Open AccessDevelopment of a competitive polymerase chain reaction assay for the ruminal bacterium Butyrivibrio fibrisolvens OB156 and its use for tracking an OB156‐derived recombinant
Author(s) -
Kobayashi Yasuo,
Forster Robert J.,
Teather Ronald M.
Publication year - 2000
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2000.tb09191.x
Subject(s) - recombinant dna , polymerase chain reaction , bacteria , microbiology and biotechnology , primer (cosmetics) , biology , dna , rumen , polymerase , biochemistry , chemistry , gene , genetics , organic chemistry , fermentation
A competitive polymerase chain reaction assay targeting the 16S rDNA was developed for quantitating the rumen bacterium Butyrivibrio fibrisolvens OB156. A competitor DNA, serving as an internal control in the competitive polymerase chain reaction reaction, was constructed by polymerase chain reaction using a looped oligo longer than the normal primer. Coamplification of the target DNA with known amounts of the competitor DNA allowed quantitation of the target DNA in both pure culture and mixed culture systems, where minimum quantifiable level of OB156 was 1.7×10 2 and 5.6×10 4 cells, respectively. When an erythromycin‐resistant recombinant derived from OB156 was inoculated into a rumen fluid culture, its numbers decreased with time. The rate of decrease measured by the competitive polymerase chain reaction assay was much slower than the rate determined by culture enumeration using erythromycin selection. The competitive polymerase chain reaction assay also showed 48 h persistence of the recombinant at 10 4 ml −1 even after disappearance of culturable recombinant, suggesting maintenance of the target DNA from uncultivable cells. In an in vivo tracking trial, the recombinant became undetectable within 72 h with either assay, indicating rapid hydrolysis and/or outflow of the cells from the rumen.