Competitive PCR assay for the measurement of ruminal bacteria count and microbial attachment

Abstract

c-PCR technique was utilized in two studies to estimate rumen microbial population change when animals received different ratio of roughage and concentrate, and incubation medium pH was different in in vitro studies. In experiment 1, a competitive PCR technique was used to enumerate the Fibrobacter succinogenes and Streptococcus bovis in the rumen of Holstein steers. An internal control DNA was constructed for use in competitive PCRs and was shown to amplify under the same reaction conditions and with the same amplification efficiency as the target DNA. DNA from a known number of F. succinogenes and S. bovis cells was coamplified with the internal control to construct a standard curve. Rumen samples were collected from two Holstein steers fed two diets in rotation: high roughage and high concentrate. DNA extracted from these and spiked with internal control DNA was amplified with the F. succinogenes and S. bovis specific primer pair. The relative intensities of the PCR products were used to quantitate the numbers of F. succinogenes and S. bovis cell equivalents to those in the rumen samples. The numbers ranged from 3.41×107 ml-1 to 5.36×107 ml-1 and from 2.03×107 to 4.47×107 ml-1, respectively. There was a trend of increased F. succinogenes with higher roughage diet, and increased S. bovis with higher concentrate diet.