PCR monitoring of Lactobacillus and Bifidobacterium dynamics in fermentations by piglet intestinal microbiota.
A new group-specific primer (Lact71R), targeting the 16S-23S rDNA intergenic spacer region of Lactobacillus, was tested in its specificity to amplify rDNA of lactobacilli from piglet intestinal origin by polymerase chain reaction (PCR). Lact71R and Lab0677F, a Lactobacillus group-specific primer targeting the 16S rDNA, generated a common amplicon by PCR with DNA from Lactobacillus and Pediococcus reference strains, but not from Weissella strains. Sequence analysis of clones obtained by PCR amplification with Lact71R and Lab0677F and total DNA isolated from the ileal, caecal and colonic contents of one piglet resulted in Lactobacillus and Lactobacillus-like sequences mainly retrieved from intestinal environments. The primer pair was further validated in a culture independent PCR-analysis to monitor broad fluctuations of lactobacilli populations in fructo-oligosaccharides (FOS) fermentations by piglet intestinal microbiota. Bifidobacterium genus-specific primers were also used for PCR titre determination throughout FOS fermentations, in parallel with lactate and short chain fatty acids (SCFA) quantification. Increases between PCR titres were correlated with lactate detection in early stages of fermentation. Based on the obtained results, a simple monitoring PCR approach is proposed, foreseeing its application to the study of the dynamics of specific bacterial populations in complex environments.
Moura
Patrícia
P
Instituto Nacional de Engenharia, Tecnologia e Inovação, Departamento de Biotecnologia, Lisboa, Portugal.
Simões
Fernanda
F
Gírio
Francisco
F
Loureiro-Dias
Maria C
MC
Esteves
M Paula
MP
D000818:Animals; D001644:Bifidobacterium / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism; D002432:Cecum / Q000382:microbiology; D003106:Colon / Q000382:microbiology; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D005285:Fermentation; D007082:Ileum / Q000382:microbiology; D007422:Intestines / Q000382:microbiology*; D007778:Lactobacillus / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity; D034421:Sus scrofa
D002264:Carboxylic Acids; D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D021903:DNA, Ribosomal Spacer; D009844:Oligosaccharides