Development And Application Of A Real Time PCR Approach For Quantification Of Rumen Microorganisms

A perfect quantitative method of rumen microorganisms was developed by real time PCR as the example of methanogens, protozoa and rumen bacteria in this paper, on the sequence of 16S /18S rDNA, ITS and the genes of own protein basis. And the detecting and quantitative reaction system of methanogens was established from the angles of species and genera, absolute and relative quantification, known and unknown copy numbers. Furthermore, the regression relation was developed on comparing the method of real time PCR with the method of most probable number. At the same time, the effect of plant oil on the population of methanogens, able-hydrogenated bacteria, ciliate protozoa and methane production was studied by real time PCR, and the approach of plant oil reducing methane production of rumen microorganisms was discussed.The method of DNA extraction by a bead-beating technique and phenol-CHCl₃ was used. The DNA extracted was longer than 20kb, OD_260nm/OD_280nm of DNA was 1.7-1.9, and the efficiency of the DNA extraction was 87.64%. we got the target 16S rDNA sequences of the PCR-amplification by primers of bacteria, archaea 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 protozoa into pieces and could meet needs for other subsequent studies.It was determined by the digestion of genomic DNA with six different restriction endonucleases: Accâ… , EcoRâ… , Pstâ… , Pvuâ…¡, Sacland Smaâ…  and southern hybridization that the copy number of M.formicicum'sl6S rDNA was two. A special probe was designed and it was approved that exclusive sequences was own by aligning in GeneBank. We optimized the reaction condition of M. formicicum. When the concentrations of its primers and probe were 0.4μmol/L, 0.2μmol/L respectively and the concentration of MgCl₂ and templates were 4mmol/L and 1260ng respectively, the real time PCR reaction system of M. formicicum was best. The lowest copy number of M. formicicum which could be detected was 30 in 25μL reaction system. The population of M. formicicum by real time PCR was lower than the population of M. formicicum by the method of most probable number, and the positive correlation between them was very high(r=0.957. P<0.01). This showed the quantitative method by real time PCR could reflect the variation of rumen microorganism's population truly.We successfully detected M. mazei by real time PCR with SYBR GreenI when the sequence of ITS was used as a target sequence, and the lowest copy number of M. formicicum which could be well and truly detected was 300 in 25μL reaction system. At the same time, we successfully developed the method of methanogens' relative quantification by real time PCR with SYBR GreenI when the gene of mcrA was used as a target sequence. Furthermore, it showed that the population of M. formicicum and methanogens from rumen fluid of steers was more than those from rumen fluid of cows by those detection methods, it maybe relative to the diets of steers in which the ratio between concentration and forage was high. We also successfully detected B.fibrivolen and R.ablus by real time PCR with SYBR...