Development And Application Of A Real Time PCR Approach For Quantification Of Remen Cellulytic Bacteria Of Cattle

Account for the complex rumen bacteria, a completely set of method that extracting the total rumen bacteria was developed on the base of previous experience in this paper. Through comparison and optimization for these methods, freeze-thaw treatment and glass bead mill homogenization method was proved that The molecular size of the DNA of rumen microbes is about 20kb using these two modified methods. Meanwhile, extracted efficiency is stabilization. We got the target 16S rDNA sequences of the PCR-amplification by primers of bacteria, archaea and fungi. This show that the two methods of DNA extraction in this paper could meet needs for other subsequent studies. Using ultrasonic lyses treatment and liquid N2 mill treatment just could get low concentration of total rumen bacteria DNA, and these two method could meet primal studies.The five representative cellulytic bacteria were choose including Anaerovibrio lipolytica, Fibrobacter Succinogenes, Ruminococcus flavefaciens, Butyrivibrio fibrisolven, Ruminococcus albus. A relative quantitative method of real time PCR on the sequence of 16S rDNA was developed.And using this real time PCR studied the effect of quantities of cattle rumen bacteria through supplemented by soybean oil and linseed oil in the diet. The result was showed that soybean oil and linseed oil in the diet restrained the production of cellulytic bacteria. A. lipolyticao,F. Succinogene,R. flavefaciens,B. fibrisolven and R. Albus was reduced by 54%, 42%, 52%, 65% and 17% in LOC1, 81%,43%,96%,57% and 5% in LOC2. The effect of linseed oil in the diet on the quantities of microbe was obvious than soybean oil. This phenomenon probably was related to high unsaturation.