Development of a competitive polymerase chain reaction assay for the ruminal bacterium Butyrivibrio fibrisolvens OB156 and its use for tracking an OB156-derived recombinant

A competitive polymerase chain reaction assay targeting the 16S rDNA was de veloped for quantitating the rumen bacterium Butyrivibrio fibrisolvens OB15 6. A competitor DNA, serving as an internal control in the competitive poly merase chain reaction reaction, was constructed by polymerase chain reactio n using a looped oligo longer than the normal primer. Coamplification of th e target DNA with known amounts of the competitor DNA allowed quantitation of the target DNA in both pure culture and mixed culture systems, where min imum quantifiable level of OB156 was 1.7 x 10(2) and 5.6 x 10(4) cells, res pectively. When an erythromycin-resistant recombinant derived from OB156 wa s inoculated into a rumen fluid culture, its numbers decreased with time. T he rate of decrease measured by the competitive polymerase chain reaction a ssay was much slower than the rate determined by culture enumeration using erythromycin selection. The competitive polymerase chain reaction assay als o showed 48 h persistence of the recombinant at 10(4) ml(-1) even after dis appearance 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 hydroly sis and/or outflow of the cells from the rumen. (C) 2000 Federation of Euro pean Microbiological Societies. Published by Elsevier Science B.V. All righ ts reserved.