定量检测瘤胃纤维降解细菌的RT-PCR法

 【目的】建立一种适用于瘤胃主要纤维降解细菌的RT-PCR定量检测方法，通过培养技术监测瘤胃纤维降解细菌在不同环境下的数量变化，为深入研究反刍动物瘤胃发酵机理以及对纤维素资源的有效利用提供技术支持。【方法】分别以Fibrobacter succinogenes、Ruminococcus flavefaciens与Ruminococcus albus 的16SrDNA为靶序列设计相应的引物，建立SYBR GreenⅠRT-PCR定量体系。【结果】本研究所采用的引物及相应的PCR反应体系具有较高的特异性和灵敏度，检测极限可达50拷贝。采用RT-PCR方法检测瘤胃纤维降解细菌的定量结果与传统计数结果具有一致的变化趋势，相关性较高（r2=0.9591，P＜0.05），表明RT-PCR定量检测方法可以较为准确、迅速的检测绵羊瘤胃纤维降解菌群的数量随环境变化的情况。【结论】本研究所建立的RT-PCR检测方法能够较准确反映瘤胃纤维降解细菌数量的变化。

Real-Time PCR Approach for Cellulolytic Bacteria Quantification in the Rumen

Objective] The aim of this study was to investigate the possibility of Real-Time PCR technique for study the mechanism of rumen fermentation. In addition, in order to monitor the change of rumen microbes under different diet, the Real-Time PCR method was applied to quantify cellulolytic bacteria in the rumen of sheep. Method] SYBR GreenⅠreal-time PCR technique was used in this experiment. According to the 16S rDNA sequence of three predominant cellulolytic bacteria, three pairs of primers were designed. Results] Primers and reaction condition applied in this study showed a high specificity and sensitivity. The same change tendency was displayed in the population of cellulolytic bacteria between the real-time PCR technique and the traditional Hungate roll tube technique. Moreover, quantification of rumen cellulolytic bacteria by real-time PCR was faster and more sensitive, as compared with traditional method. This indicated that the method of real-time PCR could accurately reflect the variation of rumen microflora , especially in monitoring the change of specified strains in amounts of samples. Conclusion]The quantitative method for cellulolytic bacteria in the rumen of sheep by real-time PCR could accurately reflect the variation of rumen cellulolytic bacteria.