Detecção de Salmonella por amplificação isotérmica mediada por loop (LAMP) e diferenciação de sorovares de interesse por qPCR em carcaças de frangos
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Data
2023-01-20
Autores
Silva, Evelyn Cristine da [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Salmonella está presente na cadeia produtiva avícola e é um grande desafio no que se refere à segurança dos alimentos e sanidade animal. Técnicas de diagnóstico molecular vêm sendo amplamente utilizadas por proporcionarem resultados rápidos e seguros no diagnóstico deste patógeno. Assim, o objetivo do estudo foi avaliar a utilização da LAMP em comparação à qPCR para o gene invA para detecção de Salmonella associada à qPCR multiplex para identificação e diferenciação simultânea de S. Enteritidis, S. Typhimurium, S. Pullorum e S. Gallinarum. Para a metodologia do estudo a técnica de LAMP foi aplicada utilizando dois kits comercias (LAMP, WarmStart® Colorimetric LAMP 2X Master Mix e WarmStart® LAMP 2X Master Mix), as três técnicas foram avaliadas quanto a especificidade para 21 sorovares diferentes de Salmonella e para 37 cepas dos sorovares de interesse. Foi preconizado a determinação do limiar de detecção das técnicas e as eficiências das reações de qPCR e qPCR multiplex. Para validação as técnicas foram aplicadas em 33 amostras de carcaças de frangos e comparadas aos resultados de microbiologia convencional. Como resultados a LAMP foi específica na detecção dos diferentes sorovares de Salmonella obtendo os mesmos resultados em ambos os kits, mas apresentou baixos limiares de detecção variando entre 101 a 104 UFC/reação. Em comparação, a qPCR para o gene invA obteve melhores limiares de detecção (100 a 102 UFC/reação), atingindo igual especificidade e melhor repetibilidade nos ensaios. A multiplexação da qPCR na identificação dos diferentes sorovares também apresentou boa especificidade, com o limiar de detecção entre 101 a 102 UFC/reação. Os resultados obtidos nas análises em carcaça de frango sugerem uma correspondência entre os resultados obtidos nos métodos moleculares e na microbiologia convencional e levantam uma discussão importante ao tema envolvido. O estudo estabeleceu ensaios padronizados no diagnóstico de Salmonella e os resultados são precursores para aplicações futuras das técnicas em análises de rotina laboratorial.
Salmonella is present in the poultry production chain and is a major challenge in terms of food safety and animal health. Molecular diagnostic techniques have been widely used for providing fast and safe results in the diagnosis of this pathogen. Thus, the aim of the study was to evaluate the use of LAMP compared to qPCR for the invA gene for detection of Salmonella associated with multiplex qPCR for simultaneous identification and differentiation of S. Enteritidis, S. Typhimurium, S. Pullorum and S. Gallinarum. For the methodology of the study, the LAMP technique was applied using two commercial kits (LAMP, WarmStart® Colorimetric LAMP 2X Master Mix and WarmStart® LAMP 2X Master Mix), the three techniques were evaluated for specificity for 21 different serovars of Salmonella and for 37 strains of the serovars of interest. The determination limit of detection of the techniques and the efficiency of the qPCR and multiplex qPCR reactions was advocated. For validation the techniques were applied to 33 samples of chicken carcasses and compared to the results of conventional microbiology. As a result, LAMP was specific in the detection of different Salmonella serovars, obtaining the same results in both kits, but presented low limit of detection ranging from 101 to 104 CFU/reaction. In comparison, qPCR obtained lower limit of limit of detection (100 to 102 CFU/reaction), reaching equal specificity and better repeatability in the assays. The qPCR multiplexing to identify the different serovars also showed good specificity, with the detection threshold between entre 101 to 102 CFU/reaction. The results obtained in the analyzes on chicken carcasses suggest a correspondence between the results obtained in molecular methods and in conventional microbiology and raise an important discussion on the subject involved. The study established standardized tests in the diagnosis of Salmonella and the results are precursors for future applications of the techniques in routine laboratory analysis.
Salmonella is present in the poultry production chain and is a major challenge in terms of food safety and animal health. Molecular diagnostic techniques have been widely used for providing fast and safe results in the diagnosis of this pathogen. Thus, the aim of the study was to evaluate the use of LAMP compared to qPCR for the invA gene for detection of Salmonella associated with multiplex qPCR for simultaneous identification and differentiation of S. Enteritidis, S. Typhimurium, S. Pullorum and S. Gallinarum. For the methodology of the study, the LAMP technique was applied using two commercial kits (LAMP, WarmStart® Colorimetric LAMP 2X Master Mix and WarmStart® LAMP 2X Master Mix), the three techniques were evaluated for specificity for 21 different serovars of Salmonella and for 37 strains of the serovars of interest. The determination limit of detection of the techniques and the efficiency of the qPCR and multiplex qPCR reactions was advocated. For validation the techniques were applied to 33 samples of chicken carcasses and compared to the results of conventional microbiology. As a result, LAMP was specific in the detection of different Salmonella serovars, obtaining the same results in both kits, but presented low limit of detection ranging from 101 to 104 CFU/reaction. In comparison, qPCR obtained lower limit of limit of detection (100 to 102 CFU/reaction), reaching equal specificity and better repeatability in the assays. The qPCR multiplexing to identify the different serovars also showed good specificity, with the detection threshold between entre 101 to 102 CFU/reaction. The results obtained in the analyzes on chicken carcasses suggest a correspondence between the results obtained in molecular methods and in conventional microbiology and raise an important discussion on the subject involved. The study established standardized tests in the diagnosis of Salmonella and the results are precursors for future applications of the techniques in routine laboratory analysis.
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Palavras-chave
Salmonella, qPCR, LAMP, Avicultura, Diagnóstico molecular, Reação em cadeia de polimerase, Frango de corte, Carcaça