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Desenvolvimento e validação de um kit qPCR liofilizado para serotipagem de STEC para Aplicações de Segurança Alimentar
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| Documento em Acesso Embargado até dia 07-06-2028. Tente solicitar cópia ao autor carregando no ficheiro | 1.76 MB | Adobe PDF |
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Abstract(s)
O presente relatório de estágio descreve o decorrer do estágio integrado no 2º ano de mestrado em Bioquímica, na Universidade da Beira Interior. Este teve a duração de nove meses no departamento de Biologia Molecular da empresa ALS Life Sciences Portugal, S.A, sediada em Tondela. O principal objetivo do estágio consistiu no desenvolvimento de um método para a deteção dos genes de virulência (stx1, stx2, eae) e dos serogrupos e serótipos (O26, O45, O103, O104:H4, O111, O121, O145, O157:H7) de E. coli através da reação em cadeia da polimerase (PCR) em tempo real com base na ISO 13136 em processo de atualização, com o intuito de ser produzido um kit liofilizado. A PCR em tempo real foi escolhida como método de diagnóstico, por ser rápida e eficaz para impedir a disseminação das infeções provocadas por estirpes de E. coli, especificamente as Escherichia coli produtoras de toxinas Shiga (STEC). As STEC são bactérias Gram-negativas existentes predominantemente no sistema digestivo dos ruminantes e podem-se tornar bactérias patogénicas quando adquirem elementos de mobilidade como os bacteriófagos. As STEC podem ser transmitidas a humanos através do contacto direto com animais, ingestão de alimentos mal cozinhados ou não pasteurizados. A sua dispersão no trato gastrointestinal dos humanos provoca sintomas como vómitos, diarreia podendo agravar-se para o Síndrome hemolítico-urémico e/ou Colite hemorrágica. De modo a desenvolverem-se e implementarem-se quatro multiplex liofilizados (stx1+stx2+eae+IAC; O157+H7+O104+H4; O26+O45+O145; O103+O111+O121) para a deteção por PCR em tempo real, otimizaram-se as concentrações dos primers e sondas que constam na ISO 13136 em processo de atualização, verificaram-se as especificidades para microrganismos específicos, determinaram-se os limites de deteção das reações líquidas e liofilizadas com DNAs plasmídicos, as respetivas eficiências das reações e testaram-se os multiplex para a identificação dos microrganismos alvo em amostras reais e artificialmente contaminadas. Foi demonstrado que os primers e sondas usados para os diferentes multiplex eram específicos, os limites de deteção eram as 3 cópias dos DNAs plasmídicos por microlitro nas reações e as eficiências variaram entre 92% e 110%. Realça-se ainda que foi possível detetar os microrganismos em amostras reais e contaminadas artificialmente, demonstrando que o método desenvolvido pode avançar para os testes das reações de PCR multiplex em tempo real liofilizadas e posteriormente ser usado de forma comercial para a análise de diversos tipos de amostras.
This internship report describes the course of the internship that is integrated in the 2nd year of the Biochemistry Master's degree at the University of Beira Interior. The internship lasted nine months in the Molecular Biology department of the ALS Life Sciences Portugal, S.A, based in Tondela. The main objective was to develop a method to detect virulence genes (stx1, stx2, eae) and serogroups, serotypes (O26, O45, O103, O104:H4, O111, O121, O145, O157:H7) of E. coli through real-time polymerase chain reaction (PCR) based on ISO 13136 in update, with the intention of producing a lyophilized kit. Real-time PCR was chosen as the diagnostic method because it is fast and effective in preventing the spread of infections by E. coli strains, specifically Shiga toxin-producing Escherichia coli (STEC). STEC are Gram-negative bacteria predominantly found in the digestive system of ruminants and can become pathogenic bacteria when they acquire mobility elements such as bacteriophages. STEC can be transmitted to humans through direct contact with animals, ingestion of undercooked or unpasteurized food. Its dispersion in the human’s gastrointestinal tract causes symptoms such as vomiting and diarrhea, which can worsen into hemolytic-uremic syndrome and/or hemorrhagic colitis. To develop and implement four multiplexes (stx1+stx2+eae+IAC; O157+H7+O104+H4; O26+O45+O145; O103+O111+O121) for detection by real-time PCR, the concentrations of the primers and probes listed in the ISO 13136, which is undergoing updates, were optimized. Specificities for specific microorganisms were verified, detection limits for liquid and lyophilized reactions for plasmid DNAs were determined, the respective efficiencies of the reactions were measured, and multiplex tests for identifying target microorganisms in real and artificially contaminated samples were conducted. It was demonstrated that the primers and probes used for the different multiplexes were specific, the detection limits were 3 copies of plasmid DNAs per microliter in the reactions, and the efficiencies varied between 92% and 110%. It is also noteworthy that it was possible to detect microorganisms in both real and artificially contaminated samples, demonstrating that the developed method can advance to the testing of lyophilized multiplex real-time PCR reactions and subsequently be used commercially for the analysis of various types of samples.
This internship report describes the course of the internship that is integrated in the 2nd year of the Biochemistry Master's degree at the University of Beira Interior. The internship lasted nine months in the Molecular Biology department of the ALS Life Sciences Portugal, S.A, based in Tondela. The main objective was to develop a method to detect virulence genes (stx1, stx2, eae) and serogroups, serotypes (O26, O45, O103, O104:H4, O111, O121, O145, O157:H7) of E. coli through real-time polymerase chain reaction (PCR) based on ISO 13136 in update, with the intention of producing a lyophilized kit. Real-time PCR was chosen as the diagnostic method because it is fast and effective in preventing the spread of infections by E. coli strains, specifically Shiga toxin-producing Escherichia coli (STEC). STEC are Gram-negative bacteria predominantly found in the digestive system of ruminants and can become pathogenic bacteria when they acquire mobility elements such as bacteriophages. STEC can be transmitted to humans through direct contact with animals, ingestion of undercooked or unpasteurized food. Its dispersion in the human’s gastrointestinal tract causes symptoms such as vomiting and diarrhea, which can worsen into hemolytic-uremic syndrome and/or hemorrhagic colitis. To develop and implement four multiplexes (stx1+stx2+eae+IAC; O157+H7+O104+H4; O26+O45+O145; O103+O111+O121) for detection by real-time PCR, the concentrations of the primers and probes listed in the ISO 13136, which is undergoing updates, were optimized. Specificities for specific microorganisms were verified, detection limits for liquid and lyophilized reactions for plasmid DNAs were determined, the respective efficiencies of the reactions were measured, and multiplex tests for identifying target microorganisms in real and artificially contaminated samples were conducted. It was demonstrated that the primers and probes used for the different multiplexes were specific, the detection limits were 3 copies of plasmid DNAs per microliter in the reactions, and the efficiencies varied between 92% and 110%. It is also noteworthy that it was possible to detect microorganisms in both real and artificially contaminated samples, demonstrating that the developed method can advance to the testing of lyophilized multiplex real-time PCR reactions and subsequently be used commercially for the analysis of various types of samples.
Description
Keywords
Diagnóstico Genes de Virulência PCR em Tempo-real Escherichia coli produtoras de toxinas Shiga - STEC Serogrupos Diagnosis Virulence Genes Real-Time PCR Serogroups