Development of a competitive polymerase chain reaction assay for the ruminal bacteriumButyrivibrio fibrisolvensOB156 and its use for tracking an OB156-derived recombinant

Abstract

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 1.7x10(2) and 5.6x10(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.