Development And Application Of A Fluorescence Quantification Real-Time PCR Approach For The Ruminal Cellulolytic Microbes

Establishment of Real-Time Fluorescence Quantitative PCR for sheep rumen microorganisms as the example of Fibrobacter succinogenes ,Rumincoccus albus ,Rumincoccus flavefaciens and rumen fungis in this paper, the results indicated:1. The method of DNA extraction by a bead-beating technique and was used. The DNA extracted was longer than 20kb, OD260/OD280 of DNA was above 1.7. we got the target 16s/18s rDNA sequences of the PCR-amplification by primers of eubacteria , archaebacteria and fungi. This showed that the method of DNA extraction in this paper could well break the cells of rumen microorganisms including bacteria, fungi and so on into pieces and could meet needs for other subsequent studies.2. The quantitaties of rumen cellulytic microbes were successfully detected by Fluorescence Quantification real-time PCR with SYBR GreenⅠwhen the sequence of 16s/18s rDNA were used as target sequence. The standerd curve for this method were successfully build,and lowest copy number was 101 per 20μL reaction system. The reaction condition of cellulolytic microbes were optimized and the detecting and quantitative reaction system of rumen cellulolytic microbes were established from the angles of species and genera, a rapid and exact absolute quantification method . Furthermore, the regression relation was developed on comparing the method of Fluorescence Quantification Real-Time PCR with the method of roll tube , and the relation ratio was 95.5%. That testified the Fluorescence Quantification Real-Time PCR was a more superiority and practicability method for quantification of rumen cellulolytic microbes.3. We used real-time PCR with SYBR GreenⅠmethod detected the cellulytic microbes in sheep rumen which under different forage to concentrate ratio. 9 sunite sheep, health, weithting about 50kg, were randomized allocated in three groups. The ration of concentrate to forage were 1:9, 3:7 and 5:5 for groupⅠ, groupⅡand groupⅢrespectively. The results obtained by Fluorescence Quantification real-time PCR method and traditional roll tube method showed the same changed trand. The result of traditional roll tube method showed signification differences between the quantity of cellulytic bacterias under different ration of concentrate to forage; but no signification differences about fungi.However the signification differences were showed in the results of Fluorescence Quantification real-time PCR method, Fibrobacter succinogenes, Ruminococcus flavefaciens and fungi except Ruminococcus albus under different ration of concentrate to forage. From the results of this paper, a conclusion was obtained: Fluorescence Quantification Real-Time PCR method has superiority on monitoring the changed of rumen microbes, like the dynamics changed in some time. Especially in monitoring the changed of spacified strains in amounts of samples.