Publicação
Development of rapid methods for the detection of pathogenic microorganisms, based on NAM-FISH technology
| Resumo: | The access of pathogenic microorganisms to the human body can compromise the health of the individual, causing several clinical manifestations. Helicobacter pylori and Campylobacter are two important gastrointestinal pathogens. H. pylori infection is one of the most common human infections, whose treatment includes the administration of antibiotics, namely fluoroquinolones (FQ). However, there has been an increasing resistance of H. pylori to FQ, which can lead to treatment failures, making it important not only to detect the bacterium but also to define its resistance profile. Campylobacter is currently considered the leading cause of bacterial foodborne illnesses, usually associated with the consumption of raw meat. The detection of pathogenic microorganisms can be achieved by conventional culture techniques or by molecular methods, namely immunological tests or nucleic acid detection. Biomode is an innovative company that develops and commercializes rapid diagnostic methods based on Nucleic Acid Mimic (NAM) – fluorescence in situ hybridization (FISH) technology, which enables the rapid detection of microorganisms, through the hybridization of complementary fluorescent probes with specific sequences present in the target microorganism. In this context, the present work focused on two applications of NAM-FISH. In the clinical area, the main objective was the development of a method for the detection of H. pylori and its resistance to FQ. For this purpose, Peptide Nucleic Acid (PNA) and Locked Nucleic Acid (LNA)/2'OMe probes were designed for the detection of mutations that cause resistance. In order to cover the most prevalent mutations, as well as the wildtype phenotype, 5 LNA/2'OMe probes were selected. In the area of food safety, the objective was the optimization of a PNA-FISH method for Campylobacter detection in food samples. A preliminary testing of the inclusivity/exclusivity of the Campylobacter probe resulted in the detection of two non-target microorganisms, H. cinaedi and H. pamatensis. To optimize the procedure, samples of raw broiler meat inoculated artificially with C. jejuni were used. Prior to PNA -FISH, a new step was introduced in which the enriched samples are subjected to a centrifugation (10 000 g), followed by resuspension in 0.1% Triton X-100, in order to reduce the strong autofluorescence shown in samples without any treatment. The possibility of a two-step sample enrichment was also tested, however, this approach did not show advantages compared to the one-step procedure. Additionally, a robustness test, required by the AOAC International to obtain product certification, was performed, which showed that the variation of the parameters of the time and temperature of hybridization influence the performance of the method, showing that the PNA-FISH conditions must be strictly controlled. The results obtained in this study showed that the PNA-FISH method is suitable for the rapid detection of Campylobacter in food samples. With this work, it is concluded that although the two NAM-FISH based methods are a promising alternative for the detection of H. pylori and Campylobacter, they both require optimization. |
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| Autores principais: | Sousa, Ana Catarina da Silva e |
| Assunto: | NAM-FISH Helicobacter pylori Antimicrobial resistance Foodborne ilnesses Campylobacter spp Enrichment Autofluorescence AOAC international |
| Ano: | 2018 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Aveiro |
| Idioma: | inglês |
| Origem: | RIA - Repositório Institucional da Universidade de Aveiro |
| Resumo: | The access of pathogenic microorganisms to the human body can compromise the health of the individual, causing several clinical manifestations. Helicobacter pylori and Campylobacter are two important gastrointestinal pathogens. H. pylori infection is one of the most common human infections, whose treatment includes the administration of antibiotics, namely fluoroquinolones (FQ). However, there has been an increasing resistance of H. pylori to FQ, which can lead to treatment failures, making it important not only to detect the bacterium but also to define its resistance profile. Campylobacter is currently considered the leading cause of bacterial foodborne illnesses, usually associated with the consumption of raw meat. The detection of pathogenic microorganisms can be achieved by conventional culture techniques or by molecular methods, namely immunological tests or nucleic acid detection. Biomode is an innovative company that develops and commercializes rapid diagnostic methods based on Nucleic Acid Mimic (NAM) – fluorescence in situ hybridization (FISH) technology, which enables the rapid detection of microorganisms, through the hybridization of complementary fluorescent probes with specific sequences present in the target microorganism. In this context, the present work focused on two applications of NAM-FISH. In the clinical area, the main objective was the development of a method for the detection of H. pylori and its resistance to FQ. For this purpose, Peptide Nucleic Acid (PNA) and Locked Nucleic Acid (LNA)/2'OMe probes were designed for the detection of mutations that cause resistance. In order to cover the most prevalent mutations, as well as the wildtype phenotype, 5 LNA/2'OMe probes were selected. In the area of food safety, the objective was the optimization of a PNA-FISH method for Campylobacter detection in food samples. A preliminary testing of the inclusivity/exclusivity of the Campylobacter probe resulted in the detection of two non-target microorganisms, H. cinaedi and H. pamatensis. To optimize the procedure, samples of raw broiler meat inoculated artificially with C. jejuni were used. Prior to PNA -FISH, a new step was introduced in which the enriched samples are subjected to a centrifugation (10 000 g), followed by resuspension in 0.1% Triton X-100, in order to reduce the strong autofluorescence shown in samples without any treatment. The possibility of a two-step sample enrichment was also tested, however, this approach did not show advantages compared to the one-step procedure. Additionally, a robustness test, required by the AOAC International to obtain product certification, was performed, which showed that the variation of the parameters of the time and temperature of hybridization influence the performance of the method, showing that the PNA-FISH conditions must be strictly controlled. The results obtained in this study showed that the PNA-FISH method is suitable for the rapid detection of Campylobacter in food samples. With this work, it is concluded that although the two NAM-FISH based methods are a promising alternative for the detection of H. pylori and Campylobacter, they both require optimization. |
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