Use of quantitative real-time PCR to assess the in vitro survival of specific DNA gene sequences of rumen microbes under simulated abomasal conditions.
The use of specific DNA sequences (DS) as a microbial marker in post-rumen digesta requires their persistence and integrity throughout gastric digestion. The aim of this study was to evaluate in vitro the survival of microbial DS during gastric digestion and the factors involved. Gastric pH had a highly significant effect on the integrity of DS. pH 4.2 allows for a significant growth of microbes in the medium, but at pH 1.2, almost all of the DS were hydrolysed. In the presence of carboxymethylcellulose, the effect of pH was reduced, pepsin activity was inhibited and gene survival increased considerably. In the simulated abomasal conditions (pH = 2.3, 2 g/l of carboxymethylcellulose, and 40-min retention time), almost all of the bacterial genes and around 78% of the protozoa gene sequences retained their molecular integrity throughout gastric digestion, although factors such as acidity and viscera retention time might compromise the utilisation of DS as a microbial marker.
Belanche
A
A
Dep. Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Zaragoza, Spain.
Erroa
I R
IR
Balcells
J
J
Calleja
L
L
D000018:Abomasum; D000818:Animals; D001419:Bacteria / Q000235:genetics*; D001483:Base Sequence; D015415:Biomarkers; D004247:DNA / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012417:Rumen / Q000382:microbiology*
D015415:Biomarkers; D004247:DNA