Sampling methods for rumen microbial counts by Real-Time PCR techniques

摘要

Abstract Fresh rumen samples were withdrawn from 4 cannulated buffalo females fed a fibrous diets in order to quantify bacteria concentration in the rumen by Real-Time PCR techniques. To obtain DNA of a good quality from whole rumen fluid, eight (M1-M8) different pre-filtration methods (cheese cloths, glass-fibre and nylon filter) in combination with various centrifugation speeds (1000, 5000 and 14,000 rpm) were tested. Genomic DNA extraction was performed either on fresh or frozen samples (-20°C). The quantitative bacteria analysis was realized according to Real-Time PCR procedure for Butyrivibrio fibrisolvens reported in literature. M5 resulted the best sampling procedure allowing to obtain a suitable genomic DNA. No differences were revealed between fresh and frozen samples.