Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen

Abstract

Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using PCR primers to target total rumen fungi and the cellulolytic bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes, is described, including design and validation. The DNA and crude protein contents with respect to the fungal biomass of both polycentric and monocentric fungal isolates were investigated across the fungal growth stages to aid in standard curve generation. The primer sets used were found to be target specific with no detectable cross-reactivity. Subsequently, the real-time PCR assay was employed in a study to detect these populations within cattle rumen. The anaerobic fungal target was observed to increase 3.6-fold from 0 to 12 h after feeding. The results also indicated a 5.4-fold increase in F. succinogenes target between 0 and 12 h after feeding, whereas R. flavefaciens was observed to maintain more or less consistent levels. This is the first report of a real-time PCR assay to estimate the rumen anaerobic fungal population.

Effect of disodium fumarate on microbial abundance, ruminal fermentation and methane emission in goats under different forage : concentrate ratios

“…Primers and real-time quantitative PCR Total DNA was extracted from rumen contents by the beadbeating method as described by Denman and McSweeney (2006). The PCR primers for methanogens, fungi, total bacteria, Ruminococus flavefaciens, F. succinogenes and S. ruminantium are listed in Table 2 (Denman and McSweeney, 2006;Denman et al, 2007;Khafipour et al, 2009).…”

“…The PCR primers for methanogens, fungi, total bacteria, Ruminococus flavefaciens, F. succinogenes and S. ruminantium are listed in Table 2 (Denman and McSweeney, 2006;Denman et al, 2007;Khafipour et al, 2009). Species of Methanobrevibacter sp.…”

Rumen-like methanogens identified from the crop of the folivorous South American bird, the hoatzin (Opisthocomus hoazin)

“…The external standards used for the real-time PCR amplifications have been previously validated for bacteria (Denman and McSweeney, 2006), ciliate protozoa (Sylvester et al, 2004) and methanogenic archaea (Denman et al, 2007), and have been discussed elsewhere (Sundset et al, 2009). Real-time PCR amplifications were carried out with the Bio-Rad iCycler in a 25 ml volume containing the following reagents: 1.0 ml template DNA (10 ng), 400 nM (final concentration) of each primer, 12.5 ml iQ SYBR Green supermix (Bio-Rad) and 9.5 ml ddH 2 O. Real-time PCR amplification was initiated by a hot start at 95 1C for 15 min, followed by 40 cycles of 95 1C for 30 s, 60 1C for 30 s and 72 1C for 60 s. A final melting curve analysis was carried out by continuously monitoring fluorescence between 60 and 95 1C with 0.5 1C increments every 10 s. Three dilutions of DNA were amplified and the C t of the most efficient PCR was recorded.…”

Technical note: use of internal transcribed spacer for ruminal yeast identification in dairy cows

“…In addition, the composition and proportion of ruminal microorganisms are influenced by external factors such as diet, feeding frequency, age (Denman and McSweeney, 2006) and have an important function in feed digestion (Oliveira et al, 2014).…”