利用实时定量PCR对瘤胃甲酸甲烷杆菌定量方法的建立与应用

 【目的】解决传统微生物评价方法存在的弊端，寻找快速、准确、灵敏的瘤胃微生物评价的新方法。【方法】利用实时定量PCR技术，以16S rDNA为靶序列，建立了瘤胃甲酸甲烷杆菌（Methanobacterium formicicum）的分子定量方法。【结果】采用本文所设计的引物、探针以及相应的PCR反应体系，检测极限可达到30拷贝（15个甲酸甲烷杆菌），与传统检测方法相比要更为迅速、灵敏。采用实时定量PCR技术所测得的细菌数量与采用传统的最大或然数记数法所测的细菌数量具有很高的相关性（r=0.957，P<0.01），表明采用实时定量PCR方法能够准确反映瘤胃微生物数量的变化。利用所建立的方法对利鲁杂交肉牛和荷斯坦奶牛瘤胃液中甲酸甲烷杆菌进行了定量检测。结果表明，利鲁杂交肉牛瘤胃液中的甲酸甲烷杆菌浓度是荷斯坦奶牛的5.6倍。【结论】利用实时定量PCR技术建立的瘤胃甲酸甲烷杆菌的分子定量方法能够准确反映瘤胃微生物数量的变化。

Development and Application of a Real-Time PCR Approach for Quantification of Methanobacterium formicium in Rumen

【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.