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Design and development of a Real Time PCR assay for the detection and quantification of Faecalibacterium Prausnitzii in the intestine of poultry (Master thesis)

Τζοβάρας-Μήτρογλου, Κωνσταντίνος-Ανδρέας/ Tzovaras-Mitroglou, Konstantinos-Andreas

Faecalibacterium prausnitzii is a non-pathogenic, gram-negative and strictly anaerobic bacterium of both the human and animal gut microbiome. It is a gut health biomarker, as it produces a significant amount of butyric acid in the gastrointestinal tract, contributing to the protection of the host against pathogenic microorganisms. The aim of this study was to design and develop a Real-time PCR assay for the molecular detection and quantification of F. prausnitzii levels. Initially, a pan-genome analysis was performed using Roary to obtain the conserved genes among F. prausnitzii strains available in the NCBI database. The genes were examined in terms of their homology with genes of other bacteria by aligning them in the nr database, using the NCBI BLAST tool. Unique genes were used to design specific primers using the PrimerBlast3 tool. Primer specificity was initially evaluated in-silico using the NCBI nr and RefSeq databases. Subsequently, the Real-time PCR assay was applied to detect F. prausnitzii in chicken intestinal samples. The results confirmed the specificity and efficacy of the novel method.