摘要
【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SYBRGreenⅠRT-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。
【Objective】The aim of this study was to investigate the possibility of Real-Time PCR method to detect the quantity changing of the cellulolytic bacteria under different diets in sheep rumen.【Method】 Three pairs of primers were designed according to the 16S rDNA sequences of Fibrobacter succinogenes,Ruminococcus flavefaciens,and Ruminococcus albus respectively in this experiment. A SYBR GreenⅠquantified system by the real-time PCR technique was developed. 【Result】Primers and reaction condition developed in this study showed a high specificity and sensitivity,and the detection-limit of this method was up to 50 copies. The dynamic patterns of cellulolytic bacteria were similar with the two methods of Real-Time PCR and the roll tube technique. The positive correlation between the two results was high （r^2=0.9591,P〈0.05） . The result indicated that the quantified system of real-time PCR could accurately detect the variation of the rumen cellulolytic bacteria,and the Real-Time PCR method was more effective than the traditional method in quantification. 【Conclusion】The quantitative method based on real-time PCR could accurately measure the variation of rumen cellulolytic bacteria in this experiment.
出处
《中国农业科学》
CAS
CSCD
北大核心
2008年第6期1795-1803,共9页
Scientia Agricultura Sinica
基金
国家自然科学基金项目(30460095)
关键词
瘤胃纤维降解菌
RT-PCR
定量
Rumen cellulolytic bacteria
Real-Time PCR
Quantification
