title: Quantification of total bacteria, enterobacteria and lactobacilli populations in pig digesta by real-time PCR.
abstract: Jejunum digesta samples were taken from weaning pigs in order to evaluate real-time PCR (qPCR) as a method for quantifying pig gut bacteria. Total bacteria, lactobacilli and enterobacteria were quantified by qPCR and the results were compared with those obtained with traditional methods: 4',6-diamidino-2-phenylindole (DAPI staining) for total bacteria, selective culture for lactobacilli and enterobacteria. Real-time PCR showed higher values in terms of 16S rRNA gene copies than DAPI counts or CFU. Despite the differences, the lactobacilli:enterobacteria ratio was similar between methods (2.5 +/- 0.58 for qPCR and 3.1 +/- 0.71 for selective culture, P = 0.39). Possible reasons for the higher PCR counts are discussed considering both an overestimation with PCR by quantification of dead bacteria or free DNA and also an underestimation with conventional methods. Inherent differences in the pre-treatment of the samples could partially explain the discrepancies observed. Regardless of the numerical differences between methods, values obtained by qPCR and traditional methods showed a significant correlation for lactobacilli and total bacteria. In the light of these results, real-time PCR seems a valid method to quantify microbial shifts in the gastrointestinal tract.
authors.affiliation: Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona 08193, Bellaterra, Barcelona, Spain.
authors.forename: Marisol
authors.initials: M
authors.lastname: Castillo
authors.forename: Susana M
authors.initials: SM
authors.lastname: Martín-Orúe
authors.forename: Edgar G
authors.initials: EG
authors.lastname: Manzanilla
authors.forename: Ignacio
authors.initials: I
authors.lastname: Badiola
authors.forename: Marga
authors.initials: M
authors.lastname: Martín
authors.forename: Josep
authors.initials: J
authors.lastname: Gasa
mesh_terms: D000818:Animals; D001419:Bacteria / Q000254:growth & development* / Q000302:isolation & purification; D015169:Colony Count, Microbial / Q000379:methods / Q000592:standards / Q000662:veterinary; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis; D004755:Enterobacteriaceae / Q000254:growth & development / Q000302:isolation & purification; D007211:Indoles / Q000378:metabolism; D007583:Jejunum / Q000382:microbiology*; D007778:Lactobacillus / Q000254:growth & development / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000592:standards / Q000662:veterinary*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013223:Statistics as Topic; D013552:Swine
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D007211:Indoles; D012336:RNA, Ribosomal, 16S; C007293:DAPI