Development of an assay to quantify rumen ciliate protozoal biomass in cows using real-time PCR

被引:325
作者
Sylvester, JT
Karnati, SKR
Yu, ZT
Morrison, M
Firkins, JL [1 ]
机构
[1] Ohio State Univ, Interdisciplinary Nutr Program, Columbus, OH 43210 USA
[2] Ohio State Univ, Dept Anim Sci, Columbus, OH 43210 USA
关键词
rumen protozoa; real-time PCR; 18S rDNA; cattle;
D O I
10.1093/jn/134.12.3378
中图分类号
R15 [营养卫生、食品卫生]; TS201 [基础科学];
学科分类号
100403 ;
摘要
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.
引用
收藏
页码:3378 / 3384
页数:7
相关论文
共 46 条
[1]  
[Anonymous], 1993, LAB MANUAL CLASSIFIC
[2]   Comparison of mechanistic rumen models on mathematical formulation of extramicrobial and microbial processes [J].
Bannink, A ;
DeVisser, H .
JOURNAL OF DAIRY SCIENCE, 1997, 80 (07) :1296-1314
[3]   Influence of the quantity of nonspecific DNA and repeated freezing and thawing of samples on the quantification of DNA by the Light Cycler® [J].
Bellete, B ;
Flori, P ;
Hafid, J ;
Raberin, H ;
Sung, RTM .
JOURNAL OF MICROBIOLOGICAL METHODS, 2003, 55 (01) :213-219
[4]   GenBank: update [J].
Benson, DA ;
Karsch-Mizrachi, I ;
Lipman, DJ ;
Ostell, J ;
Wheeler, DL .
NUCLEIC ACIDS RESEARCH, 2004, 32 :D23-D26
[5]   Quantitative determination of rumen ciliate protozoal biomass with real-time PCR [J].
Bergen, WG .
JOURNAL OF NUTRITION, 2004, 134 (12) :3223-3224
[6]   Regulatory sequences for the amplification and replication of the ribosomal DNA minichromosome in Tetrahymena thermophila [J].
Blomberg, P ;
Randolph, C ;
Yao, CH ;
Yao, MC .
MOLECULAR AND CELLULAR BIOLOGY, 1997, 17 (12) :7237-7247
[7]   MARKERS FOR QUANTIFYING MICROBIAL PROTEIN-SYNTHESIS IN THE RUMEN [J].
BRODERICK, GA ;
MERCHEN, NR .
JOURNAL OF DAIRY SCIENCE, 1992, 75 (09) :2618-2632
[8]  
CHARGAFF E, 1955, NUCL ACIDS CHEM BIOL, V1
[9]  
Coleman G. S., 1979, Tropical Animal Production, V4, P199