proteínas animais transformadas PCR em tempo real PCR digital Ruminants Bovine Spongiform Encephalopathy Processed Animal Proteins Real-Time PCR Digital PCR