RESUMO
Salmonellosis is a foodborne disease caused by Salmonella spp. Although cell culture is the gold standard for its identification, validated molecular methods are becoming an alternative, because of their rapidity, selectivity, and specificity. A simplex and duplex droplet digital polymerase chain reaction (ddPCR)-based method for the identification and quantification of Salmonella using ttr, invA, hilA, spaQ, and siiA gene sequences was validated. The method has high specificity, working interval between 8 and 8,000 cp/µL in ddPCR reaction, a limit of detection of 0.5 copies/µL, and precision ranging between 5 and 10% measured as a repeatability standard deviation. The relative standard measurement uncertainty was between 2 and 12%. This tool will improve food safety in national consumption products and will increase the competitiveness in agricultural product trade.
RESUMO
The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/genética , Enzimas/genética , Biblioteca Gênica , Metagenômica/métodos , Solo/química , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Enzimas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Microbiologia do SoloRESUMO
El presente trabajo tuvo como objetivo la bioprospección de ADN metagenómico derivado de comunidades microbianas asociadas a un agroecosistema de importancia nacional. Este análisis permitió realizar la producción, expresión, purificación y caracterización de una enzima novedosa con actividad esterasa. Esta enzima, denominada LipM, había sido previamente identificada en clones metagenómicos derivados de suelos dedicados al cultivo de papa criolla (Solanum pureja), mediante secuencia de nueva generación y análisis bioinformáticos. La secuencia codificante de la enzima fue clonada en el vector pBADgiii y expresada en E. coli como sistema de expresión, lo que permitió optimizar el proceso de producción recombinante y su posterior purificación. Funcionalmente la enzima presentó una mayor afinidad por sustratos de p-nitrofenil con ácidos grasos de cadena corta (
The present work had as a main objective to bioprospect metagenomic DNA from microbial communities associated with an agro-ecosystem of national importance. This analysis allowed the production, expression, purification and characterization of a novel enzyme with esterase activity. This enzyme, named here as LipM, was previously identified in metagenomic clones derived from soils dedicated to creole potato (Solanum pureja) crops by means of next-generation sequencing and bioinformatics analyses. The coding sequence of the enzyme was cloned into pBADgiii vector and expressed in E. coli as an expression system, allowing to optimize its recombinant production process and its further purification. The enzyme functionally showed a greater affinity for p-nitrophenyl substrates with short-chain fatty acids (