Polymerase chain reaction detection of bacterial ribosomal genes from fresh and stored animal feeds

Abstract

Feed quality can be influenced by both the microbial population levels and types of organisms that become associated with the feed matrices during processing and storage. Evaluation of microbial quality of animal feeds is difficult, because feeds generally contain a diverse bacterial population that can fluctuate widely depending on a variety of factors. Microbial diversity may be investigated in animal feed using the polymerase chain reaction (PCR) and 16S rDNA primers. In this study a bacterial isolation step involving centrifugation was combined with several DNA extraction techniques, and PCR amplicons were visualised on electrophoresis gels. Seven different animal feeds and two commonly used feed ingredients, either fresh or stored for approximately 14 months at À20°C, were chosen (18 feed sources in total) to represent a variety of different matrices, concentrations of macronutrients such as protein and fat, and particle sizes. DNA extraction involving polyethylene glycol 8000 appeared to be the most reliable protocol for the extraction of community DNA for PCR analysis of feeds. The majority of the feeds (14 out of 18 animal feeds and feed ingredients) examined in this study yielded at least one PCR-positive replicate (1.

Application of microbial analyses to feeds and potential implications for poultry nutrition

“…However, limitations for applying microbiome analyses of feeds remain to be resolved. For example, biological compounds, such as proteins and lipids, that are present in feed matrices may not entirely be separated during the DNA extraction procedure and can affect the integrity of DNA or inhibit the following PCR process ( Maciorowski et al., 2001b , 2002 ; Piskata et al., 2017 , 2019 ). These inhibitors can restrict PCR efficiency by reducing or blocking DNA polymerase activity.…”

“…(2014) found 16S rRNA gene sequencing variation when comparing different DNA extraction kits. There is no particular DNA extraction kit available for animal feed due to its variability in composition ( Maciorowski et al., 2001b , 2002 , 2005 ). Therefore, an optimized DNA extraction technique associated with feed is necessary for comparable downstream sequencing analyses.…”