关键词: 实时定量PCR, 瘤胃, 甲酸甲烷杆菌, 定量

Abstract: 【Objective】To solve the short comings of the conventional quantitative method in determination of runmen microorganisms. 【Method】A quantitative method for determination of Methanobacterium formicicum in rumen was developed by real-time PCR using 16S rDNA in this experiment. 【Results】The detection limit of this mehod was 30 copies (15 M. formicicum cell ) in 25 l reaction system with the primers, probe and reaction condition developed in this study, which is faster and more sensitive than the traditional mode for quantification of microorganisms. The positive correlation between the population of M. formicicum by real-time PCR and the population of M. formicicum by traditional mode was very high (r=0.957, P<0.01), which showed that the quantitative method by real-time PCR could accurately reflect the variation of rumen microorganism's population. The numbers of M. formicicum in rumina of steers and Holstein cows, were determined respectively. The result showed that the concentration of M. formicicum in rumen fluid of steers was 5.6 times higher than that of Holstein cows.【Conclusion】The quantitative method for determination of M. formicicum in rumen by real- time PCR could accurately reflect the variation of rumen microorganism's population.

Key words: Real-time PCR, Rumen, Methanobacterium formicicum, Quantification