期刊
JOURNAL OF NUTRITION
卷 134, 期 12, 页码 3378-3384出版社
OXFORD UNIV PRESS
DOI: 10.1093/jn/134.12.3378
关键词
rumen protozoa; real-time PCR; 18S rDNA; cattle
Currently used microbial markers cannot distinguish protozoal nitrogen (N) from bacterial N, thus limiting research on protozoal quantification in vivo by the lack of a repeatable, accurate marker for protozoal N. We report the development of a real-time PCR assay targeting the gene encoding 18S rDNA to quantify the amount of protozoal biomass in ruminal fluid and duodenal digesta. Protozoal cells were harvested from rumen fluid and concentrated for evaluation of recovery of rDNA in samples from the rumen and the duodenum. The DNA from concentrated cells was extracted with virtually 100% efficiency both before and after column purification. After serial spiking of protozoal cells into duodenal fluid over the entire range of quantification, the recovery was highly linear and constant at 81%. After serially spiking increasing quantities of protozoal rDNA into a constant volume of duodenal samples, nonlinear regression verified constant recovery of background rDNA in duodenal samples regardless of the ratio of target:nontarget rDNA. Recommendations for the procedure, including replication per sample, are described herein.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据