Application of Real-Time PCR for Quantifying Gastrointestinal Microbiota in Weaning Pigs Influenced by Dietary Feed Additive Supplementation

Abstract

To apply real-time polymerase chain reaction (PCR) to detect and quantify the gastrointestinal microbiota modulated by dietary feed additives was the objective of this study. Sixty-four three-crossbred weaning piglets at 24 days were used and randomly assigned to be fed one of the experimental diets supplemented with different feed additives (0.2% inulin, 0.1% fructooligosaccharide and sub-therapeutic antibiotics), and compared with the non-supplemented group. Comparison of microbiota enumeration by conventional culture and real-time PCR methods in response to feed additives was investigated. The microbiota enumeration by conventional culture and real-time PCR methods showed corresponding responses of selected bacterial counts (Lactobacilli, Bifidobacteria, Escherichia coli) in feces and digesta from caecum and colon to different feed additives. The inulin-treated group tended to have the highest Lactobacilli number, while the sub-therapeutic antibiotics-treated group had significantly lower number of Lactobacilli and Bifidobacteria in the fecal and digesta samples (p < 0.05) and tended to have a reduction in the population of Escherichia coli in the digesta. However, higher numbers were detected by real-time PCR due to the count of DNA copies from both non-viable and viable bacteria. The sensitivity of real-time PCR of this study could detect as low as 102 copies of specific bacteria 16S rRNA gene. Besides, the optimized real-time PCR had the advantage of being less time-consuming and less labor-intensive, and allowing samples to be stored until analysis compared to the culture method. Therefore, real-time PCR could be used as an effective method to detect microbiota changing in the GI tract. Moreover, this technique can be applied in the future for evaluation of feed quality, which affects microbiota balance.